A nonradioactive high-performance liquid chromatographic microassay for uridine 5'-monophosphate synthase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase.

PubMed

Krungkrai, J; Wutipraditkul, N; Prapunwattana, P; Krungkrai, S R; Rochanakij, S

2001-12-15

A novel nonradioactive, microassay method has been developed to determine simultaneously the two enzymatic activities of orotate phosphoribosyltransferase (OPRTase) and orotidine 5'-monophosphate decarboxylase (ODCase), either as a bifunctional protein (uridine 5'-monophosphate synthase, UMPS) or as separate enzymes. Substrates (orotate for OPRTase or orotidine 5'-monophosphate for ODCase) and a product (UMP) of the enzymatic assay were separated by high-performance liquid chromatography (HPLC) using a reversed-phase column and an ion-pairing system; the amount of UMP was quantified by dual-wavelength uv detection at 260 and 278 nm. This HPLC assay can easily detect picomole levels of UMP in enzymatic reactions using low specific activity UMPS of mammalian cell extracts, which is difficult to do with the other nonradioactive assays that have been described. The HPLC assay is suitable for use in protein purification and for kinetic study of these enzymes. (c)2001 Elsevier Science.

Real-time ESI-MS of enzymatic conversion: impact of organic solvents and multiplexing.

PubMed

Scheerle, Romy K; Grassmann, Johanna; Letzel, Thomas

2012-01-01

Different enzymatic assays were characterized systematically by real-time electrospray ionization mass spectrometry (ESI-MS) in the presence of organic solvents as well as in multiplex approaches and in a combination of both. Typically, biological enzymatic reactions are studied in aqueous solutions, since most enzymes show their full activity solely in aqueous solutions. However, in recent years, the use of organic solvents in combination with enzymatic reactions has gained increasing interest due to biotechnological advantages in chemical synthesis, development of online coupled setups screening for enzyme regulatory compounds, advantages regarding mass spectrometric detection and others. In the current study, the influence of several common organic solvents (methanol, ethanol, isopropanol, acetone, acetonitrile) on enzymatic activity (hen egg white lysozyme, chitinase, α-chymotrypsin, elastase from human neutrophils and porcine pancreas, acetylcholinesterase) was tested. Moreover, multiplexing is a promising approach enabling fast and cost-efficient screening methods, e.g. for determination of inhibitors in complex mixtures or in the field of biomedical research. Although in multiplexed setups the enzymatic activity may be affected by the presence of other substrates and/or enzymes, the expected advantages possibly will predominate. To investigate those effects, we measured multiple enzymatic assays simultaneously. For all conducted measurements, the conversion rate of the substrate(s) was calculated, which reflects the enzymatic activity. The results provide an overview about the susceptibility of the selected enzymes towards diverse factors and a reference point for many applications in analytical chemistry and biotechnology.

Mass spectrometric real-time monitoring of an enzymatic phosphorylation assay using internal standards and data-handling freeware.

PubMed

Krappmann, Michael; de Boer, Arjen R; Kool, Daniël R W; Irth, Hubertus; Letzel, Thomas

2016-04-30

Continuous-flow reaction detection systems (monitoring enzymatic reactions with mass spectrometry (MS)) lack quantitative values so far. Therefore, two independent internal standards (IS) are implemented in a way that the online system stability can be observed, quantitative conversion values for substrate and product can be obtained and they can be used as mass calibration standards for high MS accuracy. An application previously developed for the MS detection of peptide phosphorylation by cAMP-dependent protein kinase A (PKA) (De Boer et al., Anal. Bioanal. Chem. 2005, 381, 647-655) was transferred to a continuous-flow reaction detection system. This enzymatic reaction, involving enzyme activation as well as the transfer of a phosphate group from ATP to a peptide substrate, was used to prove the compatibility of a quantitative enzymatic assay in a continuous-flow real-time system (connected to MS). Moreover (using internal standards), the critical parameter reaction temperature (including solution density variations depending on temperature) was studied in the continuous-flow mixing system. Furthermore, two substrates (malantide and kemptide), two enzyme types (catalytic subunit of PKA and complete PKA) and one inhibitor were tested to determine system robustness and long-term availability. Even spraying solutions that contained significant amount of MS contaminants (e.g. the polluted catalytic subunit) resulted in quantifiable MS signal intensities. Subsequent recalculations using the internal standards led to results representing the power of this application. The presented methodology and the data evaluation with available Achroma freeware enable the direct coupling of biochemical assays with quantitative MS detection. Monitoring changes such as temperature, reaction time, inhibition, or compound concentrations can be observed quantitatively and thus enzymatic activity can be calculated. Copyright © 2016 John Wiley & Sons, Ltd.

Guaiacol Peroxidase Zymography for the Undergraduate Laboratory

ERIC Educational Resources Information Center

Wilkesman, Jeff; Castro, Diana; Contreras, Lellys M.; Kurz, Liliana

2014-01-01

This laboratory exercise presents a novel way to introduce undergraduate students to the specific detection of enzymatic activity by electrophoresis. First, students prepare a crude peroxidase extract and then analyze the homogenate via electrophoresis. Zymography, that is, a SDS-PAGE method to detect enzyme activity, is used to specifically…

Electrodeposition of flower-like platinum on electrophoretically grown nitrogen-doped graphene as a highly sensitive electrochemical non-enzymatic biosensor for hydrogen peroxide detection

NASA Astrophysics Data System (ADS)

Tajabadi, M. T.; Sookhakian, M.; Zalnezhad, E.; Yoon, G. H.; Hamouda, A. M. S.; Azarang, Majid; Basirun, W. J.; Alias, Y.

2016-11-01

An efficient non-enzymatic biosensor electrode consisting of nitrogen-doped graphene (N-graphene) and platinum nanoflower (Pt NF) with different N-graphene loadings were fabricated on indium tin oxide (ITO) glass using a simple layer-by-layer electrophoretic and electrochemical sequential deposition approach. N-graphene was synthesized by annealing graphene oxide with urea at 900 °C. The structure and morphology of the as-fabricated non-enzymatic biosensor electrodes were determined using X-ray diffraction, field emission electron microscopy, transmission electron microscopy, Raman and X-ray photoelectron spectra. The as-fabricated Pt NF-N-graphene-modified ITO electrodes with different N-graphene loadings were utilized as a non-enzymatic biosensor electrode for the detection of hydrogen peroxide (H2O2). The behaviors of the hybrid electrodes towards H2O2 reduction were assessed using chronoamperometry, cyclic voltammetry and electrochemical impedance spectroscopy analysis. The Pt NF-N-graphene-modified ITO electrode with a 0.05 mg ml-1 N-graphene loading exhibited the lowest detection limit, fastest amperometric sensing, a wide linear response range, excellent stability and reproducibility for the non-enzymatic H2O2 detection, due to the synergistic effect between the electrocatalytic activity of the Pt NF and the high conductivity and large surface area of N-graphene.

Human Prostate Cancer Infiltrating Lymphocytes: Raft Microdomains, Signaling and Activation in Organ Cultures

DTIC Science & Technology

2006-06-01

and cell growth and differentiation (14). Increased ARG activity has long been detected in pa- tients with colon, breast , lung, and prostate cancer ...activity has long been detected in patients with colon, breast , lung and prostate cancer [23] and it was advanced that this enzymatic activity...AD_________________ Award Number: DAMD 17-03-01-0032 TITLE: Human Prostate Cancer Infiltrating

Highly Sensitive Detection of Caspase-3/7 Activity in Living Mice Using Enzyme-Responsive 19F MRI Nanoprobes.

PubMed

Akazawa, Kazuki; Sugihara, Fuminori; Nakamura, Tatsuya; Mizukami, Shin; Kikuchi, Kazuya

2018-05-16

Highly sensitive imaging of enzymatic activities in the deep tissues of living mammals provides useful information about their biological functions and for developing new drugs; however, such imaging is challenging. 19 F magnetic resonance imaging (MRI) is suitable for noninvasive visualization of enzymatic activities without endogenous background signals. Although various enzyme-responsive 19 F MRI probes have been developed, most cannot be used for in vivo imaging because of their low sensitivity. Recently, we developed unique nanoparticles, called FLAMEs, that are composed of a liquid perfluorocarbon core and a robust silica shell, and demonstrated their outstanding sensitivity in vivo. Here, we report a highly functionalized nanoprobe, FLAME-DEVD 2, with an OFF/ON 19 F MRI switch for detecting caspase-3/7 activity based on the paramagnetic relaxation enhancement effect. To improve the cleavage efficiency of peptides by caspase-3, we designed a novel Gd 3+ complex-conjugated peptide, DEVD X ( X = 1, 2), which is a substrate peptide sequence tandemly repeated X times, and demonstrated that DEVD 2 showed faster cleavage kinetics than DEVD 1. By incorporating this novel concept into a signal activation strategy, FLAME-DEVD 2 showed a high 19 F MRI signal enhancement rate in response to caspase-3 activity. After intravenous injection of FLAME-DEVD 2 and an apoptosis-inducing reagent, caspase-3/7 activity in the spleen of a living mouse was successfully imaged by 19 F MRI. This imaging platform shows great potential for highly sensitive detection of enzymatic activities in vivo.

Test/QA Plan for Verification of Coliform Detection Technologies for Drinking Water

EPA Science Inventory

The coliform detection technologies to be tested use chromatogenic and fluorogenic growth media to detect coliforms and E. coli based on the enzymatic activity of these organisms. The systems consist of single-use sample containers that contain pre-measured reagents and can be u...

Construction of graphene oxide magnetic nanocomposites-based on-chip enzymatic microreactor for ultrasensitive pesticide detection.

PubMed

Liang, Ru-Ping; Wang, Xiao-Ni; Liu, Chun-Ming; Meng, Xiang-Ying; Qiu, Jian-Ding

2013-11-08

A new strategy for facile construction of graphene oxide magnetic nanocomposites (GO/Fe3O4 MNCs)-based on-chip enzymatic microreactor and ultrasensitive pesticide detection has been proposed. GO/Fe3O4 MNCs were first prepared through an in situ chemical deposition strategy. Then, acetylcholinesterase (AChE) was adsorbed onto the GO/Fe3O4 surface to form GO/Fe3O4/AChE MNCs which was locally packed into PDMS microchannel simply with the help of external magnetic field to form an on-chip enzymatic microreactor. The constructed GO/Fe3O4/AChE MNCs-based enzymatic microreactor not only have the magnetism of Fe3O4 NPs that make them conveniently manipulated by an external magnetic field, but also have the larger surface and excellent biocompatibility of graphene which can incorporate much more AChE molecules and well maintain their biological activity. On the basis of the AChE inhibition principle, a novel on-chip enzymatic microreactor was constructed for analyzing dimethoate which is usually used as a model of organophosphorus pesticides. Under optimal conditions, a linear relationship between the inhibition rates of AChE and the concentration of dimethoate from 1 to 20 μgL(-1) with a detection limit of 0.18 μgL(-1) (S/N=3) was obtained. The developed electrophoretic and magnetic-based chip exhibited excellent reproducibility and stability with no decrease in the activity of enzyme for more than 20 repeated measurements over one week period, which provided a new and promising tool for the analysis of enzyme inhibitors with low cost and excellent performance. Copyright © 2013 Elsevier B.V. All rights reserved.

Cultured astrocytes do not release adenosine during hypoxic conditions

PubMed Central

Fujita, Takumi; Williams, Erika K; Jensen, Tina K; Smith, Nathan A; Takano, Takahiro; Tieu, Kim; Nedergaard, Maiken

2012-01-01

Recent reports based on a chemiluminescent enzymatic assay for detection of adenosine conclude that cultured astrocytes release adenosine during mildly hypoxic conditions. If so, astrocytes may suppress neural activity in early stages of hypoxia. The aim of this study was to reevaluate the observation using high-performance liquid chromatography (HPLC). The HPLC analysis showed that exposure to 20 or 120 minutes of mild hypoxia failed to increase release of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine from cultured astrocytes. Similar results were obtained using a chemiluminescent enzymatic assay. Moreover, since the chemiluminescent enzymatic assay relies on hydrogen peroxide generation, release of free-radical scavengers from hypoxic cells can interfere with the assay. Accordingly, adenosine added to samples collected from hypoxic cultures could not be detected using the chemiluminescent enzymatic assay. Furthermore, addition of free-radical scavengers sharply reduced the sensitivity of adenosine detection. Conversely, use of a single-step assay inflated measured values due to the inability of the assay to distinguish adenosine and its metabolite inosine. These results show that cultured astrocytes do not release adenosine during mild hypoxia, an observation consistent with their high resistance to hypoxia. PMID:21989480

Multi-wavelength dye concentration determination for enzymatic assays: evaluation of chromogenic para-nitrophenol over a wide pH range.

PubMed

Max, Jean-Joseph; Meddeb-Mouelhi, Fatma; Beauregard, Marc; Chapados, Camille

2012-12-01

Enzymatic assays need robust, rapid colorimetric methods that can follow ongoing reactions. For this, we developed a highly accurate, multi-wavelength detection method that could be used for several systems. Here, it was applied to the detection of para-nitrophenol (pNP) in basic and acidic solutions. First, we confirmed by factor analysis that pNP has two forms, with unique spectral characteristics in the 240 to 600 nm range: Phenol in acidic conditions absorbs in the lower range, whereas phenolate in basic conditions absorbs in the higher range. Thereafter, the method was used for the determination of species concentration. For this, the intensity measurements were made at only two wavelengths with a microtiter plate reader. This yielded total dye concentration, species relative abundance, and solution pH value. The method was applied to an enzymatic assay. For this, a chromogenic substrate that generates pNP after hydrolysis catalyzed by a lipase from the fungus Yarrowia lipolytica was used. Over the pH range of 3-11, accurate amounts of acidic and basic pNP were determined at 340 and 405 nm, respectively. This method surpasses the commonly used single-wavelength assay at 405 nm, which does not detect pNP acidic species, leading to activity underestimations. Moreover, alleviation of this pH-related problem by neutralization is not necessary. On the whole, the method developed is readily applicable to rapid high-throughput of enzymatic activity measurements over a wide pH range.

Applicability of rapid and on-site measured enzyme activity for surface water quality monitoring in an agricultural catchment

NASA Astrophysics Data System (ADS)

Stadler, Philipp; Farnleitner, Andreas H.; Sommer, Regina; Kumpan, Monika; Zessner, Matthias

2014-05-01

For the near real time and on-site detection of microbiological fecal pollution of water, the measurement of beta-D- Glucuronidase (GLUC) enzymatic activity has been suggested as a surrogate parameter and has been already successfully operated for water quality monitoring of ground water resources (Ryzinska-Paier et al. 2014). Due to possible short measure intervals of three hours, this method has high potential as a water quality monitoring tool. While cultivation based standard determination takes more than one working day (Cabral 2010) the potential advantage of detecting the GLUC activity is the high temporal measuring resolution. Yet, there is still a big gap of knowledge on the fecal indication capacity of GLUC (specificity, sensitivity, persistence, etc.) in relation to potential pollution sources and catchment conditions (Cabral 2010, Ryzinska-Paier et al. 2014). Furthermore surface waters are a big challenge for automated detection devices in a technical point of view due to the high sediment load during event conditions. This presentation shows results gained form two years of monitoring in an experimental catchment (HOAL) dominated by agricultural land use. Two enzymatic measurement devices are operated parallel at the catchment outlet to test the reproducibility and precision of the method. Data from continuous GLUC monitoring under both base flow and event conditions is compared with reference samples analyzed by standardized laboratory methods for fecal pollution detection (e.g. ISO 16649-1, Colilert18). It is shown that rapid enzymatic on-site GLUC determination can successfully be operated from a technical point of view for surface water quality monitoring under the observed catchment conditions. The comparison of enzyme activity with microbiological standard analytics reveals distinct differences in the dynamic of the signals during event conditions. Cabral J. P. S. (2010) "Water Microbiology. Bacterial Pathogens and Water" International Journal of Environmental Research and Public Health 7 (10): 3657-3703. Ryzinska-Paier, G., T. Lendenfeld, K. Correa, P. Stadler, A.P. Blaschke, R. L. Mach, H. Stadler, AKT Kirschner und A.H. Farnleitner (2014) A sensitive and robust method for automated on-line monitoring of enzymatic activities in water and water resources. Water Sci. Technol. in press

Gold nanoparticles/4-aminothiophenol interfaces for direct electron transfer of horseradish peroxidase: Enzymatic orientation and modulation of sensitivity towards hydrogen peroxide detection.

PubMed

Huerta-Miranda, G A; Arrocha-Arcos, A A; Miranda-Hernández, M

2018-08-01

Hydrogen peroxide electrochemical detection by horseradish peroxidase has been widely studied. The use of gold nanoparticles to prepare electrode/enzyme bioconjugates has attracted attention due to their catalytic properties. In this work, it is reported the use of gold nanoparticles and 4-aminothiophenol as a scaffold to obtain a suitable matrix for enzyme bioconjugation with horseradish peroxidase. A critical factor in biosensors design and development is the enzymatic electrochemical activity understanding. Comparison of voltammetric studies of the heme prosthetic group showed a reversible electrochemical behavior when the enzymes were immobilized in a well-dispersed gold deposit; on the other hand, a discrete redox response was observed on a randomly deposited gold electrode. These results show that the distance between enzymes is essential. Hydrogen peroxide catalysis and the enzymatic behavior were analyzed considering two types of nanoparticles dispositions. The catalytic behavior observed in the well-dispersed nanoparticles configuration suggests a preserved enzyme folding, a decrease of steric impediments, and appears to be a better immobilization strategy. In contrast, the randomly electrodeposited gold electrode decreased the enzyme orientation and the electrochemical activity. The advantages of this methodology are the electrode fabrication affordable cost and the enzymatic direct electron transfer response improvement. Copyright © 2018 Elsevier B.V. All rights reserved.

Digital Assays Part II: Digital Protein and Cell Assays.

PubMed

Basu, Amar S

2017-08-01

A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays. Digital enzyme assays measure the kinetics of single proteins with enzymatic activity. Digital enzyme-linked immunoassays (ELISAs) quantify antigenic proteins with 2 to 3 log lower detection limit than conventional ELISA, making them well suited for low-abundance biomarkers. Digital cell assays probe single-cell genotype and phenotype, including gene expression, intracellular and surface proteins, metabolic activity, cytotoxicity, and transcriptomes (scRNA-seq). These methods exploit partitioning to 1) isolate single cells or proteins, 2) detect their activity via enzymatic amplification, and 3) tag them individually by coencapsulating them with molecular barcodes. When scaled, digital assays reveal stochastic differences between proteins or cells within a population, a key to understanding biological heterogeneity. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows.

Yeasts from sub-Antarctic region: biodiversity, enzymatic activities and their potential as oleaginous microorganisms.

PubMed

Martinez, A; Cavello, I; Garmendia, G; Rufo, C; Cavalitto, S; Vero, S

2016-09-01

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. Sixty-one yeasts strains were isolated from King George Island, Antarctica which were characterized physiologically and identified at the molecular level using the D1/D2 region of rDNA. Fifty-eight yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Rhodotorula, Guehomyces, Candida, Metschnikowia and Debaryomyces) were screened for extracellular amylolytic, proteolytic, esterasic, pectinolytic, inulolytic xylanolytic and cellulolytic activities at low and moderate temperatures. Esterase activity was the most common enzymatic activity expressed by the yeast isolates regardless the assay temperature and inulinase was the second most common enzymatic activity. No cellulolytic activity was detected. One yeast identified as Guehomyces pullulans (8E) showed significant activity across six of seven enzymes types tested. Twenty-eight yeast isolates were classified as oleaginous, being the isolate 8E the strain that accumulated the highest levels of saponifiable lipids (42 %).

Quenching of graphene quantum dots fluorescence by alkaline phosphatase activity in the presence of hydroquinone diphosphate.

PubMed

Pereira da Silva Neves, Marta Maria; González-García, María Begoña; Pérez-Junquera, Alejandro; Hernández-Santos, David; Fanjul-Bolado, Pablo

2018-05-01

In this work, a turn-off photoluminescent sensing proof-of-concept based on blue luminescent graphene quantum dots (GQDs) as the fluorescent probe was developed. For that purpose, GQDs optical response was related with the catalytic enzymatic activity of alkaline phosphatase (ALP), in the presence of hydroquinone diphosphate (HQDP). The hydrolysis of HQDP by ALP generated hydroquinone (HQ). The oxidation of HQ, enzymatically produced, to p-benzoquinone (BQ) resulted in the quenching of GQDs fluorescence (FL). Therefore, the developed luminescent sensing mechanism allowed the FL quenching with ALP activity to be related and thus quantified the concentration of ALP down to 0.5 nM of enzyme. This innovative design principle appears as a promising tool for the development of enzymatic sensors based on ALP labeling with fluorescent detection or even for direct ALP luminescent quantification in an easy, fast and sensitive manner. Copyright © 2018 John Wiley & Sons, Ltd.

Enzyme-free detection and quantification of double-stranded nucleic acids.

PubMed

Feuillie, Cécile; Merheb, Maxime Mohamad; Gillet, Benjamin; Montagnac, Gilles; Hänni, Catherine; Daniel, Isabelle

2012-08-01

We have developed a fully enzyme-free SERRS hybridization assay for specific detection of double-stranded DNA sequences. Although all DNA detection methods ranging from PCR to high-throughput sequencing rely on enzymes, this method is unique for being totally non-enzymatic. The efficiency of enzymatic processes is affected by alterations, modifications, and/or quality of DNA. For instance, a limitation of most DNA polymerases is their inability to process DNA damaged by blocking lesions. As a result, enzymatic amplification and sequencing of degraded DNA often fail. In this study we succeeded in detecting and quantifying, within a mixture, relative amounts of closely related double-stranded DNA sequences from Rupicapra rupicapra (chamois) and Capra hircus (goat). The non-enzymatic SERRS assay presented here is the corner stone of a promising approach to overcome the failure of DNA polymerase when DNA is too degraded or when the concentration of polymerase inhibitors is too high. It is the first time double-stranded DNA has been detected with a truly non-enzymatic SERRS-based method. This non-enzymatic, inexpensive, rapid assay is therefore a breakthrough in nucleic acid detection.

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography.

PubMed

Nagatsu, T; Oka, K; Kato, T

1979-07-21

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. D-Tyrosine was used for the control. alpha-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.

A CuNi/C Nanosheet Array Based on a Metal-Organic Framework Derivate as a Supersensitive Non-Enzymatic Glucose Sensor

NASA Astrophysics Data System (ADS)

Zhang, Li; Ye, Chen; Li, Xu; Ding, Yaru; Liang, Hongbo; Zhao, Guangyu; Wang, Yan

2018-06-01

Bimetal catalysts are good alternatives for non-enzymatic glucose sensors owing to their low cost, high activity, good conductivity, and ease of fabrication. In the present study, a self-supported CuNi/C electrode prepared by electrodepositing Cu nanoparticles on a Ni-based metal-organic framework (MOF) derivate was used as a non-enzymatic glucose sensor. The porous construction and carbon scaffold inherited from the Ni-MOF guarantee good kinetics of the electrode process in electrochemical glucose detection. Furthermore, Cu nanoparticles disturb the array structure of MOF derived films and evidently enhance their electrochemical performances in glucose detection. Electrochemical measurements indicate that the CuNi/C electrode possesses a high sensitivity of 17.12 mA mM-1 cm-2, a low detection limit of 66.67 nM, and a wider linearity range from 0.20 to 2.72 mM. Additionally, the electrode exhibits good reusability, reproducibility, and stability, thereby catering to the practical use of glucose sensors. Similar values of glucose concentrations in human blood serum samples are detected with our electrode and with the method involving glucose-6-phosphate dehydrogenase; the results further demonstrate the practical feasibility of our electrode.

A sensitive electrochemical biosensor for detection of protein kinase A activity and inhibitors based on Phos-tag and enzymatic signal amplification.

PubMed

Yin, Huanshun; Wang, Mo; Li, Bingchen; Yang, Zhiqing; Zhou, Yunlei; Ai, Shiyun

2015-01-15

A simple, highly sensitive and selective electrochemical assay is developed for the detection of protein kinase A (PKA) activity based on the specific recognition utility of Phos-tag for kinase-induced phosphopeptides and enzymatic signal amplification. When the substrate peptide was phosphorylated by PKA reaction, they could specifically bind with Phos-tag-biotin in the presence of Zn(2+) through the formation of a specific noncovalent complex with the phosphomonoester dianion in phosphorylated peptides. Through the further specific interaction between biotin and avidin, avidin functionalized horseradish peroxidase (HRP) can be captured on the electrode surface. Under the catalytic effect of HRP, a sensitive electrochemical signal for benzoquinone was obtained, which was related to PKA activity. Under the optimal experiment conditions, the proposed electrochemical method presented dynamic range from 0.5 to 25 unit/mL with low detection limit of 0.15 unit/mL. This new detection strategy was also successfully applied to analyze the inhibition effect of inhibitors (ellagic acid and H-89) on PKA activity and monitored the PKA activity in cell lysates. Therefore, this Phos-tag-based electrochemical assay offers an alternative platform for PKA activity assay and inhibitor screening, and thus it might be a valuable tool for development of targeted therapy and clinical diagnosis. Copyright © 2014 Elsevier B.V. All rights reserved.

One-step zymogram method for the simultaneous detection of cellulase/xylanase activity and molecular weight estimation of the enzyme.

PubMed

Cano-Ramírez, Claudia; Santiago-Hernández, Alejandro; Rivera-Orduña, Flor Nohemí; Pineda-Mendoza, Rosa María; Zúñiga, Gerardo; Hidalgo-Lara, María Eugenia

2017-02-01

Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X-100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio-active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cu0.89Zn0.11O, A New Peroxidase-Mimicking Nanozyme with High Sensitivity for Glucose and Antioxidant Detection.

PubMed

Nagvenkar, Anjani P; Gedanken, Aharon

2016-08-31

Nanomaterial-based enzyme mimetics (nanozymes) is an emerging field of research that promises to produce alternatives to natural enzymes for a variety of applications. The search for the most cost-effective and efficient inorganic nanomaterials, such as metal oxides, cannot be won by pristine CuO. However, unlike CuO, the Zn-doped CuO (Zn-CuO) nanoparticles reported in this paper reveal superior peroxidase-like enzyme activity. This places Zn-CuO in a good position to participate in a range of activities aimed at developing diverse enzyme applications. The peroxidase-like activity was tested and confirmed against various chromogenic substrates in the presence of H2O2 and obeyed the Michaelis-Menten enzymatic pathway. The mechanism of enhanced enzymatic activity was proved by employing terephthalic acid as a fluorescence probe and by electron spin resonance. The nanozyme, when tested for the detection of glucose, showed a substantial enhancement in the detection selectivity. The limit of detection (LOD) was also decreased reaching a limit as low as 0.27 ppm. Such a low LOD has not been reported so far for the metal oxides without any surface modifications. Moreover, the nanozyme (Zn-CuO) was utilized to detect the three antioxidants tannic acid, tartaric acid, and ascorbic acid and the relative strength of their antioxidant capacity was compared.

Induction of hepatic antioxidants in freshwater catfish (Channa punctatus Bloch) is a biomarker of paper mill effluent exposure.

PubMed

Ahmad, I; Hamid, T; Fatima, M; Chand, H S; Jain, S K; Athar, M; Raisuddin, S

2000-09-01

Enzymatic and non-enzymatic antioxidants serve as an important biological defense against environmental oxidative stress. Information on antioxidant defense in fish is meager despite that fish are constantly exposed to a myriad of environmental stress including the oxidants. This study, therefore, assesses the activities of antioxidant enzymes viz., glutathione peroxidase, catalase and glutathione S-transferase and the non-enzymatic antioxidants viz., glutathione and metallothionein in various tissues of freshwater fish Channa punctatus (Bloch), in response to short-term and long-term exposures to paper mill effluent. The fish were exposed to the effluent at a concentration of 1.0% (v/v) for 15, 30, 60 and 90 days. The exposure caused a time-dependent increase in glutathione level (P < 0.001), activities of glutathione peroxidase (P < 0.05 to P < 0.001), glutathione S-transferase (P < 0.001) and a marginal initial decrease in catalase activity in the liver (P < 0.01 to P < 0.001). Metallothionein was induced in liver after 60 days of exposure. Two isoforms of metallothionein were detected. Catalase activity also increased 60 days afterwards. Antioxidant pattern was different in gill and kidney showing that liver was more resistant to oxidative damage as compared to gills and kidney. Our results demonstrate a pollutant-induced adaptive response in fish. In addition, levels of enzymatic and non-enzymatic tissue antioxidants may serve as surrogate markers of exposure to oxidant pollutants in fish.

Novel enzymatic method for assaying Lp-PLA2 in serum.

PubMed

Yamaura, Saki; Sakasegawa, Shin-Ichi; Koguma, Emisa; Ueda, Shigeru; Kayamori, Yuzo; Sugimori, Daisuke; Karasawa, Ken

2018-06-01

Measurement of lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) can be used as an adjunct to traditional cardiovascular risk factors for identifying individuals at higher risk of cardiovascular events. This can be performed by quantification of the protein concentration using an ELISA platform or by measuring Lp-PLA 2 activity using platelet-activating factor (PAF) analog as substrate. Here, an enzymatic Lp-PLA 2 activity assay method using 1-O-Hexadecyl-2-acetyl-rac-glycero-3-phosphocholine (rac C 16 PAF) was developed. The newly revealed substrate specificity of lysoplasmalogen-specific phospholipase D (lysophospholipase D (LysoPLD)) was exploited. Lp-PLA 2 hydrolyzes 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C 16 PAF) to 1-O-Hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPAF). LysoPLD acted on LysoPAF, and the hydrolytically released choline was detected by choline oxidase. Regression analysis of Lp-PLA 2 activity measured by the enzymatic Lp-PLA 2 activity assay vs. two chemical Lp-PLA 2 activity assays, i.e. LpPLA 2 FS and PLAC® test, and ELISA, gave the following correlation coefficients: 0.990, 0.893 and 0.785, respectively (n = 30). Advantages of this enzymatic Lp-PLA 2 activity assay compared with chemical Lp-PLA 2 methods include the following; (i) only requires two reagents enabling a simple two-point linear calibration method with one calibrator (ii) no need for inhibitors of esterase-like activity in serum. Copyright © 2018 Elsevier B.V. All rights reserved.

Ship-borne measurements of microbial enzymatic activity: A rapid biochemical indicator for microbial water quality monitoring

NASA Astrophysics Data System (ADS)

Stadler, Philipp; Loken, Luke; Crawford, John; Schramm, Paul; Sorsa, Kirsti; Kuhn, Catherine; Savio, Domenico; Striegl, Rob; Butman, David; Stanley, Emily; Farnleitner, Andreas H.; Zessner, Matthias

2017-04-01

Contamination of aquatic ecosystems by human and animal wastes is a global concern for water quality. Disclosing fate and transport processes of fecal indicator organism (FIO) in large water bodies is a big challenge due to material intensive and time consuming methods used in microbiological water quality monitoring. In respect of utilization of large surface water resources there is a dearth of rapid microbiological methods that allow a near-real time health related water quality monitoring to be implemented into early warning systems. The detection of enzymatic activities has been proposed as a rapid surrogate for microbiological pollution monitoring of water and water resources (Cabral, 2010; Farnleitner et al., 2001, 2002). Methods such as the beta-D-Glucuronidase assay (GLUC), targeting FIO such as E. coli, were established. New automated enzymatic assays have been implemented during the last years into on-site monitoring stations, ranging from ground- to surface waters (Ryzinska-Paier et al., 2014; Stadler et al., 2017, 2016). While these automated enzymatic methods cannot completely replace assays for culture-based FIO enumeration, they yielded significant information on pollution events and temporal dynamics on a catchment specific basis, but were restricted to stationary measurements. For the first time we conducted ship-borne and automated measurements of enzymatic GLUC activity on large fresh water bodies, including the Columbia River, the Mississippi River and Lake Mendota. Not only are automated enzymatic assays technically feasible from a mobile vessel, but also can be used to localize point sources of potential microbial fecal contamination, such as tributaries or storm drainages. Spatial and temporal patterns of enzymatic activity were disclosed and the habitat specific correlation with microbiological standard assays for FIO determined due to reference samples. The integration of rapid and automated enzymatic assays into well-established systems for ship-borne measurements of physico-chemical parameters, such as the FLAMe (Crawford et al., 2015), paves new ground for data interpretation and process understanding. Cabral, J.P.S., 2010. Water Microbiology. Bacterial Pathogens and Water. Int. J. Environ. Res. Public. Health 7, 3657-3703. doi:10.3390/ijerph7103657 Crawford, J.T., Loken, L.C., Casson, N.J., Smith, C., Stone, A.G., Winslow, L.A., 2015. High-speed limnology: using advanced sensors to investigate spatial variability in biogeochemistry and hydrology. Environ. Sci. Technol. 49, 442-450. doi:10.1021/es504773x Farnleitner, A. h., Hocke, L., Beiwl, C., Kavka, G. c., Zechmeister, T., Kirschner, A. k. t., Mach, R. l., 2001. Rapid enzymatic detection of Escherichia coli contamination in polluted river water. Lett. Appl. Microbiol. 33, 246-250. doi:10.1046/j.1472-765x.2001.00990.x Farnleitner, A.H., Hocke, L., Beiwl, C., Kavka, G.G., Mach, R.L., 2002. Hydrolysis of 4-methylumbelliferyl-β-d-glucuronide in differing sample fractions of river waters and its implication for the detection of fecal pollution. Water Res. 36, 975-981. doi:10.1016/S0043-1354(01)00288-3 Ryzinska-Paier, G., Lendenfeld, T., Correa, K., Stadler, P., Blaschke, A.P., Mach, R.L., Stadler, H., Kirschner, A.K.T., Farnleitner, A.H., 2014. A sensitive and robust method for automated on-line monitoring of enzymatic activities in water and water resources. Water Sci. Technol. J. Int. Assoc. Water Pollut. Res. 69, 1349-1358. doi:10.2166/wst.2014.032 Stadler, P., Blöschl, G., Vogl, W., Koschelnik, J., Epp, M., Lackner, M., Oismüller, M., Kumpan, M., Nemeth, L., Strauss, P., Sommer, R., Ryzinska-Paier, G., Farnleitner, A.H., Zessner, M., 2016. Real-time monitoring of beta-d-glucuronidase activity in sediment laden streams: A comparison of prototypes. Water Res. 101, 252-261. doi:10.1016/j.watres.2016.05.072 Stadler, P., Farnleitner, A.H., Zessner, M., 2017. Development and evaluation of a self-cleaning custom-built auto sampler controlled by a low-cost RaspberryPi microcomputer for online enzymatic activity measurements. Talanta 162, 390-397. doi:10.1016/j.talanta.2016.10.031

Ultrasonic accelerates asparagine-glucose non-enzymatic browning reaction without acrylamide formation.

PubMed

Gao, Zhiqiang; Zheng, Junfeng; Chen, Lian

2017-01-01

Ultrasonic accelerated the asparagine-glucose non-enzymatic browning reaction with significant decrease of glucose and asparagine concentrations, and marked increase of intermediate products (UV-absorbance value at 294nm, Abs 294 ), melanoidins (UV-absorbance value at 420nm, Abs 420 ) and in vitro antioxidant activity (DPPH free radical scavenging activity). As the ultrasonic intensity was 17.83W/cm 2 , the asparagine-glucose solution's Abs 294 , Abs 420 and antioxidant activity increased from 0 to 1.26, 0.88 and 21.56%, respectively, and the glucose and asparagine concentrations of the asparagine-glucose solution reduced 58.97 and 12.57%, respectively. The high performance liquid chromatography (HPLC)-Diode Array Detector (DAD) analyses showed that no acrylamide was detected after 50-min ultrasonic reaction. This study suggested that ultrasonic at higher intensity was a potential method to accelerate the non-enzymatic browning reaction in the asparagine-glucose solution without acrylamide production. Copyright © 2016 Elsevier B.V. All rights reserved.

Novel application of digital microfluidics for the detection of biotinidase deficiency in newborns.

PubMed

Graham, Carrie; Sista, Ramakrishna S; Kleinert, Jairus; Wu, Ning; Eckhardt, Allen; Bali, Deeksha; Millington, David S; Pamula, Vamsee K

2013-12-01

Newborn screening for biotinidase deficiency can be performed using a fluorometric enzyme assay on dried blood spot specimens. As a pre-requisite to the consolidation of different enzymatic assays onto a single platform, we describe here a novel analytical method for detecting biotinidase deficiency using the same digital microfluidic cartridge that has already been demonstrated to screen for five lysosomal storage diseases (Pompe, Fabry, Gaucher, Hurler and Hunter) in a multiplex format. A novel assay to quantify biotinidase concentration in dried blood spots (DBS) was developed and optimized on the digital microfluidic platform using proficiency testing samples from the Centers for Disease Control and Prevention. The enzymatic assay uses 4-methylumbelliferyl biotin as the fluorogenic substrate. Biotinidase deficiency assays were performed on normal (n=200) and deficient (n=7) newborn DBS specimens. Enzymatic activity analysis of biotinidase deficiency revealed distinct separation between normal and affected DBS specimens using digital microfluidics and these results matched the expected activity. This study has demonstrated performance of biotinidase deficiency assays by measurement of 4-methylumbelliferyl product on a digital microfluidic platform. Due to the inherent ease in multiplexing on such a platform, consolidation of other fluorometric assays onto a single cartridge may be realized. © 2013.

A biosensor platform for rapid detection of E. coli in drinking water.

PubMed

Hesari, Nikou; Alum, Absar; Elzein, Mohamad; Abbaszadegan, Morteza

2016-02-01

There remains a need for rapid, specific and sensitive assays for the detection of bacterial indicators for water quality monitoring. In this study, a strategy for rapid detection of Escherichia coli in drinking water has been developed. This strategy is based on the use of the substrate 4-methylumbelliferyl-β-d-glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-d-glucuronidase (GUD) enzyme to yield a fluorogenic 4-methylumbelliferone (4-MU) product that can be quantified and related to the number of E. coli cells present in water samples. In this study, the detection time required for the biosensor response ranged between 20 and 120 min, depending on the number of bacteria in the sample. This approach does not need extensive sample processing with a rapid detection capability. The specificity of the MUG substrate was examined in both, pure cultures of non-target bacterial genera such as Klebsiella, Salmonella, Enterobacter and Bacillus. Non-target substrates that included 4-methylumbelliferyl-β-d-galactopyranoside (MUGal) and l-leucine β-naphthylamide aminopeptidase (LLβ-N) were also investigated to identify nonspecific patterns of enzymatic activities in E. coli. GUD activity was found to be specific for E. coli and no further enzymatic activity was detected by other species. In addition, fluorescence assays were performed for the detection of E. coli to generate standard curves; and the sensitivity of the GUD enzymatic response was measured and repeatedly determined to be less than 10 E. coli cells in a reaction vial. The applicability of the method was tested by performing multiple fluorescence assays under pure and mixed bacterial flora in environmental samples. The results of this study showed that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. Furthermore, this system can be applied independently or in conjunction with other methods as a part of an array of biochemical assays in order to reliably detect E. coli in water. Copyright © 2015 Elsevier Inc. All rights reserved.

Single-Molecule Spectroscopy and Imaging Studies of Protein Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter

2012-04-01

Enzymatic reactions and protein-protein interactions are traditionally studied at the ensemble level, despite significant static and dynamic inhomogeneities. Subtle conformational changes play a crucial role in protein functions, and these protein conformations are highly dynamic rather than being static. We applied AFM-enhanced single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of T4 lysozyme and HPPK enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation. Our new approach is applicable to a wide range of single-molecule FRET measurements for protein conformational changes under enzymatic reactions.

Catalase activity is modulated by calcium and calmodulin in detached mature leaves of sweet potato.

PubMed

Afiyanti, Mufidah; Chen, Hsien-Jung

2014-01-15

Catalase (CAT) functions as one of the key enzymes in the scavenging of reactive oxygen species and affects the H2O2 homeostasis in plants. In sweet potato, a major catalase isoform was detected, and total catalase activity showed the highest level in mature leaves (L3) compared to immature (L1) and completely yellow, senescent leaves (L5). The major catalase isoform as well as total enzymatic activity were strongly suppressed by ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). This inhibition could be specifically and significantly mitigated in mature L3 leaves by exogenous CaCl2, but not MgCl2 or CoCl2. EGTA also inhibited the activity of the catalase isoform in vitro. Furthermore, chlorpromazine (CPZ), a calmodulin (CAM) inhibitor, drastically suppressed the major catalase isoform as well as total enzymatic activity, and this suppression was alleviated by exogenous sweet potato calmodulin (SPCAM) fusion protein in L3 leaves. CPZ also inhibited the activity of the catalase isoform in vitro. Protein blot hybridization showed that both anti-catalase SPCAT1 and anti-calmodulin SPCAM antibodies detect a band at the same position, which corresponds to the activity of the major catalase isoform from unboiled, but not boiled crude protein extract of L3 leaves. An inverse correlation between the major catalase isoform/total enzymatic activity and the H2O2 level was also observed. These data suggest that sweet potato CAT activity is modulated by CaCl2 and SPCAM, and plays an important role in H2O2 homeostasis in mature leaves. Association of SPCAM with the major CAT isoform is required and regulates the in-gel CAT activity band. Copyright © 2013 Elsevier GmbH. All rights reserved.

In situ nanoplasmonic probing of enzymatic activity of monolayer-confined glucose oxidase on colloidal nanoparticles.

PubMed

He, Haili; Xu, Xiaolong; Wu, Haoxi; Zhai, Yujuan; Jin, Yongdong

2013-05-07

In situ probing protein-particle interactions and activities of proteins on colloidal nanoparticle (NP) surfaces is a long-standing key challenge in understanding the nanobio interfaces and virtually important for a variety of biological and biomedical applications. The interactions of NPs with proteins, for instance, are known to form NP bioconjugates or protein coronas; protein surface immobilization and molecular layer-by-layer deposition techniques are widely used, but a clear understanding of the confinement effect on protein activity by molecular coating, at the monolayer level, remains poorly understood. We explore here a novel approach, using colloidal plasmonic nanocomplexes coated with glucose oxidase (GOx) as self-sensing nanoprobes for in situ optical probing of surface-confined enzymatic activity, which is at least 1-2 orders of magnitude more sensitive than standard colorimetric assays for detecting GOx activity. We found that enzymatic activity of monolayer-confined GOx on colloidal NPs was significantly enhanced as compared with free GOx (also proved by conformational changes from circular dichroism studies), with a low apparent Michaelis-Menten constant Km of ~0.115 mM and high turnover kcat/Km of ~8394 M(-1)·s(-1); compared with the "anchored-type" suspending GOx, the outmost polyelectrolyte monolayer-protected "sandwiched-type" GOx exhibits significantly improved enzymatic activities toward higher temperatures and wider pH range. This finding is of fundamental important and instructive for safe use of such nanomaterials for bioapplications.

Enzymatic carotenoid cleavage in star fruit (Averrhoa carambola).

PubMed

Fleischmann, Peter; Watanabe, Naoharu; Winterhalter, Peter

2003-05-01

This paper presents the first description of an enzyme fraction exhibiting carotenoid cleavage activity isolated from fruit skin of Averrhoa carambola. Partial purification of the enzyme could be achieved by acetone precipitation, ultrafiltration (300 kDa, 50 kDa), isoelectric focusing (pH 3-10) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (7.5%). In this way, an enzymatically active protein fraction was obtained, consisting of four proteins in the molecular weight range of between 12 and 90 kDa. Using beta-carotene as substrate, the enzyme activity was detected spectrophotometrically at 505 nm. The main reaction product, detected by GC analysis, was beta-ionone. This proves that the isolated enzymes are closely related to aroma metabolism and release of star fruit. The time constant of the reaction was 16.6 min, the Michaelis Constant K(m)=3.6 micromol 1(-1) and the maximum velocity V(max)=10.5 x 10(-3) micromol l(-1) s(-1) mg((Protein))(-1). The optimum temperature was 45 degrees C.

Label-free electrochemical detection of botulinum neurotoxin type E based on its enzymatic activity using interdigitated electrodes

NASA Astrophysics Data System (ADS)

Hyun, Sang Hwa; Park, Dae Keun; Kang, Aeyeon; Kim, Soohyun; Kim, Daehee; Shin, Yu Mi; Song, Ji-Joon; Yun, Wan Soo

2016-02-01

We report a simple label-free electrochemical method of detecting low concentrations of botulinum neurotoxin type E light chain (BoNT/E LC) based on its peptide cleavage activity. Dual-mode cyclic voltammetry was employed to observe changes in the redox signal of ferri-/ferro-cyanide on interdigitated microelectrodes, whose surfaces were covered by peptides designed from synaptosomal-associated protein 25 to be cleaved by BoNT/E LC. With the introduction of BoNT/E LC, the redox signal showed a time-dependent increase due to cleavage of the immobilized peptide molecules. In addition to the increased redox signal intensity, its time-dependence can be considered as a strong evidence of BoNT/E sensing, since the time-dependent increase can only result from the enzymatic activity of BoNT/E LC. Using this method, BoNT/E LC, at concentrations as low as 5 pg/ml, was readily measurable with only an hour of incubation.

Filtrates and Residues: Optical Projection Experiments to Demonstrate New Curricula Contents.

ERIC Educational Resources Information Center

Perina, Ivo

1986-01-01

Presents background information and procedures for 12 experiments dealing with such areas as: reactivity of a homologous series of saturated monovalent alcohols; enzymatic degradation of hydrogen peroxide by catalase; effect of an activator and inhibitor on amylase activity; proving the existence of phenol in waste water; detecting common air…

Biochemical characterization of prostate-specific membrane antigen from canine prostate carcinoma cells.

PubMed

Wu, Lisa Y; Johnson, Jacqueline M; Simmons, Jessica K; Mendes, Desiree E; Geruntho, Jonathan J; Liu, Tiancheng; Dirksen, Wessel P; Rosol, Thomas J; Davis, William C; Berkman, Clifford E

2014-05-01

Prostate-specific membrane antigen (PSMA) remains an important target for diagnostic and therapeutic application for human prostate cancer. Model cell lines have been recently developed to study canine prostate cancer but their PSMA expression and enzymatic activity have not been elucidated. The present study was focused on determining PSMA expression in these model canine cell lines and the use of fluorescent small-molecule enzyme inhibitors to detect canine PSMA expression by flow cytometry. Western blot and RT-PCR were used to determine the transcriptional and translational expression of PSMA on the canine cell lines Leo and Ace-1. An endpoint HPLC-based assay was used to monitor the enzymatic activity of canine PSMA and the potency of enzyme inhibitors. Flow cytometry was used to detect the PSMA expressed on Leo and Ace-1 cells using a fluorescently tagged PSMA enzyme inhibitor. Canine PSMA expression on the Leo cell line was confirmed by Western blot and RT-PCR, the enzyme activity, and flow cytometry. Kinetic parameters Km and Vmax of PSMA enzymatic activity for the synthetic substrate (PABGγG) were determined to be 393 nM and 220 pmol min(-1)  mg protein(-1) , respectively. The inhibitor core 1 and fluorescent inhibitor 2 were found to be potent reversible inhibitors (IC50  = 13.2 and 1.6 nM, respectively) of PSMA expressed on the Leo cell line. Fluorescent labeling of Leo cells demonstrated that the fluorescent PSMA inhibitor 2 can be used for the detection of PSMA-positive canine prostate tumor cells. Expression of PSMA on Ace-1 was low and not detectable by flow cytometry. The results described herein have demonstrated that PSMA is expressed on canine prostate tumor cells and exhibits similar enzymatic characteristics as human PSMA. The findings show that the small molecule enzyme inhibitors currently being studied for use in diagnosis and therapy of human prostate cancer can also be extended to include canine prostate cancer. Importantly, the findings demonstrate that the potential of the inhibitors for use in diagnosis and therapy can be evaluated in an immunocompetent animal model that naturally develops prostate cancer before use in humans. © 2014 Wiley Periodicals, Inc.

Upregulation of microRNA-320 decreases the risk of developing steroid-induced avascular necrosis of femoral head by inhibiting CYP1A2 both in vivo and in vitro.

PubMed

Wei, Ji-Hua; Luo, Qun-Qiang; Tang, Yu-Jin; Chen, Ji-Xia; Huang, Chun-Lan; Lu, Ding-Gui; Tang, Qian-Li

2018-06-20

Steroid-induced avascular necrosis of femoral head (SANFH) occurs frequently in patients receiving high-dose steroid treatment for these underlying diseases. The target of this study is to investigate the effect of microRNA-320 (miR-320) on SANFH by targeting CYP1A2. CYP1A2 expression was detected using immunohistochemistry. Specimens were collected from patients with SANFH and femoral neck fracture. Seventy rats were assigned into seven groups. The targeting relationship between miR-320 and CYP1A2 was verified by bioinformatics website and dual luciferase reporter gene assay. RT-qPCR and Western blot analysis were used to detect miR-320 and CYP1A2 expressions. The enzymatic activity of CYP1A2 was detected by fluorescence spectrophotometry. Hemorheology and microcirculation were measured in rats. MiR-320 expression decreased and CYP1A2 expression and enzymatic activity increased in SANFH patients compared to those with femoral neck fracture. CYP1A2 was the target gene of miR-320. Hemorheology and microcirculation results showed that up-regulated expression of CYP1A2 promoted the development of SANFH while increased expression of miR-320 inhibited the development of SANFH. Compared with the SANFH group, the SANFH + miR-320 mimic group showed increased miRNA-320 expression, and decreased CYP1A2 expression and enzymatic activity. Opposite results were found in the SANFH + miR-320 inhibitor group. The SANFH + miR-320 inhibitor + pCR-CYP1A2_KO group showed decreased miRNA-320 expression and the SANFH + pCR-CYP1A2_KO group showed decreased CYP1A2 expression and enzymatic activity. Our findings provide evidences that miR-320 might inhibit the development of SANFH by targeting CYP1A2. Copyright © 2018 Elsevier B.V. All rights reserved.

Activity-Dependent Enzymatic Assay for the Detection of Toluene-Oxidizing Bacteria Capable of Trichloroethylene Degradation

NASA Astrophysics Data System (ADS)

Kauffman, M. E.; Kauffman, M. E.; Keener, W. K.; Watwood, M. E.; Lehman, R. M.

2001-12-01

Toluene-oxidizing bacteria produce enzymes that cometabolically degrade trichloroethylene (TCE). These inducible enzymes are produced only in the presence of certain aromatic substrates such as toluene or phenol. Recent laboratory studies have utilized analog chemical substrates to identify production of bacterial enzymes capable of degrading trichloroethylene. These analog substrates produce chromogenic and/or fluorescent products when biotransformed by the enzymes of interest. In this study, 3-hydroxyphenylacetylene (3-HPA) was identified as an activity-dependent enzymatic probe for the detection of three of the four known toluene oxygenase enzymes capable of TCE degradation. Laboratory studies were conducted using pure cultures of Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas putida F1. Cell cultures grown on lactate (non-enzyme inducing) or lactate and toluene (inducing) were trapped trapped on black polycarbonate filters, exposed to 3-HPA, and examined for fluorescence using an epifluorescent microscope. Additionally, B. cepacia G4 cells were grown under the same conditions, but in the presence of mineral and basalt specimens to allow for bacterial attachment. The specimens were then exposed to 3-HPA and examined under an epifluorescent microscope. Our results demonstrate that cells induced for the production of oxygenase enzymes, both unattached and attached, are able to transform 3-HPA to a fluorescent product, although cells attached to geologic materials, such as basalt, take substantially longer to transform the probe. Cells grown under non-inducing conditions do not transform the probe, regardless of their attachment status. Additionally, well water samples taken from a TCE-contaminated aquifer were successfully assayed using the 3-HPA enzymatic probe. The development of this enzyme activity-dependent enzymatic assay provides a fast and reliable method to assess the potential for TCE and aromatic contaminant bioremediation.

Fabrication and electrochemistry characteristics of nickel-doped diamond-like carbon film toward applications in non-enzymatic glucose detection

NASA Astrophysics Data System (ADS)

Liu, Chi-Wen; Chen, Wei-En; Sun, Yin Tung Albert; Lin, Chii-Ruey

2018-04-01

This research work focused on the fabrication of nickel-doped diamond-like carbon (DLC) films and their characteristics including of surface morphology, microstructure, and electrochemical aiming at applications in non-enzymatic glucose detection. Novel nanodiamond target was employed in unbalanced magnetron radio-frequency co-sputtering process to prepared high quality Ni-doped DLC thin film at room temperature. TEM analysis reveals a highly uniform distribution of Ni crystallites in amorphous carbon matrix with fraction ranged from 3 to 11.5 at.% which is considered as active sites for the glucose detection. Our cyclic voltammetry measurements using 0.1 M H2SO4 solution demonstrated that the as-prepared Ni-doped DLC films possess large electrochemical potential window of 2.12 V, and this was also observed to be significantly reduced at high Ni doping level owing to lower sp3 fraction. The non-enzymatic glucose detection investigation indicates that the Ni-doped DLC thin film electrode prepared under 7 W of DC sputtering power on Ni target possesses good detecting performance, high stability, and high sensitivity to glucose concentration up to 10 mM, even with the existence of uric acid and ascorbic acid. The peak current was observed to be proportional to glucose concentration and scanning rate, demonstrating highly reversibility redox process of the film electrode and glucose.

Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs).

PubMed

Yu, Shann S; Scherer, Randy L; Ortega, Ryan A; Bell, Charleson S; O'Neil, Conlin P; Hubbell, Jeffrey A; Giorgio, Todd D

2011-02-27

Drug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA. Monodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature. This work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug release in oxidative environments, smart theranostic applications combining drug delivery with imaging of platform localization are within reach. The modular design of these USPIO nanoclusters enables future development of platforms for imaging and drug delivery targeted towards proteolytic activity in tumors and in advanced atherosclerotic plaques.

Cystamine restores GSTA3 levels in Vanin-1 null mice.

PubMed

Di Leandro, Luana; Maras, Bruno; Schininà, M Eugenia; Dupré, Silvestro; Koutris, Ilias; Martin, Florent M; Naquet, Philippe; Galland, Franck; Pitari, Giuseppina

2008-03-15

Free cysteamine levels in mouse tissues have been strictly correlated to the presence of membrane-bound pantetheinase activity encoded by Vanin-1. Vanin-1 is involved in many biological processes in mouse, from thymus homing to sexual development. Vanin-1 -/- mice are fertile and grow and develop normally; they better control inflammation and most of the knockout effects were rescued by cystamine treatment. Gene structure analysis showed the presence of an oxidative stimuli-responsive ARE-like sequence in the promoter. In this paper we investigate antioxidant-detoxifying enzymatic activities at the tissue level, comparing Vanin-1 -/- and wild-type mice. In Vanin-1 null animals we pointed out a decrease in the Se-independent glutathione peroxidase activity. The decrease in enzymatic activity appeared to be correlated to an impairment of GST isoenzyme levels. In particular a significant drop in GSTA3 together with a minor decrement in GSTM1 and an increase in GSTP1 levels was detected in Vanin-1 -/- livers. Cystamine administration to Vanin-1 -/- mice restored specifically GSTA3 levels and the corresponding enzymatic activity without influencing protein expression. A possible role of cystamine on protein stability/folding can be postulated.

Enzyme-immobilized SiO2-Si electrode: Fast interfacial electron transfer with preserved enzymatic activity

NASA Astrophysics Data System (ADS)

Wang, Gang; Yau, Siu-Tung

2005-12-01

The enzyme, glucose oxidase (GOx), is immobilized using electrostatic interaction on the native oxide of heavily doped n-type silicon. Voltammetric measurement shows that the immobilized GOx gives rise to a very fast enzyme-silicon interfacial electron transfer rate constant of 7.9s-1. The measurement also suggests that the enzyme retains its native conformation when immobilized on the silicon surface. The preserved native conformation of GOx is further confirmed by testing the enzymatic activity of the immobilized GOx using glucose. The GOx-immobilized silicon is shown to behave as a glucose sensor that detects glucose with concentrations as low as 50μM.

A pilot study of the modulation of sirtuins on arylamine N-acetyltransferase 1 and 2 enzymatic activity.

PubMed

Turiján-Espinoza, Eneida; Salazar-González, Rául Alejandro; Uresti-Rivera, Edith Elena; Hernández-Hernández, Gloria Estela; Ortega-Juárez, Montserrat; Milán, Rosa; Portales-Pérez, Diana

2018-03-01

Arylamine N -acetyltransferase (NAT; E.C. 2.3.1.5) enzymes are responsible for the biotransformation of several arylamine and hydrazine drugs by acetylation. In this process, the acetyl group transferred to the acceptor substrate produces NAT deacetylation and, in consequence, it is susceptible of degradation. Sirtuins are protein deacetylases, dependent on nicotine adenine dinucleotide, which perform post-translational modifications on cytosolic proteins. To explore possible sirtuin participation in the enzymatic activity of arylamine NATs, the expression levels of NAT1, NAT2, SIRT1 and SIRT6 in peripheral blood mononuclear cells (PBMC) from healthy subjects were examined by flow cytometry and Western blot. The in situ activity of the sirtuins on NAT enzymatic activity was analyzed by HPLC, in the presence or absence of an agonist (resveratrol) and inhibitor (nicotinamide) of sirtuins. We detected a higher percentage of positive cells for NAT2 in comparison with NAT1, and higher numbers of SIRT1+ cells compared to SIRT6 in lymphocytes. In situ NAT2 activity in the presence of NAM inhibitors was higher than in the presence of its substrate, but not in the presence of resveratrol. In contrast, the activity of NAT1 was not affected by sirtuins. These results showed that NAT2 activity might be modified by sirtuins.

Highly selective apo-arginase based method for sensitive enzymatic assay of manganese (II) and cobalt (II) ions

NASA Astrophysics Data System (ADS)

Stasyuk, Nataliya; Gayda, Galina; Zakalskiy, Andriy; Zakalska, Oksana; Errachid, Abdelhamid; Gonchar, Mykhailo

2018-03-01

A novel enzymatic method of manganese (II) and cobalt (II) ions assay, based on using apo-enzyme of Mn2 +-dependent recombinant arginase I (arginase) and 2,3-butanedione monoxime (DMO) as a chemical reagent is proposed. The principle of the method is the evaluation of the activity of L-arginine-hydrolyzing of arginase holoenzyme after the specific binding of Mn2 + or Co2 + with apo-arginase. Urea, which is the product of enzymatic hydrolysis of L-arginine (Arg), reacts with DMO and the resulted compound is detected by both fluorometry and visual spectrophotometry. Thus, the content of metal ions in the tested samples can be determined by measuring the level of urea generated after enzymatic hydrolysis of Arg by reconstructed arginase holoenzyme in the presence of tested metal ions. The linearity range of the fluorometric apo-arginase-DMO method in the case of Mn2 + assay is from 4 pM to 1.10 nM with a limit of detection of 1 pM Mn2 +, whereas the linearity range of the present method in the case of Co2 + assay is from 8 pM to 45 nM with a limit of detection of 2.5 pM Co2 +. The proposed method being highly sensitive, selective, valid and low-cost, may be useful to monitor Mn2 + and Co2 + content in clinical laboratories, food industry and environmental control service.

Zymogram profiling of superoxide dismutase and catalase activities allows Saccharomyces and non-Saccharomyces species differentiation and correlates to their fermentation performance.

PubMed

Gamero-Sandemetrio, Esther; Gómez-Pastor, Rocío; Matallana, Emilia

2013-05-01

Aerobic organisms have devised several enzymatic and non-enzymatic antioxidant defenses to deal with reactive oxygen species (ROS) produced by cellular metabolism. To combat such stress, cells induce ROS scavenging enzymes such as catalase, peroxidase, superoxide dismutase (SOD) and glutathione reductase. In the present research, we have used a double staining technique of SOD and catalase enzymes in the same polyacrylamide gel to analyze the different antioxidant enzymatic activities and protein isoforms present in Saccharomyces and non-Saccharomyces yeast species. Moreover, we used a technique to differentially detect Sod1p and Sod2p on gel by immersion in NaCN, which specifically inhibits the Sod1p isoform. We observed unique SOD and catalase zymogram profiles for all the analyzed yeasts and we propose this technique as a new approach for Saccharomyces and non-Saccharomyces yeast strains differentiation. In addition, we observed functional correlations between SOD and catalase enzyme activities, accumulation of essential metabolites, such as glutathione and trehalose, and the fermentative performance of different yeasts strains with industrial relevance.

Impact of cysteine variants on the structure, activity, and stability of recombinant human α-galactosidase A

PubMed Central

Qiu, Huawei; Honey, Denise M; Kingsbury, Jonathan S; Park, Anna; Boudanova, Ekaterina; Wei, Ronnie R; Pan, Clark Q; Edmunds, Tim

2015-01-01

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity. PMID:26044846

Real-Time, Single-Step Bioassay Using Nanoplasmonic Resonator With Ultra-High Sensitivity

NASA Technical Reports Server (NTRS)

Zhang, Xiang (Inventor); Chen, Fanqing Frank (Inventor); Su, Kai-Hang (Inventor); Wei, Qi-Huo (Inventor); Ellman, Jonathan A. (Inventor); Sun, Cheng (Inventor)

2014-01-01

A nanoplasmonic resonator (NPR) comprising a metallic nanodisk with alternating shielding layer(s), having a tagged biomolecule conjugated or tethered to the surface of the nanoplasmonic resonator for highly sensitive measurement of enzymatic activity. NPRs enhance Raman signals in a highly reproducible manner, enabling fast detection of protease and enzyme activity, such as Prostate Specific Antigen (paPSA), in real-time, at picomolar sensitivity levels. Experiments on extracellular fluid (ECF) from paPSA-positive cells demonstrate specific detection in a complex bio-fluid background in real-time single-step detection in very small sample volumes.

Real-time, single-step bioassay using nanoplasmonic resonator with ultra-high sensitivity

DOEpatents

Zhang, Xiang; Ellman, Jonathan A; Chen, Fanqing Frank; Su, Kai-Hang; Wei, Qi-Huo; Sun, Cheng

2014-04-01

A nanoplasmonic resonator (NPR) comprising a metallic nanodisk with alternating shielding layer(s), having a tagged biomolecule conjugated or tethered to the surface of the nanoplasmonic resonator for highly sensitive measurement of enzymatic activity. NPRs enhance Raman signals in a highly reproducible manner, enabling fast detection of protease and enzyme activity, such as Prostate Specific Antigen (paPSA), in real-time, at picomolar sensitivity levels. Experiments on extracellular fluid (ECF) from paPSA-positive cells demonstrate specific detection in a complex bio-fluid background in real-time single-step detection in very small sample volumes.

In vitro anti-inflammatory effects of arctigenin, a lignan from Arctium lappa L., through inhibition on iNOS pathway.

PubMed

Zhao, Feng; Wang, Lu; Liu, Ke

2009-04-21

Arctigenin, a bioactive constituent from dried seeds of Arctium lappa L. (Compositae) which has been widely used as a Traditional Chinese Medicine for dispelling wind and heat included in Chinese Pharmacophere, was found to exhibit anti-inflammatory activities but its molecular mechanism remains unknown yet. To investigate the anti-inflammatory mechanism of arctigenin. Cultured macrophage RAW 264.7 cells and THP-1 cells were used for the experiments. Griess assay was used to evaluate the inhibitory effect of arctigenin on the overproduction of nitric oxide (NO). ELISA was used to determine the level of pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). The inhibitory effect on the enzymatic activity of cyclooxygenase-2 (COX-2) was tested by colorimetric method. Western blot was used to detect the expression of inducible nitric oxide synthase (iNOS) and COX-2. Arctigenin suppressed lipopolysaccharide (LPS)-stimulated NO production and pro-inflammatory cytokines secretion, including TNF-alpha and IL-6 in a dose-dependent manner. Arctigenin also strongly inhibited the expression of iNOS and iNOS enzymatic activity, whereas the expression of COX-2 and COX-2 enzymatic activity were not affected by arctigenin. These results indicated that potent inhibition on NO, TNF-alpha and IL-6, but not COX-2 expression and COX-2 activity, might constitute the anti-inflammatory mechanism of arctigenin. Arctigenin suppressed the overproduction of NO through down-regulation of iNOS expression and iNOS enzymatic activity in LPS-stimulated macrophage.

Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes.

PubMed

Singh, R; Chénier, D; Bériault, R; Mailloux, R; Hamel, R D; Appanna, V D

2005-09-30

We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-PAGE or SDS-PAGE enabled the rapid purification of the enzyme of interest. This technique also provides a quick and inexpensive means of quantifying these enzymatic activities in normal and stressed biological systems.

Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation.

PubMed

Sutto-Ortiz, Priscila; Camacho-Ruiz, María de Los Angeles; Kirchmayr, Manuel R; Camacho-Ruiz, Rosa María; Mateos-Díaz, Juan Carlos; Noiriel, Alexandre; Carrière, Frédéric; Abousalham, Abdelkarim; Rodríguez, Jorge A

2017-01-01

Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging to Streptomyces (73%) and Micromonospora (10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of the Streptomyces genus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl- sn -glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel PLAs with potential applications in biotechnology.

Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation

PubMed Central

Sutto-Ortiz, Priscila; Camacho-Ruiz, María de los Angeles; Kirchmayr, Manuel R.; Camacho-Ruiz, Rosa María; Mateos-Díaz, Juan Carlos; Noiriel, Alexandre; Carrière, Frédéric; Abousalham, Abdelkarim

2017-01-01

Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging to Streptomyces (73%) and Micromonospora (10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of the Streptomyces genus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel PLAs with potential applications in biotechnology. PMID:28695068

Active Site Detection by Spatial Conformity and Electrostatic Analysis—Unravelling a Proteolytic Function in Shrimp Alkaline Phosphatase

PubMed Central

Chakraborty, Sandeep; Minda, Renu; Salaye, Lipika; Bhattacharjee, Swapan K.; Rao, Basuthkar J.

2011-01-01

Computational methods are increasingly gaining importance as an aid in identifying active sites. Mostly these methods tend to have structural information that supplement sequence conservation based analyses. Development of tools that compute electrostatic potentials has further improved our ability to better characterize the active site residues in proteins. We have described a computational methodology for detecting active sites based on structural and electrostatic conformity - C ata L ytic A ctive S ite P rediction (CLASP). In our pipelined model, physical 3D signature of any particular enzymatic function as defined by its active sites is used to obtain spatially congruent matches. While previous work has revealed that catalytic residues have large pKa deviations from standard values, we show that for a given enzymatic activity, electrostatic potential difference (PD) between analogous residue pairs in an active site taken from different proteins of the same family are similar. False positives in spatially congruent matches are further pruned by PD analysis where cognate pairs with large deviations are rejected. We first present the results of active site prediction by CLASP for two enzymatic activities - β-lactamases and serine proteases, two of the most extensively investigated enzymes. The results of CLASP analysis on motifs extracted from Catalytic Site Atlas (CSA) are also presented in order to demonstrate its ability to accurately classify any protein, putative or otherwise, with known structure. The source code and database is made available at www.sanchak.com/clasp/. Subsequently, we probed alkaline phosphatases (AP), one of the well known promiscuous enzymes, for additional activities. Such a search has led us to predict a hitherto unknown function of shrimp alkaline phosphatase (SAP), where the protein acts as a protease. Finally, we present experimental evidence of the prediction by CLASP by showing that SAP indeed has protease activity in vitro. PMID:22174814

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica.

PubMed

Beneyton, Thomas; Thomas, Stéphane; Griffiths, Andrew D; Nicaud, Jean-Marc; Drevelle, Antoine; Rossignol, Tristan

2017-01-31

Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS. Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement). Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (10 5 strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.

Effect of temperature and high pressure on the activity and mode of action of fungal pectin methyl esterase.

PubMed

Duvetter, Thomas; Fraeye, Ilse; Sila, Daniel N; Verlent, Isabel; Smout, Chantal; Clynen, Elke; Schoofs, Liliane; Schols, Henk; Hendrickx, Marc; Van Loey, Ann

2006-01-01

Pectin was de-esterified with purified recombinant Aspergillus aculeatus pectin methyl esterase (PME) during isothermal-isobaric treatments. By measuring the release of methanol as a function of treatment time, the rate of enzymatic pectin conversion was determined. Elevated temperature and pressure were found to stimulate PME activity. The highest rate of PME-catalyzed pectin de-esterification was obtained when combining pressures in the range 200-300 MPa with temperatures in the range 50-55 degrees C. The mode of pectin de-esterification was investigated by characterizing the pectin reaction products by enzymatic fingerprinting. No significant effect of increasing pressure (300 MPa) and/or temperature (50 degrees C) on the mode of pectin conversion was detected.

Enzymatic activity profile of a Brazilian culture collection of Candida albicans isolated from diabetics and non-diabetics with oral candidiasis.

PubMed

Sanitá, Paula Volpato; Zago, Chaiene Evelin; Pavarina, Ana Cláudia; Jorge, Janaina Habib; Machado, Ana Lúcia; Vergani, Carlos Eduardo

2014-06-01

The secretion of hydrolytic enzymes is a fundamental virulence factor of Candida albicans to develop disease. The objective of this study was to characterise the virulence of 148 clinical isolates of C. albicans from oral candidiasis by assessing the expression of phospholipase (PL) and secreted aspartyl proteinase (SAP). Isolates were obtained from healthy subjects (HS) and diabetics (DOC) and non-diabetics with oral candidiasis (NDOC). An aliquot (5 μl) of each cell suspension was inoculated on PL and SAP agar plates and incubated. Enzymes secretion was detected by the formation of an opaque halo around the colonies and enzymatic activity (PZ) was determined by the ratio between colony diameter and colony diameter plus the halo zone. Statistical comparisons were made by a one-way anova followed by Tukey's post hoc test (α = 0.05). The clinical sources of C. albicans had significant effect (P < 0.001) on the PZ values of both enzymes. For PL, clinical isolates from NDOC and DOC had highest enzymatic activity than those from HS (P < 0.05), with no significant differences between them (P = 0.506). For SAP, C. albicans from NDOC showed the lower enzymatic activity (P < 0.001). There were no significant differences between isolates from HS and DOC (P = 0.7051). C. albicans isolates from NDOC and DOC patients showed an increased production of PL. © 2013 Blackwell Verlag GmbH.

Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling

As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-inducedmore » inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.« less

Effect of body temperature on chondroitinase ABC's ability to cleave chondroitin sulfate glycosaminoglycans.

PubMed

Tester, Nicole J; Plaas, Anna H; Howland, Dena R

2007-04-01

Chondroitinase ABC (Ch'ase ABC) is a bacterial lyase that degrades chondroitin sulfate (CS), dermatan sulfate, and hyaluronan glycosaminoglycans (GAGs). This enzyme has received significant attention as a potential therapy for promoting central nervous system and peripheral nervous system repair based on its degradation of CS GAGs. Determination of the stability of Ch'ase ABC activity at temperatures equivalent to normal (37 degrees C) and elevated (39 degrees C) body temperatures is important for optimizing its clinical usage. We report here data obtained from examining enzymatic activity at these temperatures across nine lots of commercially available protease-free Ch'ase ABC. CS GAG degrading activity was assayed by using 1) immunohistochemical detection of unsaturated disaccharide stubs generated by digestion of proteoglycans in tissue sections and 2) fluorophore-assisted carbohydrate electrophoresis (FACE) and/or high-performance liquid chromatography (HPLC) to separate and quantify unsaturated disaccharide digestion products. Our results indicate that there is a significant effect of lot and time on enzymatic thermostability. Average enzymatic activity is significantly decreased at 1 and 3 days at 39 degrees C and 37 degrees C, respectively. Furthermore, the average activity seen after 1 day was significantly different between the two temperatures. Addition of bovine serum albumin as a stabilizer significantly preserved enzymatic activity at 1 day, but not 3 days, at 39 degrees C. These results show that the CS GAG degrading activity of Ch'ase ABC is significantly decreased with incubation at body temperature over time and that all lots do not show equal thermostability. These findings are important for the design and interpretation of experimental and potential clinical studies involving Ch'ase ABC. (c) 2007 Wiley-Liss, Inc.

Distribution in microbial genomes of genes similar to lodA and goxA which encode a novel family of quinoproteins with amino acid oxidase activity.

PubMed

Campillo-Brocal, Jonatan C; Chacón-Verdú, María Dolores; Lucas-Elío, Patricia; Sánchez-Amat, Antonio

2015-03-24

L-Amino acid oxidases (LAOs) have been generally described as flavoproteins that oxidize amino acids releasing the corresponding ketoacid, ammonium and hydrogen peroxide. The generation of hydrogen peroxide gives to these enzymes antimicrobial characteristics. They are involved in processes such as biofilm development and microbial competition. LAOs are of great biotechnological interest in different applications such as the design of biosensors, biotransformations and biomedicine. The marine bacterium Marinomonas mediterranea synthesizes LodA, the first known LAO that contains a quinone cofactor. LodA is encoded in an operon that contains a second gene coding for LodB, a protein required for the post-translational modification generating the cofactor. Recently, GoxA, a quinoprotein with sequence similarity to LodA but with a different enzymatic activity (glycine oxidase instead of lysine-ε-oxidase) has been described. The aim of this work has been to study the distribution of genes similar to lodA and/or goxA in sequenced microbial genomes and to get insight into the evolution of this novel family of proteins through phylogenetic analysis. Genes encoding LodA-like proteins have been detected in several bacterial classes. However, they are absent in Archaea and detected only in a small group of fungi of the class Agaromycetes. The vast majority of the genes detected are in a genome region with a nearby lodB-like gene suggesting a specific interaction between both partner proteins. Sequence alignment of the LodA-like proteins allowed the detection of several conserved residues. All of them showed a Cys and a Trp that aligned with the residues that are forming part of the cysteine tryptophilquinone (CTQ) cofactor in LodA. Phylogenetic analysis revealed that LodA-like proteins can be clustered in different groups. Interestingly, LodA and GoxA are in different groups, indicating that those groups are related to the enzymatic activity of the proteins detected. Genome mining has revealed for the first time the broad distribution of LodA-like proteins containing a CTQ cofactor in many different microbial groups. This study provides a platform to explore the potentially novel enzymatic activities of the proteins detected, the mechanisms of post-translational modifications involved in their synthesis, as well as their biological relevance.

Methods, microfluidic devices, and systems for detection of an active enzymatic agent

DOEpatents

Sommer, Gregory J; Hatch, Anson V; Singh, Anup K; Wang, Ying-Chih

2014-10-28

Embodiments of the present invention provide methods, microfluidic devices, and systems for the detection of an active target agent in a fluid sample. A substrate molecule is used that contains a sequence which may cleave in the presence of an active target agent. A SNAP25 sequence is described, for example, that may be cleaved in the presence of Botulinum Neurotoxin. The substrate molecule includes a reporter moiety. The substrate molecule is exposed to the sample, and resulting reaction products separated using electrophoretic separation. The elution time of the reporter moiety may be utilized to identify the presence or absence of the active target agent.

Enzymatic activities produced by mixed Saccharomyces and non-Saccharomyces cultures: relationship with wine volatile composition.

PubMed

Maturano, Yolanda Paola; Assof, Mariela; Fabani, María Paula; Nally, María Cristina; Jofré, Viviana; Rodríguez Assaf, Leticia Anahí; Toro, María Eugenia; Castellanos de Figueroa, Lucía Inés; Vazquez, Fabio

2015-11-01

During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as β-glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. β-Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4-5 of the fermentation process, and highest populations were observed in MSD2 (10% S. cerevisiae/90% D. vanrijiae) and MSC1 (1% S. cerevisiae/99% C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of β-glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation.

Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs)

PubMed Central

2011-01-01

Background Drug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA. Results Monodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature. Conclusions This work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug release in oxidative environments, smart theranostic applications combining drug delivery with imaging of platform localization are within reach. The modular design of these USPIO nanoclusters enables future development of platforms for imaging and drug delivery targeted towards proteolytic activity in tumors and in advanced atherosclerotic plaques. PMID:21352596

A high-throughput assay for enzymatic polyester hydrolysis activity by fluorimetric detection.

PubMed

Wei, Ren; Oeser, Thorsten; Billig, Susan; Zimmermann, Wolfgang

2012-12-01

A fluorimetric assay for the fast determination of the activity of polyester-hydrolyzing enzymes in a large number of samples has been developed. Terephthalic acid (TPA) is a main product of the enzymatic hydrolysis of polyethylene terephthalate (PET), a synthetic polyester. Terephthalate has been quantified following its conversion to the fluorescent 2-hydroxyterephthalate by an iron autoxidation-mediated generation of free hydroxyl radicals. The assay proved to be robust at different buffer concentrations, reaction times, pH values, and in the presence of proteins. A validation of the assay was performed by analyzing TPA formation from PET films and nanoparticles catalyzed by a polyester hydrolase from Thermobifida fusca KW3 in a 96-well microplate format. The results showed a close correlation (R(2) = 0.99) with those obtained by a considerably more tedious and time-consuming HPLC method, suggesting the aptness of the fluorimetric assay for a high-throughput screening for polyester hydrolases. The method described in this paper will facilitate the detection and development of biocatalysts for the modification and degradation of synthetic polymers. The fluorimetric assay can be used to quantify the amount of TPA obtained as the final degradation product of the enzymatic hydrolysis of PET. In a microplate format, this assay can be applied for the high-throughput screening of polyester hydrolases. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Guitot, Karine; Scarabelli, Silvia; Drujon, Thierry; Bolbach, Gérard; Amoura, Mehdi; Burlina, Fabienne; Jeltsch, Albert; Sagan, Sandrine; Guianvarc'h, Dominique

2014-07-01

Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC50 for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors. Copyright © 2014 Elsevier Inc. All rights reserved.

Functionalization of monolithic and porous three-dimensional graphene by one-step chitosan electrodeposition for enzymatic biosensor.

PubMed

Liu, Jiyang; Wang, Xiaohui; Wang, Tianshu; Li, Dan; Xi, Fengna; Wang, Jin; Wang, Erkang

2014-11-26

Biological modification of monolithic and porous 3D graphene is of great significance for extending its application in fabricating highly sensitive biosensors. The present work reports on the first biofunctionalization of monolithic and freestanding 3D graphene foam for one-step preparation of reagentless enzymatic biosensors by controllable chitosan (CS) electrodeposition technology. Using a homogeneous three-component electrodeposition solution containing a ferrocene (Fc) grafted CS hybrid (Fc-CS), glucose oxidase (GOD), and single-walled carbon nanotubes (SWNTs), a homogeneous biocomposite film of Fc-CS/SWNTs/GOD was immobilized on the surface of 3D graphene foam by one-step electrodeposition. The Fc groups grafted on chitosan can be stably immobilized on the 3D graphene surface and keep their original electrochemical activity. The SWNTs doped into the Fc-CS matrix act as a nanowire to facilitate electron transfer and improve the conductivity of the biocomposite film. Combined with the extraordinary properties of 3D graphene foam including large active surface area, high conductivity, and fast mass transport dynamics, the 3D graphene based enzymatic biosensor achieved a large linear range (5.0 μM to 19.8 mM), a low detection limit (1.2 μM), and rapid response (reaching the 95% steady-state response within 8 s) for reagentless detection of glucose in the phosphate buffer solution.

A laser microsurgical method of cell wall removal allows detection of large-conductance ion channels in the guard cell plasma membrane

NASA Technical Reports Server (NTRS)

Miedema, H.; Henriksen, G. H.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

1999-01-01

Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. "Laser-assisted" patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.

Muti-component nanocomposite of nickel and manganese oxides with enhanced stability and catalytic performance for non-enzymatic glucose sensors

NASA Astrophysics Data System (ADS)

Wang, Dandan; Cai, Daoping; Wang, Chenxia; Liu, Bin; Wang, Lingling; Liu, Yuan; Li, Han; Wang, Yanrong; Li, Qiuhong; Wang, Taihong

2016-06-01

A muti-component nanocomposite of nickel and manganese oxides with a uniformly dispersed microspherical structure has been fabricated by a hydrothermal synthesis method. The as-prepared nanocomposite has been employed as a sensing material for non-enzymatic glucose detection and shown excellent electrocatalytic activity, such as high sensitivities of 82.44 μA mM-1 cm-2 and 27.92 μA mM-1 cm-2 over the linear range of 0.1-1 mM and 1-4.5 mM, respectively, a low detection limit of 0.2 μM and a fast response time of <3 s. Moreover, satisfactory specificity and excellent stability have also been achieved. The results demonstrate that a muti-component nanocomposite of nickel and manganese oxides has great potential applications as glucose sensors.

A novel SERRS sandwich-hybridization assay to detect specific DNA target.

PubMed

Feuillie, Cécile; Merheb, Maxime Mohamad; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2011-01-01

In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.

A Novel SERRS Sandwich-Hybridization Assay to Detect Specific DNA Target

PubMed Central

Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2011-01-01

In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics. PMID:21655320

Ceramic microsystem incorporating a microreactor with immobilized biocatalyst for enzymatic spectrophotometric assays.

PubMed

Baeza, Mireia; López, Carmen; Alonso, Julián; López-Santín, Josep; Alvaro, Gregorio

2010-02-01

Low-temperature cofired ceramics (LTCC) technology is a versatile fabrication technique used to construct microflow systems. It permits the integration of several unitary operations (pretreatment, separation, (bio)chemical reaction, and detection stage) of an analytical process in a modular or monolithic way. Moreover, because of its compatibility with biological material, LTCC is adequate for analytical applications based on enzymatic reactions. Here we present the design, construction, and evaluation of a LTCC microfluidic system that integrates a microreactor (internal volume, 24.28 microL) with an immobilized beta-galactosidase from Escherichia coli (0.479 activity units) and an optical flow cell to measure the product of the enzymatic reaction. The enzyme was immobilized on a glyoxal-agarose support, maintaining its activity along the time of the study. As a proof of concept, the LTCC-beta-galactosidase system was tested by measuring the conversion of ortho-nitrophenyl beta-D-galactopyranoside, the substrate usually employed for activity determinations. Once packed in a monolithically integrated microcolumn, the miniaturized flow system was characterized, the operational conditions optimized (flow rate and injection volume), and its performance successfully evaluated by determining the beta-galactosidase substrate concentration at the millimolar level.

Effect of salinity tolerant PDH45 transgenic rice on physicochemical properties, enzymatic activities and microbial communities of rhizosphere soils

PubMed Central

Sahoo, Ranjan Kumar; Tuteja, Narendra

2013-01-01

The effect of genetically modified (GM) plants on environment is now major concern worldwide. The plant roots of rhizosphere soil interact with variety of bacteria which could be influenced by the transgene in GM plants. The antibiotic resistance genes in GM plants may be transferred to soil microbes. In this study we have examined the effect of overexpression of salinity tolerant pea DNA helicase 45 (PDH45) gene on microbes and enzymatic activities in the rhizosphere soil of transgenic rice IR64 in presence and absence of salt stress in two different rhizospheric soils (New Delhi and Odisha, India). The diversity of the microbial community and soil enzymes viz., dehydrogenase, alkaline phosphatase, urease and nitrate reductase was assessed. The results revealed that there was no significant effect of transgene expression on rhizosphere soil of the rice plants. The isolated bacteria were phenotyped both in absence and presence of salt and no significant changes were found in their phenotypic characters as well as in their population. Overall, the overexpression of PDH45 in rice did not cause detectable changes in the microbial population, soil enzymatic activities and functional diversity of the rhizosphere soil microbial community. PMID:23733066

Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.

2010-01-01

In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, andmore » enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.« less

Diagnosis of Morquio Syndrome in Dried Blood Spots Based on a New MRM-MS Assay.

PubMed

Cozma, Claudia; Eichler, Sabrina; Wittmann, Gyula; Flores Bonet, Alba; Kramp, Guido Johannes; Giese, Anne-Katrin; Rolfs, Arndt

2015-01-01

Mucopolysaccharidosis IVA (MPS IVA; Morquio A disease) is an autosomal recessive disease caused and characterized by a decreased activity of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in accumulation of keratan sulfate and chondroitin-6-sulfate in tissues and secondary organ damage. Recently approved enzyme replacement therapy renders the easy and early identification of MPS IVA of out-most importance. We propose a completely new assay for the stable and reproducible detection of GALNS deficiency in dry blood spots (DBS). For the validation blood samples were taken from 59 healthy individuals and 24 randomly selected genetically confirmed MPS IVA patients. The material extracted from DBS was incubated with a 4-methylumbelliferyl-β-D-galactopyranoside-6-sulfate as a specific substrate. Final enzymatic product, 4-methylumbelliferone, obtained after adding exogenous beta-galactosidase, was quantified by LC/MRM-MS (liquid-chromatography/multiple-reaction-monitoring mass-spectrometry). 4-propyl-5-hydroxy-7-methyl-2h-chromen-2-one was used as internal standard, a compound with a similar molecular structure and fragmentation pattern in negative ion mode as 4-methylumbelliferone. The enzymatic assay yielded a positive and negative predictive value of 1.0 for genetically confirmed MPS IVA patients (GALNS activity of 0.35 ± 0.21 μmol/L/h) and for controls with normal GALNS activity (23.1 ± 5.3 μmol/L /h). With present enzymatic conditions, the reaction yield in dried blood spots is at least 20 fold higher than any previously reported data with other assays. The present LC/MRM-MS based assay for MPS IVA diagnosis provides an easy, highly-standardized, accurate and innovative quantification of the enzymatic product in vitro and distinguishes perfectly between MPS IVA affected patients and normal controls. This technique will significantly simplify the early detection of MPS IVA patients.

Enzymatic activities and effects of mycovirus infection on the virulence of Metarhizium anisopliae in Rhipicephalus microplus.

PubMed

Perinotto, Wendell M S; Golo, Patricia S; Coutinho Rodrigues, Caio J B; Sá, Fillipe A; Santi, Lucélia; Beys da Silva, Walter O; Junges, Angela; Vainstein, Marilene H; Schrank, Augusto; Salles, Cristiane M C; Bittencourt, Vânia R E P

2014-06-16

The present study aimed to evaluate the pathogenic potential of different Metarhizium anisopliae s.l. isolates and to determine whether differences in enzymatic activities of proteases, lipases and chitinases and infection with mycoviruses affect the control of Rhipicephalus microplus achieved by these fungal isolates. Engorged female ticks were exposed to fungal suspensions. The lipolytic and proteolytic activities in the isolates were evaluated using chromogenic substrates and the chitinolytic activity was determined using fluorescent substrates. A gel zymography was performed to determine the approximate size of serine proteases released by M. anisopliae isolates. To detect mycoviral infections, dsRNA was digested using both RNAse A and S1 endonuclease; samples were analyzed on an agarose gel. Four of the five isolates tested were infected with mycovirus; however, the level of control of R. microplus ticks achieved with the only isolate free of infection (isolate CG 347) was low. This finding suggests that mycoviral infection does not affect the virulence of fungi against ticks. Although all five isolates were considered pathogenic to R. microplus, the best tick control and the highest levels of enzymatic activity were achieved with the isolates CG 629 and CG 148. The in vitro activities of lipases, proteases and chitinases produced by M. anisopliae s.l. differed among isolates and may be related to their virulence. Copyright © 2014 Elsevier B.V. All rights reserved.

Interaction of on-site and near real time measured turbidity and enzyme activity in stream water.

NASA Astrophysics Data System (ADS)

Stadler, Philipp; Farnleitner, Andreas H.; Zessner, Matthias

2013-04-01

On-site and on-line systems that provide an integrated surveillance of physicochemical and microbiological parameters gain significance in water quality monitoring. Particular relating to diffuse pollution from agricultural areas and use-orientated protection of waters the detection of faecal pollution is a fundamental part. For the near real time and on-site detection of microbiological faecal pollution of water, the beta-D- Glucuronidase (GLUC) enzymatic activity has been suggested as a surrogate parameter. Due to possible short measure intervals of three hours, this method has high potential as a water quality monitoring tool. While cultivation based standard determination takes more than one working day (Cabral 2010) the potential advantage of detecting the GLUC activity is the high temporal measuring resolution. Yet, there is still a big gap of knowledge on the sensitivity and specificity concerning the faecal indication capacity of GLUC in relation to standard assays (Cabral 2010). Interference effects of physicochemical parameters on the enzymatic activity respectively fluorescence have been discussed (Molina-Munoz et al. 2007; Tryland and Fiksdal 1998, Biswal et al. 2003). Results from a monitoring of a rivulet in an agricultural catchment in Lower Austria (HOAL - Hydrological Open Air Laboratory) are presented here. The HOAL offers technical resources that allow measurements at high temporal and spatial resolution and to apply various hydrological methods in one catchment. Two automated enzymatic measuring devices (Coliguard, mbOnline, Austria) and physicochemical in-stream measurements are used, as well as in-stream spectroscopy (spectrolyser, s::can, Austria). Accuracy of both enzymatic measuring devices is compared through diverse hydrological and seasonal conditions. Reference analyses by cultivation based determination were performed. Data from Coliguard devices is combined with physicochemical and spectroscopy data to gain information about the influence of turbidity on rapid GLUC measurements of stream water. During event run off conditions with high sediment load, accuracy of the GLUC determination was assayed. Various on-site set ups were tested to ascertain the use of sample prefiltration. We would like acknowledge financial support from the Austrian Science Funds (FWF) as part of the Vienna Doctoral Programme on Water Resource Systems (DK-plus W1219-N22). References: Cabral, J. P. S. 2010. "Water Microbiology. Bacterial Pathogens and Water." International Journal of Environmental Research and Public Health 7 (10): 3657-3703. Biswal , N. , S. Gupta, N. Ghosh, and A. Pradhan 2003. "Recovery of turbidity free fluorescence from measured fluorescence: An experimental approach. Optics Express 11, (24): 3320. Molina-Munoz, M., J. M. Poyatos, R. Vilchez, E. Hontoria, B. Rodelas, and J. Gonzalez-Lopez. 2007. "Effect of the Concentration of Suspended Solids on the Enzymatic Activities and Biodiversity of a Submerged Membrane Bioreactor for Aerobic Treatment of Domestic Wastewater." Applied Microbiology and Biotechnology 73 (6): 1441-1451. Tryland, I., and L. Fiksdal. 1998. "Enzyme Characteristics of β-d-Galactosidase-and β-d-Glucuronidase-Positive Bacteria and Their Interference in Rapid Methods for Detection of Waterborne Coliforms andEscherichia Coli." Applied and Environmental Microbiology 64 (3): 1018-1023.

Profiling of urinary bile acids in piglets by a combination of enzymatic deconjugation and targeted LC-MRM-MS[S

PubMed Central

Fang, Nianbai; Yu, Shanggong; Adams, Sean H.; Ronis, Martin J. J.; Badger, Thomas M.

2016-01-01

We present a method using a combination of enzymatic deconjugation and targeted LC-multiple reaction monitoring (MRM)-MS analysis for analyzing all common bile acids (BAs) in piglet urine, and in particular, for detecting conjugated BAs either in the absence of their standards, or when present in low concentrations. Initially, before enzymatic deconjugation, 19 unconjugated BAs (FBAs) were detected where the total concentration of the detected FBAs was 9.90 μmol/l. Sixty-seven conjugated BAs were identified by LC-MRM-MS analysis before and after enzymatic deconjugation. Four enzymatic assays were used to deconjugate the BA conjugates. FBAs in urine after cholylglycine hydrolase/sulfatase treatment were 33.40 μmol/l, indicating the urinary BAs were comprised of 29.75% FBAs and 70.25% conjugated BAs in single and multiple conjugated forms. For the conjugates in single form, released FBAs from cholylglycine hydrolase deconjugation indicated that the conjugates with amino acids were 14.54% of urinary BAs, 16.27% glycosidic conjugates were found by β-glucuronidase treatment, and sulfatase with glucuronidase inhibitor treatment liberated FBAs that constituted 16.67% of urinary BAs. Notably, chenodeoxycholic acid (CDCA) was initially detected only in trace amounts in urine, but was found at significant levels after the enzymatic assays above. These results support that CDCA is a precursor of γ-muricholic acid in BA biosynthesis in piglets. PMID:27538824

Design, Synthesis, and Enzymatic Evaluation of Novel ZnO Quantum Dot-Based Assay for Detection of Proteinase 3 Activity.

PubMed

Popow-Stellmaszyk, Jadwiga; Bajorowicz, Beata; Malankowska, Anna; Wysocka, Magdalena; Klimczuk, Tomasz; Zaleska-Medynska, Adriana; Lesner, Adam

2018-05-16

Herein, the synthesis and application of functionalized quantum dot-based protease probes is described. Such probes are composed of nontoxic ZnO nanocrystals decorated by amino groups followed by linker and labeled peptide attachment. Spherical NH 2 -terminated ZnO quantum dots (QDs) with the average size ranging from 4 to 8 nm and strong emission centered at 530 nm were prepared using the sol-gel method. The fluorescence of ZnO QDs was quenched by the BHQ1 moiety present on the N-terminal amino group of the peptide. The enzymatic cleavage of the peptide mediated by the proteinase 3 (PR3) bond resulted in an increase in the QD probe fluorescence. This observation was verified using both model and biological systems; and the picomolar detection limit was found to be more than 30 times lower than that of the previously reported internally quenched peptide (a decrease in detection limit from 43 to 1.3 pmol was observed).

MnOx/C nanocomposite: An insight on high-performance supercapacitor and non-enzymatic hydrogen peroxide detection

NASA Astrophysics Data System (ADS)

Ahuja, Preety; Kumar Ujjain, Sanjeev; Kanojia, Rajni

2017-05-01

In this work, we have used microemulsion method for synthesis of MnOx/C nanocomposite and investigated its electrochemical properties via fabrication of supercapacitor and non-enzymatic hydrogen peroxide (H2O2) sensor. In-situ inclusion of conducting carbon in manganese oxide (MnOx/C) enhances the network conductivity facilitating the charge transfer process which is beneficial for supercapacitor and sensing applications. MnOx/C provides high energy and power density, 31.6 Wh kg-1 and 3.8 kW kg-1 respectively and short relaxation time ∼3 ms for fabricated cell (MnOx/C//MnOx/C) endowing excellent power delivery capacity. Furthermore, MnOx/C as sensor, exhibits excellent catalytic activity toward the oxidation of H2O2 and shows high sensitivity (0.37 mA mM-1 cm-2) with low detection limit (0.5 μM at an S/N of 3). Hence, this study provides new avenue for high performance supercapacitor and H2O2 detection.

Oxygen isotope ratios of PO4: An inorganic indicator of enzymatic activity and P metabolism and a new biomarker in the search for life

PubMed Central

Blake, Ruth E.; Alt, Jeffrey C.; Martini, Anna M.

2001-01-01

The distinctive relations between biological activity and isotopic effect recorded in biomarkers (e.g., carbon and sulfur isotope ratios) have allowed scientists to suggest that life originated on this planet nearly 3.8 billion years ago. The existence of life on other planets may be similarly identified by geochemical biomarkers, including the oxygen isotope ratio of phosphate (δ18Op) presented here. At low near-surface temperatures, the exchange of oxygen isotopes between phosphate and water requires enzymatic catalysis. Because enzymes are indicative of cellular activity, the demonstration of enzyme-catalyzed PO4–H2O exchange is indicative of the presence of life. Results of laboratory experiments are presented that clearly show that δ18OP values of inorganic phosphate can be used to detect enzymatic activity and microbial metabolism of phosphate. Applications of δ18Op as a biomarker are presented for two Earth environments relevant to the search for extraterrestrial life: a shallow groundwater reservoir and a marine hydrothermal vent system. With the development of in situ analytical techniques and future planned sample return strategies, δ18Op may provide an important biosignature of the presence of life in extraterrestrial systems such as that on Mars. PMID:11226207

Oxygen isotope ratios of PO4: an inorganic indicator of enzymatic activity and P metabolism and a new biomarker in the search for life.

PubMed

Blake, R E; Alt, J C; Martini, A M

2001-02-27

The distinctive relations between biological activity and isotopic effect recorded in biomarkers (e.g., carbon and sulfur isotope ratios) have allowed scientists to suggest that life originated on this planet nearly 3.8 billion years ago. The existence of life on other planets may be similarly identified by geochemical biomarkers, including the oxygen isotope ratio of phosphate (delta(18)O(p)) presented here. At low near-surface temperatures, the exchange of oxygen isotopes between phosphate and water requires enzymatic catalysis. Because enzymes are indicative of cellular activity, the demonstration of enzyme-catalyzed PO(4)-H(2)O exchange is indicative of the presence of life. Results of laboratory experiments are presented that clearly show that delta(18)O(P) values of inorganic phosphate can be used to detect enzymatic activity and microbial metabolism of phosphate. Applications of delta(18)O(p) as a biomarker are presented for two Earth environments relevant to the search for extraterrestrial life: a shallow groundwater reservoir and a marine hydrothermal vent system. With the development of in situ analytical techniques and future planned sample return strategies, delta(18)O(p) may provide an important biosignature of the presence of life in extraterrestrial systems such as that on Mars.

Infrared light-assisted preparation of Ag nanoparticles-reduced graphene oxide nanocomposites for non-enzymatic H{sub 2}O{sub 2} sensing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ji, Ye; State Key Laboratory of Transducer Technology, Chinese Academy of Sciences; Zhang, Yong

2015-12-15

Graphical abstract: An infrared light irradiation method has been developed for preparation of AgNPs/rGO nanocomposites for electrochemical detection of H{sub 2}O{sub 2}. - Highlights: • AgNPs/rGO nanocomposites have been prepared by photochemical method. • AgNPs/rGO nanocomposites exhibit good sensing performances for detection of H{sub 2}O{sub 2}. • The present work provides a simple and green method for preparation of rGO-based materials. - Abstract: A green method has been developed for preparation of Ag nanoparticles/reduced graphene oxide (AgNPs/rGO) nanocomposites by infrared light irradiation. The characterizations indicate the successful preparation of AgNPs/rGO nanocomposites. Most importantly, AgNPs/rGO nanocomposites exhibit excellent electrocatalytic activity formore » reduction of H{sub 2}O{sub 2}, leading to a high-performance non-enzymatic H{sub 2}O{sub 2} sensor with linear detection range and detection limit about 0.1 mM to 140 mM (r = 0.9896) and 3.0 μM, respectively.« less

A comparison of the enzymatic properties of three recombinant isoforms of thrombolytic and antibacterial protein--Destabilase-Lysozyme from medicinal leech.

PubMed

Kurdyumov, Alexey S; Manuvera, Valentin A; Baskova, Isolda P; Lazarev, Vassili N

2015-11-21

Destabilase-Lysozyme (mlDL) is a multifunctional i-type enzyme that has been found in the secretions from the salivary glands of medicinal leeches. mlDL has been shown to exhibit isopeptidase, muramidase and antibacterial activity. This enzyme attracts interest because it expresses thrombolytic activity through isopeptidolysis of the ε-(γ-Glu)-Lys bonds that cross-link polypeptide chains in stabilised fibrin. To date, three isoforms of mlDL have been identified. The enzymatic properties of pure mlDL isoforms have not yet been described because only destabilase complexes containing other proteins could be isolated from the salivary gland secretion and because low product yield from the generation of recombinant proteins has made comprehensive testing difficult. In the present study, we optimised the procedures related to the expression, isolation and purification of active mlDL isoforms (mlDL-Ds1, mlDL-Ds2, mlDL-Ds3) using an Escherichia coli expression system, and we detected and compared their muramidase, lytic, isopeptidase and antimicrobial activities. After optimisation, the product yield was 30 mg per litre of culture. The data obtained in our study led to the suggestion that the recombinant mlDL isoforms isolated from inclusion bodies form stable oligomeric complexes. Analyses of the tested activities revealed that all isoforms exhibited almost identical patterns of pH and ionic strength effects on the activities. We determined that mlDL-Ds1, 2, 3 possessed non-enzymatic antibacterial activity independent of their muramidase activity. For the first time, we demonstrated the fibrinolytic activity of the recombinant mlDL and showed that only intact proteins possessed this activity, suggesting their enzymatic nature. The recombinant Destabilase-Lysozyme isoforms obtained in our study may be considered potential thrombolytic agents that act through a mechanism different from that of common thrombolytics.

Utility of Adenosine Monophosphate Detection System for Monitoring the Activities of Diverse Enzyme Reactions.

PubMed

Mondal, Subhanjan; Hsiao, Kevin; Goueli, Said A

Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are dysregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. In this study, we demonstrate the versatility of an enzyme-coupled assay that quantifies the amount of AMP produced by any enzymatic reaction regardless of its substrates. We successfully implemented it to enzyme reactions that use adenosine triphosphate (ATP) as a substrate (aminoacyl tRNA synthetase and DNA ligase) by an elaborate strategy of removing residual ATP and converting AMP produced into ATP; so it can be detected using luciferase/luciferin and generating light. We also tested this assay to measure the activities of AMP-generating enzymes that do not require ATP as substrate, including phosphodiesterases (cyclic adenosine monophosphate) and Escherichia coli DNA ligases (nicotinamide adenine dinucleotide [NAD + ]). In a further elaboration of the AMP-Glo platform, we coupled it to E. coli DNA ligase, enabling measurement of NAD + and enzymes that use NAD + like monoadenosine and polyadenosine diphosphate-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system, we can measure the activities of sulfotransferases. Thus, by utilizing the combinations of biochemical enzymatic conversion of various cellular metabolites to AMP, we were able to demonstrate the versatility of the AMP-Glo assay.

Effect of semi-permeable cover system on the bacterial diversity during sewage sludge composting.

PubMed

Robledo-Mahón, Tatiana; Aranda, Elisabet; Pesciaroli, Chiara; Rodríguez-Calvo, Alfonso; Silva-Castro, Gloria Andrea; González-López, Jesús; Calvo, Concepción

2018-06-01

Sewage sludge composting is a profitable process economically viable and environmentally friendly. In despite of there are several kind of composting types, the use of combined system of semipermeable cover film and aeration air-floor is widely developed at industrial scale. However, the knowledge of the linkages between microbial communities structure, enzyme activities and physico-chemical factors under these conditions it has been poorly explored. Thus, the aim of this study was to investigate the bacterial dynamic and community structure using next generation sequencing coupled to analyses of microbial enzymatic activity and culturable dependent techniques in a full-scale real composting plant. Sewage sludge composting process was conducted using a semi-permeable Gore-tex cover, in combination with an air-insufflation system. The highest values of enzymatic activities such as dehydrogenase, protease and arylsulphatase were detected in the first 5 days of composting; suggesting that during this period of time a greater degrading activity of organic matter took place. Culturable bacteria identified were in agreement with the bacteria found by massive sequencing technologies. The greatest bacterial diversity was detected between days 15 and 30, with Actinomycetales and Bacillales being the predominant orders at the beginning and end of the process. Bacillus was the most representative genus during all the process. A strong correlation between abiotic factors as total organic content and organic matter and enzymatic activities such as dehydrogenase, alkaline phosphatase, and ß-glucosidase activity was found. Bacterial diversity was strongly influenced by the stage of the process, community-structure change was concomitant with a temperature rise, rendering favorable conditions to stimulate microbial activity and facilitate the change in the microbial community linked to the degradation process. Moreover, results obtained confirmed that the use of semipermeable cover in the composting of sewage sludge allow a noticeable reduction in the process-time comparing to conventional open windrows. Copyright © 2018 Elsevier Ltd. All rights reserved.

Neuraminidase as an enzymatic marker for detecting airborne Influenza virus and other viruses.

PubMed

Turgeon, Nathalie; Toulouse, Marie-Josée; Ho, Jim; Li, Dongqing; Duchaine, Caroline

2017-02-01

Little information is available regarding the effectiveness of air samplers to collect viruses and regarding the effects of sampling processes on viral integrity. The neuraminidase enzyme is present on the surface of viruses that are of agricultural and medical importance. It has been demonstrated that viruses carrying this enzyme can be detected using commercial substrates without having to process the sample by methods such as RNA extraction. This project aims at evaluating the effects of 3 aerosol-sampling devices on the neuraminidase enzyme activity of airborne viruses. The purified neuraminidase enzymes from Clostridium perfringens, a strain of Influenza A (H1N1) virus, the FluMist influenza vaccine, and the Newcastle disease virus were used as models. The neuraminidase models were aerosolized in aerosol chambers and sampled with 3 different air samplers (SKC BioSampler, 3-piece cassettes with polycarbonate filters, and Coriolis μ) to assess the effect on neuraminidase enzyme activity. Our results demonstrated that Influenza virus and Newcastle disease virus neuraminidase enzymes are resistant to aerosolization and sampling with all air samplers tested. Moreover, we demonstrated that the enzymatic neuraminidase assay is as sensitive as RT-qPCR for detecting low concentrations of Influenza virus and Newcastle disease virus. Therefore, given the sensitivity of the assay and its compatibility with air sampling methods, viruses carrying the neuraminidase enzyme can be rapidly detected from air samples using neuraminidase activity assay without having to preprocess the samples.

Enzymatic specificity of three ribosome-inactivating proteins against fungal ribosomes, and correlation with antifungal activity.

PubMed

Park, Sang-Wook; Stevens, Noah M; Vivanco, Jorge M

2002-12-01

Ribosome-inactivating proteins (RIPs) are enzymes that cleave a specific adenine base from the highly conserved sarcin/ricin (S/R) loop of the large ribosomal RNA, thus arresting protein synthesis at the translocation step. In the present study, we employed three RIPs to dissect the antifungal activity of RIPs as plant defense proteins. We measured the catalytic activity of RAT (the catalytic A-chain of ricin from Ricinus communis L.), saporin-S6 (from Saponaria officinalis L.), and ME (RIP from Mirabilis expansa R&P) against intact ribosomal substrates isolated from various pathogenic fungi. We further determined the enzymatic specificity of these three RIPs against fungal ribosomes, from Rhizoctonia solani Kuhn, Alternaria solani Sorauer, Trichoderma reesei Simmons and Candida albicans Berkhout, and correlated the data with antifungal activity. RAT showed the strongest toxicity against all tested fungal ribosomes, except for the ribosomes isolated from C. albicans, which were most susceptible to saporin. RAT and saporin showed higher enzymatic activity than ME against ribosomes from all of the fungal species assayed, but did not show detectable antifungal activity. In contrast, ME showed substantial inhibitory activity against fungal growth. Using N-hydroxysuccinimide-fluorescein labeling of RIPs and fluorescence microscopy, we determined that ME was targeted to the surface of fungal cells and transferred into the cells. Thus, ME caused ribosome depurination and subsequent fungal mortality. In contrast, saporin did not interact with fungal cells, correlating with its lack of antifungal activity.

Electrochemical detection of the MT-ND6 gene and its enzymatic digestion: application in human genomic sample.

PubMed

Mazloum-Ardakani, Mohammad; Ahmadi, Roya; Heidari, Mohammad Mehdi; Sheikh-Mohseni, Mohammad Ali

2014-06-15

A simple electrochemical biosensor was developed for the detection of the mitochondrial NADH dehydrogenase 6 gene (MT-ND6) and its enzymatic digestion by BamHI enzyme. This biosensor was fabricated by modification of a glassy carbon electrode with gold nanoparticles (AuNPs/GCE) and a probe oligonucleotide (ssDNA/AuNPs/GCE). The probe, which is a thiolated segment of the MT-ND6 gene, was deposited by self-assembling immobilization on AuNPs/GCE. Two indicators including methylene blue (MB) and neutral red (NR) were used as the electroactive indicators and the electrochemical response of the modified electrode was measured by differential pulse voltammetry. The proposed biosensor can detect the complementary sequences of the MT-ND6 gene. Also the modified electrode was used for the detection of an enzymatic digestion process by BamHI enzyme. The electrochemical biosensor can detect the MT-ND6 gene and its enzymatic digestion in polymerase chain reaction (PCR)-amplified DNA extracted from human blood. Also the biosensor was used directly for detection of the MT-ND6 gene in all of the human genome. Copyright © 2014 Elsevier Inc. All rights reserved.

Origin, enzymatic response and fate of dissolved organic matter during flood and non-flood conditions in a river-floodplain system of the Danube (Austria).

PubMed

Sieczko, Anna; Peduzzi, Peter

2014-01-01

Spectroscopic techniques and extracellular enzyme activity measurements were combined with assessments of bacterial secondary production (BSP) to elucidate flood-pulse-linked differences in carbon (C) sources and related microbial processes in a river-floodplain system near Vienna (Austria). Surface connection with the main channel significantly influenced the quantity and quality of dissolved organic matter (DOM) in floodplain backwaters. The highest values of dissolved organic carbon (DOC) and chromophoric DOM (CDOM) were observed during the peak of the flood, when DOC increased from 1.36 to 4.37 mg l -1 and CDOM from 2.94 to 14.32 m -1 . The flood introduced DOC which consisted of more allochthonously-derived, aromatic compounds. Bacterial enzymatic activity, as a proxy to track the response to changes in DOM, indicated elevated utilization of imported allochthonous material. Based on the enzyme measurements, new parameters were calculated: metabolic effort and enzymatic indices (EEA 1 and EEA 2). During connection, bacterial glucosidase and protease activity were dominant, whereas during disconnected phases a switch to lignin degradation (phenol oxidase) occurred. The enzymatic activity analysis revealed that flooding mobilized reactive DOM, which then supported bacterial metabolism. No significant differences in overall BSP between the two phases were detected, indicating that heterogeneous sources of C sufficiently support BSP. The study demonstrates that floods are important for delivering DOM, which, despite its allochthonous origin, is reactive and can be effectively utilized by aquatic bacteria in this river-floodplain systems. The presence of active floodplains, characterized by hydrological connectivity with the main channel, creates the opportunity to process allochthonous DOC. This has potential consequences for carbon flux, enhancing C sequestration and mineralization processes in this river-floodplain system.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Ning; He, Yuqing; Mao, Xun

This paper presents a novel approach to electrochemically determine enzymatically active PSA using ferrocene-functionalized helix peptide (CHSSLKQK). The principle of electrochemical measurement is based on the specific proteolytic cleavage events of the FC-peptide on the gold electrode surface in the presence of PSA, resulting the change of the current signal of the electrode. The percentage of the decreased current is linear with the concentration of active PSA at the range of 0.5-40 ng/mL with a detection limit of 0.2 ng/mL. The direct transduction of peptide cleavage events into an electrical signal provides a simple, sensitive method for detecting the enzymaticmore » activity of PSA and determining the active PSA concentration.« less

A novel multicomponent redox polymer nanobead based high performance non-enzymatic glucose sensor.

PubMed

Gopalan, A I; Muthuchamy, N; Komathi, S; Lee, K-P

2016-10-15

The fabrication of a highly sensitive electrochemical non-enzymatic glucose sensor based on copper nanoparticles (Cu NPs) dispersed in a graphene (G)-ferrocene (Fc) redox polymer multicomponent nanobead (MCNB) is reported. The preparation of MCNB involves three major steps, namely: i) the preparation of a poly(aniline-co-anthranilic acid)-grafted graphene (G-PANI(COOH), ii) the covalent linking of ferrocene to G-PANI(COOH) via a polyethylene imine (PEI), and iii) the electrodeposition of Cu NPs. The prepared MCNB (designated as G-PANI(COOH)-PEI-Fc/Cu-MCNB), contains a conductive G-PANI(COOH), electron mediating Fc, and electrocatalytic Cu NPs that make it suitable for ultrasensitive non-enzymatic electrochemical sensing. The morphology, structure, and electro activities of MCNB were characterized. Electrochemical measurements showed that the G-PANI(COOH)-PEI-Fc/Cu-MCNB/GCE modified electrode exhibited good electrocatalytic behavior towards the detection of glucose in a wide linear range (0.50 to 15mM), with a low detection limit (0.16mM) and high sensitivity (14.3µAmM(-1)cm(-2)). Besides, the G-PANI(COOH)-PEI-Fc/Cu-MCNB/GCE sensor electrode did not respond to the presence of electroactive interferrants (such as uric acid, ascorbic acid, and dopamine) and saccharides or carbohydrates (fructose, lactose, d-isoascorbic acid, and dextrin), demonstrating its selectivity towards glucose. The fabricated NEG sensor exhibited high precision for measuring glucose in serum samples, with an average RSD of 4.3% and results comparable to those of commercial glucose test strips. This reliability and stability of glucose sensing indicates that G-PANI(COOH)-PEI-Fc/Cu-MCNB/GCE would be a promising material for the non-enzymatic detection of glucose in physiological fluids. Copyright © 2015 Elsevier B.V. All rights reserved.

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP).

PubMed

Ma, Hongyan; Delafield, Daniel G; Wang, Zhe; You, Jianlan; Wu, Si

2017-04-01

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion. Graphical Abstract ᅟ.

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

NASA Astrophysics Data System (ADS)

Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si

2017-04-01

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

Amperometric biosensors for the determination of heavy metals

NASA Astrophysics Data System (ADS)

Compagnone, Dario; Palleschi, Giuseppe; Varallo, Giuseppe; Imperiali, PierLuigi

1995-10-01

A bioelectrochemical method for the determination of heavy metal ions has been developed. This method is based on the inhibition effect of metal ions on the enzymatic activity of oxidase enzymes. The enzymatic activity was determined with an amperometric hydrogen peroxide probe. The inhibition effect on enzymes in solution and covalently immobilized on polymeric supports has been evaluated. Hg(II) was the metal ion that inhibited almost all the enzymes, particularly glycerol-3-P oxidase. Hg(II) was detected in the 0.05/0.5 ppm range with the enzyme in solution. Calibration curves for Hg(II) were also obtained with the other oxidase enzymes in the 0.5/10 ppm range. The other metal ions tested inhibited the enzymes more specifically. The metal ion/enzyme systems which gave the best inhibition were Se(IV)/glutathione oxidase, Ni(II)/sarcosine oxidase, V(V)/glutathione oxidase, Cu(II)/alcohol oxidase from Pichia Pastoris and Cd(II)/D-aminoacid oxidase. All these metal ions were detected in the 0.1/10 ppm range using the enzymes in solution or covalently immobilized.

High-throughput selection for cellulase catalysts using chemical complementation.

PubMed

Peralta-Yahya, Pamela; Carter, Brian T; Lin, Hening; Tao, Haiyan; Cornish, Virginia W

2008-12-24

Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases, however, is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Because of the large number of enzyme variants that selections can now test as compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity.

A High-throughput Selection for Cellulase Catalysts Using Chemical Complementation

PubMed Central

Peralta-Yahya, Pamela; Carter, Brian T.; Lin, Hening; Tao, Haiyan; Cornish, Virginia W.

2010-01-01

Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases however is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Due to the large number of enzyme variants selections can test compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity. PMID:19053460

Development of digestive enzyme activity in larvae of spotted sand bass Paralabrax maculatofasciatus. 1. Biochemical analysis.

PubMed

Alvarez-González, C A; Moyano-López, F J; Civera-Cerecedo, R; Carrasco-Chávez, V; Ortiz-Galindo, J L; Dumas, S

2008-12-01

Spotted sand bass Paralabrax maculatofasciatus is a potential aquaculture species in Northwest Mexico. In the last few years it has been possible to close its life cycle and to develop larviculture technology at on pilot scale using live food, however survival values are low (11%) and improvements in growth and survival requires the study of the morpho-physiological development during the initial ontogeny. In this research digestive activity of several enzymes were evaluated in larvae, from hatching to 30 days after hatching (dah), and in live prey (rotifers and Artemia), by use of biochemical and electrophoretic techniques. This paper, is the first of two parts, and covers only the biochemical analysis. All digestive enzyme activities were detected from mouth opening; however the, maximum activities varied among different digestive enzymes. For alkaline protease and trypsin the maximum activities were detected from 12 to 18 dah. Acid protease activity was observed from day 12 onwards. The other digestive enzymes appear between days 4 and 18 after hatching, with marked fluctuations. These activities indicate the beginning of the juvenile stage and the maturation of the digestive system, in agreement with changes that occur during morpho-physiological development and food changes from rotifers to Artemia. All enzymatic activities were detected in rotifers and Artemia, and their contribution to enhancement the digestion capacity of the larvae appears to be low, but cannot be minimised. We concluded that the enzymatic equipment of P. maculatofasciatus larvae is similar to that of other marine fish species, that it becomes complete between days 12 and 18 after hatching, and that it is totally efficient up to 25 dah.

Enzymatic properties and localization of motopsin (PRSS12), a protease whose absence causes mental retardation.

PubMed

Mitsui, Shinichi; Yamaguchi, Nozomi; Osako, Yoji; Yuri, Kazunari

2007-03-09

Motopsin (PRSS12) is a mosaic protease expressed in the central nervous system. Truncation of the human motopsin gene causes nonsyndromic mental retardation. Understanding the enzymatic properties and localization of motopsin protein in the central nervous system will help identify the molecular mechanism by which the loss of motopsin function causes mental retardation. Recombinant motopsin showed amidolytic activity against the synthetic substrate benzyloxycarbonyl-l-phenylalanyl-l-arginine 4-methyl-coumaryl-7-amide. Motopsin activated the single-chain tissue plasminogen activator precursor and exhibited gelatinolytic activity. This enzymatic activity was inhibited by typical serine protease inhibitors such as aprotinin, leupeptin, and (4-amidinophenyl) methanesulfonyl fluoride. Immunocytochemistry using anti-motopsin IgG revealed that both human and mouse motopsin proteins were distributed in discrete puncta along the dendrites and soma as well as axons in cultured hippocampal neurons. In the limbic system, including the cingulate and hippocampal pyramidal neurons and piriform cortex, high level of motopsin protein was expressed at postnatal day 10, but a very low level at 10-week-old mice. Motopsin and tissue plasminogen activator were co-expressed in the cingulate pyramidal neurons at postnatal day 10 and were distributed along dendrites of cultured pyramidal neurons. In cranial nuclei, a moderate level of motopsin protein was detected independently on the developmental stage. Our results suggest that motopsin has multiple functions, such as axon outgrowth, arranging perineuronal environment, and maintaining neuronal plasticity, partly in coordination with other proteases including tissue plasminogen activator.

Solvent-assisted morphology confinement of a nickel sulfide nanostructure and its application for non-enzymatic glucose sensor.

PubMed

Kim, Soochan; Lee, Sang Ha; Cho, Misuk; Lee, Youngkwan

2016-11-15

Morphology-controlled synthesis of nickel sulfide (Ni3S2) was performed directly on Ni foam using thioacetamide as a sulfur ion source. Various morphologies of nickel sulfide were fabricated using a hydrothermal process by adjusting the solvent composition of ethanol and water. In the water-dominant condition, a dendrite structure was obtained; otherwise, a flaky structure was achieved. A hierarchical cauliflower-like structure was obtained at a solvent mixture composition of 1:1 and was used as non-enzymatic glucose sensor. The hierarchical Ni3S2 electrode showed a high level of electro-catalytic activity toward the oxidation of glucose (16,460μAmM(-1)cm(-2)) over a wide range of detection (0.0005-3mM) and a low detection limit (0.82μM) with excellent selectivity in the presence of several electroactive species. Copyright © 2016 Elsevier B.V. All rights reserved.

Layer-by-Layer Assembly of Glucose Oxidase on Carbon Nanotube Modified Electrodes.

PubMed

Suroviec, Alice H

2017-01-01

The use of enzymatically modified electrodes for the detection of glucose or other non-electrochemically active analytes is becoming increasingly common. Direct heterogeneous electron transfer to glucose oxidase has been shown to be kinetically difficult, which is why electron transfer mediators or indirect detection is usually used for monitoring glucose with electrochemical sensors. It has been found, however, that electrodes modified with single or multi-walled carbon nanotubes (CNTs) demonstrate fast heterogeneous electron transfer kinetics as compared to that found for traditional electrodes. Incorporating CNTs into the assembly of electrochemical glucose sensors, therefore, affords the possibility of facile electron transfer to glucose oxidase, and a more direct determination of glucose. This chapter describes the methods used to use CNTs in a layer-by-layer structure along with glucose oxidase to produce an enzymatically modified electrode with high turnover rates, increased stability and shelf-life.

Flexible, Low-Cost Sensor Based on Electrolyte Gated Carbon Nanotube Field Effect Transistor for Organo-Phosphate Detection

PubMed Central

Bhatt, Vijay Deep; Joshi, Saumya; Becherer, Markus; Lugli, Paolo

2017-01-01

A flexible enzymatic acetylcholinesterase biosensor based on an electrolyte-gated carbon nanotube field effect transistor is demonstrated. The enzyme immobilization is done on a planar gold gate electrode using 3-mercapto propionic acid as the linker molecule. The sensor showed good sensing capability as a sensor for the neurotransmitter acetylcholine, with a sensitivity of 5.7 μA/decade, and demonstrated excellent specificity when tested against interfering analytes present in the body. As the flexible sensor is supposed to suffer mechanical deformations, the endurance of the sensor was measured by putting it under extensive mechanical stress. The enzymatic activity was inhibited by more than 70% when the phosphate-buffered saline (PBS) buffer was spiked with 5 mg/mL malathion (an organophosphate) solution. The biosensor was successfully challenged with tap water and strawberry juice, demonstrating its usefulness as an analytical tool for organophosphate detection. PMID:28524071

Self-powered gustation electronic skin for mimicking taste buds based on piezoelectric-enzymatic reaction coupling process

NASA Astrophysics Data System (ADS)

Zhao, Tianming; Fu, Yongming; He, Haoxuan; Dong, Chuanyi; Zhang, Linlin; Zeng, Hui; Xing, Lili; Xue, Xinyu

2018-02-01

A new self-powered wearable gustation electronic skin for mimicking taste buds has been realized based on enzyme-modified/ZnO nanowire arrays on patterned-electrode flexible substrate. The e-skin can actively taste beverages or fruits without any external electric power. Through the piezoelectric-enzymatic reaction coupling effect, the nanowires can harvest the mechanical energy of body movement and output piezoelectric signal. The piezoelectric output is significantly dependent on the concentration of target analyte. The response for detecting 2 × 10-2 M ascorbic acid (ascorbate acid oxidase@ZnO) is up to 171.747, and the selectivity is high. The response for detecting 50% alcohol (alcohol oxidase@ZnO) is up to 45.867. Our results provide a new research direction for the development of multifunctional e-skin and expand the study scope for self-powered bionic systems.

Ag electrode modified with polyhexamethylene biguanide stabilized silver nanoparticles: a new type of SERS substrates for detection of enzymatically generated thiocholine

NASA Astrophysics Data System (ADS)

Tepanov, A. A.; Nechaeva, N. L.; Prokopkina, T. A.; Kudrinskiy, A. A.; Kurochkin, I. N.; Lisichkin, G. V.

2015-11-01

The detection of thiocholine is one of the most widespread techniques for estimation of the cholinesterase activity - acetylcholinesterase and butyrylcholinesterase. Both cholinesterases can be inhibited by organophosphates and carbamates and accordingly can be considered for estimation of these pollutants in the environment. In the current work, SERS spectroscopy was applied for the thiocholine detection. The Ag electrodes modified with silver nanoparticles stabilized by polyhexamethylene biguanide were for the first time suggested as SERS-substrates for that purpose. Such electrodes can be applicable for SERS detection of submicromolar concentrations of thiocholine.

Profiling of urinary bile acids in piglets by a combination of enzymatic deconjugation and targeted LC-MRM-MS.

PubMed

Fang, Nianbai; Yu, Shanggong; Adams, Sean H; Ronis, Martin J J; Badger, Thomas M

2016-10-01

We present a method using a combination of enzymatic deconjugation and targeted LC-multiple reaction monitoring (MRM)-MS analysis for analyzing all common bile acids (BAs) in piglet urine, and in particular, for detecting conjugated BAs either in the absence of their standards, or when present in low concentrations. Initially, before enzymatic deconjugation, 19 unconjugated BAs (FBAs) were detected where the total concentration of the detected FBAs was 9.90 μmol/l. Sixty-seven conjugated BAs were identified by LC-MRM-MS analysis before and after enzymatic deconjugation. Four enzymatic assays were used to deconjugate the BA conjugates. FBAs in urine after cholylglycine hydrolase/sulfatase treatment were 33.40 μmol/l, indicating the urinary BAs were comprised of 29.75% FBAs and 70.25% conjugated BAs in single and multiple conjugated forms. For the conjugates in single form, released FBAs from cholylglycine hydrolase deconjugation indicated that the conjugates with amino acids were 14.54% of urinary BAs, 16.27% glycosidic conjugates were found by β-glucuronidase treatment, and sulfatase with glucuronidase inhibitor treatment liberated FBAs that constituted 16.67% of urinary BAs. Notably, chenodeoxycholic acid (CDCA) was initially detected only in trace amounts in urine, but was found at significant levels after the enzymatic assays above. These results support that CDCA is a precursor of γ-muricholic acid in BA biosynthesis in piglets. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

INACTIVATION OF E. COLI PYRUVATE FORMATE-LYASE: ROLE OF AdhE AND SMALL MOLECULES

PubMed Central

Nnyepi, Mbako R.; Peng, Yi; Broderick, Joan B.

2007-01-01

E. coli AdhE has been reported to harbor three distinct enzymatic activities: alcohol dehydrogenase, acetaldehyde-CoA dehydrogenase, and pyruvate formate-lyase (PFL) deactivase. Herein we report on the cloning, expression, and purification of E. coli AdhE, and the re-investigation of its purported enzymatic activities. While both the alcohol dehydrogenase and acetaldehyde-CoA dehydrogenase activities were readily detectible, we were unable to obtain any evidence for catalytic deactivation of PFL by AdhE, regardless of whether the reported cofactors for deactivation (Fe(II), NAD, and CoA) were present. Our results demonstrate that AdhE is not a PFL deactivating enzyme. We have also examined the potential for deactivation of active PFL by small-molecule thiols. Both β-mercaptoethanol and dithiothreitol deactivate PFL efficiently, with the former providing quite rapid deactivation. PFL deactivated by these thiols can be reactivated, suggesting that this deactivation is non-destructive transfer of an H atom equivalent to quench the glycyl radical. PMID:17280641

Polyphenol oxidase and peroxidase expression in four pineapple varieties (Ananas comosus L.) after a chilling injury.

PubMed

Raimbault, Astrid-Kim; Marie-Alphonsine, Paul-Alex; Horry, Jean-Pierre; Francois-Haugrin, Madlyn; Romuald, Karell; Soler, Alain

2011-01-12

Pineapple internal browning (IB) is a chilling injury that produces enzymatic browning associated with flesh translucency. Pineapple biodiversity allowed the investigation of how polyphenol oxidase (PPO) and peroxidase (POD) activities with their different isoforms are involved in the IB mechanism. Fruits of four varieties that expressed IB symptoms differently, Smooth Cayenne (SCay) and the hybrids MD2, Flhoran 41 (Flh 41), and Flhoran 53 (Flh 53), were stressed by cold. The susceptible varieties showed classical brown spots but different patterns of IB, whereas MD2 and controls showed no IB. Enzymatic activities were measured on fruit protein extracts and PPO and POD isoforms separated on mini-gels (PhastSystem). Only PPO activity was significantly enhanced in the presence of IB. Up to six PPO isoforms were identified in the susceptible varieties. PPO was barely detectable in the nonsusceptible variety MD2 and in controls. The number of PPO isoforms and the total PPO activity after chilling are varietal characteristics.

Dual functional rhodium oxide nanocorals enabled sensor for both non-enzymatic glucose and solid-state pH sensing.

PubMed

Dong, Qiuchen; Huang, Yikun; Song, Donghui; Wu, Huixiang; Cao, Fei; Lei, Yu

2018-07-30

Both pH-sensitive and glucose-responsive rhodium oxide nanocorals (Rh 2 O 3 NCs) were synthesized through electrospinning followed by high-temperature calcination. The as-prepared Rh 2 O 3 NCs were systematically characterized using various advanced techniques including scanning electron microscopy, X-ray powder diffraction and Raman spectroscopy, and then employed as a dual functional nanomaterial to fabricate a dual sensor for both non-enzymatic glucose sensing and solid-state pH monitoring. The sensing performance of the Rh 2 O 3 NCs based dual sensor toward pH and glucose was evaluated using open circuit potential, cyclic voltammetry and amperometric techniques, respectively. The results show that the as-prepared Rh 2 O 3 NCs not only maintain accurate and reversible pH sensitivity of Rh 2 O 3 , but also demonstrate a good electrocatalytic activity toward glucose oxidation in alkaline medium with a sensitivity of 11.46 μA mM -1 cm -2 , a limit of detection of 3.1 μM (S/N = 3), and a reasonable selectivity against various interferents in non-enzymatic glucose detection. Its accuracy in determining glucose in human serum samples was further demonstrated. These features indicate that the as-prepared Rh 2 O 3 NCs hold great promise as a dual-functional sensing material in the development of a high-performance sensor forManjakkal both solid-state pH and non-enzymatic glucose sensing. Copyright © 2018 Elsevier B.V. All rights reserved.

Plasmodium falciparum SERA5 plays a non-enzymatic role in the malarial asexual blood-stage lifecycle

PubMed Central

Stallmach, Robert; Kavishwar, Manoli; Withers-Martinez, Chrislaine; Hackett, Fiona; Collins, Christine R; Howell, Steven A; Yeoh, Sharon; Knuepfer, Ellen; Atid, Avshalom J; Holder, Anthony A; Blackman, Michael J

2015-01-01

The malaria parasite Plasmodium falciparum replicates in an intraerythrocytic parasitophorous vacuole (PV). The most abundant P. falciparum PV protein, called SERA5, is essential in blood stages and possesses a papain-like domain, prompting speculation that it functions as a proteolytic enzyme. Unusually however, SERA5 possesses a Ser residue (Ser596) at the position of the canonical catalytic Cys of papain-like proteases, and the function of SERA5 or whether it performs an enzymatic role is unknown. In this study, we failed to detect proteolytic activity associated with the Ser596-containing parasite-derived or recombinant protein. However, substitution of Ser596 with a Cys residue produced an active recombinant enzyme with characteristics of a cysteine protease, demonstrating that SERA5 can bind peptides. Using targeted homologous recombination in P. falciparum, we substituted Ser596 with Ala with no phenotypic consequences, proving that SERA5 does not perform an essential enzymatic role in the parasite. We could also replace an internal segment of SERA5 with an affinity-purification tag. In contrast, using almost identical targeting constructs, we could not truncate or C-terminally tag the SERA5 gene, or replace Ser596 with a bulky Arg residue. Our findings show that SERA5 plays an indispensable but non-enzymatic role in the P. falciparum blood-stage life cycle. PMID:25599609

Release of Active Peptidyl Arginine Deiminases by Neutrophils Can Explain Production of Extracellular Citrullinated Autoantigens in Rheumatoid Arthritis Synovial Fluid

PubMed Central

Spengler, Julia; Lugonja, Božo; Jimmy Ytterberg, A.; Zubarev, Roman A.; Creese, Andrew J.; Pearson, Mark J.; Grant, Melissa M.; Milward, Michael; Lundberg, Karin; Buckley, Christopher D.; Filer, Andrew; Raza, Karim; Cooper, Paul R.; Chapple, Iain L.

2015-01-01

Objective In the majority of patients with rheumatoid arthritis (RA), antibodies specifically recognize citrullinated autoantigens that are generated by peptidylarginine deiminases (PADs). Neutrophils express high levels of PAD and accumulate in the synovial fluid (SF) of RA patients during disease flares. This study was undertaken to test the hypothesis that neutrophil cell death, induced by either NETosis (extrusion of genomic DNA–protein complexes known as neutrophil extracellular traps [NETs]) or necrosis, can contribute to production of autoantigens in the inflamed joint. Methods Extracellular DNA was quantified in the SF of patients with RA, patients with osteoarthritis (OA), and patients with psoriatic arthritis (PsA). Release of PAD from neutrophils was investigated by Western blotting, mass spectrometry, immunofluorescence staining, and PAD activity assays. PAD2 and PAD4 protein expression, as well as PAD enzymatic activity, were assessed in the SF of patients with RA and those with OA. Results Extracellular DNA was detected at significantly higher levels in RA SF than in OA SF (P < 0.001) or PsA SF (P < 0.05), and its expression levels correlated with neutrophil concentrations and PAD activity in RA SF. Necrotic neutrophils released less soluble extracellular DNA compared to NETotic cells in vitro (P < 0.05). Higher PAD activity was detected in RA SF than in OA SF (P < 0.05). The citrullinated proteins PAD2 and PAD4 were found attached to NETs and also freely diffused in the supernatant. PAD enzymatic activity was detected in supernatants of neutrophils undergoing either NETosis or necrosis. Conclusion Release of active PAD isoforms into the SF by neutrophil cell death is a plausible explanation for the generation of extracellular autoantigens in RA. PMID:26245941

Solid-State Treatment of Castor Cake Employing the Enzymatic Cocktail Produced from Pleurotus djamor Fungi.

PubMed

Sánchez-Cantú, Manuel; Ortiz-Moreno, Liliana; Ramos-Cassellis, María E; Marín-Castro, Marco; De la Cerna-Hernández, C

2018-06-01

In this work, the enzymatic cocktail produced by Pleurotus djamor fungi extracted at pH of 4.8 and 5.3 was employed for castor cake solid-state treatment. Proximal, X-ray powder diffraction and scanning electron microscopy analysis of the pristine castor cake were carried out. First, Pleurotus djamor stain was inoculated in castor cake for the enzymatic production and the enzymatic activity was determined. The maximum enzymatic activity was identified at days 14 (65.9 UI/gss) and 11 (140.3 UI/gss) for the enzymatic cocktail obtained at pH 5.3 and 4.8, respectively. Then, the enzymatic cocktail obtained at the highest enzymatic activity days was employed directly over castor cake. Lignin was degraded throughout incubation time achieving a 47 and 45% decrease for the cocktail produced at pH 4.8 and 5.3, correspondingly. These results were corroborated by the SEM and XRD analysis where a higher porosity and xylan degradation were perceived throughout the enzymatic treatment.

Cobalt oxide acicular nanorods with high sensitivity for the non-enzymatic detection of glucose.

PubMed

Kung, Chung-Wei; Lin, Chia-Yu; Lai, Yi-Hsuan; Vittal, R; Ho, Kuo-Chuan

2011-09-15

Acicular cobalt oxide nanorods (CoONRs) were prepared for the non-enzymatic detection of glucose, first by directly growing layered cobalt carbonate hydroxide (LCCH) on a conducting fluorine-doped tin oxide (FTO) substrate using a simple chemical bath deposition (CBD) technique and then by transforming the LCCH into CoONRs through pyrolysis. The composition and grain size of the films of LCCH and CoONRs were verified by X-ray diffraction (XRD); their morphologies were examined by scanning electron microscopic (SEM) and transmission electron microscopic (TEM) images. CoONRs showed high electrocatalytic activity for the electro-oxidation of glucose in alkaline media, and the activity was strongly influenced by NaOH concentration, annealing temperature of CoONRs, and thickness of CoONRs film. The pertinent sensor could be successfully used for the quantification of glucose by amperometric method. The sensing parameters include wide linear range up to 3.5 mM, a high sensitivity of 571.8 μA/(cm(2) mM), and a remarkable low detection limit of 0.058 μM. The CoONRs modified electrode exhibited a high selectivity for glucose in human serum, against ascorbic acid, uric acid, and acetaminophen. Copyright © 2011 Elsevier B.V. All rights reserved.

A liquid crystal-based sensor for the simple and sensitive detection of cellulase and cysteine.

PubMed

Wang, Yi; Hu, Qiongzheng; Tian, Tongtong; Gao, Yan'an; Yu, Li

2016-11-01

A liquid crystal (LC)-based sensor, which is capable of monitoring enzymatic activity at the aqueous/LC interface and detecting cellulase and cysteine (Cys), was herein reported. When functionalized with a surfactant, dodecyl β-d-glucopyranoside, the 4-cyano-4'-pentylbiphenyl (5CB) displays a dark-to-bright transition in the optical appearance for cellulase. We attribute this change to the orientational transition of LCs, as a result of enzymatic hydrolysis between cellulase and surfactant. Furthermore, by adding cellulase and Cu(2+), our surfactant-LCs system performs an interesting ability to detect Cys, even though Cys could not interact with surfactant or LC directly. Alternatively, through the strong binding between Cys and Cu(2+), cellulase was able to hydrolyze surfactant in the presence of Cu(2+), leading to the transition of LCs from dark to bright. The detection limit of the LC sensor was around 1×10(-5)mg/mL and 82.5μM for cellulase and Cys, respectively. The LC-based sensor may contribute to the development of low-cost, expedient, and label-free detection for cellulase and Cys and the design strategy may also provide a novel way for detecting multiple analytes. Copyright © 2016 Elsevier B.V. All rights reserved.

Functional and Structural Characterization of PaeM, a Colicin M-like Bacteriocin Produced by Pseudomonas aeruginosa *

PubMed Central

Barreteau, Hélène; Tiouajni, Mounira; Graille, Marc; Josseaume, Nathalie; Bouhss, Ahmed; Patin, Delphine; Blanot, Didier; Fourgeaud, Martine; Mainardi, Jean-Luc; Arthur, Michel; van Tilbeurgh, Herman; Mengin-Lecreulx, Dominique; Touzé, Thierry

2012-01-01

Colicin M (ColM) is the only enzymatic colicin reported to date that inhibits cell wall peptidoglycan biosynthesis. It catalyzes the specific degradation of the lipid intermediates involved in this pathway, thereby provoking lysis of susceptible Escherichia coli cells. A gene encoding a homologue of ColM was detected within the exoU-containing genomic island A carried by certain pathogenic Pseudomonas aeruginosa strains. This bacteriocin (pyocin) that we have named PaeM was crystallized, and its structure with and without an Mg2+ ion bound was solved. In parallel, site-directed mutagenesis of conserved PaeM residues from the C-terminal domain was performed, confirming their essentiality for the protein activity both in vitro (lipid II-degrading activity) and in vivo (cytotoxicity against a susceptible P. aeruginosa strain). Although PaeM is structurally similar to ColM, the conformation of their active sites differs radically; in PaeM, residues essential for enzymatic activity and cytotoxicity converge toward a same pocket, whereas in ColM they are spread along a particularly elongated active site. We have also isolated a minimal domain corresponding to the C-terminal half of the PaeM protein and exhibiting a 70-fold higher enzymatic activity as compared with the full-length protein. This isolated domain of the PaeM bacteriocin was further shown to kill E. coli cells when addressed to the periplasm of these bacteria. PMID:22977250

Procongopain from Trypanosoma congolense is processed at basic pH: an unusual feature among cathepsin L-like cysteine proteases.

PubMed

Serveau, Carole; Boulangé, Alain; Lecaille, Fabien; Gauthier, Francis; Authié, Edith; Lalmanach, Gilles

2003-06-01

Congopain, the major cysteine protease from Trypanosoma congolense, is synthesized as an inactive zymogen, and further converted into its active form after removal of the proregion, most probably via an autocatalytic mechanism. Processing of recombinant procongopain occurs via an apparent one-step or a multistep mechanism depending on the ionic strength. The auto-activation is pH-dependent, with an optimum at pH 4.0, and no activation observed at pH 6.0. After addition of dextran sulfate (10 microg/ml), an approx. 20-fold increase of processing (expressed as enzymatic activity) is observed. Furthermore, in the presence of dextran sulfate, procongopain can be processed at pH 8.0, an unusual feature among papain-like enzymes. Detection of procongopain and trypanosomal enzymatic activity in the plasma of T. congolense-infected cattle, together with the capacity of procongopain to be activated at weakly basic pH, suggest that procongopain may be extracellularly processed in the presence of blood vessel glycosaminoglycans, supporting the hypothesis that congopain acts as a pathogenic factor in host-parasite relationships.

White grape pomace extracts, obtained by a sequential enzymatic plus ethanol-based extraction, exert antioxidant, anti-tyrosinase and anti-inflammatory activities.

PubMed

Ferri, Maura; Rondini, Greta; Calabretta, Maria Maddalena; Michelini, Elisa; Vallini, Veronica; Fava, Fabio; Roda, Aldo; Minnucci, Giordano; Tassoni, Annalisa

2017-10-25

The present work aimed at optimizing a two-step enzymatic plus solvent-based process for the recovery of bioactive compounds from white grape (Vitis vinifera L., mix of Trebbiano and Verdicchio cultivars) pomace, the winemaking primary by-product. Phenolic compounds solubilised by water enzyme-assisted and ethanol-based extractions of wet (WP) and dried (DP) pomace were characterised for composition and tested for antioxidant, anti-tyrosinase and anti-inflammatory bioactivities. Ethanol treatment led to higher phenol yields than water extraction, while DP samples showed the highest capacity of releasing polyphenols, most probably as a positive consequence of the pomace drying process. Different compositions and bioactivities were observed between water and ethanol extracts and among different treatments and for the first time the anti-tyrosinase activity of V. vinifera pomace extracts, was here reported. Enzymatic treatments did not significantly improve the total amount of solubilised compounds; Celluclast in DP led to the recovery of extracts enriched in specific compounds, when compared to control. The best extracts (enzymatic plus ethanol treatment total levels) were obtained from DP showing significantly higher amounts of polyphenols, flavonoids, flavanols and tannins and exerted higher antioxidant and anti-tyrosinase activities than WP total extracts. Conversely, anti-inflammatory capacity was only detected in water (with and without enzyme) extracts, with WP samples showing on average a higher activity than DP. The present findings demonstrate that white grape pomace constitute a sustainable source for the extraction of phytochemicals that might be exploited as functional ingredients in the food, nutraceutical, pharmaceutical or cosmetic industries. Copyright © 2017 Elsevier B.V. All rights reserved.

Time Resolved Microfluorescence In Biomedical Diagnosis

NASA Astrophysics Data System (ADS)

Schneckenburger, Herbert

1985-12-01

A measuring system combining subnanosecond laser-induced fluorescence with microscopic signal detection was installed and used for diverse projects in the biomedical and environmental fields. These projects range from tumor diagnosis and enzymatic analysis to measurements of the activity of methanogenic bacteria, which affect biogas production and waste water cleaning. The advantages of this method and its practical applicability are discussed.

Electrochemical enzymatic biosensors using carbon nanofiber nanoelectrode arrays

NASA Astrophysics Data System (ADS)

Li, Jun; Li, Yi-fen; Swisher, Luxi Z.; Syed, Lateef U.; Prior, Allan M.; Nguyen, Thu A.; Hua, Duy H.

2012-10-01

The reduction of electrode size down to nanometers could dramatically enhance detection sensitivity and temporal resolution. Nanoelectrode arrays (NEAs) are of particular interest for ultrasensitive biosensors. Here we report the study of two types of biosensors for measuring enzyme activities using NEAs fabricated with vertically aligned carbon nanofibers (VACNFs). VACNFs of ~100 nm in average diameter and 3-5 μm in length were grown on conductive substrates as uniform vertical arrays which were then encapsulated in SiO2 matrix leaving only the tips exposed. We demonstrate that such VACNF NEAs can be used in profiling enzyme activities through monitoring the change in electrochemical signals induced by enzymatic reactions to the peptides attached to the VACNF tip. The cleavage of the tetrapeptide with a ferrocene tag by a cancerrelated protease (legumain) was monitored with AC voltammetry. Real-time electrochemical impedance spectroscopy (REIS) was used for fast label-free detection of two reversible processes, i.e. phosphorylation by c-Src tyrosine kinase and dephosphorylation by protein tyrosine phosphatase 1B (PTP1B). The REIS data of phosphorylation were slow and unreliable, but those of dephosphorylation showed large and fast exponential decay due to much higher activity of phosphatase PTP1B. The kinetic data were analyzed with a heterogeneous Michaelis-Menten model to derive the "specificity constant" kcat/Km, which is 8.2x103 M-1s-1 for legumain and (2.1 ± 0.1) x 107 M-1s-1 for phosphatase (PTP1B), well consistent with literature. It is promising to develop VACNF NEA based electrochemical enzymatic biosensors as portable multiplex electronic techniques for rapid cancer diagnosis and treatment monitoring.

Enzymatic activity inside and outside of water-stable aggregates in soils under different land use

NASA Astrophysics Data System (ADS)

Garbuz, S. A.; Yaroslavtseva, N. V.; Kholodov, V. A.

2016-03-01

A method is presented for assessing the distribution of enzymatic activity inside and outside of water-stable aggregates. Two samples of water-stable aggregates >1 mm have been isolated from dry aggregates of 1-2 mm. To determine the enzymatic activity, a substrate has been added to one of the samples without disaggregation; the other sample has been preliminarily disaggregated. Enzymatic activity within waterstable aggregates has been assessed from the difference between the obtained results under the supposition that the penetration of substrate within the water-saturated aggregates is hampered, and enzymatic reactions occur only at the periphery. The levels and distributions of enzymatic (peroxidase, polyphenol oxidase, and catalase) activities in water-stable aggregates of soddy-podzolic soils under forest and plowland and typical chernozems of long-term field experiments have been studied. The peroxidase, polyphenol oxidase, and catalase activities of water-stable aggregates vary from 6 to 23, from 7 to 30, and from 5 to 7 mmol/(g h), respectively. The ratio between the enzymatic activities inside and outside of soil aggregates showed a higher dependence on soil type and land use, as well as on the input of organic matter and the structural state, than the general activity level in water-stable aggregates.

A Cuprous Oxide Thin Film Non-Enzymatic Glucose Sensor Using Differential Pulse Voltammetry and Other Voltammetry Methods and a Comparison to Different Thin Film Electrodes on the Detection of Glucose in an Alkaline Solution

PubMed Central

Molazemhosseini, Alireza; Liu, Chung Chiun

2018-01-01

A cuprous oxide (Cu2O) thin layer served as the base for a non-enzymatic glucose sensor in an alkaline medium, 0.1 NaOH solution, with a linear range of 50–200 mg/dL using differential pulse voltammetry (DPV) measurement. An X-ray photoelectron spectroscopy (XPS) study confirmed the formation of the cuprous oxide layer on the thin gold film sensor prototype. Quantitative detection of glucose in both phosphate-buffered saline (PBS) and undiluted human serum was carried out. Neither ascorbic acid nor uric acid, even at a relatively high concentration level (100 mg/dL in serum), interfered with the glucose detection, demonstrating the excellent selectivity of this non-enzymatic cuprous oxide thin layer-based glucose sensor. Chronoamperometry and single potential amperometric voltammetry were used to verify the measurements obtained by DPV, and the positive results validated that the detection of glucose in a 0.1 M NaOH alkaline medium by DPV measurement was effective. Nickel, platinum, and copper are commonly used metals for non-enzymatic glucose detection. The performance of these metal-based sensors for glucose detection using DPV were also evaluated. The cuprous oxide (Cu2O) thin layer-based sensor showed the best sensitivity for glucose detection among the sensors evaluated. PMID:29316652

Hierarchical CuInS2-based heterostructure: Application for photocathodic bioanalysis of sarcosine.

PubMed

Jiang, Xin-Yuan; Zhang, Ling; Liu, Yi-Li; Yu, Xiao-Dong; Liang, Yan-Yu; Qu, Peng; Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

2018-06-01

In this study, on the basis of hierarchical CuInS 2 -based heterostructure, a novel cathodic photoelectrochemical (PEC) enzymatic bioanalysis of the sarcosine detection was reported. Specifically, heterostructured CuInS 2 /NiO/ITO photocathode was prepared and sarcosine oxidases (SOx) were integrated for the construction of the enzymatic biosensor. In the bioanalysis, the O 2 -dependent suppression of the cathodic photocurrent can be observed due to the competition between the as-fabricated O 2 -sensitive photocathode and the SOx-catalytic event toward O 2 reduction. Based on the sarcosine-controlled O 2 concentration, a novel photocathodic enzymatic biosensor could be realized for the sensitive and specific sarcosine detection. This work manifested the great potential of CuInS 2 -based heterostructure as a novel platform for future PEC bioanalytical development and also a PEC method for sarcosine detection, which could be easily extended to numerous other enzymatic systems and to our knowledge has not been reported. This work is expected to stimulate more interest in the design and implementation of numerous CuInS 2 -based heterostructured photocathodic enzymatic sensing. Copyright © 2018 Elsevier B.V. All rights reserved.

Detection of carbapenemases and other mechanisms of enzymatic resistance to β-lactams in Enterobacteriaceae with diminished susceptibility to carbapenems in a tertiary care hospital.

PubMed

Gómara, Marta; López-Calleja, Ana Isabel; Iglesia, Berta María Pilar Vela; Cerón, Isabel Ferrer; López, Antonio Rezusta; Pinilla, María José Revillo

2018-05-01

Our objective was to characterize the enzymatic β-lactam resistance in clinical Enterobacteriaceae isolates with diminished susceptibility to carbapenems from 2013 to 2014 at Hospital Universitario Miguel Servet. A total of 63 clinical isolates were analyzed for the presence of carbapenemases (KPC, OXA-48 and MBL), ESBLs and AmpC enzymes by combined disk methods and PCR detection of carbapenemase-encoding and beta-lactamase-encoding genes. Fifteen isolates had a phenotypic test compatible with carbapenemase production; two of these were confirmed by PCR as OXA-48 producers. ESBL detection was positive in 27 isolates (43%); plasmid-mediated AmpC was detected in nine isolates (14.2%) and derepressed AmpC β-lactamase was present in 18 isolates (28%). During the study period, the decreased susceptibility to carbapenems in Enterobacteriaceae in our area was not due to true carbapenemases but rather to β-lactamase activity (82.5% were ESBL or AmpC producers), probably in combination with decreased permeability of the outer membrane. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

N-doped graphene-carbon nanotube hybrid networks attaching with gold nanoparticles for glucose non-enzymatic sensor.

PubMed

Jeong, Hun; Nguyen, Dang Mao; Lee, Min Sang; Kim, Hong Gun; Ko, Sang Cheol; Kwac, Lee Ku

2018-09-01

Herein, we successfully developed a novel three dimensional (3D) opened networks based on nitrogen doped graphene‑carbon nanotubes attaching with gold nanoparticles (N-GR-CNTs/AuNPs) to apply for non-enzymatic glucose determination. It was demonstrated that the N-GR-CNTs/AuNPs modified electrode exhibited good behavior for glucose detection with a long linear range of 2 μM to 19.6 mM, high sensitivity of 0.9824 μA·mM -1 ·cm -2 , low detection limit of 500 nM, and negligible interference effect. The high performance of the N-GR-CNTs/AuNPs based sensor was assumed due to the outstanding catalytic activity of AuNPs well dispersing on N-GR-CNTs networks, which exhibited as a perfect supporting scaffold due to the enhanced electrical conductivity and large surface area. The obtained results indicated that the N-GR-CNTs/AuNPs hybrid is highly promising for sensitive and selective detection of glucose in sensor application. Copyright © 2018 Elsevier B.V. All rights reserved.

The effects of xylitol and sorbitol on lysozyme- and peroxidase-related enzymatic and candidacidal activities.

PubMed

Kim, Bum-Soo; Chang, Ji-Youn; Kim, Yoon-Young; Kho, Hong-Seop

2015-07-01

To investigate whether xylitol and sorbitol affect enzymatic and candidacidal activities of lysozyme, the peroxidase system, and the glucose oxidase-mediated peroxidase system. Xylitol and sorbitol were added to hen egg-white lysozyme, bovine lactoperoxidase, glucose oxidase-mediated peroxidase, and whole saliva in solution and on hydroxyapatite surfaces. The enzymatic activities of lysozyme, peroxidase, and glucose oxidase-mediated peroxidase were determined by the turbidimetric method, the NbsSCN assay, and production of oxidized o-dianisidine, respectively. Candidacidal activities were determined by comparing colony forming units using Candida albicans ATCC strains 10231, 11006, and 18804. While xylitol and sorbitol did not affect the enzymatic activity of hen egg-white lysozyme both in solution and on hydroxyapatite surfaces, they did inhibit the enzymatic activity of salivary lysozyme significantly in solution, but not on the surfaces. Xylitol and sorbitol enhanced the enzymatic activities of both bovine lactoperoxidase and salivary peroxidase significantly in a dose-dependent manner in solution, but not on the surfaces. Sorbitol, but not xylitol, inhibited the enzymatic activity of glucose oxidase-mediated peroxidase significantly. Both xylitol and sorbitol did not affect candidacidal activities of hen egg-white lysozyme, the bovine lactoperoxidase system, or the glucose oxidase-mediated bovine lactoperoxidase system. Xylitol and sorbitol inhibited salivary lysozyme activity, but enhanced both bovine lactoperoxidase and salivary peroxidase activities significantly in solution. Xylitol and sorbitol did not augment lysozyme- and peroxidase-related candidacidal activities. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enzymatic electrochemical detection coupled to multivariate calibration for the determination of phenolic compounds in environmental samples.

PubMed

Hernandez, Silvia R; Kergaravat, Silvina V; Pividori, Maria Isabel

2013-03-15

An approach based on the electrochemical detection of the horseradish peroxidase enzymatic reaction by means of square wave voltammetry was developed for the determination of phenolic compounds in environmental samples. First, a systematic optimization procedure of three factors involved in the enzymatic reaction was carried out using response surface methodology through a central composite design. Second, the enzymatic electrochemical detection coupled with a multivariate calibration method based in the partial least-squares technique was optimized for the determination of a mixture of five phenolic compounds, i.e. phenol, p-aminophenol, p-chlorophenol, hydroquinone and pyrocatechol. The calibration and validation sets were built and assessed. In the calibration model, the LODs for phenolic compounds oscillated from 0.6 to 1.4 × 10(-6) mol L(-1). Recoveries for prediction samples were higher than 85%. These compounds were analyzed simultaneously in spiked samples and in water samples collected close to tanneries and landfills. Published by Elsevier B.V.

Enzymatic Activity of Free-Prostate-Specific Antigen (f-PSA) Is Not Required for Some of its Physiological Activities

PubMed Central

Chadha, Kailash C.; Nair, Bindukumar B.; Chakravarthi, Srikant; Zhou, Rita; Godoy, Alejandro; Mohler, James L.; Aalinkeel, Ravikumar; Schwartz, Stanley A.; Smith, Gary J.

2015-01-01

BACKGROUND Prostate specific antigen (PSA) is a well known biomarker for early diagnosis and management of prostate cancer. Furthermore, PSA has been documented to have anti-angiogenic and anti-tumorigenic activities in both in vitro and in vivo studies. However, little is known about the molecular mechanism(s) involved in regulation of these processes, in particular the role of the serine-protease enzymatic activity of PSA. METHODS Enzymatic activity of PSA isolated directly from seminal plasma was inhibited specifically (>95%) by incubation with zinc2+. Human umbilical vein endothelial cells (HUVEC) were utilized to compare/contrast the physiological effects of enzymatically active versus inactive PSA. RESULTS Equimolar concentrations of enzymatically active PSA and PSA enzymatically inactivated by incubation with Zn2+ had similar physiological effects on HUVEC, including inhibiting the gene expression of pro-angiogenic growth factors, like VEGF and bFGF, and up-regulation of expression of the anti-angiogenic growth factor IFN-γ; suppression of mRNA expression for markers of blood vessel development, like FAK, FLT, KDR, TWIST-1; P-38; inhibition of endothelial tube formation in the in vitro Matrigel Tube Formation Assay; and inhibition of endothelial cell invasion and migration properties. DISCUSSION Our data provides compelling evidence that the transcriptional regulatory and the anti-angiogenic activities of human PSA are independent of the innate enzymatic activity PMID:21446007

Identification of a preferred substrate peptide for transglutaminase 3 and detection of in situ activity in skin and hair follicles.

PubMed

Yamane, Asaka; Fukui, Mina; Sugimura, Yoshiaki; Itoh, Miho; Alea, Mileidys Perez; Thomas, Vincent; El Alaoui, Said; Akiyama, Masashi; Hitomi, Kiyotaka

2010-09-01

Transglutaminases (TGases) are a family of enzymes that catalyze cross-linking reactions between proteins. During epidermal differentiation, these enzymatic reactions are essential for formation of the cornified envelope, which consists of cross-linked structural proteins. Two main transglutaminases isoforms, epidermal-type (TGase 3) and keratinocyte-type (TGase 1), are cooperatively involved in this process of differentiating keratinocytes. Information regarding their substrate preference is of great importance to determine the functional role of these isozymes and clarify their possible co-operative action. Thus far, we have identified highly reactive peptide sequences specifically recognized by TGases isozymes such as TGase 1, TGase 2 (tissue-type isozyme) and the blood coagulation isozyme, Factor XIII. In this study, several substrate peptide sequences for human TGase 3 were screened from a phage-displayed peptide library. The preferred substrate sequences for TGase 3 were selected and evaluated as fusion proteins with mutated glutathione S-transferase. From these studies, a highly reactive and isozyme-specific sequence (E51) was identified. Furthermore, this sequence was found to be a prominent substrate in the peptide form and was suitable for detection of in situ TGase 3 activity in the mouse epidermis. TGase 3 enzymatic activity was detected in the layers of differentiating keratinocytes and hair follicles with patterns distinct from those of TGase 1. Our findings provide new information on the specific distribution of TGase 3 and constitute a useful tool to clarify its functional role in the epidermis.

Fluorescent Probes for Single-Step Detection and Proteomic Profiling of Histone Deacetylases.

PubMed

Xie, Yusheng; Ge, Jingyan; Lei, Haipeng; Peng, Bo; Zhang, Huatang; Wang, Danyang; Pan, Sijun; Chen, Ganchao; Chen, Lanfang; Wang, Yi; Hao, Quan; Yao, Shao Q; Sun, Hongyan

2016-12-07

Histone deacetylases (HDACs) play important roles in regulating various physiological and pathological processes. Developing fluorescent probes capable of detecting HDAC activity can help further elucidate the roles of HDACs in biology. In this study, we first developed a set of activity-based fluorescent probes by incorporating the Kac residue and the O-NBD group. Upon enzymatic removal of the acetyl group in the Kac residue, the released free amine reacted intramolecularly with the O-NBD moiety, resulting in turn-on fluorescence. These designed probes are capable of detecting HDAC activity in a continuous fashion, thereby eliminating the extra step of fluorescence development. Remarkably, the amount of turn-on fluorescence can be as high as 50-fold, which is superior to the existing one-step HDAC fluorescent probes. Inhibition experiments further proved that the probes can serve as useful tools for screening HDAC inhibitors. Building on these results, we moved on and designed a dual-purpose fluorescent probe by introducing a diazirine photo-cross-linker into the probe. The resulting probe was not only capable of reporting enzymatic activity but also able to directly identify and capture the protein targets from the complex cellular environment. By combining a fluorometric method and in-gel fluorescence scanning technique, we found that epigenetic readers and erasers can be readily identified and differentiated using a single probe. This is not achievable with traditional photoaffinity probes. In light of the prominent properties and the diverse functions of this newly developed probe, we envision that it can provide a robust tool for functional analysis of HDACs and facilitate future drug discovery in epigenetics.

Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.

PubMed

Cheng, Rui; Tao, Mangjuan; Shi, Zhilu; Zhang, Xiafei; Jin, Yan; Li, Baoxin

2015-11-15

Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research. Copyright © 2015 Elsevier B.V. All rights reserved.

Determination of myoglobin based on its enzymatic activity by stopped-flow spectrophotometry

NASA Astrophysics Data System (ADS)

Zheng, Qi; Liu, Zhihong; Cai, Ruxiu

2005-04-01

A new method has been developed for the determination of myoglobin (Mb) based on its enzymatic activity for the oxidation of o-phenylenediamine (OPDA) with hydrogen peroxide. Stopped-flow spectrophotometry was used to study the kinetic behavior of the oxidation reaction. The catalytic activity of Mb was compared to other three kinds of catalyst. The time dependent absorbance of the reaction product, 2,3-diamimophenazine (DAPN), at a wavelength of 426 nm was recorded. The initial reaction rate obtained at 40 °C was found to be proportional to the concentration of Mb in the range of 1.0 × 10 -6 to 4.0 × 10 -9 mol L -1. The detection limit of Mb was found to be 9.93 × 10 -10 mol L -1. The relative standard deviations were within 5% for the determination of different concentrations of Mb. Excess of bovine serum albumin (BSA), Ca(II), Mg(II), Cu(II), glucose, caffeine, lactose and uric acid did not interfere.

Characterization of a PEGylated protein therapeutic by ion exchange chromatography with on-line detection by native ESI MS and MS/MS.

PubMed

Muneeruddin, K; Bobst, C E; Frenkel, R; Houde, D; Turyan, I; Sosic, Z; Kaltashov, I A

2017-01-16

Detailed profiling of both enzymatic (e.g., glycosylation) and non-enzymatic (e.g., oxidation and deamidation) post-translational modifications (PTMs) is frequently required for the quality assessment of protein-based drugs. Challenging as it is, this task is further complicated for the so-called second-generation biopharmaceuticals, which also contain "designer PTMs" introduced to either enhance their pharmacokinetic profiles (e.g., PEGylated proteins) or endow them with therapeutic activity (e.g., protein-drug conjugates). Such modifications of protein covalent structure can dramatically increase structural heterogeneity, making the very notion of "molecular mass" meaningless, as ions representing different glycoforms of a PEGylated protein may have nearly identical distributions of ionic current as a function of m/z, making their contributions to the mass spectrum impossible to distinguish. In this work we demonstrate that a combination of ion exchange chromatography (IXC) with on-line detection by electrospray ionization mass spectrometry (ESI MS) and methods of ion manipulation in the gas phase (limited charge reduction and collision-induced dissociation) allows meaningful structural information to be obtained on a structurally heterogeneous sample of PEGylated interferon β-1a. IXC profiling of the protein sample gives rise to a convoluted chromatogram with several partially resolved peaks which can represent both deamidation and different glycosylation patterns within the protein, as well as varying extent of PEGylation. Thus, profiling the protein with on-line IXC/ESI/MS/MS allows it to be characterized by providing information on three different types of PTMs (designer, enzymatic and non-enzymatic) within a single protein therapeutic.

Magnetic Enzymatic Platform for Organophosphate Pesticide Detection Using Boron-doped Diamond Electrodes.

PubMed

Pino, Flavio; Ivandini, Tribidasari A; Nakata, Kazuya; Fujishima, Akira; Merkoçi, Arben; Einaga, Yasuaki

2015-01-01

A simple and reliable enzymatic system for organophosporus pesticide detection was successfully developed, by exploiting the synergy between the magnetic beads collection capacity and the outstanding electrochemistry property of boron-doped diamond electrodes. The determination of an organophosphate pesticide, chlorpyrifos (CPF), was performed based on the inhibition system of the enzyme acetylcholinesterase bonded to magnetic beads through a biotin-streptavidin complex system. A better sensitivity was found for a system with magnetic beads in the concentration range of 10(-9) to 10(-5) M. The estimated limits of detection based on IC10 (10% acetylcholinesterase (AChE) inhibition) have been detected and optimized to be 5.7 × 10(-10) M CPF. Spiked samples of water of Yokohama (Japan) have been measured to validate the efficiency of the enzymatic system. The results suggested that the use of magnetic beads to immobilize biomolecules or biosensing agents is suitable to maintain the superiority of BDD electrodes.

Time-Resolved Microfluorescence In Biomedical Diagnosis

NASA Astrophysics Data System (ADS)

Schneckenburger, Herbert

1985-02-01

A measuring system combining subnanosecond laser-induced fluorescence with microscopic signal detection was installed and used for diverse projects in the biomedical and environmental field. These projects are ranging from tumor diagnosis and enzymatic analysis to measurements of the activity of methanogenic bacteria which effect biogas production and waste water cleaning. The advantages of this method and its practical applicability are discussed.

Magnetic Graphene Nanosheet-Based Microfluidic Device for Homogeneous Real-Time Electronic Monitoring of Pyrophosphatase Activity Using Enzymatic Hydrolysate-Induced Release of Copper Ion.

PubMed

Lin, Youxiu; Zhou, Qian; Li, Juan; Shu, Jian; Qiu, Zhenli; Lin, Yuping; Tang, Dianping

2016-01-05

A novel flow-through microfluidic device based on a magneto-controlled graphene sensing platform was designed for homogeneous electronic monitoring of pyrophosphatase (PPase) activity; enzymatic hydrolysate-induced release of inorganic copper ion (Cu(2+)) from the Cu(2+)-coordinated pyrophosphate ions (Cu(2+)-PPi) complex was assessed to determine enzyme activity. Magnetic graphene nanosheets (MGNS) functionalized with negatively charged Nafion were synthesized by using the wet-chemistry method. The Cu(2+)-PPi complexes were prepared on the basis of the coordination reaction between copper ion and inorganic pyrophosphate ions. Upon target PPase introduction into the detection system, the analyte initially hydrolyzed pyrophosphate ions into phosphate ions and released the electroactive copper ions from Cu(2+)-PPi complexes. The released copper ions could be readily captured through the negatively charged Nafion on the magnetic graphene nanosheets, which could be quantitatively monitored by using the stripping voltammetry on the flow-through detection cell with an external magnet. Under optimal conditions, the obtained electrochemical signal exhibited a high dependence on PPase activity within a dynamic range from 0.1 to 20 mU mL(-1) and allowed the detection at a concentration as low as 0.05 mU mL(-1). Coefficients of variation for reproducibility of the intra-assay and interassay were below 7.6 and 9.8%, respectively. The inhibition efficiency of sodium fluoride (NaF) also received good results in pyrophosphatase inhibitor screening research. In addition, the methodology afforded good specificity and selectivity, simplification, and low cost without the need of sample separations and multiple washing steps, thus representing a user-friendly protocol for practical utilization in a quantitative PPase activity.

Biocompatible enzymatic roller pens for direct writing of biocatalytic materials: "do-it-yourself" electrochemical biosensors.

PubMed

Bandodkar, Amay J; Jia, Wenzhao; Ramírez, Julian; Wang, Joseph

2015-06-03

The development of enzymatic-ink-based roller pens for direct drawing of biocatalytic sensors, in general, and for realizing renewable glucose sensor strips, in particular, is described. The resulting enzymatic-ink pen allows facile fabrication of high-quality inexpensive electrochemical biosensors of any design by the user on a wide variety of surfaces having complex textures with minimal user training. Unlike prefabricated sensors, this approach empowers the end user with the ability of "on-demand" and "on-site" designing and fabricating of biocatalytic sensors to suit their specific requirement. The resulting devices are thus referred to as "do-it-yourself" sensors. The bio-active pens produce highly reproducible biocatalytic traces with minimal edge roughness. The composition of the new enzymatic inks has been optimized for ensuring good biocatalytic activity, electrical conductivity, biocompati-bility, reproducible writing, and surface adherence. The resulting inks are characterized using spectroscopic, viscometric, electrochemical, thermal and microscopic techniques. Applicability to renewable blood glucose testing, epidermal glucose monitoring, and on-leaf phenol detection are demonstrated in connection to glucose oxidase and tyrosinase-based carbon inks. The "do-it-yourself" renewable glucose sensor strips offer a "fresh," reproducible, low-cost biocatalytic sensor surface for each blood test. The ability to directly draw biocatalytic conducting traces even on unconventional surfaces opens up new avenues in various sensing applications in low-resource settings and holds great promise for diverse healthcare, environmental, and defense domains. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Towards practical time-of-flight secondary ion mass spectrometry lignocellulolytic enzyme assays

PubMed Central

2013-01-01

Background Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is a surface sensitive mass spectrometry technique with potential strengths as a method for detecting enzymatic activity on solid materials. In particular, ToF-SIMS has been applied to detect the enzymatic degradation of woody lignocellulose. Proof-of-principle experiments previously demonstrated the detection of both lignin-degrading and cellulose-degrading enzymes on solvent-extracted hardwood and softwood. However, these preliminary experiments suffered from low sample throughput and were restricted to samples which had been solvent-extracted in order to minimize the potential for mass interferences between low molecular weight extractive compounds and polymeric lignocellulose components. Results The present work introduces a new, higher-throughput method for processing powdered wood samples for ToF-SIMS, meanwhile exploring likely sources of sample contamination. Multivariate analysis (MVA) including Principal Component Analysis (PCA) and Multivariate Curve Resolution (MCR) was regularly used to check for sample contamination as well as to detect extractives and enzyme activity. New data also demonstrates successful ToF-SIMS analysis of unextracted samples, placing an emphasis on identifying the low-mass secondary ion peaks related to extractives, revealing how extractives change previously established peak ratios used to describe enzyme activity, and elucidating peak intensity patterns for better detection of cellulase activity in the presence of extractives. The sensitivity of ToF-SIMS to a range of cellulase doses is also shown, along with preliminary experiments augmenting the cellulase cocktail with other proteins. Conclusions These new procedures increase the throughput of sample preparation for ToF-SIMS analysis of lignocellulose and expand the applications of the method to include unextracted lignocellulose. These are important steps towards the practical use of ToF-SIMS as a tool to screen for changes in plant composition, whether the transformation of the lignocellulose is achieved through enzyme application, plant mutagenesis, or other treatments. PMID:24034438

Enzymatic properties of venoms from Brazilian scorpions of Tityus genus and the neutralisation potential of therapeutical antivenoms.

PubMed

Venancio, Emerson J; Portaro, Fernanda C V; Kuniyoshi, Alexandre K; Carvalho, Daniela Cajado; Pidde-Queiroz, Giselle; Tambourgi, Denise V

2013-07-01

Tityus scorpion stings are an important public health problem in Brazil, where the incidence of such stings exceeds the incidence of the health problems caused by other venomous animals, including snakes. In this study, we have analysed specific enzymatic activities of the venom from the Brazilian scorpions of Tityus genus, i.e., Tityus serrulatus, Tityus bahiensis and Tityus stigmurus. The data presented here revealed that Tityus spp. venoms exhibited significant hyaluronidase activity but no phospholipase activity. All the venom samples exhibited the ability to hydrolyse Abz-FLRRV-EDDnp and dynorphin 1-13 substrates. These activities were inhibited by 1,10-phenanthroline but not by PMSF, indicating the presence of metalloproteinases in the Tityus spp. venoms. The venom peptidase activity on Abz-FLRRV-EDDnp and on dynorphin 1-13 was partially inhibited by therapeutic Brazilian anti-scorpion and anti-arachnidic antivenoms. Dynorphin 1-13 (YGGFLRRIRPKLK) contains two scissile bonds between the residues Leu-Arg and Arg-Arg that are susceptible to cleavage by the Tityus venom metallopeptidase(s). Their cleavage releases leu-enkephalin, an important bioactive peptide. The detection of metalloproteinase(s) with specificity for both dynorphin 1-13 degradation and leu-enkephalin releasing can be important for the mechanistic understanding of hypotension and bradycardia induction in cases of scorpion stings, whereas hyaluronidases might contribute to the diffusion of the toxins present in these venoms. Furthermore, the limited inhibition of the toxic enzymatic activities by commercial antivenoms illustrates the necessity of improvements in current antivenom preparation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Binding of NAD+-Glycohydrolase to Streptolysin O Stabilizes Both Toxins and Promotes Virulence of Group A Streptococcus

PubMed Central

Velarde, Jorge J.; O’Seaghdha, Maghnus; Baddal, Buket; Bastiat-Sempe, Benedicte

2017-01-01

ABSTRACT The globally dominant, invasive M1T1 strain of group A Streptococcus (GAS) harbors polymorphisms in the promoter region of an operon that contains the genes encoding streptolysin O (SLO) and NAD+-glycohydrolase (NADase), resulting in high-level expression of these toxins. While both toxins have been shown experimentally to contribute to pathogenesis, many GAS isolates lack detectable NADase activity. DNA sequencing of such strains has revealed that reduced or absent enzymatic activity can be associated with a variety of point mutations in nga, the gene encoding NADase; a commonly observed polymorphism associated with near-complete abrogation of activity is a substitution of aspartic acid for glycine at position 330 (G330D). However, nga has not been observed to contain early termination codons or mutations that would result in a truncated protein, even when the gene product contains missense mutations that abrogate enzymatic activity. It has been suggested that NADase that lacks NAD-glycohydrolase activity retains an as-yet-unidentified inherent cytotoxicity to mammalian cells and thus is still a potent virulence factor. We now show that expression of NADase, either enzymatically active or inactive, augments SLO-mediated toxicity for keratinocytes. In culture supernatants, SLO and NADase are mutually interdependent for protein stability. We demonstrate that the two proteins interact in solution and that both the translocation domain and catalytic domain of NADase are required for maximal binding between the two toxins. We conclude that binding of NADase to SLO stabilizes both toxins, thereby enhancing GAS virulence. PMID:28900022

Jasmonic acid-amino acid conjugation enzyme assays.

PubMed

Rowe, Martha L; Staswick, Paul E

2013-01-01

Jasmonic acid (JA) is activated for signaling by its conjugation to isoleucine (Ile) through an amide linkage. The Arabidopsis thaliana JASMONIC ACID RESISTANT1 (JAR1) enzyme carries out this Mg-ATP-dependent reaction in two steps, adenylation of the free carboxyl of JA, followed by condensation of the activated group to Ile. This chapter details the protocols used to detect and quantify the enzymatic activity obtained from a glutathione-S-transferase:JAR1 fusion protein produced in Escherichia coli, including an isotope exchange assay for the adenylation step and assays for the complete reaction that involve the high-performance liquid chromatography quantitation of adenosine monophosphate, a stoichiometric by-product of the reaction, and detection of the conjugation product by thin-layer chromatography or gas -chromatography/mass spectrometry.

Enhanced non-enzymatic glucose sensing based on copper nanoparticles decorated nitrogen-doped graphene.

PubMed

Jiang, Ding; Liu, Qian; Wang, Kun; Qian, Jing; Dong, Xiaoya; Yang, Zhenting; Du, Xiaojiao; Qiu, Baijing

2014-04-15

Copper nanoparticles (NPs) decorated nitrogen-doped graphene (Cu-N-G) was prepared by a facile thermal treatment, and further employed as a novel sensing material for fabricating the sensitive non-enzymatic glucose sensor. Compared with pure Cu NPs, the Cu-N-G showed enhanced electrocatalytic activity to glucose oxidation due to the integration of N-G, which exhibited the oxidation peak current of glucose ca. 23-fold higher than that of pure Cu NPs. The presented sensor showed excellent performances for glucose detection including wide linear range of 0.004-4.5 mM, low detection limit (1.3 μM, S/N=3), high sensitivity (48.13 μA mM(-1)), fast response time (<5 s), good selectivity to the general coexisted interferences, etc. Such properties would promote the potential application of the nitrogen-doped graphene as enhanced materials in fabricating sensors for chemical and biochemical analysis. © 2013 Published by Elsevier B.V.

Interaction of aurintricarboxylic acid (ATA) with four nucleic acid binding proteins DNase I, RNase A, reverse transcriptase and Taq polymerase

NASA Astrophysics Data System (ADS)

Ghosh, Utpal; Giri, Kalyan; Bhattacharyya, Nitai P.

2009-12-01

In the investigation of interaction of aurintricarboxylic acid (ATA) with four biologically important proteins we observed inhibition of enzymatic activity of DNase I, RNase A, M-MLV reverse transcriptase and Taq polymerase by ATA in vitro assay. As the telomerase reverse transcriptase (TERT) is the main catalytic subunit of telomerase holoenzyme, we also monitored effect of ATA on telomerase activity in vivo and observed dose-dependent inhibition of telomerase activity in Chinese hamster V79 cells treated with ATA. Direct association of ATA with DNase I ( Kd = 9.019 μM)), RNase A ( Kd = 2.33 μM) reverse transcriptase ( Kd = 0.255 μM) and Taq polymerase ( Kd = 81.97 μM) was further shown by tryptophan fluorescence quenching studies. Such association altered the three-dimensional conformation of DNase I, RNase A and Taq polymerase as detected by circular dichroism. We propose ATA inhibits enzymatic activity of the four proteins through interfering with DNA or RNA binding to the respective proteins either competitively or allosterically, i.e. by perturbing three-dimensional structure of enzymes.

In situ enzymatic activity of transglutaminase isoforms on brain tissue sections of rodents: A new approach to monitor differences in post-translational protein modifications during neurodegeneration.

PubMed

Schulze-Krebs, Anja; Canneva, Fabio; Schnepf, Rebecca; Dobner, Julia; Dieterich, Walburga; von Hörsten, Stephan

2016-01-15

Mammalian transglutaminases (TGs) catalyze the irreversible post-translational modifications of proteins, the most prominent of which is the calcium-dependent formation of covalent acyl transfers between the γ-carboxamide group of glutamine and the ε-amino-group of lysine (GGEL-linkage). In the central nervous system, at least four TG isoforms are present and some of them are differentially expressed under pathological conditions in human patients. However, the precise TG-isoform-dependent enzymatic activities in the brain as well as their anatomical distribution are unknown. Specificity of the used biotinylated peptides was analyzed using an in vitro assay. Isoform-specific TG activity was evaluated in in vitro and in situ studies, using brain extracts and native brain tissue obtained from rodents. Our method allowed us to reveal in vitro and in situ TG-isoform-dependent enzymatic activity in brain extracts and tissue of rats and mice, with a specific focus on TG6. In situ activity of this isoform varied between BACHD mice in comparison to their wt controls. TG isozyme-specific activity can be detected by isoform-specific biotinylated peptides in brain tissue sections of rodents to reveal differences in the anatomical and/or subcellular distribution of TG activity. Our findings yield the basis for a broader application of this method for the screening of pathological expression and activity of TGs in a variety of animal models of human diseases, as in the case of neurodegenerative conditions such as Huntington׳s, Parkinson׳s and Alzheimer׳s, where protein modification is involved as a key mechanism of disease progression. Copyright © 2015 Elsevier B.V. All rights reserved.

Increased expression of native cytosolic Cu/Zn superoxide dismutase and ascorbate peroxidase improves tolerance to oxidative and chilling stresses in cassava (Manihot esculenta Crantz)

PubMed Central

2014-01-01

Background Cassava (Manihot esculenta Crantz) is a tropical root crop, and is therefore, extremely sensitive to low temperature; its antioxidative response is pivotal for its survival under stress. Timely turnover of reactive oxygen species (ROS) in plant cells generated by chilling-induced oxidative damages, and scavenging can be achieved by non-enzymatic and enzymatic reactions in order to maintain ROS homeostasis. Results Transgenic cassava plants that co-express cytosolic superoxide dismutase (SOD), MeCu/ZnSOD, and ascorbate peroxidase (APX), MeAPX2, were produced and tested for tolerance against oxidative and chilling stresses. The up-regulation of MeCu/ZnSOD and MeAPX2 expression was confirmed by the quantitative reverse transcriptase-polymerase chain reaction, and enzymatic activity analyses in the leaves of transgenic cassava plant lines with a single-transgene integration site. Upon exposure to ROS-generating agents, 100 μM ROS-generating reagent methyl viologen and 0.5 M H2O2, higher levels of enzymatic activities of SOD and APX were detected in transgenic plants than the wild type. Consequently, the oxidative stress parameters, such as lipid peroxidation, chlorophyll degradation and H2O2 synthesis, were lower in the transgenic lines than the wild type. Tolerance to chilling stress at 4°C for 2 d was greater in transgenic cassava, as observed by the higher levels of SOD, catalase, and ascorbate-glutathione cycle enzymes (e.g., APX, monodehydroascorbate reductase, dehydroascorbate reducatase and glutathione reductase) and lower levels of malondialdehyde content. Conclusions These results suggest that the expression of native cytosolic SOD and APX simultaneously activated the antioxidative defense mechanisms via cyclic ROS scavenging, thereby improving its tolerance to cold stress. PMID:25091029

Increased expression of native cytosolic Cu/Zn superoxide dismutase and ascorbate peroxidase improves tolerance to oxidative and chilling stresses in cassava (Manihot esculenta Crantz).

PubMed

Xu, Jia; Yang, Jun; Duan, Xiaoguang; Jiang, Yueming; Zhang, Peng

2014-08-05

Cassava (Manihot esculenta Crantz) is a tropical root crop, and is therefore, extremely sensitive to low temperature; its antioxidative response is pivotal for its survival under stress. Timely turnover of reactive oxygen species (ROS) in plant cells generated by chilling-induced oxidative damages, and scavenging can be achieved by non-enzymatic and enzymatic reactions in order to maintain ROS homeostasis. Transgenic cassava plants that co-express cytosolic superoxide dismutase (SOD), MeCu/ZnSOD, and ascorbate peroxidase (APX), MeAPX2, were produced and tested for tolerance against oxidative and chilling stresses. The up-regulation of MeCu/ZnSOD and MeAPX2 expression was confirmed by the quantitative reverse transcriptase-polymerase chain reaction, and enzymatic activity analyses in the leaves of transgenic cassava plant lines with a single-transgene integration site. Upon exposure to ROS-generating agents, 100 μM ROS-generating reagent methyl viologen and 0.5 M H₂O₂, higher levels of enzymatic activities of SOD and APX were detected in transgenic plants than the wild type. Consequently, the oxidative stress parameters, such as lipid peroxidation, chlorophyll degradation and H₂O₂ synthesis, were lower in the transgenic lines than the wild type. Tolerance to chilling stress at 4°C for 2 d was greater in transgenic cassava, as observed by the higher levels of SOD, catalase, and ascorbate-glutathione cycle enzymes (e.g., APX, monodehydroascorbate reductase, dehydroascorbate reducatase and glutathione reductase) and lower levels of malondialdehyde content. These results suggest that the expression of native cytosolic SOD and APX simultaneously activated the antioxidative defense mechanisms via cyclic ROS scavenging, thereby improving its tolerance to cold stress.

Mapping of human autoantibody epitopes on aromatic L-amino acid decarboxylase.

PubMed

Candeloro, Paola; Voltattorni, Carla Borri; Perniola, Roberto; Bertoldi, Mariarita; Betterle, Corrado; Mannelli, Massimo; Giordano, Roberta; De Bellis, Annamaria; Tiberti, Claudio; Laureti, Stefano; Santeusanio, Fausto; Falorni, Alberto

2007-03-01

Aromatic l-amino acid decarboxylase (AADC) is target of autoantibodies in autoimmune polyendocrine syndrome I (APS I), especially in patients with autoimmune hepatitis. Little information is currently available on AADC autoantibody epitopes and on the interrelation between autoantibody-mediated inhibition of enzymatic activity and epitope specificity. We tested the immunoreactivity of full-length porcine AADC and of eight fragments of the enzyme with human serum from 18 patients with APS I, 199 with non-APS I autoimmune Addison's disease, 124 with type 1 diabetes mellitus, 36 with Graves' disease, and 141 healthy control subjects, and we evaluated the autoantibody-mediated enzymatic inhibition. AADC antibodies (Ab) were detected in 12 of 18 (67%) APS I patients and in six of 199 (3%) autoimmune Addison's disease patients. Four patients with autoimmune hepatitis were all positive for AADCAb. None of the 141 healthy control subjects, 82 patients with nonautoimmune adrenal insufficiency, 124 with type 1 diabetes mellitus, and 36 with Graves' disease were found positive. Two epitope regions, corresponding to amino acids 274-299 (E1) and 380-471 (E2) were identified. Localization of E1 was confirmed by displacement studies with synthetic peptides corresponding to peptides of porcine AADC. All 12 AADCAb-positive APS I sera reacted with E1, and seven of 12 (58%) reacted also with E2. E2-specific, but not E1-specific, autoantibodies were associated with a significant inhibition of in vitro AADC enzymatic activity. We mapped the human AADCAb epitopes to the middle and COOH-terminal regions of the enzyme. Autoantibodies to the COOH-terminal region induce a significant inhibition of enzymatic activity.

Glucose-6-phosphate dehydrogenase deficiency among Yemeni children residing in malaria-endemic areas of Hodeidah governorate and evaluation of a rapid diagnostic test for its detection.

PubMed

Abdul-Ghani, Rashad; Mahdy, Mohammed A K; Saif-Ali, Reyadh; Alkubati, Sameer A; Alqubaty, Abdulhabib R; Al-Mikhlafy, Abdullah A; Al-Eryani, Samira M; Al-Mekhlafi, Abdusalam M; Alhaj, Ali

2016-06-21

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common genetic enzymopathy worldwide, is associated with an acute haemolytic anaemia in individuals exposed to primaquine. The present study aimed to determine G6PD deficiency among Yemeni children in malaria-endemic areas as well as to assess the performance of the CareStart™ G6PD rapid diagnostic test (RDT) for its detection. A cross-sectional study recruiting 400 children from two rural districts in Hodeidah governorate was conducted. Socio-demographic data and blood samples were collected and G6PD deficiency was qualitatively detected in fresh blood in the field using the CareStart™ G6PD RDT, while the enzymatic assay was used to quantitatively measure enzyme activity. Performance of the CareStart™ G6PD RDT was assessed by calculating its sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) against the reference enzymatic assay. The ranges of enzyme activity were 0.14-18.45 and 0.21-15.94 units/g haemoglobin (U/gHb) for males and females, respectively. However, adjusted male median G6PD activity was 5.0 U/gHb. Considering the adjusted male median as representing 100 % normal enzyme activity, the prevalence rates of G6PD deficiency were 12.0 and 2.3 % at the cut-off activities of ≤60 and ≤10 %, respectively. Multivariable analysis showed that gender, district of residence and consanguinity between parents were independent risk factors for G6PD deficiency at the cut-off activity of ≤30 % of normal. The CareStart™ G6PD RDT showed 100 % sensitivity and NPV for detecting G6PD deficiency at the cut-off activities of ≤10 and ≤20 % of normal activity compared to the reference enzymatic method. However, it showed specificity levels of 90.0 and 95.4 % as well as positive/deficient predictive values (PPVs) of 18.0 and 66.0 % at the cut-off activities of ≤10 and ≤20 %, respectively, compared to the reference method. G6PD deficiency with enzyme activity of ≤60 % of normal is prevalent among 12.0 % of children residing in malaria-endemic areas of Hodeidah governorate, with 2.3 % having severe G6PD deficiency. Gender, district of residence and consanguinity between parents are significant independent predictors of G6PD deficiency at the cut-off activity of ≤30 % of normal among children in malaria-endemic areas of Hodeidah. The CareStart™ G6PD RDT proved reliable as a point-of-care test to screen for severely G6PD-deficient patients, with 100 % sensitivity and NPV, and it can be used for making clinical decisions prior to the administration of primaquine in malaria elimination strategies.

Characterization of four arginine kinases in the ciliate Paramecium tetraurelia: Investigation on the substrate inhibition mechanism.

PubMed

Yano, Daichi; Suzuki, Takaya; Hirokawa, Saki; Fuke, Kyoko; Suzuki, Tomohiko

2017-08-01

The ciliate Paramecium tetraurelia contains four arginine kinase genes (AK1-4). We detected cDNA for only three of the AKs (AK1-3) via PCR. Recombinant AK1-4 were expressed in Escherichia coli and their kinetics parameters determined. AK3 showed typical substrate inhibition toward arginine, and enzymatic activity markedly decreased when arginine concentration increased. This is the first example of substrate inhibition in wild-type phosphagen kinases. To explore the substrate inhibition mechanism, site-directed mutations were generated, targeting the amino acid sequence D-D-S-Q-V at positions 77-81 in P. tetraurelia AK3. Among the mutants, substrate inhibition was lost remarkably in the S79A mutant. In spite of high amino acid sequence identity (91%) between P. tetraurelia AK3 and AK4, the enzymatic activity of AK4 was less by 3% than that of AK3. We noticed that the conservative G298 was unusually replaced by R in P. tetraurelia AK4, and we constructed two mutants, R298G/AK4 and G298R/AK3. Enzymatic activity of the former mutant was comparable with that of the wild-type AK3, whereas that of the latter mutant was dramatically reduced. Thus, we concluded that the significantly low activity of P. tetraurelia AK4 is due to the residue R298. Copyright © 2017 Elsevier B.V. All rights reserved.

Highly sensitive on-site detection of glucose in human urine with naked eye based on enzymatic-like reaction mediated etching of gold nanorods.

PubMed

Zhang, Zhiyang; Chen, Zhaopeng; Cheng, Fangbin; Zhang, Yaowen; Chen, Lingxin

2017-03-15

Based on enzymatic-like reaction mediated etching of gold nanorods (GNRs), an ultrasensitive visual method was developed for on-site detection of urine glucose. With the catalysis of MoO 4 2 - , GNRs were efficiently etched by H 2 O 2 which was generated by glucose-glucose oxidase enzymatic reaction. The etching of GNRs lead to a blue-shift of logitudinal localized surface plasmon resonance of GNRs, accompanied by an obvious color change from blue to red. The peak-shift and the color change can be used for detection of glucose by the spectrophotometer and the naked eyes. Under optimal condition, an excellent sensitivity toward glucose is obtained with a detection limit of 0.1μM and a visual detection limit of 3μM in buffer solution. Benefiting from the high sensitivity, the successful colorimetric detection of glucose in original urine samples was achieved, which indicates the practical applicability to the on-site determination of urine glucose. Copyright © 2016 Elsevier B.V. All rights reserved.

Control of enzymatic browning in potato (Solanum tuberosum L.) by sense and antisense RNA from tomato polyphenol oxidase.

PubMed

Coetzer, C; Corsini, D; Love, S; Pavek, J; Tumer, N

2001-02-01

Polyphenol oxidase (PPO) activity of Russet Burbank potato was inhibited by sense and antisense PPO RNAs expressed from a tomato PPO cDNA under the control of the 35S promoter from the cauliflower mosaic virus. Transgenic Russet Burbank potato plants from 37 different lines were grown in the field. PPO activity and the level of enzymatic browning were measured in the tubers harvested from the field. Of the tubers from 28 transgenic lines that were sampled, tubers from 5 lines exhibited reduced browning. The level of PPO activity correlated with the reduction in enzymatic browning in these lines. These results indicate that expression of tomato PPO RNA in sense or antisense orientation inhibits PPO activity and enzymatic browning in the major commercial potato cultivar. Expression of tomato PPO RNA in sense orientation led to the greatest decrease in PPO activity and enzymatic browning, possibly due to cosuppression. These results suggest that expression of closely related heterologous genes can be used to prevent enzymatic browning in a wide variety of food crops without the application of various food additives.

Improved antimelanogenesis and antioxidant effects of polysaccharide from Cuscuta chinensis Lam seeds after enzymatic hydrolysis.

PubMed

Liu, Zi-Jun; Wang, Ya-Lan; Li, Qi-Ling; Yang, Liu

2018-01-01

Cuscuta chinensis polysaccharide (CPS) was extracted using hot water and enzymatically hydrolyzed C. chinensis polysaccharide (ECPS) was produced by the mannase enzymatic hydrolysis process. The purpose of this research was to investigate the antimelanogenic activity of ECPS and CPS in B16F10 melanoma cells. The in vitro antioxidant activity was assessed by their ferric iron reducing power and DPPH free radical scavenging activities. The molecular mass distribution of polysaccharides was determined using SEC-MALLS-RI. CPS was successfully enzymatically degraded using mannase and the weighted average molecular weights of CPS and ECPS were 434.6 kDa and 211.7 kDa. The results of biological activity assays suggested that the enzymatically hydrolyzed polysaccharide had superior antimelanogenic activity and antioxidant effect than the original polysaccharide. ECPS exhibited antimelanogenic activity by down-regulating the expression of tyrosinase, MITF, and TRP-1 without cytotoxic effects in B16F10 melanoma cells. In conclusion, ECPS have the potential to become a skin whitening product.

Antimicrobial and enzymatic activity of anemophilous fungi of a public university in Brazil.

PubMed

Sobral, Laureana V; Melo, Kelly N; Souza, Cleciana M; Silva, Sílvio F; Silva, Gilvania L R; Silva, Andressa L F; Wanderley, Katharine A A; Oliveira, Idjane S; Cruz, Roberta

2017-01-01

To the fungal microbiota the UFPE and biotechnological potential enzymatic and antimicrobial production. Air conditioned environments were sampled using a passive sedimentation technique, the air I ratio and the presence of aflatoxigenic strains evaluated for ANVISA. Icelles were to determine the enzymatic activity of lipase, amylase and protease metabolic liquids to determine antimicrobial activity. Diversity was observed in all CAV environments, CFU/m3 ranged from 14 to 290 and I/E ratio from 0.1 to 1.5. The of the fungal genera were: Aspergillus (50%), Penicillium (21%), Talaromyces (14%), Curvularia and Paecilomyces (7% each). Aspergillus sydowii (Bainier & Sartory) Thom & Church presented enzymatic activity and the Talaromyces purpureogenus Samson, Yilmaz, Houbraken, Spierenb., Seifert, Peterson, Varga & Frisvad presented antibacterial activity against all bacteria that all environments present fungal species biodiversity no toxigenic or pathogenic fungi were found, according to ANVISA legislation for conditioned environments and airborne filamentous fungi present potential for enzymatic and antimicrobial activity.

Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces

PubMed Central

2015-01-01

Ribonuclease A (RNase A) is a small globular enzyme that lyses RNA. The remarkable solution stability of its structure and enzymatic activity has led to its investigation to develop a new class of drugs for cancer chemotherapeutics. However, the successful clinical application of RNase A has been reported to be limited by insufficient stability and loss of enzymatic activity when it was coupled with a biomaterial carrier for drug delivery. The objective of this study was to characterize the structural stability and enzymatic activity of RNase A when it was adsorbed on different surface chemistries (represented by fused silica glass, high-density polyethylene, and poly(methyl-methacrylate)). Changes in protein structure were measured by circular dichroism, amino acid labeling with mass spectrometry, and in vitro assays of its enzymatic activity. Our results indicated that the process of adsorption caused RNase A to undergo a substantial degree of unfolding with significant differences in its adsorbed structure on each material surface. Adsorption caused RNase A to lose about 60% of its native-state enzymatic activity independent of the material on which it was adsorbed. These results indicate that the native-state structure of RNase A is greatly altered when it is adsorbed on a wide range of surface chemistries, especially at the catalytic site. Therefore, drug delivery systems must focus on retaining the native structure of RNase A in order to maintain a high level of enzymatic activity for applications such as antitumor chemotherapy. PMID:25420087

Nanoribbon field-effect transistors as direct and label-free sensors of enzyme-substrate interactions

NASA Astrophysics Data System (ADS)

Mu, Luye; Droujinine, Ilia; Rajan, Nitin; Sawtelle, Sonya; Reed, Mark

2015-03-01

The ability to measure enzyme-substrate interactions is essential in areas such as diagnostics, treatment, and biochemical screens. Many enzymatic reactions alter the pH of its environment, suggesting of a simple and direct method for detection. We show the ability of Al2O3-coated Si nanoribbon field-effect transistor biosensors to sensitively measure various aspects of enzyme-substrate interactions through measuring the pH. Urea in phosphate buffered saline (PBS) and penicillinase in PBS and urine were measured to limits of <200 μM and 0.02 units/mL, respectively. We also show the ability to extract accurate kinetics from the interaction of acetylcholine and its esterase. Prior work on FET sensors has been limited by the use of surface functionalization, which not only alters enzyme-substrate affinity, but also makes enzyme activity quantification difficult. Our method involves direct detection of reactions in solution without requiring alteration to the reactants, allowing us to obtain repeatable results and sensitive limits of detection. This method is a simple, inexpensive, and effective platform for detection of enzymatic reactions, and can be readily generalized to many unrelated classes of reactants. This work was supported in part by U.S. Army Research Office and Air Force Research Laboratory.

Effect of posttranslational modifications on enzyme function and assembly.

PubMed

Ryšlavá, Helena; Doubnerová, Veronika; Kavan, Daniel; Vaněk, Ondřej

2013-10-30

The detailed examination of enzyme molecules by mass spectrometry and other techniques continues to identify hundreds of distinct PTMs. Recently, global analyses of enzymes using methods of contemporary proteomics revealed widespread distribution of PTMs on many key enzymes distributed in all cellular compartments. Critically, patterns of multiple enzymatic and nonenzymatic PTMs within a single enzyme are now functionally evaluated providing a holistic picture of a macromolecule interacting with low molecular mass compounds, some of them being substrates, enzyme regulators, or activated precursors for enzymatic and nonenzymatic PTMs. Multiple PTMs within a single enzyme molecule and their mutual interplays are critical for the regulation of catalytic activity. Full understanding of this regulation will require detailed structural investigation of enzymes, their structural analogs, and their complexes. Further, proteomics is now integrated with molecular genetics, transcriptomics, and other areas leading to systems biology strategies. These allow the functional interrogation of complex enzymatic networks in their natural environment. In the future, one might envisage the use of robust high throughput analytical techniques that will be able to detect multiple PTMs on a global scale of individual proteomes from a number of carefully selected cells and cellular compartments. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Copyright © 2013 Elsevier B.V. All rights reserved.

Occupational airborne contamination in South Brazil: 2. Oxidative stress detected in the blood of workers of incineration of hospital residues.

PubMed

Possamai, F P; Avila, S; Budni, P; Backes, P; Parisotto, E B; Rizelio, V M; Torres, M A; Colepicolo, P; Wilhelm Filho, D

2009-11-01

One of the most useful methods for elimination of solid residues of health services (SRHS) is incineration. However, it also provokes the emission of several hazardous air pollutants such as heavy metals, furans and dioxins, which produce reactive oxygen species and oxidative stress. The present study, which is parallel to an accompanied paper (Avila Jr. et al., this issue), investigated several enzymatic and non-enzymatic biomarkers of oxidative stress in the blood (contents of vitamin E, lipoperoxidation = TBARS, reduced glutathione = GSH, oxidized glutathione = GSSG, and activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in three different groups (n = 20 each) exposed to airborne contamination associated with incineration of SRHS: workers directly (ca. 100 m from the incinerator) and indirectly exposed (residents living ca. 5 km the incineration site), and controls (non-exposed subjects). TBARS and GSSG levels were increased whilst GSH, TG and alpha-tocopherol contents were decreased in workers and residents compared to controls. Increased GST and CAT activities and decreased GPx activities were detected in exposed subjects compared to controls, while GR did not show any difference among the groups. In conclusion, subjects directly or indirectly exposed to SRHS are facing an oxidative insult and health risk regarding fly ashes contamination from SRHS incineration.

Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

PubMed Central

Adam Smith, R; Sewell, Sarah L; Giorgio, Todd D

2008-01-01

The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure. PMID:18488420

MICROCHIP ENZYMATIC ASSAY OF ORGANOPHOSPHATE NERVE AGENTS. (R830900)

EPA Science Inventory

An on-chip enzymatic assay for screening organophosphate (OP) nerve agents, based on a pre-column reaction of organophosphorus hydrolase (OPH), electrophoretic separation of the phosphonic acid products, and their contactless-conductivity detection, is described. Factors affec...

Pressure and Temperature Effects on the Activity and Structure of the Catalytic Domain of Human MT1-MMP

PubMed Central

Decaneto, Elena; Suladze, Saba; Rosin, Christopher; Havenith, Martina; Lubitz, Wolfgang; Winter, Roland

2015-01-01

Membrane type 1-matrix metalloproteinase (MT1-MMP or MMP-14) is a zinc-transmembrane metalloprotease involved in the degradation of extracellular matrix and tumor invasion. While changes in solvation of MT1-MMP have been recently studied, little is known about the structural and energetic changes associated with MT1-MMP while interacting with substrates. Steady-state kinetic and thermodynamic data (including activation energies and activation volumes) were measured over a wide range of temperatures and pressures by means of a stopped-flow fluorescence technique. Complementary temperature- and pressure-dependent Fourier-transform infrared measurements provided corresponding structural information of the protein. MT1-MMP is stable and active over a wide range of temperatures (10–55°C). A small conformational change was detected at 37°C, which is responsible for the change in activity observed at the same temperature. Pressure decreases the enzymatic activity until complete inactivation occurs at 2 kbar. The inactivation is associated with changes in the rate-limiting step of the reaction caused by additional hydration of the active site upon compression and/or minor conformational changes in the active site region. Based on these data, an energy and volume diagram could be established for the various steps of the enzymatic reaction. PMID:26636948

Utilizing a CdTe quantum dots-enzyme hybrid system for the determination of both phenolic compounds and hydrogen peroxide.

PubMed

Yuan, Jipei; Guo, Weiwei; Wang, Erkang

2008-02-15

In this paper, we attempt to construct a simple and sensitive detection method for both phenolic compounds and hydrogen peroxide, with the successful combination of the unique property of quantum dots and the specificity of enzymatic reactions. In the presence of H2O2 and horseradish peroxidase, phenolic compounds can quench quantum dots' photoluminescence efficiently, and the extent of quenching is severalfold to more than 100-fold increase. Quinone intermediates produced from the enzymatic catalyzed oxidation of phenolic compounds were believed to play the main role in the photoluminescence quenching. Using a quantum dots-enzyme system, the detection limits for phenolic compounds and hydrogen peroxide were detected to be approximately 10(-7) mol L(-1). The coupling of efficient quenching of quantum dot photoluminescence by quinone and the effective enzymatic reactions make this a simple and sensitive method for phenolic compound detection and great potential in the development of H2O2 biosensors for various analytes.

Discriminating a Single Nucleotide Difference for Enhanced miRNA Detection Using Tunable Graphene and Oligonucleotide Nanodevices.

PubMed

Robertson, Neil M; Hizir, Mustafa Salih; Balcioglu, Mustafa; Wang, Rui; Yavuz, Mustafa Selman; Yumak, Hasan; Ozturk, Birol; Sheng, Jia; Yigit, Mehmet V

2015-09-15

In this study we have reported our efforts to address some of the challenges in the detection of miRNAs using water-soluble graphene oxide and DNA nanoassemblies. Purposefully inserting mismatches at specific positions in our DNA (probe) strands shows increasing specificity against our target miRNA, miR-10b, over miR-10a which varies by only a single nucleotide. This increased specificity came at a loss of signal intensity within the system, but we demonstrated that this could be addressed with the use of DNase I, an endonuclease capable of cleaving the DNA strands of the RNA/DNA heteroduplex and recycling the RNA target to hybridize to another probe strand. As we previously demonstrated, this enzymatic signal also comes with an inherent activity of the enzyme on the surface-adsorbed probe strands. To remove this activity of DNase I and the steady nonspecific increase in the fluorescence signal without compromising the recovered signal, we attached a thermoresponsive PEGMA polymer (poly(ethylene glycol) methyl ether methacrylate) to nGO. This smart polymer is able to shield the probes adsorbed on the nGO surface from the DNase I activity and is capable of tuning the detection capacity of the nGO nanoassembly with a thermoswitch at 39 °C. By utilizing probes with multiple mismatches, DNase I cleavage of the DNA probe strands, and the attachment of PEGMA polymers to graphene oxide to block undesired DNase I activity, we were able to detect miR-10b from liquid biopsy mimics and breast cancer cell lines. Overall we have reported our efforts to improve the specificity, increase the sensitivity, and eliminate the undesired enzymatic activity of DNase I on surface-adsorbed probes for miR-10b detection using water-soluble graphene nanodevices. Even though we have demonstrated only the discrimination of miR-10b from miR-10a, our approach can be extended to other short RNA molecules which differ by a single nucleotide.

Paraoxonase 1 and oxidative stress in paediatric non-alcoholic steatohepatitis.

PubMed

Desai, Sonal; Baker, Susan S; Liu, Wensheng; Moya, Diana A; Browne, Richard W; Mastrandrea, Lucy; Baker, Robert D; Zhu, Lixin

2014-01-01

Non-alcoholic steatohepatitis (NASH) in children is a significant public health concern. Oxidative stress is an important component in the pathophysiology of NASH. Several enzymatic antioxidant mechanisms protect the liver from oxidative injury. Examination of the expression of these enzymes in NASH livers may provide insight on the roles for these antioxidant mechanisms in the pathophysiology of NASH. The mRNA expression of catalase, glutathione peroxidase 1 (GPX1), glutathione reductase (GSR), paraoxonase 1 (PON1) and other reactive oxygen species-related genes was evaluated by microarray and quantitative real-time PCR analyses. The PON1 protein levels were evaluated in liver and serum by Western blot analyses. Serum enzymatic activities of GPX, GSR and PON1 (paraoxonase and arylesterase activities) were examined. NASH livers exhibited elevated mRNA expression of catalase and PON1, but not GPX1 or GSR. No difference in serum GPX or GSR activity was detected between NASH patients and controls. Elevated expression of PON1 mRNA and protein was detected in NASH livers, but serum PON1 protein and activities were not elevated. Elevated expression of catalase and PON1 suggests protective roles for these antioxidants in NASH livers. Given the importance of oxidative stress in the pathophysiology of NASH, future studies focusing on these enzymes could identify important targets for therapeutic or preventive interventions for NASH patients. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Imaging trypsin activity through changes in the orientation of liquid crystals coupled to the interactions between a polyelectrolyte and a phospholipid layer.

PubMed

Hu, Qiong-Zheng; Jang, Chang-Hyun

2012-03-01

In this study, we developed a new type of liquid crystal (LC)-based sensor for the real-time and label-free monitoring of enzymatic activity through changes in the orientation of LCs coupled to the interactions between polyelectrolyte and phospholipid. The LCs changed from dark to bright after an aqueous solution of poly-l-lysine (PLL) was transferred onto a self-assembled monolayer of the phospholipid, dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), at the aqueous/LC interface. Interactions between the positively charged PLL and the negatively charged DOPG drove the reorganization of the phospholipid membrane, which induced an orientational transition in the LCs from a homeotropic to planar state. Since the serine endopeptidase trypsin can enzymatically catalyze the hydrolysis of PLL, the dark-to-bright shift in the optical response was not observed after transferring a mixed solution of PLL and trypsin onto the DOPG-decorated LC interface, indicating that no orientational transitions in the LCs occurred. However, the optical response from dark to bright was observed when the mixture in the optical cell was replaced by an aqueous solution of PLL. Control experiments with trypsin or an aqueous mixture of PLL and deactivated trypsin further confirmed the feasibility of this approach. The detection limit of trypsin was determined to be ~1 μg/mL. This approach holds great promise for use in the development of LC-based sensors for the detection of enzymatic reactions in cases where the biological polyelectrolyte substrates of enzymes could disrupt the organization of the membrane and induce orientational transitions of LCs at the aqueous/LC interface. © 2012 American Chemical Society

Enzymatic vegetable organic extracts as soil biochemical biostimulants and atrazine extenders.

PubMed

García-Martínez, Ana María; Tejada, Manuel; Díaz, Ana Isabel; Rodríguez-Morgado, Bruno; Bautista, Juan; Parrado, Juan

2010-09-08

The purpose of this study was to gather information on the potential effects of organic biostimulants on soil activity and atrazine biodegradation. Carob germ enzymatic extract (CGEE) and wheat condensed distiller solubles enzymatic extract (WCDS-EE) have been obtained using an enzymatic process; their main organic components are soluble carbohydrates and proteins in the form of peptides and free amino acids. Their application to soil results in high biostimulation, rapidly increased dehydrogenase, phosphatase and glucosidase activities, and an observed atrazine extender capacity due to inhibition of its mineralization. The extender capacity of both extracts is proportional to the protein/carbohydrate ratio content. As a result, these enzymatic extracts are highly microbially available, leading to two independent phenomena, fertility and an atrazine persistence that is linked to increased soil activity.

Electrochemical Nanoparticle-Enzyme Sensors for Screening Bacterial Contamination in Drinking Water

PubMed Central

Chen, Juhong; Jiang, Ziwen; Ackerman, Jonathan D.; Yazdani, Mahdieh; Hou, Singyuk

2015-01-01

Traditional plating and culturing methods used to quantify bacteria commonly require hours to days from sampling to results. We present here a simple, sensitive and rapid electrochemical method for bacteria detection in drinking water based on gold nanoparticle-enzyme complexes. The gold nanoparticles were functionalized with positively charged quaternary amine headgroups that could bind to enzymes through electrostatic interactions, resulting in inhibition of enzymatic activity. In the presence of bacteria, the nanoparticles released from the enzymes and preferentially bound to the bacteria, resulting in an increase in enzyme activity, releasing a redox-active phenol from the substrate. We employed this strategy for the electrochemical sensing of Escherichia coli and Staphylococcus aureus, resulting in a rapid detection (<1h) with high sensitivity (102 CFU·mL−1). PMID:26042607

Enzymatically Regulated Peptide Pairing and Catalysis for the Bioanalysis of Extracellular Prometastatic Activities of Functionally Linked Enzymes

NASA Astrophysics Data System (ADS)

Li, Hao; Huang, Yue; Yu, Yue; Li, Tianqi; Li, Genxi; Anzai, Jun-Ichi

2016-05-01

Diseases such as cancer arise from systematical reconfiguration of interactions of exceedingly large numbers of proteins in cell signaling. The study of such complicated molecular mechanisms requires multiplexed detection of the inter-connected activities of several proteins in a disease-associated context. However, the existing methods are generally not well-equipped for this kind of application. Here a method for analyzing functionally linked protein activities is developed based on enzyme controlled pairing between complementary peptide helix strands, which simultaneously enables elaborate regulation of catalytic activity of the paired peptides. This method has been used to detect three different types of protein modification enzymes that participate in the modification of extracellular matrix and the formation of invasion front in tumour. In detecting breast cancer tissue samples using this method, up-regulated activity can be observed for two of the assessed enzymes, while the third enzyme is found to have a subtle fluctuation of activity. These results may point to the application of this method in evaluating prometastatic activities of proteins in tumour.

Bioluminescence methods for enzymatic determinations

DOEpatents

Bostick, William D.; Denton, Mark S.; Dinsmore, Stanley R.

1982-01-01

An enzymatic method for continuous, on-line and rapid detection of diagnostically useful biomarkers, which are symptomatic of disease or trauma-related tissue damage, is disclosed. The method is characterized by operability on authentic samples of complex biological fluids which contain the biomarkers.

Non-Enzymatic Glucose Sensor Composed of Carbon-Coated Nano-Zinc Oxide

PubMed Central

Chung, Ren-Jei; Wang, An-Ni; Liao, Qing-Liang; Chuang, Kai-Yu

2017-01-01

Nowadays glucose detection is of great importance in the fields of biological, environmental, and clinical analyzes. In this research, we report a zinc oxide (ZnO) nanorod powder surface-coated with carbon material for non-enzymatic glucose sensor applications through a hydrothermal process and chemical vapor deposition method. A series of tests, including crystallinity analysis, microstructure observation, and electrochemical property investigations were carried out. For the cyclic voltammetric (CV) glucose detection, the low detection limit of 1 mM with a linear range from 0.1 mM to 10 mM was attained. The sensitivity was 2.97 μA/cm2mM, which is the most optimized ever reported. With such good analytical performance from a simple process, it is believed that the nanocomposites composed of ZnO nanorod powder surface-coated with carbon material are promising for the development of cost-effective non-enzymatic electrochemical glucose biosensors with high sensitivity. PMID:28336869

Continuous adsorption and biotransformation of micropollutants by granular activated carbon-bound laccase in a packed-bed enzyme reactor.

PubMed

Nguyen, Luong N; Hai, Faisal I; Dosseto, Anthony; Richardson, Christopher; Price, William E; Nghiem, Long D

2016-06-01

Laccase was immobilized on granular activated carbon (GAC) and the resulting GAC-bound laccase was used to degrade four micropollutants in a packed-bed column. Compared to the free enzyme, the immobilized laccase showed high residual activities over a broad range of pH and temperature. The GAC-bound laccase efficiently removed four micropollutants, namely, sulfamethoxazole, carbamazepine, diclofenac and bisphenol A, commonly detected in raw wastewater and wastewater-impacted water sources. Mass balance analysis showed that these micropollutants were enzymatically degraded following adsorption onto GAC. Higher degradation efficiency of micropollutants by the immobilized compared to free laccase was possibly due to better electron transfer between laccase and substrate molecules once they have adsorbed onto the GAC surface. Results here highlight the complementary effects of adsorption and enzymatic degradation on micropollutant removal by GAC-bound laccase. Indeed laccase-immobilized GAC outperformed regular GAC during continuous operation of packed-bed columns over two months (a throughput of 12,000 bed volumes). Copyright © 2016 Elsevier Ltd. All rights reserved.

Trypanosoma brucei Metacaspase 4 Is a Pseudopeptidase and a Virulence Factor*

PubMed Central

Proto, William R.; Castanys-Munoz, Esther; Black, Alana; Tetley, Laurence; Moss, Catherine X.; Juliano, Luiz; Coombs, Graham H.; Mottram, Jeremy C.

2011-01-01

Metacaspases are caspase family cysteine peptidases found in plants, fungi, and protozoa but not mammals. Trypanosoma brucei is unusual in having five metacaspases (MCA1–MCA5), of which MCA1 and MCA4 have active site substitutions, making them possible non-enzymatic homologues. Here we demonstrate that recombinant MCA4 lacks detectable peptidase activity despite maintaining a functional peptidase structure. MCA4 is expressed primarily in the bloodstream form of the parasite and associates with the flagellar membrane via dual myristoylation/palmitoylation. Loss of function phenotyping revealed critical roles for MCA4; rapid depletion by RNAi caused lethal disruption to the parasite's cell cycle, yet the generation of MCA4 null mutant parasites (Δmca4) was possible. Δmca4 had normal growth in axenic culture but markedly reduced virulence in mice. Further analysis revealed that MCA4 is released from the parasite and is specifically processed by MCA3, the only metacaspase that is both palmitoylated and enzymatically active. Accordingly, we have identified that the multiple metacaspases in T. brucei form a membrane-associated proteolytic cascade to generate a pseudopeptidase virulence factor. PMID:21949125

High-performance non-enzymatic catalysts based on 3D hierarchical hollow porous Co3O4 nanododecahedras in situ decorated on carbon nanotubes for glucose detection and biofuel cell application.

PubMed

Wang, Shiyue; Zhang, Xiaohua; Huang, Junlin; Chen, Jinhua

2018-03-01

In this work, high-performance non-enzymatic catalysts based on 3D hierarchical hollow porous Co 3 O 4 nanododecahedras in situ decorated on carbon nanotubes (3D Co 3 O 4 -HPND/CNTs) were successfully prepared via direct carbonizing metal-organic framework-67 in situ grown on carbon nanotubes. The morphology, microstructure, and composite of 3D Co 3 O 4 -HPND/CNTs were characterized by scanning electron microscopy, transmission electron microscopy, micropore and chemisorption analyzer, and X-ray diffraction. The electrochemical characterizations indicated that 3D Co 3 O 4 -HPND/CNTs present considerably catalytic activity toward glucose oxidation and could be promising for constructing high-performance electrochemical non-enzymatic glucose sensors and glucose/O 2 biofuel cell. When used for non-enzymatic glucose detection, the 3D Co 3 O 4 -HPND/CNTs modified glassy carbon electrode (3D Co 3 O 4 -HPND/CNTs/GCE) exhibited excellent analytical performance with high sensitivity (22.21 mA mM -1  cm -2 ), low detection limit of 0.35 μM (S/N = 3), fast response (less than 5 s) and good stability. On the other hand, when the 3D Co 3 O 4 -HPND/CNTs/GCE worked as an anode of a biofuel cell, a maximum power density of 210 μW cm -2 at 0.15 V could be obtained, and the open circuit potential was 0.68 V. The attractive 3D hierarchical porous structural features, the large surface area, and the excellent conductivity based on the continuous and effective electron transport network in 3D Co 3 O 4 -HPND/CNTs endow 3D Co 3 O 4 -HPND/CNTs with the enhanced electrochemical performance and promising applications in electrochemical sensing, biofuel cell, and other energy storage and conversion devices such as supercapacitor. Graphical abstract High-performance non-enzymatic catalysts for enzymeless glucose sensing and biofuel cell based on 3D hierarchical hollow porous Co 3 O 4 nanododecahedras anchored on carbon nanotubes were successfully prepared via direct carbonizing metal-organic framework-67 in situ grown on carbon nanotubes.

Validation of amino-acids measurement in dried blood spot by FIA-MS/MS for PKU management.

PubMed

Bruno, C; Dufour-Rainfray, D; Patin, F; Vourc'h, P; Guilloteau, D; Maillot, F; Labarthe, F; Tardieu, M; Andres, C R; Emond, P; Blasco, H

2016-09-01

Phenylketonuria (PKU) is a metabolic disorder leading to high concentrations of phenylalanine (Phe) and low concentrations of tyrosine (Tyr) in blood and brain that may be neurotoxic. This disease requires a regular monitoring of plasma Phe and Tyr as well as branched-chain amino-acids concentrations to adapt the Phe-restricted diet and other therapy that may be prescribed in PKU. We validated a Flow Injection Analysis tandem Mass Spectrometry (FIA-MS/MS) to replace the enzymatic method routinely used for neonatal screening in order to monitor in parallel to Phe, Tyr and branched-chain amino-acids not detected by the enzymatic method. We ascertained the performances of the method: linearity, detection and quantification limits, contamination index, accuracy. We cross validated the FIA-MS/MS and enzymatic methods and we evaluated our own reference ranges to monitor Phe, Tyr, Leu, Val on 59 dried blood spots of normal controls. We also evaluated Tyr, Leu and Val concentrations in PKU patients to detect some potential abnormalities, not evaluated by the enzymatic method. We developed a rapid method with excellent performances including precision and accuracy <15%. We noted an excellent correlation of Phe concentrations between FIA-MS/MS and enzymatic methods (p<0.0001) based on our database which are similar to references ranges published. We observed that 50% of PKU patients had lower concentrations of Tyr, Leu and/or Val that could not be detected by the enzymatic method. Based on laboratory accreditation recommendations, we validated a robust, rapid and reliable FIA-MS/MS method to monitor plasma Phe concentrations but also Tyr, Leu and Val concentrations, suitable for PKU management. We evaluated our own reference ranges of concentration for a routine application of this method. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Hierarchical 3-dimensional nickel-iron nanosheet arrays on carbon fiber paper as a novel electrode for non-enzymatic glucose sensing.

PubMed

Kannan, Palanisamy; Maiyalagan, Thandavarayan; Marsili, Enrico; Ghosh, Srabanti; Niedziolka-Jönsson, Joanna; Jönsson-Niedziolka, Martin

2016-01-14

Three-dimensional nickel-iron (3-D/Ni-Fe) nanostructures are exciting candidates for various applications because they produce more reaction-active sites than 1-D and 2-D nanostructured materials and exhibit attractive optical, electrical and catalytic properties. In this work, freestanding 3-D/Ni-Fe interconnected hierarchical nanosheets, hierarchical nanospheres, and porous nanospheres are directly grown on a flexible carbon fiber paper (CFP) substrate by a single-step hydrothermal process. Among the nanostructures, 3-D/Ni-Fe interconnected hierarchical nanosheets show excellent electrochemical properties because of its high conductivity, large specific active surface area, and mesopores on its walls (vide infra). The 3-D/Ni-Fe hierarchical nanosheet array modified CFP substrate is further explored as a novel electrode for electrochemical non-enzymatic glucose sensor application. The 3-D/Ni-Fe hierarchical nanosheet arrays exhibit significant catalytic activity towards the electrochemical oxidation of glucose, as compared to the 3-D/Ni-Fe hierarchical nanospheres, and porous nanospheres. The 3-D/Ni-Fe hierarchical nanosheet arrays can access a large amount of glucose molecules on their surface (mesopore walls) for an efficient electrocatalytic oxidation process. Moreover, 3-D/Ni-Fe hierarchical nanosheet arrays showed higher sensitivity (7.90 μA μM(-1) cm(-2)) with wide linear glucose concentration ranging from 0.05 μM to 0.2 mM, and the low detection limit (LOD) of 0.031 μM (S/N = 3) is achieved by the amperometry method. Further, the 3-D/Ni-Fe hierarchical nanosheet array modified CFP electrode can be demonstrated to have excellent selectivity towards the detection of glucose in the presence of 500-fold excess of major important interferents. All these results indicate that 3-D/Ni-Fe hierarchical nanosheet arrays are promising candidates for non-enzymatic glucose sensing.

THE LOCALIZATION OF CHOLINESTERASE ACTIVITY IN RAT CARDIAC MUSCLE BY ELECTRON MICROSCOPY

PubMed Central

Karnovsky, Morris J.

1964-01-01

A method has been developed for localizing sites of cholinesterase activity in rat cardiac muscle by electron microscopy. The method utilizes thiocholine esters as substrates, and is believed to be dependent on the reduction of ferricyanide to ferrocyanide by thiocholine released by enzymatic activity. The ferrocyanide thus formed is captured by copper to form fine, electron-opaque deposits of copper ferrocyanide, which sharply delineate sites of enzymatic activity at the ultrastructural level. Cholinesterase activity in formalin-fixed heart muscle was localized: (a) in longitudinal elements of the sarcoplasmic reticulum, but not in the T, or transverse, elements; and (b) in the A band, with virtually no activity noted in the M band, or in the H zone. The I band was also negative. No activity was detected in the sarcolemma, or in invaginations of the sarcolemma at the level of the Z band. The perinuclear element of the sarcoplasmic (endoplasmic) reticulum was frequently strongly positive. Activity at all sites was completely abolished by omitting the substrates, or by inhibition with eserine 10-4 M and diisopropylfluorophosphate 10-5 M. Eserine 10-5 M completely inhibited reaction in the sarcoplasmic reticulum, and virtually abolished that in the A band. These observations, together with the use of the relatively specific substrates and suitable controls to eliminate non-enzymatic staining, indicate that cholinesterase activity was being demonstrated. The activity in rat heart against different substrates was that of non-specific cholinesterases, in accordance with biochemical data. The activity in the A band was considered to be probably due to myosincholinesterase. It is proposed that the localization of cholinesterases in myocardium at the ultrastructural level should be taken into account in considering the possible functions of these myocardial enzymes, and it is hoped that knowledge of their localization will open up new avenues of approach in considering their physiological role in myocardium, which at present is not definitely known. PMID:14222810

Fabrication of sensitive enzymatic biosensor based on multi-layered reduced graphene oxide added PtAu nanoparticles-modified hybrid electrode

PubMed Central

Hossain, Md Faruk; Park, Jae Y.

2017-01-01

A highly sensitive amperometric glucose sensor was developed by immobilization of glucose oxidase (GOx) onto multi-layer reduced graphene oxide (MRGO) sheets decorated with platinum and gold flower-like nanoparticles (PtAuNPs) modified Au substrate electrode. The fabricated MRGO/PtAuNPs modified hybrid electrode demonstrated high electrocatalytic activities toward oxidation of H2O2, to which it had a wide linear response that ranged from 0.5 to 8 mM (R2 = 0.997), and high sensitivity of 506.25 μA/mMcm2. Furthermore, glucose oxidase-chitosan composite and cationic polydiallyldimethylammonium chloride (PDDA) were assembled by a casting method on the surface of MRGO/PtAuNPs modified electrode. This as-fabricated hybrid biosensor electrode exhibited high electrocatalytic activity for the detection of glucose in PBS. It demonstrated good analytical properties in terms of a low detection limit of 1 μM (signal-to-noise ratio of 3), short response time (3 s), high sensitivity (17.85 μA/mMcm2), and a wide linear range (0.01–8 mM) for glucose sensing. These results reveal that the newly developed sensing electrode offers great promise for new type enzymatic biosensor applications. PMID:28333943

Non-enzymatic browning kinetics analysed through water-solids interactions and water mobility in dehydrated potato.

PubMed

Acevedo, Nuria C; Schebor, Carolina; Buera, Pilar

2008-06-01

Non-enzymatic browning (NEB) development was studied in dehydrated potato at 70°C. It was related to the macroscopic and molecular properties and to water-solid interactions over a wide range of water activities. Time resolved (1)H NMR, thermal transitions and water sorption isotherms were evaluated. Although non-enzymatic browning could be detected in the glassy state; colour development was higher in the supercooled state. The reaction rate increased up to a water content of 26g/100g of solids (aw=0.84) and then decreased at higher water contents, concomitantly with the increase of water proton mobility. The joint analyses of NEB kinetics, water sorption isotherm and proton relaxation behaviour made it evident that the point at which the reaction rate decreased, after a maximum value, could be related to the appearance of highly mobile water. The results obtained in this work indicate that the prediction of chemical reaction kinetics can be performed through the integrated analysis of water sorption, water and solids mobility and the physical state of the matrix. Copyright © 2007 Elsevier Ltd. All rights reserved.

Management of enzyme diversity in high-performance cellulolytic cocktails.

PubMed

Reyes-Sosa, Francisco Manuel; López Morales, Macarena; Platero Gómez, Ana Isabel; Valbuena Crespo, Noelia; Sánchez Zamorano, Laura; Rocha-Martín, Javier; Molina-Heredia, Fernando P; Díez García, Bruno

2017-01-01

Modern biorefineries require enzymatic cocktails of improved efficiency to generate fermentable sugars from lignocellulosic biomass. Cellulolytic fungi, among other microorganisms, have demonstrated the highest potential in terms of enzymatic productivity, complexity and efficiency. On the other hand, under cellulolytic-inducing conditions, they often produce a considerable diversity of carbohydrate-active enzymes which allow them to adapt to changing environmental conditions. However, industrial conditions are fixed and adjusted to the optimum of the whole cocktail, resulting in underperformance of individual enzymes. One of these cellulolytic cocktails from Myceliophthora thermophila has been analyzed here by means of LC-MS/MS. Pure GH6 family members detected have been characterized, confirming previous studies, and added to whole cocktails to compare their contribution in the hydrolysis of industrial substrates. Finally, independent deletions of two GH6 family members, as an example of the enzymatic diversity management, led to the development of a strain producing a more efficient cellulolytic cocktail. These data indicate that the deletion of noncontributive cellulases (here EG VI) can increase the cellulolytic efficiency of the cocktail, validating the management of cellulase diversity as a strategy to obtain improved fungal cellulolytic cocktails.

Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: The N-terminal region is more important than enzymatic activity

PubMed Central

Rouault, Morgane; Rash, Lachlan D.; Escoubas, Pierre; Boilard, Eric; Bollinger, James; Lomonte, Bruno; Maurin, Thomas; Guillaume, Carole; Canaan, Stéphane; Deregnaucourt, Christiane; Schrével, Joseph; Doglio, Alain; Gutiérrez, José María; Lazdunski, Michel; Gelb, Michael H.; Lambeau, Gérard

2009-01-01

Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, an homologous but non toxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1–22) of OS2, but not the central one (residues 58–89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102–119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity. PMID:16669624

Development of a common biosensor format for an enzyme based biosensor array to monitor fruit quality.

PubMed

Jawaheer, Shobha; White, S F; Rughooputh, S D D V; Cullen, David C

2003-10-15

Individual enzyme-based biosensors involving three-electrode systems were developed for the detection of analytes comprising markers of the stage of maturity and quality in selected fruits of economic importance to tropical countries. Importantly, a common fabrication format has been developed to simplify manufacture and allow future integration of the individual sensors into a single multi-sensor array. Specifically, sensors for beta-D-glucose, total D-glucose, sucrose and ascorbic acid have been developed. Pectin, a natural polysaccharide present in plant cells, was used as a novel matrix to enhance enzyme entrapment and stabilisation in the sensors. Except for ascorbic acid, all the sensors function via the detection of enzymatically generated H2O2 at rhodinised carbon electrodes. Since ascorbic acid is electrochemically active at the working potential chosen (+350 mV vs. Ag/AgCl), it was measured directly. Enzyme sensors demonstrated expected response with respect to their substrates, typically 0-0.8 microA/20 mm2 electrode area response over analyte ranges of 0-7 mM. Interferences related to electrochemically active compounds present in fruits under study were significantly reduced by inclusion of a suitable cellulose acetate (CA) membrane or by enzymatic inactivation with ascorbate oxidase. Initial development was carried out into production of biosensor arrays. CA membranes were used to improve the linear range of the sensors, producing up to a fivefold improvement in the detection range compared to sensors without an additional diffusion barrier.

Inhibition of venom serine proteinase and metalloproteinase activities by Renealmia alpinia (Zingiberaceae) extracts: comparison of wild and in vitro propagated plants.

PubMed

Patiño, Arley Camilo; Benjumea, Dora María; Pereañez, Jaime Andrés

2013-09-16

The plant Renealmia alpinia has been used in folk medicine to treat snakebites in the northwest region of Colombia. In addition, it has been shown to neutralize edema-forming, hemorrhagic, lethal, and defibrin(ogen)ating activities of Bothrops asper venom. In this work, extracts of Renealmia alpinia obtained by micropropagation (in vitro) and from specimens collected in the wild were tested and compared in their capacity to inhibit enzymatic and toxic activities of a snake venom metalloproteinase isolated from Bothrops atrox (Batx-I) venom and a serine proteinase (Cdc SII) from Crotalus durissus cumanensis venom. We have investigated the inhibition capacity of Renealmia alpinia extracts on enzymatic and toxic actions of isolated toxins, a metalloproteinase and a serine proteinase. The protocols investigated included inhibition of proteolytic activity on azocasein, inhibition of proteolytic activity on fibrinogen, inhibition of pro-coagulant activity, inhibition of hemorrhagic activity and inhibition of edema-forming activity. Colorimetric assays detected the presence of terpenoids, flavonoids, tannins and coumarins in Renealmia alpinia extracts. Renealmia alpinia extracts inhibited the enzymatic, hemorrhagic and fibrinogenolytic activities of Batx-I. Extracts also inhibited coagulant, defibrin(ogen)ating and edema-forming activities of Cdc SII. Results highlight that Renealmia alpinia in vitro extract displayed comparable inhibitory capacity on venom proteinases that Renealmia alpinia wild extract. No alteration was observed in the electrophoretic pattern of venom proteinases after incubation with Renealmia alpinia extracts, thus excluding proteolytic degradation or protein denaturation/precipitation as a mechanism of inhibition. Our results showed that Renealmia alpinia wild and in vitro extracts contain compounds that neutralize metallo- and serine proteinases present in snake venoms. The mechanism of inhibition is not related to proteolytic degradation of the enzymes nor protein aggregation, but is likely to depend on molecular interactions of secondary metabolites in the plant with these venom proteinases. Crown Copyright © 2013 Published by Elsevier Ireland Ltd. All rights reserved.

Activation of RIG-I-like Receptor Signal Transduction

PubMed Central

Bruns, Annie; Horvath, Curt M.

2011-01-01

Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral response. Pattern recognition receptor proteins detect molecular signatures of virus infection and activate antiviral signaling cascades. The RIG-I-like receptors are cytoplasmic DExD/H box proteins that can specifically recognize virus-derived RNA species as a molecular feature discriminating the pathogen from the host. The RIG-I-like receptor family is composed of three homologous proteins, RIG-I, MDA5, and LGP2. All of these proteins can bind double-stranded RNA species with varying affinities via their conserved DExD/H box RNA helicase domains and C-terminal regulatory domains. The recognition of foreign RNA by the RLRs activates enzymatic functions and initiates signal transduction pathways resulting in the production of antiviral cytokines and the establishment of a broadly effective cellular antiviral state that protects neighboring cells from infection and triggers innate and adaptive immune systems. The propagation of this signal via the interferon antiviral system has been studied extensively, while the precise roles for enzymatic activities of the RNA helicase domain in antiviral responses are only beginning to be elucidated. Here, current models for RLR ligand recognition and signaling are reviewed. PMID:22066529

Allergenicity, trypsin inhibitor activity and nutritive quality of enzymatically modified soy proteins.

PubMed

De La Barca, Ana María Calderón; Wall, Abraham; López-Díaz, José Alberto

2005-05-01

Two ultrafiltered soy flour protein fractions were evaluated; the first was obtained by hydrolysis (0.5-3 kDa, F(0.5-3)), and the second was an enzymatically methionine-enriched fraction (1-10 kDa, F(1-10)E). Amino acid profiles, protein quality, allergenicity (against soy-sensitive infant sera) and trypsin inhibitor activity were determined. Fraction F(1-10)E fulfilled amino acid requirements for infants, whereas the F(0.5-3) fraction was methionine deficient. Both fractions were similar in net protein utilization, and F(1-10)E digestibility was comparable with casein and higher (P?

Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

NASA Astrophysics Data System (ADS)

Ioanna Skilitsi, Anastasia; Turko, Timothé; Cianfarani, Damien; Barre, Sophie; Uhring, Wilfried; Hassiepen, Ulrich; Léonard, Jérémie

2017-09-01

Time-resolved fluorescence detection for robust sensing of biomolecular interactions is developed by implementing time-correlated single photon counting in high-throughput conditions. Droplet microfluidics is used as a promising platform for the very fast handling of low-volume samples. We illustrate the potential of this very sensitive and cost-effective technology in the context of an enzymatic activity assay based on fluorescently-labeled biomolecules. Fluorescence lifetime detection by time-correlated single photon counting is shown to enable reliable discrimination between positive and negative control samples at a throughput as high as several hundred samples per second.

Digestive enzymatic activity on tropical gar (Atractosteus tropicus) larvae fed different diets.

PubMed

Aguilera, Carlos; Mendoza, Roberto; Iracheta, Israel; Marquez, Gabriel

2012-06-01

Digestive enzymatic activity and growth performance on tropical gar (Atractosteus tropicus) larvae fed Artemia nauplii (LF), frozen adult Artemia (AB), an artificial diet (AF) with 46% protein and 16% lipids and a starvation group (SG) from first feeding (5 days after hatching-5 DAH) to 34 DAH were studied. All larvae under starvation (SG) died at 15 DAH. By the end of the experimental period, morphological variables (total length, wet weight and specific growth rate) were significant in larvae fed AF compared to LF and AB. All enzymes studied in the experiment were present since the start of exogenous feeding (including pepsin) and the enzymatic activity varied with the diets. Low levels of enzymatic activity were observed until the 29 DAH; however, after this moment, there was a significant increase (eightfold), particularly for the AF treatment. In vitro protein digestibility tests performed with enzymatic extracts showed that artificial diets with 52% protein and 14% lipids were better digested by larvae before 30 DAH, while diets with 45% protein and 11% lipids were better digested after this age. Taking into account the better growth performance, higher enzymatic activity and better protein digestibility obtained, artificial diets can be used since the start of exogenous feeding on tropical gar larvae, as in other lepisosteids.

Deletion mutation analysis on C-terminal domain of plant vacuolar H(+)-pyrophosphatase.

PubMed

Lin, Hsin Hung; Pan, Yih Jiuan; Hsu, Shen Hsing; Van, Ru Chuan; Hsiao, Yi Yuong; Chen, Jiun Hsien; Pan, Rong Long

2005-10-15

Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton-translocase; it contains a single type of polypeptide of approximately 81kDa. A line of evidence demonstrated that the carboxyl terminus of V-PPase is relatively conserved in various plant V-PPases and presumably locates in the vicinity of the catalytic site. In this study, we attempt to identify the roles of the C-terminus of V-PPase by generating a series of C-terminal deletion mutants over-expressed in Saccharomyces cerevisiae, and determining their enzymatic and proton translocating reactions. Our results showed that the deletion mutation at last 5 amino acids in the C-terminus (DeltaC5) induced a dramatic decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase; but the mutant lacking last 10 amino acids (DeltaC10) retained about 60-70% of the enzymatic activity of wild-type. Truncation of the C-terminus by more than 10 amino acids completely abolished the enzymatic activity and proton translocation of V-PPase. Furthermore, the DeltaC10 mutant displayed a shift in T(1/2) (pretreatment temperature at which half enzymatic activity is observed) but not the optimal pH for PP(i) hydrolytic activity. The deletion of the C-terminus substantially modified apparent K(+) binding constant, but exert no significant changes in the Na(+)-, F(-)-, and Ca(2+)-inhibition of the enzymatic activity of V-PPase. Taken together, we speculate that the C-terminus of V-PPase may play a crucial role in sustaining enzymatic activity and is likely involved in the K(+)-regulation of the enzyme in an indirect manner.

Non-enzymatic detection of glucose using poly(azure A)-nickel modified glassy carbon electrode.

PubMed

Liu, Tong; Luo, Yiqun; Zhu, Jiaming; Kong, Liyan; Wang, Wen; Tan, Liang

2016-08-15

A simple, sensitive and selective non-enzymatic glucose sensor was constructed in this paper. The poly(azure A)-nickel modified glassy carbon electrode was successfully fabricated by the electropolymerization of azure A and the adsorption of Ni(2+). The Ni modified electrode, which was characterized by scanning electron microscope, cyclic voltammetry, electrochemical impedance spectra and X-ray photoelectron spectroscopy measurements, respectively, displayed well-defined current responses of the Ni(III)/Ni(II) couple and showed a good activity for electrocatalytic oxidation of glucose in alkaline medium. Under the optimized conditions, the developed sensor exhibited a broad linear calibration range of 5 μM-12mM for quantification of glucose and a low detection limit of 0.64μM (3σ). The excellent analytical performance including simple structure, fast response time, good anti-interference ability, satisfying stability and reliable reproducibility were also found from the proposed amperometric sensor. The results were satisfactory for the determination of glucose in human serum samples as comparison to those from a local hospital. Copyright © 2016 Elsevier B.V. All rights reserved.

Nickel-copper oxide nanowires for highly sensitive sensing of glucose

NASA Astrophysics Data System (ADS)

Bai, Xiaofang; Chen, Wei; Song, Yanfang; Zhang, Jiazhou; Ge, Ruipeng; Wei, Wei; Jiao, Zheng; Sun, Yuhan

2017-10-01

Accurate determination of glucose is of considerable importance in diverse fields such as clinical diagnostics, biotechnology, and food industry. A low-cost and easy to scale-up approach has been developed for the preparation of nickel-copper oxide nanowires (Ni-CuO NWs) with hierarchical structures comprising porous NiO substrate and CuO nanowires. The successfully prepared Ni-CuO NWs were exploited as non-enzymatic electrochemical sensing probes for the reliable detection of glucose. Electrochemical measurements such as cyclic voltammetry (CV) and chronoamperometry (CA) illustrated that the Ni-CuO NWs exhibited excellent electrochemical performance toward glucose oxidation with a superior sensitivity of 5610.6 μA mM-1 cm-2, a low detection limit of 0.07 μM, a wide linear range from 0.2 to 3.0 mM, and a good selectivity. This was attributed to the synergetic effect of the hierarchical structures and active Ni(OH)2 surface species in Ni-CuO NWs. The rational design of the metal oxide composites provided an efficient strategy for the fabrication of electrochemical non-enzymatic sensors.

Quantitation of Na+, K+-atpase Enzymatic Activity in Tissues of the Mammalian Vestibular System

NASA Technical Reports Server (NTRS)

Kerr, T. P.

1985-01-01

In order to quantify vestibular Na(+), K(+)-ATPase, a microassay technique was developed which is sufficiently sensitive to measure the enzymatic activity in tissue from a single animal. The assay was used to characterize ATPase in he vestibular apparatus of the Mongolian gerbil. The quantitative procedure employs NPP (5 mM) as synthetic enzyme substrate. The assay relies upon spectrophotometric measurement (410 nm) of nitrophenol (NP) released by enzymatic hydrolysis of the substrate. Product formation in the absence of ouabain reflects both specific (Na(+), K(+)-ATPase) and non-specific (Mg(++)-ATPase) enzymatic activity. By measuring the accumulation of reaction product (NP) at three-minute intervals during the course of incubation, it is found that the overall enzymatic reaction proceeds linearly for at least 45 minutes. It is therefore possible to determine two separate reaction rates from a single set of tissues. Initial results indicate that total activity amounts to 53.3 + or - 11.2 (S.E.M.) nmol/hr/mg dry tissue, of which approximately 20% is ouabain-sensitive.

Effect of restricted motion in high temperature on enzymatic activity of the pancreas

NASA Technical Reports Server (NTRS)

Abdusattarov, A.; Smirnova, G. I.

1980-01-01

Effects of 30 day hypodynamia coupled with high temperature (35-36 C) on enzymatic activity of the pancreas of male adult rats were studied. The test animals were divided into four groups. Group one served as controls (freedom of movement and a temperature of 25-26 C, considered optimal). The remaining animals were divided into three additional groups: Group two freedom of movement but high temperature (35-36 C); group three hypodynamia but an optimal temperature; group four hypodynamia and 35-36 C. Considerable change in the enzymatic activity in the pancreas of the four groups is observed in three experimental groups (two, three, and four) as compared to the control (group one). The results indicate that adaption of the organism to the thermal factor and restricted movement is accompanied by a change in the enzymatic spectrum of the pancreas. With the combined effect of these two stresses under conditions of the adaption of the organism especially sharp shifts occur in the enzymatic activity.

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues.

PubMed

Molenaar, Remco J; Khurshed, Mohammed; Hira, Vashendriya V V; Van Noorden, Cornelis J F

2018-05-26

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation. We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P) + and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase's activity. The formazan's absorbance is therefore a direct measure of the dehydrogenase's activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays. Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Rondonin an antifungal peptide from spider (Acanthoscurria rondoniae) haemolymph

PubMed Central

Riciluca, K.C.T.; Sayegh, R.S.R.; Melo, R.L.; Silva, P.I.

2012-01-01

Antimicrobial activities were detected in the haemolymph of the spider Acanthoscurrria rondoniae. A novel antifungal peptide, rondonin, was purified by reverse phase high performance liquid chromatography (RP-HPLC). Rondonin has an amino acid sequence of IIIQYEGHKH and a molecular mass of 1236.776 Da. This peptide has identity to a C-terminal fragment of the “d” subunit of haemocyanin from the spiders Eurypelma californicum and Acanthoscurria gomesiana. A synthetic peptide mimicking rondonin had identical characteristics to those of the isolated material, confirming its sequence. The synthetic peptide was active only against fungus. These data led us to conclude that the antifungal activity detected in the plasma of these spiders is the result of enzymatic processing of a protein that delivers oxygen in the haemolymph of many chelicerate. Several studies have suggested that haemocyanins are involved in the arthropod immune system, and the activity of this haemocyanin fragment reinforces this idea. PMID:24371568

Novel, dually radiolabeled peptides for simultaneous monitoring of enzymatic activity and protein targets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Efrem Mebrahtu, Suzanne Lapi

2012-12-13

This application investigated a novel imaging approach to develop methods to incorporate multiple radionuclides into a single peptide at chemoselective sites for simultaneous monitoring of cell-bound protein targets as well as specific enzymatic activity, both of which are associated with enhanced tumor growth and metastasis. This imaging construct was synthesized in such a manner so that the PET radionuclide will remain associated with the tumor cells and the SPECT radionuclide was cleaved from the imaging agent. Measurement of the PET agent only will yield information about the tumor marker density while measurement of the amount of co-localization and mismatch ofmore » the two radionuclides will yield information about the enzymatic activity. This coincident measuring technique using both PET and SPECT agents allows us to draw correlations involving the interactions of enzymes (cathepsin, serine-protease urokinase (uPA) and matrix metalloproteases) and other cellular proteins which play a role in cancer growth and metastasis. This technique will allow for studies in xenograft or genetic models of cancer in the same animal at the same time, thus eliminating problems that may occur when trying to invoke comparisons across animals or timepoints. By using radionuclide imaging as opposed to other imaging modalities, this technique has the potential to be translatable and can exploit the high specific activity probes which can be generated with radiotracers. The proof of principle test of this system investigated simultaneous monitoring of matrix metalloprotease (MMP) activity in the extracellular matrix (ECM) as well as density of integrins on the cell surface, both of which can serve as tumor markers. The outcomes/deliverables of this project were as follows: 1. Peptides were synthesized dually labeled at chemospecific sites with PET and SPECT agents. 2. Stability (intrinsic and to radiolysis) and specific activity of these labeled compounds were determined. 3. The feasibility of using these agents for simultaneous monitoring of MMP-2 enzymatic activity and ²3 integrin density was demonstrated in several in vitro assays Radiotracers can be detected at concentrations up to 1000 fold lower than those labeled with non-radioactive markers (e.g. MRI contrast agents), thus using this technique has the advantage of very high sensitivity to measure these processes in vivo. Hence, the development of an efficient approach to the dual labeling of these molecular probes is embodied within this project, with the end result yielding a molecular imaging probe with the highest specific activity possible. An advantage to this dual labeling approach is the ability to measure two different biochemical processes at the same time, a benefit which is not possible in scans involving protocols utilizing two different radiolabeled agents injected sequentially. Another advantage to this technique is the ability to measure enzymatic activity in the form of substrate cleavage. This can only be achieved with a dually labeled compound as has been demonstrated in the case of FRET1. To our knowledge this is the first instance of a measurement of enzymatic substrate cleavage by a dually labeled PET/SPECT radionuclide imaging agent.« less

Review and drug therapy implications of glucose-6-phosphate dehydrogenase deficiency.

PubMed

Belfield, Kristen D; Tichy, Eric M

2018-02-01

The pathophysiology, diagnosis, and medication-use implications of glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common enzyme deficiency in humans, are reviewed. Originally identified as favism in patients who experienced hemolysis after ingestion of fava beans, G6PD deficiency results from an X-linked chromosomal mutation that leads to reduced activity of the enzyme responsible for the final step of the pentose phosphate pathway, through which reduced nicotinamide adenine dinucleotide phosphate required for protection of cells from oxidative stress is produced. G6PD deficiency affects about 400 million people worldwide. Diagnosis of G6PD can be made through detection of enzymatic activity (by spectrophotometric testing, fluorescence testing, or formazan-based spot testing) or molecular analysis to detect known mutations of the gene encoding G6PD. Most individuals with G6PD deficiency are asymptomatic throughout life. Symptoms of acute hemolysis associated with G6PD deficiency include anemia, fatigue, back or abdominal pain, jaundice, and hemoglobinuria. The most common precipitators of oxidative stress and hemolysis in G6PD deficiency include medication use and infection. G6PD deficiency should be considered in patients who experience acute hemolysis after exposure to known oxidative medications, infection, or ingestion of fava beans. A diagnosis of G6PD deficiency is most often made through enzymatic activity detection, but molecular analysis may be required in females heterozygous for the disorder. When clinically feasible, rasburicase, primaquine, dapsone, pegloticase, and methylene blue should not be used until a G6PD diagnostic test has been performed. Copyright © 2018 by the American Society of Health-System Pharmacists, Inc. All rights reserved.

"Signal-on" photoelectrochemical biosensor for sensitive detection of human T-Cell lymphotropic virus type II DNA: dual signal amplification strategy integrating enzymatic amplification with terminal deoxynucleotidyl transferase-mediated extension.

PubMed

Shen, Qingming; Han, Li; Fan, Gaochao; Zhang, Jian-Rong; Jiang, Liping; Zhu, Jun-Jie

2015-01-01

A novel "signal-on" photoelectrochemical (PEC) biosensor for sensitive detection of human T-cell lymphotropic virus type II (HTLV-II) DNA was developed on the basis of enzymatic amplification coupled with terminal deoxynucleotidyl transferase (TdT)-mediated extension strategy. The intensity of the photocurrent signal was proportional to the concentration of the HTLV-II DNA-target DNA (tDNA) by dual signal amplification. In this protocol, GR-CdS:Mn/ZnS nanocomposites were used as photoelectric conversion material, while pDNA was used as the tDNA recognizing unit. Moreover, the TdT-mediated extension and the enzymatic signal amplification technique were used to enhance the sensitivity of detection. Using this novel dual signal amplification strategy, the prototype of PEC DNA sensor can detect as low as ∼0.033 fM of HTLV-II DNA with a linear range of 0.1-5000 fM, with excellent differentiation ability even for single-base mismatches. This PEC DNA assay opens a promising platform to detect various DNA targets at ultralow levels for early diagnoses of different diseases.

IDENTIFICATION OF DIMETHYLTHIOARSINIC ACID BY ICP-MS AND IC-ESI-MS/MS IN RICE SAMPLES

EPA Science Inventory

Recently, sulfur analogs of well known arsenicals have been identified in biological and dietary matrices. In this presentation, the detection and identification of dimethylthioarsinic acid (DMTA) will be reported in rice samples after an enzymatic extraction. The enzymatic ext...

High-κ GdTixOy sensing membrane-based electrolyte-insulator-semiconductor with magnetic nanoparticles as enzyme carriers for protein contamination-free glucose biosensing.

PubMed

Wu, Min-Hsien; Yang, Hung-Wei; Hua, Mu-Yi; Peng, Yen-Bo; Pan, Tung-Ming

2013-09-15

This paper reports an electrolyte-insulator-semiconductor (EIS) device featuring a novel high-κ GdTixOy sensing membrane for high-performance pH sensing and glucose biosensing. The effect of the annealing temperature (700, 800, or 900°C) on the sensing properties of the GdTixOy membranes was investigated. The GdTixOy EIS device annealed at 900°C exhibited the greatest pH sensing performance, including the highest sensitivity (62.12mV/pH), the smallest hysteresis voltage (5mV), and the lowest drift rate (0.4mV/h), presumably because of its well-crystallized GdTixOy structure. To overcome the problems typically encountered during the practical application of biosensors (e.g., protein adsorption; preservation of enzymatic activity), we employed Fe3O4-based magnetic nanoparticles (MNPs) as enzyme carriers. The adsorption of serum protein on the unmodified sensing membrane led to poor EIS-based pH sensing (r(2)=0.71); the performance was greatly improved, however, after attaching the MNPs to the sensing membrane, thereby blocking protein adsorption significantly (by 98%) and allowing excellent pH sensing (r(2)=0.99). Moreover, we prepared a hybrid configuration of the proposed GdTixOy membrane-EIS, with magnetically attached glucose oxidase-immobilized MNPs, for glucose biosensing. The use of MNPs as enzyme carriers effectively preserved the enzymatic activity of glucose oxidase, with 45.3% of the original enzymatic activity retained after 120h of storage at 4°C (compared with complete loss of the free enzyme's activity under the same storage conditions). In addition, the proposed biosensor exhibited superior detection sensitivity of 11.03mV/mM relative to that (8.17mV/mM) obtained using the conventional enzyme immobilization method. Finally, we established the accuracy of the proposed method for blood glucose measurement; gratifyingly, blood glucose detection was comparable with the high-sensitivity glucose quantification obtained using a commercial glucose assay kit. Copyright © 2013 Elsevier B.V. All rights reserved.

Intraspecific Variation of Centruroides Edwardsii Venom from Two Regions of Colombia

PubMed Central

Estrada-Gómez, Sebastián; Cupitra, Nelson Ivan; Arango, Walter Murillo; Vargas Muñoz, Leidy Johana

2014-01-01

We report the first description studies, partial characterization, and intraspecific difference of Centruroides edwardsii, Gervais 1843, venom. C. edwardsii from two Colombian regions (Antioquia and Tolima) were evaluated. Both venoms showed hemolytic activity, possibly dependent of enzymatic active phospholipases, and neither coagulant nor proteolytic activities were observed. Venom electrophoretic profile showed significant differences between C. edwardsii venom from both regions. A high concentration of proteins with molecular masses between 31 kDa and 97.4 kDa, and an important concentration close or below 14.4 kDa were detected. RP-HPLC retention times between 38.2 min and 42.1 min, showed bands close to 14.4 kDa, which may correspond to phospholipases. RP-HPLC venom profile showed a well conserved region in both venoms between 7 and 17 min, after this, significant differences were detected. From Tolima region venom, 50 well-defined peaks were detected, while in the Antioquia region venom, 55 well-defined peaks were detected. Larvicidal activity was only detected in the C. edwardsii venom from Antioquia. No antimicrobial activity was observed using complete venom or RP-HPLC collected fractions of both venoms. Lethally activity (carried out on female albino swiss mice) was detected at doses over 19.2 mg/kg of crude venom. Toxic effects included distress, excitability, eye irritation and secretions, hyperventilation, ataxia, paralysis, and salivation. PMID:25025710

Use of enzymatic tools for biomonitoring inorganic pollution in aquatic sediments: a case study (Bor, Serbia)

PubMed Central

2013-01-01

Background Sediment bacterial communities are key players in biogeochemical cycling of elements in the aquatic environment. Copper mining, smelting, and processing operations located in Bor area (Serbia) are major environmental hot spots in the lower Danube Basin and Western Balkans. In the present study, we evaluate the influence of trace element (TE) concentration in sediments and physico-chemical properties of water on sediment microbial communities in water streams adjacent to the Copper Smelter Complex Bor (RTB Bor, Serbia). The degree to which metabolic activities of bacterial biota inhabiting differently polluted sites is inhibited by inorganic pollution were compared using selected enzymatic bioindicators. Results Cu, Zn, Pb, and As concentrations systematically exceeded the target values for metal loadings in aquatic sediments. Water electrical conductivity (WEC) followed the same pattern of spatial variation, irrespective of season. Interestingly, the most intense enzymatic activity occurred at the reference site although this site showed the greatest TE levels in aquatic sediments. Catalase activity (CA), potential dehydrogenase activity (PDA), actual dehydrogenase activity (ADA), urease activity (UA), and phosphatase activity (PA) in aquatic sediments displayed heterogeneous patterns of spatio-temporal variation. Inorganic pollution greatly affected CA, ADA, and PDA, but much less so UA and PA. Canonical correlation analysis showed that pH and WEC were the strongest determinants of enzymatic activity in bacterial biota, with the latter variable being reversely correlated with the enzymatic indicator of sediment quality (EISQ). The median values of EISQ increased with distance from the major sources of pollution. In addition, it was found that sites with different degrees of inorganic pollution can be appropriately classified by applying cluster analysis to EISQ, TE levels in sediments, and physico-chemical properties of water. Conclusions Because EISQ can precisely identify changes in overall enzymatic activity of sediment bacterial communities, this enzymatic bioindicator has a great potential for biomonitoring the current status of inorganic pollution in aquatic ecosystems. PMID:23536970

Chitosan-induced Au/Ag nanoalloy dispersed in IL and application in fabricating an ultrasensitive glucose biosensor based on luminol-H₂O₂-Cu²⁺/IL chemiluminescence system.

PubMed

Chaichi, M J; Alijanpour, S O

2014-11-01

A novel glucose biosensor based on the chemiluminescence (CL) detection of enzymatically generated hydrogen peroxide (H₂O₂) was constructed by one covalent immobilization of glucose oxidase (GOD) in glutaraldehyde-functionalized glass cell. In following, chitosan-induced Au/Ag nanoparticles dispersed in ion liquid (IL) were synthesised and immobilized on it. Herein, chitosan molecules acted as both the reducing and stabilizing agent for the preparation of NPs and also, as a coupling agent GOD and Au/Ag alloy NPs. In addition to catalyze luminol CL reaction, these NPs offered excellent catalytic activity toward hydrogen peroxide generation in enzymatic reaction between GOD and glucose. The used IL in fabrication of biosensor increased its stability. Also, IL alongside Cu(2+) accelerated enzymatic and CL reaction kinetic, and decreased luminol CL reaction optimum pH to 7.5 which would enable sensitive and precision determination of glucose. Under optimum condition, linear response range of glucose was found to be 1.0 × 10(-6)-7.5 × 10(-3)M, and detection limit was 4.0 × 10(-7)M. The CL biosensor exhibited good storage stability, i.e., 90% of its initial response was retained after 2 months storage at pH 7.0. The present CL biosensor has been applied satisfactory to analysis of glucose in real serum and urine samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Bridging Enzymatic Structure Function via Mechanics: A Coarse-Grain Approach.

PubMed

Sacquin-Mora, S

2016-01-01

Flexibility is a central aspect of protein function, and ligand binding in enzymes involves a wide range of structural changes, ranging from large-scale domain movements to small loop or side-chain rearrangements. In order to understand how the mechanical properties of enzymes, and the mechanical variations that are induced by ligand binding, relate to enzymatic activity, we carried out coarse-grain Brownian dynamics simulations on a set of enzymes whose structures in the unbound and ligand-bound forms are available in the Protein Data Bank. Our results show that enzymes are remarkably heterogeneous objects from a mechanical point of view and that the local rigidity of individual residues is tightly connected to their part in the protein's overall structure and function. The systematic comparison of the rigidity of enzymes in their unbound and bound forms highlights the fact that small conformational changes can induce large mechanical effects, leading to either more or less flexibility depending on the enzyme's architecture and the location of its ligand-biding site. These mechanical variations target a limited number of specific residues that occupy key locations for enzymatic activity, and our approach thus offers a mean to detect perturbation-sensitive sites in enzymes, where the addition or removal of a few interactions will lead to important changes in the proteins internal dynamics. © 2016 Elsevier Inc. All rights reserved.

Hydrothermal synthesis of NiWO4 crystals for high performance non-enzymatic glucose biosensors

NASA Astrophysics Data System (ADS)

Mani, Sivakumar; Vediyappan, Veeramani; Chen, Shen-Ming; Madhu, Rajesh; Pitchaimani, Veerakumar; Chang, Jia-Yaw; Liu, Shang-Bin

2016-04-01

A facile hydrothermal route for the synthesis of ordered NiWO4 nanocrystals, which show promising applications as high performance non-enzymatic glucose sensor is reported. The NiWO4-modified electrodes showed excellent sensitivity (269.6 μA mM-1 cm-2) and low detection limit (0.18 μM) for detection of glucose with desirable selectivity, stability, and tolerance to interference, rendering their prospective applications as cost-effective, enzyme-free glucose sensors.

Hydrothermal synthesis of NiWO4 crystals for high performance non-enzymatic glucose biosensors.

PubMed

Mani, Sivakumar; Vediyappan, Veeramani; Chen, Shen-Ming; Madhu, Rajesh; Pitchaimani, Veerakumar; Chang, Jia-Yaw; Liu, Shang-Bin

2016-04-18

A facile hydrothermal route for the synthesis of ordered NiWO4 nanocrystals, which show promising applications as high performance non-enzymatic glucose sensor is reported. The NiWO4-modified electrodes showed excellent sensitivity (269.6 μA mM(-1 )cm(-2)) and low detection limit (0.18 μM) for detection of glucose with desirable selectivity, stability, and tolerance to interference, rendering their prospective applications as cost-effective, enzyme-free glucose sensors.

Enzyme-Initiated Quinone-Chitosan Conjugation Chemistry: Toward A General in Situ Strategy for High-Throughput Photoelectrochemical Enzymatic Bioanalysis.

PubMed

Wang, Guang-Li; Yuan, Fang; Gu, Tiantian; Dong, Yuming; Wang, Qian; Zhao, Wei-Wei

2018-02-06

Herein we report a general and novel strategy for high-throughput photoelectrochemical (PEC) enzymatic bioanalysis on the basis of enzyme-initiated quinone-chitosan conjugation chemistry (QCCC). Specifically, the strategy was illustrated by using a model quinones-generating oxidase of tyrosinase (Tyr) to catalytically produce 1,2-bezoquinone or its derivative, which can easily and selectively be conjugated onto the surface of the chitosan deposited PbS/NiO/FTO photocathode via the QCCC. Upon illumination, the covalently attached quinones could act as electron acceptors of PbS quantum dots (QDs), improving the photocurrent generation and thus allowing the elegant probing of Tyr activity. Enzyme cascades, such as alkaline phosphatase (ALP)/Tyr and β-galactosidase (Gal)/Tyr, were further introduced into the system for the successful probing of the corresponding targets. This work features not only the first use of QCCC in PEC bioanalysis but also the separation of enzymatic reaction from the photoelectrode as well as the direct signal recording in a split-type protocol, which enables quite convenient and high-throughput detection as compared to previous formats. More importantly, by using numerous other oxidoreductases that involve quinones as reactants/products, this protocol could serve as a common basis for the development of a new class of QCCC-based PEC enzymatic bioanalysis and further extended for general enzyme-labeled PEC bioanalysis of versatile targets.

Biochemical characterization and antioxidant and antiproliferative activities of different Ganoderma collections.

PubMed

Saltarelli, Roberta; Ceccaroli, Paola; Buffalini, Michele; Vallorani, Luciana; Casadei, Lucia; Zambonelli, Alessandra; Iotti, Mirco; Badalyan, Susanna; Stocchi, Vilberto

2015-01-01

The aim of this study was to conduct a molecular and biochemical characterization and to compare the antioxidant and antiproliferative activities of four Ganoderma isolates belonging to Ganoderma lucidum (Gl-4, Gl-5) and Ganoderma resinaceum (F-1, F-2) species. The molecular identification was performed by ITS and IGS sequence analyses and the biochemical characterization by enzymatic and proteomic approaches. The antioxidant activity of the ethanolic extracts was compared by three different methods and their flavonoid contents were also analyzed by high-performance liquid chromatography. The antiproliferative effect on U937 cells was determined by MTT assay. The studied mycelia differ both in the enzymatic activities and protein content. The highest content in total phenol and the highest antioxidant activity for DPPH free radical scavenging and chelating activity on Fe(2+) were observed with the Gl-4 isolate of G. lucidum. The presence of quercetin, rutin, myricetin, and morin as major flavonoids with effective antioxidant activity was detected. The ethanolic extracts from mycelia of G. lucidum isolates possess a substantial antiproliferative activity against U937 cells in contrast to G. resinaceum in which the antiproliferative effects were insignificant. This study provides a comparison between G. lucidum and G. resinaceum mycelial strains, and shows that G. resinaceum could be utilized to obtain several bioactive compounds. © 2015 S. Karger AG, Basel.

Phylogenetic and enzymatic diversity of deep subseafloor aerobic microorganisms in organics- and methane-rich sediments off Shimokita Peninsula.

PubMed

Kobayashi, Tohru; Koide, Osamu; Mori, Kozue; Shimamura, Shigeru; Matsuura, Takae; Miura, Takeshi; Takaki, Yoshihiro; Morono, Yuki; Nunoura, Takuro; Imachi, Hiroyuki; Inagaki, Fumio; Takai, Ken; Horikoshi, Koki

2008-07-01

"A meta-enzyme approach" is proposed as an ecological enzymatic method to explore the potential functions of microbial communities in extreme environments such as the deep marine subsurface. We evaluated a variety of extra-cellular enzyme activities of sediment slurries and isolates from a deep subseafloor sediment core. Using the new deep-sea drilling vessel "Chikyu", we obtained 365 m of core sediments that contained approximately 2% organic matter and considerable amounts of methane from offshore the Shimokita Peninsula in Japan at a water depth of 1,180 m. In the extra-sediment fraction of the slurry samples, phosphatase, esterase, and catalase activities were detected consistently throughout the core sediments down to the deepest slurry sample from 342.5 m below seafloor (mbsf). Detectable enzyme activities predicted the existence of a sizable population of viable aerobic microorganisms even in deep subseafloor habitats. The subsequent quantitative cultivation using solid media represented remarkably high numbers of aerobic, heterotrophic microbial populations (e.g., maximally 4.4x10(7) cells cm(-3) at 342.5 mbsf). Analysis of 16S rRNA gene sequences revealed that the predominant cultivated microbial components were affiliated with the genera Bacillus, Shewanella, Pseudoalteromonas, Halomonas, Pseudomonas, Paracoccus, Rhodococcus, Microbacterium, and Flexibacteracea. Many of the predominant and scarce isolates produced a variety of extra-cellular enzymes such as proteases, amylases, lipases, chitinases, phosphatases, and deoxyribonucleases. Our results indicate that microbes in the deep subseafloor environment off Shimokita are metabolically active and that the cultivable populations may have a great potential in biotechnology.

Constraints imposed by transmembrane domains affect enzymatic activity of membrane-associated human CD39/NTPDase1 mutants.

PubMed

Musi, Elgilda; Islam, Naziba; Drosopoulos, Joan H F

2007-05-01

Human CD39/NTPDase1 is an endothelial cell membrane-associated nucleotidase. Its large extracellular domain rapidly metabolizes nucleotides, especially ADP released from activated platelets, inhibiting further platelet activation/recruitment. Previous studies using our recombinant soluble CD39 demonstrated the importance of residues S57, D54, and D213 for enzymatic/biological activity. We now report effects of S57A, D54A, and D213A mutations on full-length (FL)CD39 function. Enzymatic activity of alanine modified FLCD39s was less than wild-type, contrasting the enhanced activity of their soluble counterparts. Furthermore, conservative substitutions D54E and D213E led to enzymes with activities greater than the alanine modified FLCD39s, but less than wild-type. Reductions in mutant activities were primarily associated with reduced catalytic rates. Differences in enzymatic activity were not attributable to gross changes in the nucleotide binding pocket or the enzyme's ability to multimerize. Thus, composition of the active site of wild-type CD39 appears optimized for ADPase function in the context of the transmembrane domains.

A probe for NADH and H2O2 amperometric detection at low applied potential for oxidase and dehydrogenase based biosensor applications.

PubMed

Ricci, Francesco; Amine, Aziz; Moscone, Danila; Palleschi, Giuseppe

2007-01-15

Modified screen-printed electrodes for amperometric detection of H(2)O(2) and nicotinamide adenine dinucleotide (NADH) at low applied potential are presented in this paper. The sensors are obtained by modifying the working electrode surface with Prussian Blue, a well known electrochemical mediator for H(2)O(2) reduction. The coupling of this sensor with phenazine methosulfate (PMS) in the working solution gives the possibility of measuring both NAD(P)H and H(2)O(2). PMS reacts with NADH producing PMSH, which in the presence of oxygen, gives an equimolar amount of H(2)O(2). This allows the measurement of both analytes with similar sensitivity (357 mA mol(-1)L cm(-2) for H(2)O(2) and 336 mA mol(-1)L cm(-2) for NADH) and LOD (5x10(-7)mol L(-1) for H(2)O(2) and NADH) and opens the possibility of a whole series of biosensor applications. In this paper, results obtained with a variety of dehydrogenase enzymes (alcohol, malic, lactate, glucose, glycerol and glutamate) for the detection of enzymatic substrates or enzymatic activity are presented demonstrating the suitability of the proposed method for future biosensor applications.

Printable organic thin film transistors for glucose detection incorporating inkjet-printing of the enzyme recognition element

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elkington, D., E-mail: Daniel.Elkington@newcastle.edu.au; Wasson, M.; Belcher, W.

The effect of device architecture upon the response of printable enzymatic glucose sensors based on poly(3-hexythiophene) (P3HT) organic thin film transistors is presented. The change in drain current is used as the basis for glucose detection and we show that significant improvements in drain current response time can be achieved by modifying the design of the sensor structure. In particular, we show that eliminating the dielectric layer and reducing the thickness of the active layer reduce the device response time considerably. The results are in good agreement with a diffusion based model of device operation, where an initial rapid dedopingmore » process is followed by a slower doping of the P3HT layer from protons that are enzymatically generated by glucose oxidase (GOX) at the Nafion gate electrode. The fitted diffusion data are consistent with a P3HT doping region that is close to the source-drain electrodes rather than located at the P3HT:[Nafion:GOX] interface. Finally, we demonstrate that further improvements in sensor structure and morphology can be achieved by inkjet-printing the GOX layer, offering a pathway to low-cost printed biosensors for the detection of glucose in saliva.« less

Amorphous Ni(OH)2/CQDs microspheres for highly sensitive non-enzymatic glucose detection prepared via CQDs induced aggregation process

NASA Astrophysics Data System (ADS)

Zhu, Jiajie; Yin, Haoyong; Cui, Zhenzhen; Qin, Dongyu; Gong, Jianying; Nie, Qiulin

2017-10-01

Non-enzymatic electrochemical sensors for the detection of glucose were designed based on amorphous Ni(OH)2/CQDs microspheres. The amorphous Ni(OH)2/CQDs microspheres were prepared by a CQDs assistant crystallization inhibition process. The morphologies and composition of the microspheres were characterized by SEM, TEM, XRD, EDS, and TG/DSC. The results showed that the microspheres had uniform heterogeneous phases with amorphous Ni(OH)2 and CQDs. The sensor based on amorphous Ni(OH)2/CQDs microspheres showed remarkable electrocatalytic activity towards glucose oxidation comparing to the conventional crystalline Ni(OH)2, which included two linear range (20 μM-350 μM and 0.45mM-2.5 mM) with high selectivity of 2760.05 and 1853.64 μA mM-1cm-2. Moreover, the interference from the commonly interfering species such as urea, ascorbic acid, NaCl, L-proline and L-Valine, can be effectively avoided. The high sensitivity, wide glucose detection range and good selectivity of the electrode may be due to their synergistic effect of amorphous phase and CQDs incorporation. These findings may promote the application of amorphous Ni(OH)2 as advanced electrochemical glucose sensing materials.

Designing and facilely synthesizing a series of cobalt nitride (Co4N) nanocatalysts as non-enzymatic glucose sensors: A comparative study toward the influences of material structures on electrocatalytic activities.

PubMed

Liu, Tingting; Li, Mian; Guo, Liping

2018-05-01

Designing high-efficiency electrocatalysts for glucose concentration detection plays a pivotal role in developing various non-enzymatic glucose detection devices. Herein, we have successfully designed and synthesized various cobalt nitrides (Co 4 N) by using different weak bases (i.e. hexamethylenetetramine (HMT), urea, and ammonium hydroxide (AH)) through nitridation treatment in ammonia (NH 3 ) atmosphere. Physical characterization results demonstrate that Co 4 N-NSs (nanosheets) with vast meso/macropores and large BET surface are successfully constructed once adding carbon paper and HMT into precursors. As the synergistic effect of metallic character of Co 4 N phase , excellent electroconductibility of pyrolytic carbon, and large surface area, Co 4 N-NSs surfaces can form more Co 4+ active sites in electrochemical reaction processes. Meanwhile, the abundant meso/macroporous structures constructed in Co 4 N-NSs further promoted its mass transfer ability. Benefitting from the above mentioned advantages, Co 4 N-NSs therefore exhibit more excellent glucose oxidation ability than another three control samples (i.e. Co 4 N-HMT, Co 4 N-Urea and Co 4 N-AH). When used for glucose detection, the optimal Co 4 N-NSs display excellent detection parameters as well, such as: a wide linear range of 0.6-10.0mM, a large sensitivity of 1137.2uAcm -2 mM -1 glucose, a low detection limit of 0.1µM, a small response time of 1.7s, good reproducibility and stability, and the excellent anti-interference to other electroactive molecules and Cl - . Upon utilized for measuring glucose concentrations in human blood serum samples, the detection results on Co 4 N-NSs are accurate and satisfying as well. This work opens a new possibility for boosting electrochemical catalysis abilities of Co 4 N samples by the structure design. Copyright © 2017 Elsevier B.V. All rights reserved.

AN ENVIRONMENTAL TECHNOLOGY VERIFICATION (ETV) TESTING OF ENZYMATIC TEST KITS FOR WARFARE AGENTS AND PESTICIDES IN DRINKING WATER

EPA Science Inventory

Enzymatic test kits, generally designed to be handheld and portable, detect the presence of chemical agents, carbamate pesticides, and/or organophosphate pesticides by relying on the reaction of the cholinesterase enzyme. Under normal conditions, the enzyme reacts as expected wi...

Toxin detection using a tyrosinase-coupled oxygen electrode.

PubMed

Smit, M H; Rechnitz, G A

1993-02-15

An enzyme-based "electrochemical canary" is described for the detection of cyanide. The sensing system imitates cyanide's site of toxicity in the mitochondria. The terminal sequence of electron transfer in aerobic respiration is mimicked by mediator coupling of tyrosinase catalysis to an electro-chemical system. An enzyme-coupled oxygen electrode is created which is sensitive to selective poisoning. Biocatalytic reduction of oxygen is promoted by electrochemically supplying tyrosinase with electrons. Thus, ferrocyanide is generated at a cathode and mediates the enzymatic reduction of oxygen to water. An enzyme-dependent reductive current can be monitored which is inhibited by cyanide in a concentration-dependent manner. Oxygen depletion in the reaction layer can be minimized by addressing enzyme activity using a potential pulsing routine. Enzyme activity is electrochemically initiated and terminated and the sensor becomes capable of continuous monitoring. Cyanide poisoning of the biological component is reversible, and it can be reused after rinsing. The resulting sensor detects cyanide based on its biological activity rather than its physical or chemical properties.

Immune effects of HFO on European sea bass, Dicentrarchus labrax, and Pacific oyster, Crassostrea gigas.

PubMed

Bado-Nilles, Anne; Quentel, Claire; Auffret, Michel; Le Floch, Stéphane; Gagnaire, Béatrice; Renault, Tristan; Thomas-Guyon, Hélène

2009-07-01

The European sea bass, Dicentrarchus labrax, and the Pacific oyster, Crassostrea gigas, were exposed to a soluble fraction of heavy fuel oil for 5 and 9 days, respectively. The organisms were then transferred to non-contaminated seawater for 1 month. The bioaccumulation and elimination of PAHs in contaminated tissues were dissimilar between species. In fish, acenaphthene and naphthalene were detected and naphthalene was still detectable 30 days after the beginning of the recovery period. In oysters, on the other hand, pyrene and phenanthrene were bioaccumulated and 14 days after exposure no more PAHs were detected. Concerning innate immune parameters, the increase of haemolytic activity of the alternative complement pathway in fish and the reduction of phenoloxidase activity in oysters endured, respectively, 1 and 2 weeks in contaminated organisms. This indicates that these two enzymatic cascades could be quite useful for monitoring pollution by oil.

Screening of plants used in the European traditional medicine to treat memory disorders for acetylcholinesterase inhibitory activity and anti amyloidogenic activity.

PubMed

Lobbens, Eva S B; Vissing, Karina J; Jorgensen, Lene; van de Weert, Marco; Jäger, Anna K

2017-03-22

Plants used in the traditional medicine of Europe to treat memory dysfunction and/or to enhance memory were investigated for activity against the underlying mechanisms of Alzheimer's disease. To investigate 35 ethanolic extracts of plants, selected using an ethnopharmacological approach, for anti-amyloidogenic activity as well as an ability to inhibit the enzymatic activity of acetylcholinesterase. The anti-amyloidogenic activity of the extracts against amyloid beta was investigated by Thioflavin T fibrillation assays and the ability to inhibit the enzymatic activity of acetylcholinesterase was evaluated monitoring the hydrolysis of acetylthiocholine RESULTS: Under the experimental conditions investigated, extracts of two plants, Carum carvi and Olea sylvestris, inhibited amyloid beta fibrillation considerably, eight plant extracts inhibited amyloid beta fibrillation to some extent, 16 plant extracts had no effect on amyloid beta fibrillation and nine extracts accelerated fibrillation of amyloid beta. Furthermore, five plant extracts from Corydalis species inhibited the enzymatic activity of acetylcholinesterase considerably, one plant extract inhibited the enzymatic activity of acetylcholinesterase to some extent and 29 plant extract had no effect on the enzymatic activity of acetylcholinesterase. An optimal extract in this study would possess acetylcholinesterase inhibitory activity as well as anti-amyloidogenic activity in order to address multiple facets of Alzheimer's disease, until the molecular origin of the disease is unraveled. Unfortunately no such extract was found. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Non-invasive imaging and cellular tracking of pulmonary emboli by near-infrared fluorescence and positron-emission tomography

PubMed Central

Page, Michael J.; Lourenço, André L.; David, Tovo; LeBeau, Aaron M.; Cattaruzza, Fiore; Castro, Helena C.; VanBrocklin, Henry F.; Coughlin, Shaun R.; Craik, Charles S.

2015-01-01

Functional imaging of proteolytic activity is an emerging strategy to quantify disease and response to therapy at the molecular level. We present a new peptide-based imaging probe technology that advances these goals by exploiting enzymatic activity to deposit probes labelled with near-infrared (NIR) fluorophores or radioisotopes in cell membranes of disease-associated proteolysis. This strategy allows for non-invasive detection of protease activity in vivo and ex vivo by tracking deposited probes in tissues. We demonstrate non-invasive detection of thrombin generation in a murine model of pulmonary embolism using our protease-activated peptide probes in microscopic clots within the lungs with NIR fluorescence optical imaging and positron-emission tomography. Thrombin activity is imaged deep in tissue and tracked predominantly to platelets within the lumen of blood vessels. The modular design of our probes allows for facile investigation of other proteases, and their contributions to disease by tailoring the protease activation and cell-binding elements. PMID:26423607

Effects of organic carbon sequestration strategies on soil enzymatic activities

NASA Astrophysics Data System (ADS)

Puglisi, E.; Suciu, N.; Botteri, L.; Ferrari, T.; Coppolecchia, D.; Trevisan, M.; Piccolo, A.

2009-04-01

Greenhouse gases emissions can be counterbalanced with proper agronomical strategies aimed at sequestering carbon in soils. These strategies must be tested not only for their ability in reducing carbon dioxide emissions, but also for their impact on soil quality: enzymatic activities are related to main soil ecological quality, and can be used as early and sensitive indicators of alteration events. Three different strategies for soil carbon sequestration were studied: minimum tillage, protection of biodegradable organic fraction by compost amendment and oxidative polimerization of soil organic matter catalyzed by biometic porfirins. All strategies were compared with a traditional agricultural management based on tillage and mineral fertilization. Experiments were carried out in three Italian soils from different pedo-climatic regions located respectively in Piacenza, Turin and Naples and cultivated with maize or wheat. Soil samples were taken for three consecutive years after harvest and analyzed for their content in phosphates, ß-glucosidase, urease and invertase. An alteration index based on these enzymatic activities levels was applied as well. The biomimetic porfirin application didn't cause changes in enzymatic activities compared to the control at any treatment or location. Enzymatic activities were generally higher in the minimum tillage and compost treatment, while differences between location and date of samplings were limited. Application of the soil alteration index based on enzymatic activities showed that soils treated with compost or subjected to minimum tillage generally have a higher biological quality. The work confirms the environmental sustainability of the carbon sequestering agronomical practices studied.

Ultra-high throughput detection of single cell β-galactosidase activity in droplets using micro-optical lens array

NASA Astrophysics Data System (ADS)

Lim, Jiseok; Vrignon, Jérémy; Gruner, Philipp; Karamitros, Christos S.; Konrad, Manfred; Baret, Jean-Christophe

2013-11-01

We demonstrate the use of a hybrid microfluidic-micro-optical system for the screening of enzymatic activity at the single cell level. Escherichia coli β-galactosidase activity is revealed by a fluorogenic assay in 100 pl droplets. Individual droplets containing cells are screened by measuring their fluorescence signal using a high-speed camera. The measurement is parallelized over 100 channels equipped with microlenses and analyzed by image processing. A reinjection rate of 1 ml of emulsion per minute was reached corresponding to more than 105 droplets per second, an analytical throughput larger than those obtained using flow cytometry.

Graphene quantum dots for ultrasensitive detection of acetylcholinesterase and its inhibitors

NASA Astrophysics Data System (ADS)

Li, Nan; Wang, Xuewan; Chen, Jie; Sun, Lei; Chen, Peng

2015-09-01

Graphene quantum dots (GQDs) are emerging zero-dimensional materials promising a wide spectrum of novel applications including development of optical sensors. Herein, a GQD-based fluorometric sensor is devised to detect acetylcholinesterase (AChE, a critical enzyme in central nervous system and neuromuscular junctions) with an ultralow detection limit (0.58 pM with S/N of 5.0), using a photoluminescence ‘turn-off’ mechanism. This simple ‘mix-and-detect’ platform can also be employed to sense a variety of compounds that can directly or indirectly inhibit the enzymatic activities of AChE, such as nerve gases, pesticides, and therapeutic drugs. As the proof-of-concept demonstrations, we show the sensitive detection of paraoxon (a pesticide), tacrine (a drug to treat Alzheimer’s disease), and dopamine (an important neurotransmitter).

Screen-printed electrode modified with carbon black and chitosan: a novel platform for acetylcholinesterase biosensor development.

PubMed

Talarico, Daria; Arduini, Fabiana; Amine, Aziz; Cacciotti, Ilaria; Moscone, Danila; Palleschi, Giuseppe

2016-10-01

We report a screen-printed electrode (SPE) modified with a dispersion of carbon black (CB) and chitosan by drop casting. A cyclic voltammetry technique towards ferricyanide, caffeic acid, hydroquinone, and thiocholine was performed and an improvement of the electrochemical response with respect to bare SPE as well as SPE modified only with chitosan was observed. The possibility to detect thiocholine at a low applied potential with high sensitivity was exploited and an acetylcholinesterase (AChE) biosensor was developed. A dispersion of CB, chitosan, and AChE was used to fabricate this biosensor in one step by drop casting. The enzymatic activity of the immobilized AChE was determined measuring the enzymatic product thiocholine at +300 mV. Owing to the capability of organophosphorus pesticides to inhibit AChE, this biosensor was used to detect these pollutants, and paraoxon was taken as model compound. The enzyme inhibition was linearly related to the concentration of paraoxon up to 0.5 μg L(-1), and a low detection limit equal to 0.05 μg L(-1) (calculated as 10% of inhibition) was achieved. This biosensor was challenged for paraoxon detection in drinking waters with satisfactory recovery values. The use of AChE embedded in a dispersion of CB and chitosan allowed an easy and fast production of a sensitive biosensor suitable for paraoxon detection in drinking waters at legal limit levels. Graphical Abstract Biosensors based on screen-printed electrodes modified with Acetylcholinesterase, Carbon Black, and Chitosan for organophosphorus pesticide detection.

Evaluation of fluorescence in situ hybridization techniques to study long non-coding RNA expression in cultured cells

PubMed Central

Soares, Ricardo J; Maglieri, Giulia; Gutschner, Tony; Lund, Anders H; Nielsen, Boye S

2018-01-01

Abstract Deciphering the functions of long non-coding RNAs (lncRNAs) is facilitated by visualization of their subcellular localization using in situ hybridization (ISH) techniques. We evaluated four different ISH methods for detection of MALAT1 and CYTOR in cultured cells: a multiple probe detection approach with or without enzymatic signal amplification, a branched-DNA (bDNA) probe and an LNA-modified probe with enzymatic signal amplification. All four methods adequately stained MALAT1 in the nucleus in all of three cell lines investigated, HeLa, NHDF and T47D, and three of the methods detected the less expressed CYTOR. The sensitivity of the four ISH methods was evaluated by image analysis. In all three cell lines, the two methods involving enzymatic amplification gave the most intense MALAT1 signal, but the signal-to-background ratios were not different. CYTOR was best detected using the bDNA method. All four ISH methods showed significantly reduced MALAT1 signal in knock-out cells, and siRNA-induced knock-down of CYTOR resulted in significantly reduced CYTOR ISH signal, indicating good specificity of the probe designs and detection systems. Our data suggest that the ISH methods allow detection of both abundant and less abundantly expressed lncRNAs, although the latter required the use of the most specific and sensitive probe detection system. PMID:29059327

Biochemical, histological and functional correction of mucopolysaccharidosis type IIIB by intra-cerebrospinal fluid gene therapy.

PubMed

Ribera, Albert; Haurigot, Virginia; Garcia, Miguel; Marcó, Sara; Motas, Sandra; Villacampa, Pilar; Maggioni, Luca; León, Xavier; Molas, Maria; Sánchez, Víctor; Muñoz, Sergio; Leborgne, Christian; Moll, Xavier; Pumarola, Martí; Mingozzi, Federico; Ruberte, Jesús; Añor, Sònia; Bosch, Fatima

2015-04-01

Gene therapy is an attractive tool for the treatment of monogenic disorders, in particular for lysosomal storage diseases (LSD) caused by deficiencies in secretable lysosomal enzymes in which neither full restoration of normal enzymatic activity nor transduction of all affected cells are necessary. However, some LSD such as Mucopolysaccharidosis Type IIIB (MPSIIIB) are challenging because the disease's main target organ is the brain and enzymes do not efficiently cross the blood-brain barrier even if present at very high concentration in circulation. To overcome these limitations, we delivered AAV9 vectors encoding for α-N-acetylglucosaminidase (NAGLU) to the Cerebrospinal Fluid (CSF) of MPSIIIB mice with the disease already detectable at biochemical, histological and functional level. Restoration of enzymatic activity in Central Nervous System (CNS) resulted in normalization of glycosaminoglycan content and lysosomal physiology, resolved neuroinflammation and restored the pattern of gene expression in brain similar to that of healthy animals. Additionally, transduction of the liver due to passage of vectors to the circulation led to whole-body disease correction. Treated animals also showed reversal of behavioural deficits and extended lifespan. Importantly, when the levels of enzymatic activity were monitored in the CSF of dogs following administration of canine NAGLU-coding vectors to animals that were either naïve or had pre-existing immunity against AAV9, similar levels of activity were achieved, suggesting that CNS efficacy would not be compromised in patients seropositive for AAV9. Our studies provide a strong rationale for the clinical development of this novel therapeutic approach as the treatment for MPSIIIB. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Primary hyperoxaluria type 1 in the Canary Islands: A conformational disease due to I244T mutation in the P11L-containing alanine:glyoxylate aminotransferase

PubMed Central

Santana, A.; Salido, E.; Torres, A.; Shapiro, L. J.

2003-01-01

Primary hyperoxaluria type 1 (PH1) is an inborn error of metabolism resulting from a deficiency of alanine:glyoxylate aminotransferase (AGXT; EC 2.6.1.44). Most of the PH1 alleles detected in the Canary Islands carry the Ile-244 → Thr (I244T) mutation in the AGXT gene, with 14 of 16 patients homozygous for this mutation. Four polymorphisms within AGXT and regional microsatellites also were shared in their haplotypes (AGXT*LTM), consistent with a founder effect. The consequences of these amino acid changes were investigated. Although I244T alone did not affect AGXT activity or subcellular localization, when present in the same protein molecule as Leu-11 → Pro (L11P), it resulted in loss of enzymatic activity in soluble cell extracts. Like its normal counterpart, the AGXT*LTM protein was present in the peroxisomes but it was insoluble in detergent-free buffers. The polymorphism L11P behaved as an intragenic modifier of the I244T mutation, with the resulting protein undergoing stable interaction with molecular chaperones and aggregation. This aggregation was temperature-sensitive. AGXT*LTM expressed in Escherichia coli, as a GST-fusion protein, and in insect cells could be purified and retained enzymatic activity. Among various chemical chaperones tested in cell culture, betaine substantially improved the solubility of the mutant protein and the enzymatic activity in cell lysates. In summary, I244T, the second most common mutation responsible for PH1, is a protein conformational disease that may benefit from new therapies with pharmacological chaperones or small molecules to minimize protein aggregation. PMID:12777626

Primary hyperoxaluria type 1 in the Canary Islands: a conformational disease due to I244T mutation in the P11L-containing alanine:glyoxylate aminotransferase.

PubMed

Santana, A; Salido, E; Torres, A; Shapiro, L J

2003-06-10

Primary hyperoxaluria type 1 (PH1) is an inborn error of metabolism resulting from a deficiency of alanine:glyoxylate aminotransferase (AGXT; EC 2.6.1.44). Most of the PH1 alleles detected in the Canary Islands carry the Ile-244 --> Thr (I244T) mutation in the AGXT gene, with 14 of 16 patients homozygous for this mutation. Four polymorphisms within AGXT and regional microsatellites also were shared in their haplotypes (AGXT*LTM), consistent with a founder effect. The consequences of these amino acid changes were investigated. Although I244T alone did not affect AGXT activity or subcellular localization, when present in the same protein molecule as Leu-11 --> Pro (L11P), it resulted in loss of enzymatic activity in soluble cell extracts. Like its normal counterpart, the AGXT*LTM protein was present in the peroxisomes but it was insoluble in detergent-free buffers. The polymorphism L11P behaved as an intragenic modifier of the I244T mutation, with the resulting protein undergoing stable interaction with molecular chaperones and aggregation. This aggregation was temperature-sensitive. AGXT*LTM expressed in Escherichia coli, as a GST-fusion protein, and in insect cells could be purified and retained enzymatic activity. Among various chemical chaperones tested in cell culture, betaine substantially improved the solubility of the mutant protein and the enzymatic activity in cell lysates. In summary, I244T, the second most common mutation responsible for PH1, is a protein conformational disease that may benefit from new therapies with pharmacological chaperones or small molecules to minimize protein aggregation.

Combined Inhibition of the Renin-Angiotensin System and Neprilysin Positively Influences Complex Mitochondrial Adaptations in Progressive Experimental Heart Failure

PubMed Central

Reinders, Jörg; Schröder, Josef; Dietl, Alexander; Schmid, Peter M.; Jungbauer, Carsten; Resch, Markus; Maier, Lars S.; Luchner, Andreas; Birner, Christoph

2017-01-01

Background Inhibitors of the renin angiotensin system and neprilysin (RAS-/NEP-inhibitors) proved to be extraordinarily beneficial in systolic heart failure. Furthermore, compelling evidence exists that impaired mitochondrial pathways are causatively involved in progressive left ventricular (LV) dysfunction. Consequently, we aimed to assess whether RAS-/NEP-inhibition can attenuate mitochondrial adaptations in experimental heart failure (HF). Methods and Results By progressive right ventricular pacing, distinct HF stages were induced in 15 rabbits, and 6 animals served as controls (CTRL). Six animals with manifest HF (CHF) were treated with the RAS-/NEP-inhibitor omapatrilat. Echocardiographic studies and invasive blood pressure measurements were undertaken during HF progression. Mitochondria were isolated from LV tissue, respectively, and further worked up for proteomic analysis using the SWATH technique. Enzymatic activities of citrate synthase and the electron transfer chain (ETC) complexes I, II, and IV were assessed. Ultrastructural analyses were performed by transmission electron microscopy. During progression to overt HF, intricate expression changes were mainly detected for proteins belonging to the tricarboxylic acid cycle, glucose and fat metabolism, and the ETC complexes, even though ETC complex I, II, or IV enzymatic activities were not significantly influenced. Treatment with a RAS-/NEP-inhibitor then reversed some maladaptive metabolic adaptations, positively influenced the decline of citrate synthase activity, and altered the composition of each respiratory chain complex, even though this was again not accompanied by altered ETC complex enzymatic activities. Finally, ultrastructural evidence pointed to a reduction of autophagolytic and degenerative processes with omapatrilat-treatment. Conclusions This study describes complex adaptations of the mitochondrial proteome in experimental tachycardia-induced heart failure and shows that a combined RAS-/NEP-inhibition can beneficially influence mitochondrial key pathways. PMID:28076404

Detection of choline and phosphatidic acid (PA) catalyzed by phospholipase D (PLD) using MALDI-QIT-TOF/MS with 9-aminoacridine matrix.

PubMed

Park, Kyung-Eui; Kim, Jun-Dal; Nagashima, Yusuke; Kako, Koichiro; Daitoku, Hiroaki; Matsui, Motoki; Park, Gwi Gun; Fukamizu, Akiyoshi

2014-01-01

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), the most abundant phospholipids of plasma membrane, resulting in the production of choline and phosphatidic acid (PA). Choline is a precursor of the neurotransmitter acetylcholine, whereas PA functions as an intracellular lipid mediator of diverse biological functions. For assessing PLD activity in vitro, PLD-derived choline has been often analyzed with radioactive or non-radioactive methods. In this study, we have developed a new method for detecting choline and PA with MALDI-QIT-TOF/MS by using 9-aminoacridine as a matrix. The standard calibration curves showed that choline and PA could be detected with linearity over the range from 0.05 and 1 pmol, respectively. Importantly, this method enables the concomitant detection of choline and PA as a reaction product of PC hydrolysis by PLD2 proteins. Thus, our simple and direct method would be useful to characterize the enzymatic properties of PLD, thereby providing insight into mechanisms of PLD activation.

Highly Sensitive Biosensing with Solid-State Nanopores Displaying Enzymatically Reconfigurable Rectification Properties.

PubMed

Pérez-Mitta, Gonzalo; Peinetti, Ana S; Cortez, M Lorena; Toimil-Molares, María Eugenia; Trautmann, Christina; Azzaroni, Omar

2018-05-09

Molecular design of biosensors based on enzymatic processes taking place in nanofluidic elements is receiving increasing attention by the scientific community. In this work, we describe the construction of novel ultrasensitive enzymatic nanopore biosensors employing "reactive signal amplifiers" as key elements coupled to the transduction mechanism. The proposed framework offers innovative design concepts not only to amplify the detected ionic signal and develop ultrasensitive nanopore-based sensors but also to construct nanofluidic diodes displaying specific chemo-reversible rectification properties. The integrated approach is demonstrated by electrostatically assembling poly(allylamine) on the anionic pore walls followed by the assembly of urease. We show that the cationic weak polyelectrolyte acts as a "reactive signal amplifier" in the presence of local pH changes induced by the enzymatic reaction. These bioinduced variations in proton concentration ultimately alter the protonation degree of the polyamine resulting in amplifiable, controlled, and reproducible changes in the surface charge of the pore walls, and consequently on the generated ionic signals. The "iontronic" response of the as-obtained devices is fully reversible, and nanopores are reused and assayed with different urea concentrations, thus ensuring reliable design. The limit of detection (LOD) was 1 nM. To the best of our knowledge, this value is the lowest LOD reported to date for enzymatic urea detection. In this context, we envision that this approach based on the use of "reactive signal amplifiers" into solid-state nanochannels will provide new alternatives for the molecular design of highly sensitive nanopore biosensors as well as (bio)chemically addressable nanofluidic elements.

Enzyme catalysis-electrophoresis titration for multiplex enzymatic assay via moving reaction boundary chip.

PubMed

Zhong, Ran; Xie, Haiyang; Kong, Fanzhi; Zhang, Qiang; Jahan, Sharmin; Xiao, Hua; Fan, Liuyin; Cao, Chengxi

2016-09-21

In this work, we developed the concept of enzyme catalysis-electrophoresis titration (EC-ET) under ideal conditions, the theory of EC-ET for multiplex enzymatic assay (MEA), and a related method based on a moving reaction boundary (MRB) chip with a collateral channel and cell phone imaging. As a proof of principle, the model enzymes horseradish peroxidase (HRP), laccase and myeloperoxidase (MPO) were chosen for the tests of the EC-ET model. The experiments revealed that the EC-ET model could be achieved via coupling EC with ET within a MRB chip; particularly the MEA analyses of catalysis rate, maximum rate, activity, Km and Kcat could be conducted via a single run of the EC-ET chip, systemically demonstrating the validity of the EC-ET theory. Moreover, the developed method had these merits: (i) two orders of magnitude higher sensitivity than a fluorescence microplate reader, (ii) simplicity and low cost, and (iii) fairly rapid (30 min incubation, 20 s imaging) analysis, fair stability (<5.0% RSD) and accuracy, thus validating the EC-ET method. Finally, the developed EC-ET method was used for the clinical assay of MPO activity in blood samples; the values of MPO activity detected via the EC-ET chip were in agreement with those obtained by a traditional fluorescence microplate reader, indicating the applicability of the EC-ET method. The work opens a window for the development of enzymatic research, enzyme assay, immunoassay, and point-of-care testing as well as titration, one of the oldest methods of analysis, based on a simple chip.

Crystal structure of β1→6-galactosidase from Bifidobacterium bifidum S17: trimeric architecture, molecular determinants of the enzymatic activity and its inhibition by α-galactose.

PubMed

Godoy, Andre Schutzer; Camilo, Cesar Moises; Kadowaki, Marco Antonio; Muniz, Heloisa Dos S; Espirito Santo, Melissa; Murakami, Mario Tyago; Nascimento, Alessandro S; Polikarpov, Igor

2016-11-01

In a search for better comprehension of β-galactosidase function and specificity, we solved the crystal structures of the GH42 β-galactosidase BbgII from Bifidobacterium bifidum S17, a well-adapted probiotic microorganism from the human digestive tract, and its complex with d-α-galactose. BbgII is a three-domain molecule that forms barrel-shaped trimers in solution. BbgII interactions with d-α-galactose, a competitive inhibitor, showed a number of residues that are involved in the coordination of ligands. A combination of site-directed mutagenesis of these amino acid residues with enzymatic activity measurements confirmed that Glu161 and Glu320 are fundamental for catalysis and their substitution by alanines led to catalytically inactive mutants. Mutation Asn160Ala resulted in a two orders of magnitude decrease of the enzyme k cat without significant modification in its K m , whereas mutations Tyr289Phe and His371Phe simultaneously decreased k cat and increased K m values. Enzymatic activity of Glu368Ala mutant was too low to be detected. Our docking and molecular dynamics simulations showed that the enzyme recognizes and tightly binds substrates with β1→6 and β1→3 bonds, while binding of the substrates with β1→4 linkages is less favorable. Structural data are available in the PDB under the accession numbers 4UZS and 4UCF. © 2016 Federation of European Biochemical Societies.

Ultrasensitive detection of superoxide anion released from living cells using a porous Pt-Pd decorated enzymatic sensor.

PubMed

Zhu, Xiang; Liu, Tingting; Zhao, Hongli; Shi, Libo; Li, Xiaoqing; Lan, Minbo

2016-05-15

Considering the critical roles of superoxide anion (O2(∙-)) in pathological conditions, it is of great urgency to establish a reliable and durable approach for real-time determination of O2(∙-). In this study, we propose a porous Pt-Pd decorated superoxide dismutase (SOD) sensor for qualitative and quantitative detection O2(∙-). The developed biosensor exhibits a fast, selective and linear amperometric response upon O2(∙-) in the concentration scope of 16 to 1,536 μM (R(2)=0.9941), with a detection limit of 0.13 μM (S/N=3) and a low Michaelis-Menten constant of 1.37 μM which indicating a high enzymatic activity and affinity to O2(∙-). Inspiringly, the proposed sensor possesses an ultrahigh sensitivity of 1270 μA mM(-1)cm(-2). In addition, SOD/porous Pt-Pd sensor exhibits excellent anti-interference property, reproducibility and long-term storage stability. Beyond our expectation, the trace level of O2(∙-) released from living cells has also been successfully captured. These satisfactory results are mainly ascribed to (1) the porous interface with larger surface area and more active sites to provide a biocompatible environment for SOD (2) the specific biocatalysis of SOD towards O2(∙-) and (3) porous Pt-Pd nanomaterials fastening the electron transfer. The superior electrochemical performance makes SOD/porous Pt-Pd sensor a promising candidate for monitoring the dynamic changes of O2(∙-)in vivo. Copyright © 2015 Elsevier B.V. All rights reserved.

Enzymatic Activity of Candida spp. from Oral Cavity and Urine in Children with Nephrotic Syndrome.

PubMed

Olczak-Kowalczyk, Dorota; Roszkowska-Blaim, Maria; Dąbkowska, Maria; Swoboda-Kopeć, Ewa; Gozdowski, Dariusz; Mizerska-Wasiak, Małgorzata; Demkow, Urszula; Pańczyk-Tomaszewska, Małgorzata

2017-01-01

Oral colonization with Candida spp. is not synonymous with a systemic active infection. The aim of the study was to evaluate enzymatic activity of Candida strains isolated from the oral cavity in patients with nephrotic syndrome (NS) and to compare it with the activity determined in urine. We studied 32 children with NS and 26 control healthy children. Children with NS were treated with glucocorticosteroids, cyclosporin A, mycophenolate mofetil or azathioprine. In all children, API-ZYM enzymatic tests were performed to evaluate hydrolytic enzymes of Candida isolated from the oral cavity and in urine. Candida spp. were isolated from the oral cavity in 11 patients with NS (34.4%), all receiving immunosuppressive treatment. All strains produced valine arylamidase, 9 alpha-glucosidase (E16), and 9 N-acetyl-beta-glucosaminidase (E18). A positive correlation between the presence of Candida in the oral cavity and E16 and E18 enzymatic activity in both oral cavity and urine was found. A dose of cyclosporin A had an effect on the enzymatic activity (p < 0.05). We conclude that immunosuppressive treatment of NS in children may predispose to systemic Candida invasion. The results of this study suggest that oral candida infection should be monitored in children with nephrotic syndrome, particularly those treated with immunosuppressive agents.

Maturity assessment of compost from municipal solid waste through the study of enzyme activities and water-soluble fractions.

PubMed

Castaldi, Paola; Garau, Giovanni; Melis, Pietro

2008-01-01

In this work the dynamics of biochemical (enzymatic activities) and chemical (water-soluble fraction) parameters during 100 days of municipal solid wastes composting were studied to evaluate their suitability as tools for compost characterization. The hydrolase (protease, urease, cellulase, beta-glucosidase) and dehydrogenase activities were characterized by significant changes during the first 2 weeks of composting, because of the increase of easily decomposable organic compounds. After the 4th week a "maturation phase" was identified in which the enzymatic activities tended to gently decrease, suggesting the stabilisation of organic matter. Also the water-soluble fractions (water-soluble carbon, nitrogen, carbohydrates and phenols), which are involved in many degradation processes, showed major fluctuations during the first month of composting. The results obtained showed that the hydrolytic activities and the water-soluble fractions did not vary statistically during the last month of composting. Significant correlations between the enzymatic activities, as well as between enzyme activities and water-soluble fractions, were also highlighted. These results highlight the suitability of both enzymatic activities and water soluble fractions as suitable indicators of the state and evolution of the organic matter during composting. However, since in the literature the amount of each activity or fraction at the end of composting depends on the raw material used for composting, single point determinations appear inadequate for compost characterization. This emphasizes the importance of the characterization of the dynamics of enzymatic activities and water-soluble fractions during the process.

A Survey of Enzymatic Activity in Commercially Available Pool and Spa Products

USDA-ARS?s Scientific Manuscript database

Many pool water treatment products currently available commercially claim that they work effectively by possessing enzyme activity (specifically lipase) that degrades common oil (lipid) contaminants found in pool water. Currently, there is no standard in measuring the enzymatic activity of these enz...

Extraction and Inhibition of Enzymatic Activity of Botulinum Neurotoxins/A1, /A2, and /A3 by a Panel of Monoclonal Anti-BoNT/A Antibodies

DTIC Science & Technology

2009-04-01

triplicate and results were averaged. MS Detection A master mix was created consisting of 9 parts matrix solution (alpha-cyano-4-hydroxy cinnamic acid ...thus, do not inhibit the catalytic activity. Another feature of BoNT/A is that it exhibits genetic and amino acid variance within the toxin type, or...less amino acid variance [23] and this variance has been reported to affect binding of the toxin to anti-BoNT/A mAbs [24]. For these reasons, it is

Serological, biochemical and enzymatic alterations in rodents after experimental envenomation with Hadruroides lunatus scorpion venom.

PubMed

Costal-Oliveira, F; Guerra-Duarte, C; Castro, K L P; Tintaya, B; Bonilla, C; Silva, W; Yarlequé, A; Fujiwara, R; Melo, M M; Chávez-Olórtegui, C

2015-09-01

Toxic effects of Peruvian Hadruroides lunatus scorpion venom on different biochemical and enzymatic parameters in blood serum of Wistar rats and Swiss mice were determined after experimental envenomation. An increase in enzymatic activities of Aspartate Aminotransferase (AST), Lactate Dehydrogenase (LDH) and levels of serum protein and albumin were observed while a decrease in creatinine level in serum was perceived after 30 min of envenomation. No alterations in urea levels and in kidney histology were detected in the envenomed rats. The global leukocytes count was diminished, with decrease in lymphocytes, eosinophils and neutrophils levels in the bloodstream, while no alterations were found in hematological parameters of red series in rats injected with H. lunatus venom. IL-2, IL-4, IL-6, INF-γ, TNF, IL-17A and IL-10 levels were evaluated 0.5, 3 and 6 h after experimental envenomation of mice with H. lunatus venom. From all the analyzed cytokines, only IL-6 showed an increase in serum levels. Taken together, these results point out that envenomation by H. lunatus can impair hematological and immunological parameters and therefore might be monitored in accidents involving this species. Copyright © 2015 Elsevier Ltd. All rights reserved.

Volatile Compound, Physicochemical, and Antioxidant Properties of Beany Flavor-Removed Soy Protein Isolate Hydrolyzates Obtained from Combined High Temperature Pre-Treatment and Enzymatic Hydrolysis

PubMed Central

Yoo, Sang-Hun; Chang, Yoon Hyuk

2016-01-01

The present study investigated the volatile compound, physicochemical, and antioxidant properties of beany flavor-removed soy protein isolate (SPI) hydrolyzates produced by combined high temperature pre-treatment and enzymatic hydrolysis. Without remarkable changes in amino acid composition, reductions of residual lipoxygenase activity and beany flavor-causing volatile compounds such as hexanol, hexanal, and pentanol in SPI were observed after combined heating and enzymatic treatments. The degree of hydrolysis, emulsion capacity and stability, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, and superoxide radical scavenging activity of SPI were significantly increased, but the magnitudes of apparent viscosity, consistency index, and dynamic moduli (G′, G″) of SPI were significantly decreased after the combined heating and enzymatic treatments. Based on these results, it was suggested that the enzymatic hydrolysis in combination with high temperature pre-treatment may allow for the production of beany flavor-removed SPI hydrolyzates with superior emulsifying and antioxidant functionalities. PMID:28078256

Volatile Compound, Physicochemical, and Antioxidant Properties of Beany Flavor-Removed Soy Protein Isolate Hydrolyzates Obtained from Combined High Temperature Pre-Treatment and Enzymatic Hydrolysis.

PubMed

Yoo, Sang-Hun; Chang, Yoon Hyuk

2016-12-01

The present study investigated the volatile compound, physicochemical, and antioxidant properties of beany flavor-removed soy protein isolate (SPI) hydrolyzates produced by combined high temperature pre-treatment and enzymatic hydrolysis. Without remarkable changes in amino acid composition, reductions of residual lipoxygenase activity and beany flavor-causing volatile compounds such as hexanol, hexanal, and pentanol in SPI were observed after combined heating and enzymatic treatments. The degree of hydrolysis, emulsion capacity and stability, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, and superoxide radical scavenging activity of SPI were significantly increased, but the magnitudes of apparent viscosity, consistency index, and dynamic moduli (G', G″) of SPI were significantly decreased after the combined heating and enzymatic treatments. Based on these results, it was suggested that the enzymatic hydrolysis in combination with high temperature pre-treatment may allow for the production of beany flavor-removed SPI hydrolyzates with superior emulsifying and antioxidant functionalities.

Pulse electrodeposited nickel-indium tin oxide nanocomposite as an electrocatalyst for non-enzymatic glucose sensing.

PubMed

Sivasakthi, P; Ramesh Bapu, G N K; Chandrasekaran, Maruthai

2016-01-01

Nickel and nickel-ITO nanocomposite on mild steel substrate were prepared by pulse electrodeposition method from nickel sulphamate electrolyte and were examined as electrocatalysts for non-enzymatic glucose sensing. The surface morphology, chemical composition, preferred orientation and oxidation states of the nickel metal ion in the deposits were characterized by SEM, EDAX, XRD and XPS. Electrochemical sensing of glucose was studied by cyclic voltammetry and amperometry. The modified Ni-ITO nanocomposite electrode showed higher electrocatalytic activity for the oxidation of glucose in alkaline medium and exhibited a linear range from 0.02 to 3.00 mM with a limit of detection 3.74 μM at a signal-to-noise ratio of 3. The higher selectivity, longer stability and better reproducibility of this electrode compared to nickel in the sensing of glucose are pointers for exploitation in practical clinical applications. Copyright © 2015 Elsevier B.V. All rights reserved.

An enzymatic fluorescent assay for the quantification of phosphite in a microtiter plate format.

PubMed

Berkowitz, Oliver; Jost, Ricarda; Pearse, Stuart J; Lambers, Hans; Finnegan, Patrick M; Hardy, Giles E St J; O'Brien, Philip A

2011-05-01

A sensitive fluorometric assay for the quantification of phosphite has been developed. The assay uses the enzymatic oxidation of phosphite to phosphate by a recombinant phosphite dehydrogenase with NAD(+) as cosubstrate to produce the highly fluorescent reaction product resorufin. The optimized assay can be carried out in a 96-well microtiter plate format for high-throughput screening purposes and has a detection limit of 0.25 nmol phosphite. We used the method to quantify phosphite levels in plant tissue extracts and to determine phosphite dehydrogenase activity in transgenic plants. The assay is suitable for other biological or environmental samples. Because phosphite is a widely used fungicide to protect plants from pathogenic oomycetes, the assay provides a cost-effective and easy-to-use method to monitor the fate of phosphite following application. Copyright © 2011 Elsevier Inc. All rights reserved.

Luciferase Protein Complementation Assays for Bioluminescence Imaging of Cells and Mice

PubMed Central

Luker, Gary D.; Luker, Kathryn E.

2015-01-01

Summary Protein fragment complementation assays (PCAs) with luciferase reporters currently are the preferred method for detecting and quantifying protein-protein interactions in living animals. At the most basic level, PCAs involve fusion of two proteins of interest to enzymatically inactive fragments of luciferase. Upon association of the proteins of interest, the luciferase fragments are capable of reconstituting enzymatic activity to generate luminescence in vivo. In addition to bi-molecular luciferase PCAs, unimolecular biosensors for hormones, kinases, and proteases also have been developed using target peptides inserted between inactive luciferase fragments. Luciferase PCAs offer unprecedented opportunities to quantify dynamics of protein-protein interactions in intact cells and living animals, but successful use of luciferase PCAs in cells and mice involves careful consideration of many technical factors. This chapter discusses the design of luciferase PCAs appropriate for animal imaging, including construction of reporters, incorporation of reporters into cells and mice, imaging techniques, and data analysis. PMID:21153371

Characterization of functionalized multiwalled carbon nanotubes for use in an enzymatic sensor.

PubMed

Guadarrama-Fernández, Leonor; Chanona-Pérez, Jorge; Manzo-Robledo, Arturo; Calderón-Domínguez, Georgina; Martínez-Rivas, Adrián; Ortiz-López, Jaime; Vargas-García, Jorge Roberto

2014-10-01

Carbon nanotubes (CNT) have proven to be materials with great potential for the construction of biosensors. Development of fast, simple, and low cost biosensors to follow reactions in bioprocesses, or to detect food contaminants such as toxins, chemical compounds, and microorganisms, is presently an important research topic. This report includes microscopy and spectroscopy to characterize raw and chemically modified multiwall carbon nanotubes (MWCNTs) synthesized by chemical vapor deposition with the intention of using them as the active transducer in bioprocessing sensors. MWCNT were simultaneously purified and functionalized by an acid mixture involving HNO3-H2SO4 and amyloglucosidase attached onto the chemically modified MWCNT surface. A 49.0% decrease in its enzymatic activity was observed. Raw, purified, and enzyme-modified MWCNTs were analyzed by scanning and transmission electron microscopy and Raman and X-ray photoelectron spectroscopy. These studies confirmed purification and functionalization of the CNTs. Finally, cyclic voltammetry electrochemistry was used for electrical characterization of CNTs, which showed promising results that can be useful for construction of electrochemical biosensors applied to biological areas.

pH-Responsive Fluorescence Enhancement in Graphene Oxide-Naphthalimide Nanoconjugates: A Fluorescence Turn-On Sensor for Acetylcholine.

PubMed

Mangalath, Sreejith; Abraham, Silja; Joseph, Joshy

2017-08-22

A pH-sensitive, fluorescence "turn-on" sensor based on a graphene oxide-naphthalimide (GO-NI) nanoconjugate for the detection of acetylcholine (ACh) by monitoring the enzymatic activity of acetylcholinesterase (AChE) in aqueous solution is reported. These nanoconjugates were synthesized by covalently anchoring picolyl-substituted NI derivatives on the GO/reduced GO surface through a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide coupling strategy, and the morphological and photophysical properties were studied in detail. Synergistic effects of π-π interactions between GO and the NI chromophore, and efficient photoinduced electron- and energy-transfer processes, were responsible for the strong quenching of fluorescence of these nanoconjugates, which were perturbed under acidic pH conditions, leading to significant enhancement of fluorescence emission. This nanoconjugate was successfully employed for the efficient sensing of pH changes caused by the enzymatic activity of AChE, thereby demonstrating its utility as a fluorescence turn-on sensor for ACh in the neurophysiological range. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transglutaminase-mediated protein immobilization to casein nanolayers created on a plastic surface.

PubMed

Kamiya, Noriho; Doi, Satoshi; Tominaga, Jo; Ichinose, Hirofumi; Goto, Masahiro

2005-01-01

An enzymatic method for covalent and site-specific immobilization of recombinant proteins on a plastic surface was explored. Using Escherichia coli alkaline phosphatase (AP) with a specific peptide tag (MKHKGS) genetically incorporated at the N-terminus as a model (NK-AP), microbial transglutaminase (MTG)-mediated protein immobilization was demonstrated. To generate a reactive surface for MTG, a 96-well polystyrene microtiter plate was physically coated with casein, a good MTG substrate. Successful immobilization of recombinant AP to the nanolayer of casein on the surface of the microtiter plate was verified by the detection of enzymatic activity. Since little activity was observed when wild-type AP was used, immobilization of NK-AP was likely directed by the specific peptide tag. When polymeric casein prepared by MTG was used as a matrix on the plate, the loading capacity of AP was increased about 2-fold compared to when casein was used as the matrix. Transglutaminase-mediated site-specific posttranslational modification of proteins offers one way of generating a variety of protein-based solid formulations for biotechnological applications.

Study on beta-galactosidase enzymatic activity of herbal yogurt.

PubMed

Chowdhury, Banani Ray; Chakraborty, Runu; Raychaudhuri, Utpal

2008-03-01

Different types of herbal yogurts were developed by mixing standardized milk with pretreated herbs, namely tulsi leaf (Ocimum sanctum), pudina leaf (Mentha arvensis) and coriander leaf (Coriandrum sativum), with leaves separately and a 1:1 (v/v) mixture of the strains of lactic starter cultures---Lactobacillus acidophilus (NCIM 2903) and Lactobacillus plantarum (NCIM 2083)-followed by incubation at 40 degrees C for 6 h. The beta-galactosidase enzymatic activity of the abovementioned herbal yogurts was determined and interestingly noted to exhibit higher enzymatic activity compared with the control yogurt (without any herbs). Among all herbal yogurts, tulsi yogurt had the maximum beta-galactosidase activity.

Photoreactive “Nanorulers” Detect a Novel Conformation of Full length HDAC3-SMRT Complex in Solution

PubMed Central

Abdelkarim, Hazem; Brunsteiner, Michael; Neelarapu, Raghupathi; Bai, He; Madriaga, Antonett; van Breemen, Richard B.; Blond, Sylvie Y.; Gaponenko, Vadim; Petukhov, Pavel A.

2013-01-01

Histone deacetylase 3 (HDAC3) is a promising epigenetic drug target for multiple therapeutic applications. Direct interaction between the Deacetylase Activating Domain of the silencing mediator for retinoid or thyroid hormone receptors (SMRT-DAD) is required for activation of enzymatic activity of HDAC3. The structure of this complex and the nature of interactions with HDAC inhibitors in solution are unknown. Using novel photoreactive HDAC probes – “nanorulers”, we determined the distance between the catalytic site of the full-length HDAC3 and SMRT-DAD in solution at physiologically relevant conditions and found it to be substantially different from that predicted by the X-ray model with a Δ379-428aa truncated HDAC3. Further experiments indicated that in solution this distance might change in response to chemical stimuli, while the enzymatic activity remained unaffected. These observations were further validated by Saturation Transfer Difference (STD) NMR experiments. We propose that the observed changes in the distance are an important part of the histone code that remains to be explored. Mapping direct interactions and distances between macromolecules with such “nanorulers” as a function of cellular events facilitates better understanding of basic biology and ways for its manipulation in cell and tissue specific manner. PMID:24010878

Cold ischemia contributes to the development of chronic rejection and mitochondrial injury after cardiac transplantation.

PubMed

Schneeberger, Stefan; Amberger, Albert; Mandl, Julia; Hautz, Theresa; Renz, Oliver; Obrist, Peter; Meusburger, Hugo; Brandacher, Gerald; Mark, Walter; Strobl, Daniela; Troppmair, Jakob; Pratschke, Johann; Margreiter, Raimund; Kuznetsov, Andrey V

2010-12-01

Chronic rejection (CR) remains an unsolved hurdle for long-term heart transplant survival. The effect of cold ischemia (CI) on progression of CR and the mechanisms resulting in functional deficit were investigated by studying gene expression, mitochondrial function, and enzymatic activity. Allogeneic (Lew→F344) and syngeneic (Lew→Lew) heart transplantations were performed with or without 10 h of CI. After evaluation of myocardial contraction, hearts were excised at 2, 10, 40, and 60 days for investigation of vasculopathy, gene expression, enzymatic activities, and mitochondrial respiration. Gene expression studies identified a gene cluster coding for subunits of the mitochondrial electron transport chain regulated in response to CI and CR. Myocardial performance, mitochondrial function, and mitochondrial marker enzyme activities declined in all allografts with time after transplantation. These declines were more rapid and severe in CI allografts (CR-CI) and correlated well with progression of vasculopathy and fibrosis. Mitochondria related gene expression and mitochondrial function are substantially compromised with the progression of CR and show that CI impacts on progression, gene profile, and mitochondrial function of CR. Monitoring mitochondrial function and enzyme activity might allow for earlier detection of CR and cardiac allograft dysfunction. © 2010 The Authors. Journal compilation © 2010 European Society for Organ Transplantation.

Rosmarinic acid and antioxidant enzyme activities in Lavandula vera MM cell suspension culture: a comparative study.

PubMed

Georgiev, Milen; Abrashev, Radoslav; Krumova, Ekaterina; Demirevska, Klimentina; Ilieva, Mladenka; Angelova, Maria

2009-11-01

The growth and intracellular protein content of lavender (Lavandula vera MM) cell suspension culture was followed along with some antioxidant defense system members-non-enzymatic (rosmarinic acid) and enzymatic [superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6)]. It was found that the media content and the cultivation mode strongly influenced the production of plant defense compounds as well as the ratio between non-enzymatic and enzymatic ones. The bioreactor culture contains about two times more rosmarinic acid, superoxide dismutase, and catalase compared to the shake-flask cultivation. These findings are discussed with respect to the relative stress levels and plant antioxidant orchestra system. It was concluded that investigated defense system components (enzymatic and non-enzymatic) were closely associated in a complex balance. The three isoenzyme forms of SOD (Cu/ZnSOD, FeSOD, and MnSOD) in the cells of Lavandula vera were revealed by polyacrylamide gel electrophoresis analysis, and the FeSOD isoform exhibited highest activity.

Subunit association of gamma-glutamyltranspeptidase of Escherichia coli K-12.

PubMed

Hashimoto, W; Suzuki, H; Nohara, S; Tachi, H; Yamamoto, K; Kumagai, H

1995-12-01

gamma-Glutamyltranspeptidase [EC 2.3.2.2] of Escherichia coli K-12 consists of one large subunit and one small subunit, which can be separated from each other by high-performance liquid chromatography. Using ion spray mass spectrometry, the masses of the large and the small subunit were determined to be 39,207 and 20,015, respectively. The large subunit exhibited no gamma-glutamyltranspeptidase activity and the small subunit had little enzymatic activity, but a mixture of the two subunits showed partial recovery of the enzymatic activity. The results of native-polyacrylamide gel electrophoresis suggested that they could partially recombine, and that the recombined dimer exhibited enzymatic activity. The gene of gamma-glutamyltranspeptidase encoded a signal peptide, and the large and small subunits in a single open reading frame in that order. Two kinds of plasmid were constructed encoding the signal peptide and either the large or the small subunit. A gamma-glutamyltranspeptidase-less mutant of E. coli K-12 was transformed with each plasmid or with both of them. The strain harboring the plasmid encoding each subunit produced a small amount of the corresponding subunit protein in the periplasmic space but exhibited no enzymatic activity. The strain transformed with both plasmids together exhibited the enzymatic activity, but its specific activity was approximately 3% of that of a strain harboring a plasmid encoding the intact structural gene. These results indicate that a portion of the separated large and small subunits can be reconstituted in vitro and exhibit the enzymatic activity, and that the expressed large and small subunits independently are able to associate in vivo and be folded into an active structure, though the specific activity of the associated subunits was much lower than that of native enzyme. This suggests that the synthesis of gamma-glutamyltranspeptidase in a single precursor polypeptide and subsequent processing are more effective to construct the intact structure of gamma-glutamyltranspeptidase than the association of the separated large and small subunits.

Detecting and Imaging of γ-Glutamytranspeptidase Activity in Serum, Live Cells, and Pathological Tissues with a High Signal-Stability Probe by Releasing a Precipitating Fluorochrome.

PubMed

Ou-Yang, Juan; Li, Yong-Fei; Wu, Ping; Jiang, Wen-Li; Liu, Hong-Wen; Li, Chun-Yan

2018-06-20

γ-Glutamytranspeptidase (GGT) is a significant tumor-related biomarker that overexpresses in several tumor cells. Accurate detection and imaging of GGT activity in serum, live cells, and pathological tissues hold great significance for cancer diagnosis, treatment, and management. Recently developed small molecule fluorescent probes for GGT tend to diffuse to the whole cytoplasm and then translocate out of live cells after enzymatic reaction, which make them fail to provide high spatial resolution and long-term imaging in biological systems. To address these problems, a novel fluorescent probe (HPQ-PDG) which releases a precipitating fluorochrome upon the catalysis of GGT is designed and synthesized. HPQ-PDG is able to detect GGT activity with high spatial resolution and good signal-stability. The large Stokes shift of the probe enables it to detect the activity of GGT in serum samples with high sensitivity. To our delight, the probe is used for imaging GGT activity in live cells with the ability of discriminating cancer cells from normal cells. What's more, we successfully apply it for pathological tissues imaging, with the results indicating that the potential application of HPQ-PDG in histopathological examination. All these results demonstrate the potential application of HPQ-PDG in the clinic.

Using an enzymatic galactose assay to detect lactose glycation extents of two proteins caseinate and soybean protein isolate via the Maillard reaction.

PubMed

Wang, Xiao-Peng; Zhao, Xin-Huai

2017-06-01

Glycation of food proteins via the Maillard reaction has been widely studied in the recent years; however, the amount of saccharide connected to proteins is usually not determined. An enzymatic galactose assay was proposed firstly in this study to detect lactose glycation extents of caseinate and soybean protein isolate (SPI) during the Maillard reaction at two temperatures and different times. The separated glycated proteins were hydrolysed to release galactose necessary for the enzymatic assay and glycation calculation. Caseinate and SPI both obtained the highest lactose glycation extents at 100 °C or 121 °C by a reaction time of 180 or 20 min. Short- and long-time reaction resulted in lower glycation extents. During the reaction, three chemical indices (absorbences at 294/490 nm and fluorescence intensities) of reaction mixtures increased continually, but another index reactable NH 2 of glycated proteins showed the opposite trend. In general, changing profiles of the four indices were inconsistent with those profiles of lactose glycation extents of glycated proteins, implying practical limitation of the four indices in studies. This proposed enzymatic assay could directly detect lactose glycation of the two proteins, and thus was more useful than the four chemical indices to monitor glycation of the two proteins. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases

NASA Astrophysics Data System (ADS)

Cesaro, Patrizia; Cattaneo, Chiara; Bona, Elisa; Berta, Graziella; Cavaletto, Maria

2015-09-01

Enzymatic reduction of arsenate to arsenite is the first known step in arsenate metabolism in all organisms. Although the presence of one mRNA arsenate reductase (PvACR2) has been characterized in gametophytes of P. vittata, no arsenate reductase protein has been directly observed in this arsenic hyperaccumulating fern, yet. In order to assess the possible presence of arsenate reductase in P. vittata, two recombinant proteins, ACR2-His6 and Trx-His6-S-Pv2.5-8 were prepared in Escherichia coli, purified and used to produce polyclonal antibodies. The presence of these two enzymes was evaluated by qRT-PCR, immunoblotting and direct MS analysis. Enzymatic activity was detected in crude extracts. For the first time we detected and identified two arsenate reductase proteins (PvACR2 and Pv2.5-8) in sporophytes and gametophytes of P. vittata. Despite an increase of the mRNA levels for both proteins in roots, no difference was observed at the protein level after arsenic treatment. Overall, our data demonstrate the constitutive protein expression of PvACR2 and Pv2.5-8 in P. vittata tissues and propose their specific role in the complex metabolic network of arsenic reduction.

Sequential Injection/Electrochemical Immunoassay for Quantifying the Pesticide Metabolite 3, 5, 6-Trichloro-2-Pyridinol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Guodong; Riechers, Shawn L.; Timchalk, Chuck

2005-12-04

An automated and sensitive sequential injection electrochemical immunoassay was developed to monitor a potential insecticide biomarker, 3, 5, 6-trichloro-2-pyridinol. The current method involved a sequential injection analysis (SIA) system equipped with a thin-layer electrochemical flow cell and permanent magnet, which was used to fix 3,5,6-trichloro-2-pyridinol (TCP) antibody coated magnetic beads (TCP-Ab-MBs) in the reaction zone. After competitive immunoreactions among TCP-Ab-MBs, TCP analyte, and horseradish peroxidase (HRP) labeled TCP, a 3, 3?, 5, 5?-tetramethylbenzidine dihydrochloride and hydrogen peroxide (TMB-H2O2) substrate solution was injected to produce an electroactive enzymatic product. The activity of HRP tracers was monitored by a square wave voltammetricmore » scanning electroactive enzymatic product in the thin-layer flow cell. The voltammetric characteristics of the substrate and the enzymatic product were investigated under batch conditions, and the parameters of the immunoassay were optimized in the SIA system. Under the optimal conditions, the system was used to measure as low as 6 ng L-1 (ppt) TCP, which is around 50-fold lower than the value indicated by the manufacturer of the TCP RaPID Assay? kit (0.25 ug/L, colorimetric detection). The performance of the developed immunoassay system was successfully evaluated on tap water and river water samples spiked with TCP. This technique could be readily used for detecting other environmental contaminants by developing specific antibodies against contaminants and is expected to open new opportunities for environmental and biological monitoring.« less

Effects of lead contamination on soil enzymatic activities, microbial biomass, and rice physiological indices in soil-lead-rice (Oryza sativa L.) system.

PubMed

Zeng, Lu S; Liao, Min; Chen, Cheng L; Huang, Chang Y

2007-05-01

The effect of lead (Pb) treatment on the soil enzymatic activities, soil microbial biomass, rice physiological indices and rice biomass were studied in a greenhouse pot experiment. Six levels of Pb viz. 0(CK), 100, 300, 500, 700, 900 mg/kg soil were applied in two types of paddy soils. The results showed that Pb treatment had a stimulating effect on soil enzymatic activities and microbial biomass carbon (Cmic) at low concentration and an inhibitory influence at higher concentration. The degree of influence on enzymatic activities and Cmic by Pb was related to the clay and organic matter contents of the soils. When the Pb treatment was raised to the level of 500 mg/kg, ecological risk appeared both to soil microorganisms and plants. The results also revealed a consistent trend of increased chlorophyll contents and rice biomass initially, maximum at a certain Pb treatment, and then decreased gradually with the increase in Pb concentration. Pb was effective in inducing proline accumulation and its toxicity causes oxidative stress in rice plants. Therefore, it was concluded that soil enzymatic activities, Cmic and rice physiological indices, could be sensitive indicators to reflect environmental stress in soil-lead-rice system.

Development of Engineered Bacteriophages for Escherichia coli Detection and High-Throughput Antibiotic Resistance Determination.

PubMed

Chen, Juhong; Alcaine, Samuel D; Jackson, Angelyca A; Rotello, Vincent M; Nugen, Sam R

2017-04-28

T7 bacteriophages (phages) have been genetically engineered to carry the lacZ operon, enabling the overexpression of beta-galactosidase (β-gal) during phage infection and allowing for the enhanced colorimetric detection of Escherichia coli (E. coli). Following the phage infection of E. coli, the enzymatic activity of the released β-gal was monitored using a colorimetric substrate. Compared with a control T7 phage, our T7 lacZ phage generated significantly higher levels of β-gal expression following phage infection, enabling a lower limit of detection for E. coli cells. Using this engineered T7 lacZ phage, we were able to detect E. coli cells at 10 CFU·mL -1 within 7 h. Furthermore, we demonstrated the potential for phage-based sensing of bacteria antibiotic resistance profiling using our T7 lacZ phage, and subsequent β-gal expression to detect antibiotic resistant profile of E. coli strains.

Enzymatic Basis for Differentiation of Rhizobium into Fast- and Slow-Growing Groups

PubMed Central

Drets, G. Martinez-De; Arias, A.

1972-01-01

Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and other enzymes related to carbohydrate metabolism were studied in rhizobia. A nicotinamide adenine dinucleotide phosphate-6-phosphogluconate dehydrogenase was detected in strains of the fast-growing group of Rhizobium but not in strains of the slow-growing group. An enzymatic differentiation of rhizobia was established. PMID:4400417

Detection of trace heavy metal ions in water by nanostructured porous Si biosensors.

PubMed

Shtenberg, Giorgi; Massad-Ivanir, Naama; Segal, Ester

2015-07-07

A generic biosensing platform, based on nanostructured porous Si (PSi), Fabry-Pérot thin films, for label-free monitoring of heavy metal ions in aqueous solutions by enzymatic activity inhibition, is described. First, we show a general detection assay by immobilizing horseradish peroxidase (HRP) within the oxidized PSi nanostructure and monitor its catalytic activity in real time by reflective interferometric Fourier transform spectroscopy. Optical studies reveal the high specificity and sensitivity of the HRP-immobilized PSi towards three metal ions (Ag(+) > Pb(2+) > Cu(2+)), with a detection limit range of 60-120 ppb. Next, we demonstrate the concept of specific detection of Cu(2+) ions (as a model heavy metal) by immobilizing Laccase, a multi-copper oxidase, within the oxidized PSi. The resulting biosensor allows for specific detection and quantification of copper ions in real water samples by monitoring the Laccase relative activity. The optical biosensing results are found to be in excellent agreement with those obtained by the gold standard analytical technique (ICP-AES) for all water samples. The main advantage of the presented biosensing concept is the ability to detect heavy metal ions at environmentally relevant concentrations using a simple and portable experimental setup, while the specific biosensor design can be tailored by varying the enzyme type.

Characterization of the endogenous enzymatic hydrolyses of Petroselinum crispum glycosides: determined by chromatography upon their sugar and flavonoid products.

PubMed

Boldizsár, Imre; Füzfai, Zsófia; Molnár-Perl, Ibolya

2013-06-07

The behavior of the flavonoid diglycosides, relevant constituents of parsley (Petroselinum crispum) fruit (PFr) and leaf (PLe) samples was characterized upon their enzymatic hydrolyses applying complementary liquid chromatography-ultraviolet (LC-UV) and gas chromatography mass selective (GC-MS) detections. Analyses were performed in quantitative manner, from the same extracts as a function of hydrolysis times. Both in fruit and leaf tissue extracts, in intact and in enzyme hydrolyzed ones, apigenin, chrysoeriol, their glycosides, sugars, sugar alcohols, carboxylic acids and phytosterols, in total 17 constituents were identified and quantified. Based primarily on the selective mass fragmentation properties of the trimethylsilyl (oxime) ether/ester derivatives of constituents, we confirmed several novelties to the field. (i) It was shown for the first time that in parsley tissues different types of glycosidase enzyme are active. In PFr samples, both the stepwise and disaccharide specific endogenous mechanisms were certified, quantifying simultaneously the continuous release of apigenin, chrysoeriol, 2-O-apiosyl-apiose, apiose and glucose. (ii) 2-O-Apiosyl-glucose was demonstrated as disaccharide due to its formation under derivatization conditions from parsley glycosides. (iii) Both in PFr and in PLe samples even the invertase enzyme activity was attainable: sucrose decomposition in both tissues was going on with the same intensity. Three different types of enzymatic glycosidase processes were followed with their specific hydrolysis products by means of HPLC-UV and GC-MS, simultaneously. Copyright © 2013 Elsevier B.V. All rights reserved.

Self-Powered Implantable Skin-Like Glucometer for Real-Time Detection of Blood Glucose Level In Vivo

NASA Astrophysics Data System (ADS)

Zhang, Wanglinhan; Zhang, Linlin; Gao, Huiling; Yang, Wenyan; Wang, Shuai; Xing, Lili; Xue, Xinyu

2018-06-01

Implantable bioelectronics for analyzing physiological biomarkers has recently been recognized as a promising technique in medical treatment or diagnostics. In this study, we developed a self-powered implantable skin-like glucometer for real-time detection of blood glucose level in vivo. Based on the piezo-enzymatic-reaction coupling effect of GOx@ZnO nanowire, the device under an applied deformation can actively output piezoelectric signal containing the glucose-detecting information. No external electricity power source or battery is needed for this device, and the outputting piezoelectric voltage acts as both the biosensing signal and electricity power. A practical application of the skin-like glucometer implanted in mouse body for detecting blood glucose level has been simply demonstrated. These results provide a new technique path for diabetes prophylaxis and treatment.

DNA damage induced by the direct effect of radiation

NASA Astrophysics Data System (ADS)

Yokoya, A.; Shikazono, N.; Fujii, K.; Urushibara, A.; Akamatsu, K.; Watanabe, R.

2008-10-01

We have studied the nature of DNA damage induced by the direct effect of radiation. The yields of single- (SSB) and double-strand breaks (DSB), base lesions and clustered damage were measured using the agarose gel electrophoresis method after exposing to various kinds of radiations to a simple model DNA molecule, fully hydrated closed-circular plasmid DNA (pUC18). The yield of SSB does not show significant dependence on linear energy transfer (LET) values. On the other hand, the yields of base lesions revealed by enzymatic probes, endonuclease III (Nth) and formamidopyrimidine DNA glycosylase (Fpg), which excise base lesions and leave a nick at the damage site, strongly depend on LET values. Soft X-ray photon (150 kVp) irradiation gives a maximum yield of the base lesions detected by the enzymatic probes as SSB and clustered damage, which is composed of one base lesion and proximate other base lesions or SSBs. The clustered damage is visualized as an enzymatically induced DSB. The yields of the enzymatically additional damages strikingly decrease with increasing levels of LET. These results suggest that in higher LET regions, the repair enzymes used as probes are compromised because of the dense damage clustering. The studies using simple plasmid DNA as a irradiation sample, however, have a technical difficulty to detect multiple SSBs in a plasmid DNA. To detect the additional SSBs induced in opposite strand of the first SSB, we have also developed a novel technique of DNA-denaturation assay. This allows us to detect multiply induced SSBs in both strand of DNA, but not induced DSB.

Rational enhancement of enzyme performance in organic solvents. Final technical report, 1992--1996

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klibanov, A.M.

1996-12-31

This research focused on the following: the dependence of enzymatic activity of several model hydrolases in nonaqueous solvents; control of substrate selectivity of the protease subtilisin Carlsberg by the solvent; control of catalytic activity and enantioselectivity of this enzyme in organic solvents by immobilization support; lipase-catalyzed acylation of sugars in anhydrous hydrophobic media; the possibility of accelerating enzymatic processes in organic solvents by certain cosolvents; whether lipase catalysis in organic solvents can be enhanced by introducing interfaces in the in the reaction medium; the structure of proteins suspended in organic solvents; improving enzymatic enantioselectivity in organic solvents; analyzing the plungemore » in enzymatic activity upon replacing water with organic solvents; and the structural basis for the phenomenon of molecular memory of imprinted proteins in organic solvents.« less

Cuticle-degrading proteases and toxins as virulence markers of Beauveria bassiana (Balsamo) Vuillemin.

PubMed

Cito, Annarita; Barzanti, Gian Paolo; Strangi, Agostino; Francardi, Valeria; Zanfini, Assunta; Dreassi, Elena

2016-09-01

Beauveria bassiana is one of the most known entomopathogenic fungal species and its entomopathogenic mechanism involves several bioactive metabolites, mainly cuticle-degrading enzymes and toxic molecules, which are predicted to play a key role as virulence factors. In this study six Beauveria bassiana strains (B 13/I03, B 13/I11, B 13/I49, B 13/I57, B 13/I63, and B 13/I64) were assayed against Tenebrio molitor larvae. Enzymatic activity of total proteases and specifically Pr 1 and Pr 2, as well as the production of toxic compounds were investigated in each fungal strain. Toxins were detected both in vitro-in medium filtrates and mycelia-and in vivo-in Tenebrio molitor larvae infected by the fungal strains tested. B 13/I11 and B 13/I63 strains showed the most significant entomopathogenic activity against Tenebrio molitor larvae (cumulative mortality rate 100 and 97%, respectively; average survival time 5.85 and 6.74 days, respectively). A widely variable and fungal strain-dependent enzymatic activity of total proteases, Pr 1 and Pr 2 was found. Beauvericin, beauvericin A and bassianolide resulted the most prevalent toxins detected in the substrates analyzed. It has been found that an increase of beauvericin content in vivo resulted significantly correlated to a decrease of Tenebrio molitor larvae average survival time in entomopathogenic bioassay (inverse correlation). The involvement of beauvericin in B. bassiana entomopathogenic process is confirmed; in vitro analysis of cuticle degrading proteases activity and toxins production in relation to the methods adopted resulted insufficient for a rapid screening to determine the virulence of B. bassiana strains against Tenebrio molitor larvae. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isolation of an invertebrate-type lysozyme from the nephridia of the echiura, Urechis unicinctus, and its recombinant production and activities.

PubMed

Oh, Hye Young; Kim, Chan-Hee; Go, Hye-Jin; Park, Nam Gyu

2018-05-09

Invertebrates, unlike vertebrates which have adaptive immune system, rely heavily on the innate immune system for the defense against pathogenic bacteria. Lysozymes, along with other immune effectors, are regarded as an important group in this defense. An invertebrate-type (i-type) lysozyme, designated Urechis unicinctus invertebrate-type lysozyme, Uu-ilys, has been isolated from nephridia of Urechis unicinctus using a series of high performance liquid chromatography (HPLC), and ultrasensitive radial diffusion assay (URDA) as a bioassay system. Analyses of the primary structure and cDNA cloning revealed that Uu-ilys was approximately 14 kDa and composed of 122 amino acids (AAs) of which the precursor had a total of 160 AAs containing a signal peptide of 18 AAs and a pro-sequence of 20 AAs encoded by the nucleotide sequence of 714 bp that comprises a 5' untranslated region (UTR) of 42 bp, an open reading frame (ORF) of 483 bp, and a 3' UTR of 189 bp. Multiple sequence alignment showed Uu-ilys has high homology to i-type lysozymes from several annelids. Relatively high transcriptional expression levels of Uu-ilys was detected in nephridia, anal vesicle, and intestine. The native Uu-ilys exhibited comparable lysozyme enzymatic and antibacterial activities to hen egg white lysozyme. Collectively, these data suggest that Uu-ilys, the isolated antibacterial protein, plays a role in the immune defense mechanism of U. unicinctus. Recombinant Uu-ilys (rUu-ilys) produced in a bacterial expression system showed significantly decreased lysozyme lytic activity from that of the native while its potency on radial diffusion assay detecting antibacterial activity was retained, which may indicate the non-enzymatic antibacterial capacity of Uu-ilys. Copyright © 2018. Published by Elsevier Ltd.

Dual chronoamperometric detection of enzymatic biomarkers using magnetic beads and a low-cost flow cell.

PubMed

Moral-Vico, Javier; Barallat, Jaume; Abad, Llibertat; Olivé-Monllau, Rosa; Muñoz-Pascual, Francesc Xavier; Galán Ortega, Amparo; del Campo, F Javier; Baldrich, Eva

2015-07-15

In this work we report on the production of a low cost microfluidic device for the multiplexed electrochemical detection of magneto bioassays. As a proof of concept, the device has been used to detect myeloperoxidase (MPO), a cardiovascular biomarker. With this purpose, two bioassays have been optimized in parallel onto magnetic beads (MBs) for the simultaneous detection of MPO endogenous peroxidase activity and quantification of total MPO. Since the two bioassays produced signals of different magnitude for each concentration of MPO tested, two detection strategies have been compared, which entailed registering steady state currents (Iss) under substrate flow, and measuring the peak currents (Ip) produced in a stopped flow approach. As it will be shown, appropriate tuning of the detection and flow conditions can provide extremely sensitive detection, but also allow simultaneous detection of assays or parameters that would produce signals of different orders of magnitude when measured by a single detection strategy. In order to demonstrate the feasibility of the detection strategy reported, a dual MPO mass and activity assay has been finally applied to the study of 10 real plasma samples, allowing patient classification according to the risk of suffering a cardiovascular event. Copyright © 2015 Elsevier B.V. All rights reserved.

Enzymatic digestion and selective quantification of underivatised delta-9-tetrahydrocannabinol and cocaine in human hair using gas chromatography-mass spectrometry.

PubMed

Breidi, Salah Eddine; Barker, James; Petróczi, Andrea; Naughton, Declan P

2012-01-01

Gas chromatography-mass spectrometric (GC-MS) methods for drug analysis routinely employ derivatising reagents. The aim of this paper was to develop a method for the analysis of two recreational drugs, delta-9-tetrahydrocannabinol (Δ(9)-THC) and cocaine in hair samples using GC-MS, without prior derivatisation, thus allowing the sample to be reanalysed in its original form. An enzymatic digestion technique was also developed. Ten hair samples, that were known positive for either Δ(9)-THC and/or cocaine, were enzymatically digested, extracted, and then analysed by GC-MS. All samples measured contained Δ(9)-THC and one sample contained cocaine. The limits of detection (LOD) and quantification (LOQ) were 0.02 ng/mg and 0.05 ng/mg, respectively, for cocaine and 0.015 ng/mg and 0.02 ng/mg, respectively, for Δ(9)-THC. The wide detection window, ease of direct analysis by GC-MS, lower detection limits of underivatised samples, and the stability of drugs using this technique may offer an improved method of analysis.

Enzymatic Digestion and Selective Quantification of Underivatised Delta-9-Tetrahydrocannabinol and Cocaine in Human Hair Using Gas Chromatography-Mass Spectrometry

PubMed Central

Breidi, Salah Eddine; Barker, James; Petróczi, Andrea; Naughton, Declan P.

2012-01-01

Gas chromatography-mass spectrometric (GC-MS) methods for drug analysis routinely employ derivatising reagents. The aim of this paper was to develop a method for the analysis of two recreational drugs, delta-9-tetrahydrocannabinol (Δ9-THC) and cocaine in hair samples using GC-MS, without prior derivatisation, thus allowing the sample to be reanalysed in its original form. An enzymatic digestion technique was also developed. Ten hair samples, that were known positive for either Δ9-THC and/or cocaine, were enzymatically digested, extracted, and then analysed by GC-MS. All samples measured contained Δ9-THC and one sample contained cocaine. The limits of detection (LOD) and quantification (LOQ) were 0.02 ng/mg and 0.05 ng/mg, respectively, for cocaine and 0.015 ng/mg and 0.02 ng/mg, respectively, for Δ9-THC. The wide detection window, ease of direct analysis by GC-MS, lower detection limits of underivatised samples, and the stability of drugs using this technique may offer an improved method of analysis. PMID:22567573

Enzymatic reactivity of glucose oxidase confined in nanochannels.

PubMed

Yu, Jiachao; Zhang, Yuanjian; Liu, Songqin

2014-05-15

The construction of nanodevices coupled with an integrated real-time detection system for evaluation of the function of biomolecules in biological processes, and enzymatic reaction kinetics occurring at the confined space or interface is a significant challenge. In this work, a nanochannel-enzyme system in which the enzymatic reaction could be investigated with an electrochemical method was constructed. The model system was established by covalently linking glucose oxidase (GOD) onto the inner wall of the nanochannels of the porous anodic alumina (PAA) membrane. An Au disc was attached at the end of the nanochannels of the PAA membrane as the working electrode for detection of H2O2 product of enzymatic reaction. The effects of ionic strength, amount of immobilized enzyme and pore diameter of the nanochannels on the enzymatic reaction kinetics were illustrated. The GOD confined in nanochannels showed high stability and reactivity. Upon addition of glucose to the nanochannel-enzyme system, the current response had a calibration range span from 0.005 to 2 mM of glucose concentration. The apparent Michaelis-Menten constant (K(m)(app)) of GOD confined in nanochannel was 0.4 mM. The presented work provided a platform for real-time monitoring of the enzyme reaction kinetics confined in nanospaces. Such a nanochannel-enzyme system could also help design future biosensors and enzyme reactors with high sensitivity and efficiency. Copyright © 2013 Elsevier B.V. All rights reserved.

A novel enzymatic glucose sensor based on Pt nanoparticles-decorated hollow carbon spheres-modified glassy carbon electrode

NASA Astrophysics Data System (ADS)

Luhana, Charles; Bo, Xiang-Jie; Ju, Jian; Guo, Li-Ping

2012-10-01

A new glucose biosensor was developed based on hollow carbon spheres decorated with platinum nanoparticles (Pt/HCSs)-modified glassy carbon electrode immobilized with glucose oxidase (GOx) with the help of Nafion. The Pt nanoparticles were well dispersed on the HCSs with an average size of 2.29 nm. The detection of glucose was achieved via electrochemical detection of the enzymatically liberated H2O2 at +0.5 V versus Ag/AgCl at physiologic pH of 7.4. The Pt/HCSs-modified electrode exhibited excellent electrocatalytic activities toward both the oxidation and reduction of H2O2. The glucose biosensor showed good electrocatalytic performance in terms of high sensitivity (4.1 μA mM-1), low detection limit (1.8 μM), fast response time <3 s, and wide linear range (0.04-8.62 mM). The apparent Michaelis-Menten constant ( K m) and the maximum current density ( i max) values for the biosensor were 10.94 mM and 887 μA cm-2 respectively. Furthermore, this biosensor showed an acceptable reproducibility and high stability. The interfering signals from ascorbic acid and uric acid at concentration levels normally found in human blood were not much compared with the response to glucose. Blood serum samples were also tested with this biosensor and a good recovery was achieved for the two spiked serum samples.

Highly sensitive glucose biosensor based on the effective immobilization of glucose oxidase/carbon-nanotube and gold nanoparticle in nafion film and peroxyoxalate chemiluminescence reaction of a new fluorophore.

PubMed

Zargoosh, Kiomars; Chaichi, Mohammad Javad; Shamsipur, Mojtaba; Hossienkhani, Saman; Asghari, Sakineh; Qandalee, Mohammad

2012-05-15

A novel glucose biosensor based on the chemiluminescence (CL) detection of enzymatically generated H(2)O(2) was constructed by the effective immobilization of glucose oxidase (GOD)/carbon-nanotubes (CNTs)/gold nanoparticles (GNPs) in nafion film on graphite support. The influences of various experimental parameters such as solution pH, the action time of the enzyme, interferents and the concentration of CL reagents were investigated. Carbon nanotubes and gold nanoparticles offer excellent catalytic activity toward hydrogen peroxide generation in enzymatic reaction between glucose oxidase and glucose, which would enable sensitive determination of glucose. Under the optimum condition, the linear response range of glucose was found to be 2.25 × 10(-6) to 1.75 × 10(-4 ) mol L(-1), and the detection limit (defined as the concentration that could be detected at the signal-to-noise ratio of 3) was 1.00 × 10(-6) mol L(-1). The CL biosensor exhibited good storage stability, i.e., 80% of its initial response was retained after 10 days storage at pH 7.0. The present CL biosensor has been used to determine the glucose concentrations in real serum and urine samples with satisfactory results. Copyright © 2011 Elsevier B.V. All rights reserved.

Ambient Ionization Mass Spectrometry Measurement of Aminotransferase Activity

NASA Astrophysics Data System (ADS)

Yan, Xin; Li, Xin; Zhang, Chengsen; Xu, Yang; Cooks, R. Graham

2017-06-01

A change in enzyme activity has been used as a clinical biomarker for diagnosis and is useful in evaluating patient prognosis. Current laboratory measurements of enzyme activity involve multi-step derivatization of the reaction products followed by quantitative analysis of these derivatives. This study simplified the reaction systems by using only the target enzymatic reaction and directly detecting its product. A protocol using paper spray mass spectrometry for identifying and quantifying the reaction product has been developed. Evaluation of the activity of aspartate aminotransferase (AST) was chosen as a proof-of-principle. The volume of sample needed is greatly reduced compared with the traditional method. Paper spray has a desalting effect that avoids sprayer clogging problems seen when examining serum samples by nanoESI. This very simple method does not require sample pretreatment and additional derivatization reactions, yet it gives high quality kinetic data, excellent limits of detection (60 ppb from serum), and coefficients of variation <10% in quantitation. [Figure not available: see fulltext.

Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase.

PubMed

Yoshida, Wataru; Kezuka, Aki; Murakami, Yoshiyuki; Lee, Jinhee; Abe, Koichi; Motoki, Hiroaki; Matsuo, Takafumi; Shimura, Nobuaki; Noda, Mamoru; Igimi, Shizunobu; Ikebukuro, Kazunori

2013-11-01

An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268-luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF-luciferase fusion protein. By means of the automatic analyzer with ZF-luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0×10 to 1.0×10(6) copies. Copyright © 2013 Elsevier B.V. All rights reserved.

Micromechanical Modeling Study of Mechanical Inhibition of Enzymatic Degradation of Collagen Tissues

PubMed Central

Tonge, Theresa K.; Ruberti, Jeffrey W.; Nguyen, Thao D.

2015-01-01

This study investigates how the collagen fiber structure influences the enzymatic degradation of collagen tissues. We developed a micromechanical model of a fibrous collagen tissue undergoing enzymatic degradation based on two central hypotheses. The collagen fibers are crimped in the undeformed configuration. Enzymatic degradation is an energy activated process and the activation energy is increased by the axial strain energy density of the fiber. We determined the intrinsic degradation rate and characteristic energy for mechanical inhibition from fibril-level degradation experiments and applied the parameters to predict the effect of the crimped fiber structure and fiber properties on the degradation of bovine cornea and pericardium tissues under controlled tension. We then applied the model to examine the effect of the tissue stress state on the rate of tissue degradation and the anisotropic fiber structures that developed from enzymatic degradation. PMID:26682825

Effects of chromium picolinate on fat deposition, activity and genetic expression of lipid metabolism-related enzymes in 21 day old Ross broilers

PubMed Central

Chen, Guangxin; Gao, Zhenhua; Chu, Wenhui; Cao, Zan; Li, Chunyi

2018-01-01

Objective This experiment was conducted to investigate the effects of chromium picolinate (CrP) on fat deposition, genetic expression and enzymatic activity of lipid metabolism-related enzymes. Methods Two hundred forty one-day-old Ross broilers were randomly divided into 5 groups with 4 replicates per group and 12 Ross broiler chicks per replicate. The normal control group was fed a basal diet, and the other groups fed the same basal diet supplemented with 0.1, 0.2, 0.4, and 0.8 mg/kg CrP respectively. The experiment lasted for 21 days. Results Added CrP in the basal diet decreased the abdominal fat, had no effects on subcutaneous fat thickness and inter-muscular fat width; 0.2 mg/kg CrP significantly decreased the fatty acid synthase (FAS) enzymatic (p<0.05); acetyl-CoA carboxylase (ACC) enzymatic activity decreased in all CrP groups (p<0.05); hormone-sensitive lipase (HSL) enzymatic activity also decreased, but the change was not significant (p>0.05); 0.4 mg/kg CrP group significantly decreased the lipoprotein lipase (LPL) enzymatic activity. FAS mRNA expression increased in all experimental groups, and the LPL mRNA expression significantly increased in all experimental groups (p<0.05), but not 0.2 mg/kg CrP group. Conclusion The results indicated that adding CrP in basal diet decreased the abdominal fat percentage, had no effects on subcutaneous fat thickness and inter-muscular fat width, decreased the enzymatic activity of FAS, ACC, LPL and HSL and increased the genetic expression levels of FAS and LPL. PMID:28830127

Effects of chromium picolinate on fat deposition, activity and genetic expression of lipid metabolism-related enzymes in 21 day old Ross broilers.

PubMed

Chen, Guangxin; Gao, Zhenhua; Chu, Wenhui; Cao, Zan; Li, Chunyi; Zhao, Haiping

2018-04-01

This experiment was conducted to investigate the effects of chromium picolinate (CrP) on fat deposition, genetic expression and enzymatic activity of lipid metabolism-related enzymes. Two hundred forty one-day-old Ross broilers were randomly divided into 5 groups with 4 replicates per group and 12 Ross broiler chicks per replicate. The normal control group was fed a basal diet, and the other groups fed the same basal diet supplemented with 0.1, 0.2, 0.4, and 0.8 mg/kg CrP respectively. The experiment lasted for 21 days. Added CrP in the basal diet decreased the abdominal fat, had no effects on subcutaneous fat thickness and inter-muscular fat width; 0.2 mg/kg CrP significantly decreased the fatty acid synthase (FAS) enzymatic (p<0.05); acetyl-CoA carboxylase (ACC) enzymatic activity decreased in all CrP groups (p<0.05); hormone-sensitive lipase (HSL) enzymatic activity also decreased, but the change was not significant (p>0.05); 0.4 mg/kg CrP group significantly decreased the lipoprotein lipase (LPL) enzymatic activity. FAS mRNA expression increased in all experimental groups, and the LPL mRNA expression significantly increased in all experimental groups (p<0.05), but not 0.2 mg/kg CrP group. The results indicated that adding CrP in basal diet decreased the abdominal fat percentage, had no effects on subcutaneous fat thickness and inter-muscular fat width, decreased the enzymatic activity of FAS, ACC, LPL and HSL and increased the genetic expression levels of FAS and LPL.

[Pancreatic functional status after wedge resection of the duodenal wall and para-pancreatic micro-irrigation].

PubMed

Voskanian, S E; Naĭdenov, E V

2011-01-01

To study influence parapancreatic microirrigation on morphological and functional condition of a pancreas and transformations of enzymatic activity of blood serum and enzymatic activity of lymph of a chest lymphatic channel after an operative trauma of a duodenum. Research is executed on 140 not purebred dogs which have been divided into six groups and united in two series. In the first series (30 dogs) were studied changes pancreatic exosecretion in the postoperative period of resection of duodenum (group 1.1), in the postoperative period of resection of duodenum with preliminary infiltration of a parapancreatic tissue of 0.5% by a solution of Novocain (group 1.2) and after resection of duodenum with application parapancreatic microirrigation (group 1.3). In the second series (110 dogs) were studied frequency of development of acute pancreatitis, enzymatic activity of blood serum and enzymatic activity of lymph of thoracal lymphatic duct after resection of duodenum (group 2.1) and in the postoperative period of resection of duodenum with preliminary infiltration of a parapancreatic tissue of 0.5% by a solution of Novocain (group 2.2) and after resection of duodenum with application parapancreatic microirrigation (group 2.3). Application parapancreatic microirrigation does not lead to oppression pancreatic exosecretion at the first o'clock after duodenotomy, and substantially reduces the pancreatic hypersecretion observed in the postoperative period of resection of a duodenum. In addition, application parapancreatic microirrigation reduces frequency of development of acute pancreatitis and promotes less expressed increase enzymatic activity of blood serum and enzymatic activity of lymph thoracal lymphatic duct at development of the given complication after operational trauma of duodenum in comparison with resection of duodenum and after a resection of a duodenum executed against infiltration of a parapancreatic tissue of 0.5% by a solution of Novocain.

Multi-Scale Computational Enzymology: Enhancing Our Understanding of Enzymatic Catalysis

PubMed Central

Gherib, Rami; Dokainish, Hisham M.; Gauld, James W.

2014-01-01

Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific enzymatic properties. Small or large density functional theory models allow the comparison of a plethora of mechanistic reactive species and divergent catalytic pathways. Molecular docking can model different substrate conformations embedded within enzyme active sites and determine those with optimal binding affinities. Molecular dynamics simulations provide insights into the dynamics and roles of active site components as well as the interactions between substrate and enzymes. Hybrid quantum mechanical/molecular mechanical (QM/MM) can model reactions in active sites while considering steric and electrostatic contributions provided by the surrounding environment. Using previous studies done within our group, on OvoA, EgtB, ThrRS, LuxS and MsrA enzymatic systems, we will review how these methods can be used either independently or cooperatively to get insights into enzymatic catalysis. PMID:24384841

PKCalpha regulates phosphorylation and enzymatic activity of cPLA2 in vitro and in activated human monocytes.

PubMed

Li, Qing; Subbulakshmi, Venkita; Oldfield, Claudine M; Aamir, Rozina; Weyman, Crystal M; Wolfman, Alan; Cathcart, Martha K

2007-02-01

Phospholipases A(2) (PLA(2)) are potent regulators of the inflammatory response. We have observed that Group IV cPLA(2) activity is required for the production of superoxide anion (O(2)(-)) in human monocytes [Li Q., Cathcart M.K. J. Biol. Chem. 272 (4) (1997) 2404-2411.]. We have previously identified PKCalpha as a kinase pathway required for monocyte O(2)(-) production [Li Q., Cathcart M.K. J. Biol. Chem. 269 (26) (1994) 17508-17515.]. We therefore investigated the potential interaction between PKCalpha and cPLA(2) by evaluating the requirement for specific PKC isoenzymes in the process of activating cPLA(2) enzymatic activity and protein phosphorylation upon monocyte activation. We first showed that general PKC inhibitors and antisense oligodeoxyribonucleotides (ODN) to the cPKC group of PKC enzymes inhibited cPLA(2) activity. To distinguish between PKCalpha and PKCbeta isoenzymes in regulating cPLA(2) protein phosphorylation and enzymatic activity, we employed our previously characterized PKCalpha or PKCbeta isoenzyme-specific antisense ODN [Li Q., Subbulakshmi V., Fields A.P., Murray, N.R., Cathcart M.K., J. Biol. Chem. 274 (6) (1999) 3764-3771]. Suppression of PKCalpha expression, but not PKCbeta expression, inhibited cPLA(2) protein phosphorylation and enzymatic activity. Additional studies ruled out a contribution by Erk1/2 to cPLA(2) phosphorylation and activation. We also found that cPLA(2) co-immunoprecipitated with PKCalpha and vice versa. In vitro studies demonstrated that PKCalpha could directly phosphorylate cPLA(2).and enhance enzymatic activity. Finally, we showed that addition of arachidonic acid restored the production of O(2)(-) in monocytes defective in either PKCalpha or cPLA(2) expression. Taken together, our data suggest that PKCalpha, but not PKCbeta, is the predominant cPKC isoenzyme required for cPLA(2) protein phosphorylation and maximal induction of cPLA(2) enzymatic activity upon activation of human monocytes. Our data also support the concept that the requirements for PKCalpha and cPLA(2) in O(2)(-) generation are solely due to their seminal role in generating arachidonic acid.

Non-Enzymatic Detection of Bacterial Genomic DNA Using the Bio-Barcode Assay

PubMed Central

Hill, Haley D.; Vega, Rafael A.; Mirkin, Chad A.

2011-01-01

The detection of bacterial genomic DNA through a non-enzymatic nanomaterials based amplification method, the bio-barcode assay, is reported. The assay utilizes oligonucleotide functionalized magnetic microparticles to capture the target of interest from the sample. A critical step in the new assay involves the use of blocking oligonucleotides during heat denaturation of the double stranded DNA. These blockers bind to specific regions of the target DNA upon cooling, and prevent the duplex DNA from re-hybridizing, which allows the particle probes to bind. Following target isolation using the magnetic particles, oligonucleotide functionalized gold nanoparticles act as target recognition agents. The oligonucleotides on the nanoparticle (barcodes) act as amplification surrogates. The barcodes are then detected using the Scanometric method. The limit of detection for this assay was determined to be 2.5 femtomolar, and this is the first demonstration of a barcode type assay for the detection of double stranded, genomic DNA. PMID:17927207

Enzymatic signal amplification for sensitive detection of intracellular antigens by flow cytometry.

PubMed

Karkmann, U; Radbruch, A; Hölzel, V; Scheffold, A

1999-11-19

Flow cytometry is the method of choice for the analysis of single cells with respect to the expression of specific antigens. Antigens can be detected with specific antibodies either on the cell surface or within the cells, after fixation and permeabilization of the cell membrane. Using conventional fluorochrome-labeled antibodies several thousand antigens are required for clear-cut separation of positive and negative cells. More sensitive reagents, e.g., magnetofluorescent liposomes conjugated to specific antibodies permit the detection of less than 200 molecules per cell but cannot be used for the detection of intracellular antigens. Here, we describe an enzymatic amplification technique (intracellular tyramine-based signal amplification, ITSA) for the sensitive cytometric analysis of intracellular cytokines by immunofluorescence. This approach results in a 10 to 15-fold improvement of the signal-to-noise ratio compared to conventional fluorochrome labeled antibodies and permits the detection of as few as 300-400 intracellular antigens per cell.

Effect of enzymatic mash treatment and storage on phenolic composition, antioxidant activity, and turbidity of cloudy apple juice.

PubMed

Oszmiański, Jan; Wojdylo, Aneta; Kolniak, Joanna

2009-08-12

The effects of different commercial enzymatic mash treatments on yield, turbidity, color, and polyphenolic and sediment of procyanidins content of cloudy apple juice were studied. Addition of pectolytic enzymes to mash treatment had positive effect on the production of cloud apple juices by improving polyphenolic contents, especially procyanidins and juice yields (68.3% in control samples to 77% after Pectinex Yield Mash). As summary of the effect of enzymatic mash treatment, polyphenol contents in cloudy apple juices significantly increased after Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL maceration were applied but no effect was observed after Pectinex Ultra-SPL I Panzym XXL use, compared to the control samples. The content of polymeric procyanidins represented 50-70% of total polyphenols, but in the present study, polymeric procyanidins were significantly lower in juices than in fruits and also affected by enzymatic treatment (Pectinex AFP L-4 and Panzym Yield Mash) compared to the control samples. The enzymatic treatment decreased procyanidin content in most sediment with the exception of Pectinex Smash XXL and Pectinex AFP L-4. Generally in samples that were treated by pectinase, radical scavenging activity of cloudy apple juices was increased compared to the untreated reference samples. The highest radical scavenging activity was associated with Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL enzyme and the lowest activity with Pectinex Ultra SP-L and Pectinex APFL-4. However, in the case of enzymatic mash treatment cloudy apple juices showed instability of turbidity and low viscosity. These results must be ascribed to the much higher hydrolysis of pectin by enzymatic preparation which is responsible for viscosity. During 6 months of storage at 4 degrees C small changes in analyzed parameters of apple juices were observed.

Co-composting of organic fraction of municipal solid waste mixed with different bulking waste: characterization of physicochemical parameters and microbial enzymatic dynamic.

PubMed

Awasthi, Mukesh Kumar; Pandey, Akhilesh Kumar; Bundela, Pushpendra Singh; Khan, Jamaluddin

2015-04-01

The effect of various bulking waste such as wood shaving, agricultural and yard trimming waste combined with organic fraction of municipal solid waste (OFMSW) composting was investigated through assessing their influence on microbial enzymatic activities and quality of finished compost. All three piles of OFMSW with different bulking waste were inoculated with microbial consortium. The results revealed that OFMSW combined with wood shaving and microbial consortium (Phanerochaete chrysosporium, Trichoderma viride and Pseudomonas aeruginosa) were helpful tool to facilitate the enzymatic activity and shortened composting period within 4 weeks. Maximum enzymatic activity were observed in pile 1 and 3 during the first 3 weeks, while in pile 2 relatively very low. But phosphatase activity was relatively higher in all piles until the end of the process. Maturity parameters of compost quality also favored the pile 1 as the best formulation for OFMSW composting. Copyright © 2015 Elsevier Ltd. All rights reserved.

Changes in the amino acid profiles and free radical scavenging activities of Tenebrio molitor larvae following enzymatic hydrolysis.

PubMed

Tang, Yujiao; Debnath, Trishna; Choi, Eun-Ju; Kim, Young Wook; Ryu, Jung Pyo; Jang, Sejin; Chung, Sang Uk; Choi, Young-Jin; Kim, Eun-Kyung

2018-01-01

Tenebrio molitor (T. molitor) larvae provide food at low environmental cost and contribute positively to livelihoods. In this research, we compared the amino acids compositions and antioxidant activities of various extracts of T. molitor to enhance their quality as food. For the comparison, distilled water extracts, enzymatic hydrolysates, and condensed enzymatic hydrolysates of T. molitor larvae were prepared. Their amino acids (AAs) profiles and antioxidant activities, including ferric-reducing antioxidant power, oxygen radical absorption capacity, and DPPH, hydroxyl radical, and hydrogen peroxide radical scavenging properties assay were analyzed. DW extracts had the lowest AAs contents and antioxidant activity compared with enzymatic extracts. Condensed hydrolysates with a combination of alcalase and flavourzyme (C-A+F) exhibited the highest levels of total free AAs (11.1759 g/100 g). C-A+F produced higher total hydrolyzed AAs (32.5292 g/100 g) compared with the other groups. The C-A+F possessed the strongest antioxidant activity. Notably, the antioxidant activities of the hydrolysates and the total hydrolyzed AAs amount were correlated. Taken together, our findings showed that C-A+F was a promising technique for obtaining extracts of T. molitor larvae with antioxidant activity as potential nutritious functional food.

Chemical redox modulated fluorescence of nitrogen-doped graphene quantum dots for probing the activity of alkaline phosphatase.

PubMed

Liu, JingJing; Tang, Duosi; Chen, Zhitao; Yan, Xiaomei; Zhong, Zhou; Kang, Longtian; Yao, Jiannian

2017-08-15

Alkaline phosphatase (ALP) as an essential enzyme plays an important role in clinical diagnoses and biomedical researches. Hence, the development of convenient and sensitivity assay for monitoring ALP is extremely important. In this work, on the basis of chemical redox strategy to modulate the fluorescence of nitrogen-doped graphene quantum dots (NGQDs), a novel label-free fluorescent sensing system for the detection of alkaline phosphatase (ALP) activity has been developed. The fluorescence of NGQDs is firstly quenched by ultrathin cobalt oxyhydroxide (CoOOH) nanosheets, and then restored by ascorbic acid (AA), which can reduce CoOOH to Co 2+ , thus the ALP can be monitored based on the enzymatic hydrolysis of L-ascorbic acid-2-phosphate (AAP) by ALP to generate AA. Quantitative evaluation of ALP activity in a range from 0.1 to 5U/L with the detection limit of 0.07U/L can be realized in this sensing system. Endowed with high sensitivity and selectivity, the proposed assay is capable of detecting ALP in biological system with satisfactory results. Meanwhile, this sensing system can be easily extended to the detection of various AA-involved analytes. Copyright © 2017 Elsevier B.V. All rights reserved.

Biocolloids with ordered urease multilayer shells as enzymatic reactors.

PubMed

Lvov, Y; Caruso, F

2001-09-01

The preparation of biocolloids with organized enzyme-containing multilayer shells for exploitation as colloidal enzymatic nanoreactors is described. Urease multilayers were assembled onto submicrometer-sized polystyrene spheres by the sequential adsorption of urease and polyelectrolyte, in a predetermined order, utilizing electrostatic interactions for layer growth. The catalytic activity of the biocolloids increased proportionally with the number of urease layers deposited on the particles, demonstrating that biocolloid particles with tailored enzymatic activities can be produced. It was further found that precoating the latex spheres with nanoparticles (40-nm silica or 12-nm magnetite) enhanced both the stability (with respect to adsorption) and enzymatic activity of the urease multilayers. The presence of the magnetite nanoparticle coating also provided a magnetic function that allowed the biocolloids to be easily and rapidly separated with a permanent magnet. The fabrication of such colloids opens new avenues for the application of bioparticles and represents a promising route for the creation of complex catalytic particles.

Specific inflammatory response of Anemonia sulcata (Cnidaria) after bacterial injection causes tissue reaction and enzymatic activity alteration.

PubMed

Trapani, M R; Parisi, M G; Parrinello, D; Sanfratello, M A; Benenati, G; Palla, F; Cammarata, M

2016-03-01

The evolution of multicellular organisms was marked by adaptations to protect against pathogens. The mechanisms for discriminating the ''self'' from ''non-self" have evolved into a long history of cellular and molecular strategies, from damage repair to the co-evolution of host-pathogen interactions. We investigated the inflammatory response in Anemonia sulcata (Cnidaria: Anthozoa) following injection of substances that varied in type and dimension, and observed clear, strong and specific reactions, especially after injection of Escherichia coli and Vibrio alginolyticus. Moreover, we analyzed enzymatic activity of protease, phosphatase and esterase, showing how the injection of different bacterial strains alters the expression of these enzymes and suggesting a correlation between the appearance of the inflammatory reaction and the modification of enzymatic activities. Our study shows for the first time, a specific reaction and enzymatic responses following injection of bacteria in a cnidarian. Copyright © 2016 Elsevier Inc. All rights reserved.

CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to Deepwater Horizon Mississippi Canyon 252 oil

USGS Publications Warehouse

Alexander, Courtney R.; Hooper, Michael J.; Cacela, Dave; Smelker, Kim D.; Calvin, Caleshia S.; Dean, Karen M.; Bursian, Steve J.; Cunningham, Fred L.; Hanson-Dorr, Katie C.; Horak, Katherine E.; Isanhart, John P.; Link, Jane E.; Shriner, Susan A.; Godard-Codding, Céline A.J.

2017-01-01

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20 ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5 ml/kg bw oil or 10 ml/kg bw oil, 21 day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10 ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

Protein folding on Biosensor tips: Folding of Maltodextrin glucosidase monitored by its interactions with GroEL

PubMed Central

Pastor, Ashutosh; Singh, Amit K.; Fisher, Mark T.; Chaudhuri, Tapan K.

2016-01-01

Protein folding has been extensively studied for past four decades by employing solution based experiments such as solubility, enzymatic activity, secondary structure analysis, and analytical methods like FRET, NMR and HD exchange. However, for rapid analysis of the folding process, solution based approaches are often plagued with aggregation side reactions resulting in poor yields. In this work we demonstrate that a Bio-Layer Interferometry (BLI) chaperonin detection system can be potentially applied to identify superior refolding conditions for denatured proteins. The degree of immobilized protein folding as a function of time can be detected by monitoring the binding of the high-affinity nucleotide-free form of the chaperonin GroEL. GroEL preferentially interacts with proteins that have hydrophobic surfaces exposed in their unfolded or partially folded form so a decrease in GroEL binding can be correlated with burial of hydrophobic surfaces as folding progresses. The magnitude of GroEL binding to the protein immobilized on Bio-layer interferometry biosensor inversely reflects the extent of protein folding and hydrophobic residue burial. We demonstrate conditions where accelerated folding can be observed for the aggregation prone protein Maltodextrin glucosidase (MalZ). Superior immobilized folding conditions identified on the Bio-layer interferometry biosensor surface were reproduced on Ni-NTA sepharose bead surfaces and resulted in significant improvement in folding yields of released MalZ (measured by enzymatic activity) compared to bulk refolding conditions in solution. PMID:27367928

Comparative Proteomic Analyses of Human Adipose Extracellular Matrices Decellularized Using Alternative Procedures.

PubMed

Thomas-Porch, Caasy; Li, Jie; Zanata, Fabiana; Martin, Elizabeth C; Pashos, Nicholas; Genemaras, Kaylynn; Poche, J Nicholas; Totaro, Nicholas P; Bratton, Melyssa R; Gaupp, Dina; Frazier, Trivia; Wu, Xiying; Ferreira, Lydia Masako; Tian, Weidong; Wang, Guangdi; Bunnell, Bruce A; Flynn, Lauren; Hayes, Daniel; Gimble, Jeffrey M

2018-04-25

Decellularized human adipose tissue has potential clinical utility as a processed biological scaffold for soft tissue cosmesis, grafting and reconstruction. Adipose tissue decellularization has been accomplished using enzymatic-, detergent-, and/or solvent-based methods. To examine the hypothesis that distinct decellularization processes may yield scaffolds with differing compositions, the current study employed mass spectrometry to compare the proteomes of human adipose-derived matrices generated through three independent methods combining enzymatic-, detergent-, and/or solvent-based steps. In addition to protein content, bioscaffolds were evaluated for DNA depletion, ECM composition, and physical structure using optical density, histochemical staining, and scanning electron microscopy (SEM). Mass spectrometry (MS) based proteomic analyses identified 25 proteins (having at least two peptide sequences detected) in the scaffolds generated with an enzymatic approach, 143 with the detergent approach, and 102 with the solvent approach, as compared to 155 detected in unprocessed native human fat. Immunohistochemical detection confirmed the presence of the structural proteins actin, collagen type VI, fibrillin, laminin, and vimentin. Subsequent in vivo analysis of the predominantly enzymatic- and detergent-based decellularized scaffolds following subcutaneous implantation in GFP + transgenic mice demonstrated that the matrices generated with both approaches supported the ingrowth of host-derived adipocyte progenitors and vasculature in a time dependent manner. Together, these results determine that decellularization methods influence the protein composition of adipose tissue-derived bioscaffolds. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

PubMed Central

Lazar, Iulia M.; Deng, Jingren; Smith, Nicole

2016-01-01

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW < 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 ºC. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively. PMID:27078683

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor.

PubMed

Lazar, Iulia M; Deng, Jingren; Smith, Nicole

2016-04-06

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW < 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 ºC. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

Bubble electrodeposition of gold porous nanocorals for the enzymatic and non-enzymatic detection of glucose.

PubMed

Sanzó, Gabriella; Taurino, Irene; Antiochia, Riccarda; Gorton, Lo; Favero, Gabriele; Mazzei, Franco; De Micheli, Giovanni; Carrara, Sandro

2016-12-01

Au nanocorals are grown on gold screen-printed electrodes (SPEs) by using a novel and simple one-step electrodeposition process. Scanning electron microscopy was used for the morphological characterization. The devices were assembled on a three-electrode SPE system, which is flexible and mass producible. The electroactive surface area, determined by cyclic voltammetry in sulphuric acid, was found to be 0.07±0.01cm(2) and 35.3±2.7cm(2) for bare Au and nanocoral Au, respectively. The nanocoral modified SPEs were used to develop an enzymatic glucose biosensor based on H2O2 detection. Au nanocoral electrodes showed a higher sensitivity of 48.3±0.9μA/(mMcm(2)) at +0.45V vs Ag|AgCl compared to a value of 24.6±1.3μA/(mMcm(2)) at +0.70V vs Ag|AgCl obtained with bare Au electrodes. However, the modified electrodes have indeed proven to be extremely powerful for the direct detection of glucose with a non-enzymatic approach. The results confirmed a clear peak observed by using nanocoral Au electrode even in the presence of chloride ions at physiological concentration. Amperometric study carried out at +0.15V vs Ag|AgCl in the presence of 0.12M NaCl showed a linear range for glucose between 0.1 and 13mM. Copyright © 2016. Published by Elsevier B.V.

Identification and characterization of Clonorchis sinensis cathepsin B proteases in the pathogenesis of clonorchiasis.

PubMed

Chen, Wenjun; Ning, Dan; Wang, Xiaoyun; Chen, Tingjin; Lv, Xiaoli; Sun, Jiufeng; Wu, De; Huang, Yan; Xu, Jin; Yu, Xinbing

2015-12-21

Human clonorchiasis is a prevailing food-borne disease caused by Clonorchis sinensis infection. Functional characterizations of key molecules from C. sinensis could facilitate the intervention of C. sinensis associated diseases. In this study, immunolocalization of C. sinensis cathepsin B proteases (CsCBs) in C. sinensis worms was investigated. Four CsCBs were expressed in Pichia pastoris yeast cells. Purified yCsCBs were measured for enzymatic and hydrolase activities in the presence of various host proteins. Cell proliferation, wound-healing and transwell assays were performed to show the effect of CsCBs on human cells. CsCBs were localized in the excretory vesicle, oral sucker and intestinal tract of C. sinensis. Recombinant yCsCBs from yeast showed active enzymatic activity at pH 5.0-5.5 and at 37-42 °C. yCsCBs can degrade various host proteins including human serum albumin, human fibronectin, human hemoglobin and human IgG. CsCBs were detected in liver tissues of mice and cancer patients afflicted with clonorchiasis. Various bioassays collectively demonstrated that CsCBs could promote cell proliferation, migration and invasion of human cancer cells. Our results demonstrated that CsCBs can degrade various human proteins and we proved that the secreted CsCBs are involved in the pathogenesis of clonorchiasis.

Electrodepositable alginate membranes for enzymatic sensors: An amperometric glucose biosensor for whole blood analysis.

PubMed

Márquez, A; Jiménez-Jorquera, C; Domínguez, C; Muñoz-Berbel, X

2017-11-15

Simple and disposable point of care systems are usually the best solution for chronic patients to get a rapid diagnosis in home care context. However, their main drawback relies on the poor reliability derived from the low stability of the bio-recognition elements and low quality of the transducers. In the current work, we study the use of electrodeposited calcium alginate hydrogels as a biocompatible matrix in the development of enzymatic amperometric biosensors for whole blood analysis, to enhance the enzymes stability and to protect the transducer from biofouling. The alginate electrodeposition involves the controlled Ca 2+ release, so the gel thickness can be modulated. In the biosensor, horseradish peroxidase (HRP) and glucose oxidase (GOD) were electrodeposited within the hydrogel and the activity of the bi-enzymatic system was analyzed chronoamperometrically using 3,3',5,5'-Tetramethylbenzidine (TMB) as the mediator. Besides enzyme entrapment, the obtained gels protected the transducer from biofouling, enabling the reuse of the transducer after hydrogel removal and re-electrodeposition. The biosensors showed good analytical characteristics to glucose determination in whole blood samples, discriminating among healthy and hyperglycemic samples, with good sensitivity (- 0.27µAcm -2 mM -1 ), low limit of detection (126µM) and long lineal range (2-12mM). Copyright © 2017. Published by Elsevier B.V.

Fusion proteins useful for producing pinene

DOEpatents

Peralta-Yahya, Pamela P.; Keasling, Jay D

2016-06-28

The present invention provides for a modified host cell comprising a heterologous pinene synthase (PS), or enzymatically active fragment or variant thereof, and optionally a geranyl pyrophosphate synthase (GPPS), or enzymatically active fragment or variant thereof, or a fusion protein comprising: (a) a PS and (b) a GPPS linked by a linker.

Antagonists' impact on enzymatic response in wilt infected cotton plants

USDA-ARS?s Scientific Manuscript database

A number of PR-proteins possess enzymatic activity. As such, these proteins maybe indicators of defensive response of plants. Thus, we have conducted a comparative analysis of beta-1,3-glucanase, peroxidase and xylanase activity in cotton plants to determine how these enzymes are affected by the pat...

In vitro antioxidant, lipoxygenase and xanthine oxidase inhibitory activities of fractions from Cienfuegosia digitata Cav., Sida alba L. and Sida acuta Burn f. (Malvaceae).

PubMed

Konaté, K; Souza, A; Coulibaly, A Y; Meda, N T R; Kiendrebeogo, M; Lamien-Meda, A; Millogo-Rasolodimby, J; Lamidi, M; Nacoulma, O G

2010-11-15

In this study polyphenol content, antioxidant activity, lipoxygenase (LOX) and Xanthine Oxidase (XO) inhibitory effects of n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of aqueous acetone extracts from S. alba L., S. acuta Burn f and Cienfuegosia digitata Cav. were investigated. The total phenolics, flavonoids, flavonols and total tannins were determined by spectrophotometric methods using Folin-ciocalteu, AlCl3 reagents and tannic acid, respectively. The antioxidant potential was evaluated using three methods: inhibition of free radical 2,2-diphenyl-1-picrylhydramzyl (DPPH), ABTS radical cation decolorization assay and Iron (III) to iron (II) reduction activity (FRAP). For enzymatic activity, lipoxygenase and xanthine oxidase inhibitory activities were used. This study shows a relationship between polyphenol contents, antioxidant and enzymatic activities. Present results showed that ethyl acetate and dichloromethane fractions elicit the highest polyphenol content, antioxidant and enzymatic activities.

ROLE OF THE SARCOPLASMIC RETICULUM IN GLYCOGEN METABOLISM

PubMed Central

Wanson, Jean-Claude; Drochmans, Pierre

1972-01-01

Sarcoplasmic vesicles and β-glycogen particles 30–40 mµ in diameter were isolated from perfused rabbit skeletal muscle by the differential precipitation-centrifugation method. This microsomal fraction was subjected to zonal centrifugation on buffered sucrose gradients, in a B XIV Anderson type rotor, for 15 hr at 45,000 rpm in order to separate the two cytoplasmic organelles. Zonal profiles of absorbance at 280 mµ, proteins, glycogen, and enzymatic activities (phosphorylase b kinase, phosphorylase b, and glycogen synthetase) were performed. Whereas the entire synthetase activity was found combined with the glycogen particles, 39% of phosphorylase and 53% of phosphorylase b kinase activities, present in the microsomal fraction, were recovered in the purified vesicular fraction (d = 1.175). This latter fraction consists of vesicles, derived from the sarcoplasmic reticulum, and of small particles 10–20 mµ in diameter attached to the outer surface of the membranes. These particles disappear after α-amylase treatment. Incubation of the sarcovesicular fraction with 14C-labeled glucose-1-phosphate confirms the localization of a polysaccharide synthesis at the level of the membranes. "Flash activation" of phosphorylase b, i.e. Ca "activation" of phosphorylase kinase followed by a conversion of phosphorylase b into a, was demonstrated in the purified sarcovesicular fraction. Moreover, the active enzymatic sites were detected on the membranes by electron microscopy. The presence of binding sites between the membranes of the sarcoplasmic vesicles and a glycogen-enzyme complex suggests that this association plays a role in the glycogenolysis during muscle contraction. PMID:5040859

Analysis of phosphorothionate pesticides using a chloroperoxidase pretreatment and acetylcholinesterase biosensor detection.

PubMed

Roepcke, Clarisse B S; Muench, Susanne B; Schulze, Holger; Bachmann, Till T; Bachmann, Till; Schmid, Rolf D; Hauer, Bernhard

2010-08-11

Acetylcholinesterase (AChE) is responsible for the hydrolysis of acetylcholine in the nervous system. It is inhibited by organophosphate and carbamate pesticides. However, this enzyme is only slightly inhibited by organophosphorothionates, which makes the detection of these pesticides analytically very difficult. A new enzymatic method for the activation and detection of phosphorothionates was developed with the capability to be used directly in food samples without the need of laborious solvent extraction steps. Chloroperoxidase (CPO) from Caldariomyces fumago was combined with tert-butyl hydroperoxide and two halides. Chlorpyrifos and triazophos were completely oxidized. Fenitrothion, methidathion and parathion methyl showed conversion rates between 54 and 61%. Furthermore, the oxidized solution was submitted to an AChE biosensor assay. Chlorpyrifos spiked in organic orange juice was oxidized, where its oxon product was detected in concentrations down to 5 microg/L (final concentration food sample: 25 microg/L). The complete duration of the method takes about 2 h.

Decomposition rate and enzymatic activity of composted municipal waste and poultry manure in the soil in a biofuel crops field.

PubMed

Cordovil, Cláudia Marques-Dos-Santos; de Varennes, Amarilis; Pinto, Renata Machado Dos Santos; Alves, Tiago Filipe; Mendes, Pedro; Sampaio, Sílvio César

2017-05-01

Biofuel crops are gaining importance because of the need to replace non-renewable sources. Also, due to the increasing amounts of wastes generated, there is the need to recycle them to the soil, both to fertilize crops and to improve soil physical properties through organic matter increase and microbiological changes in the rhizosphere. We therefore studied the influence of six biofuel crops (elephant grass, giant cane, sugarcane, blue gum, black cottonwood, willow) on the decomposition rate and enzymatic activity of composted municipal solid waste and poultry manure. Organic amendments were incubated in the field (litterbag method), buried near each plant or bare soil. Biomass decrease and dehydrogenase, urease and acid phosphatase level in amendments was monitored over a 180-day period. Soil under the litterbags was analysed for the same enzymatic activity and organic matter fractions (last sampling). After 365 days, a fractionation of organic matter was carried out in both amendments and soil under the litterbags. For compost, willow and sugarcane generally led to the greatest enzymatic activity, at the end of the experiment. For manure, dehydrogenase activity decreased sharply with time, the smallest value near sugarcane, while phosphatase and urease generally presented the highest values, at the beginning or after 90 days' incubation. Clustering showed that plant species could be grouped based on biomass and enzymes measured over time. Plant species influenced the decomposition rate and enzymatic activities of the organic amendments. Overall, mineralization of both amendments was associated with a greater urease activity in soils. Dehydrogenase activity in manure was closely associated with urease activity. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Degalactosylated/desialylated human serum containing GcMAF induces macrophage phagocytic activity and in vivo antitumor activity.

PubMed

Kuchiike, Daisuke; Uto, Yoshihiro; Mukai, Hirotaka; Ishiyama, Noriko; Abe, Chiaki; Tanaka, Daichi; Kawai, Tomohito; Kubo, Kentaro; Mette, Martin; Inui, Toshio; Endo, Yoshio; Hori, Hitoshi

2013-07-01

The group-specific component protein-derived macrophage-activating factor (GcMAF) has various biological activities, such as macrophage activation and antitumor activity. Clinical trials of GcMAF have been carried out for metastatic breast cancer, prostate cancer, and metastatic colorectal cancer. In this study, despite the complicated purification process of GcMAF, we used enzymatically-treated human serum containing GcMAF with a considerable macrophage-stimulating activity and antitumor activity. We detected GcMAF in degalactosylated/desialylated human serum by western blotting using an anti-human Gc globulin antibody, and Helix pomatia agglutinin lectin. We also found that GcMAF-containing human serum significantly enhanced the phagocytic activity of mouse peritoneal macrophages and extended the survival time of mice bearing Ehrlich ascites tumors. We demonstrated that GcMAF-containing human serum can be used as a potential macrophage activator for cancer immunotherapy.

CD73 promotes proliferation and migration of human cervical cancer cells independent of its enzyme activity.

PubMed

Gao, Zhao-Wei; Wang, Hui-Ping; Lin, Fang; Wang, Xi; Long, Min; Zhang, Hui-Zhong; Dong, Ke

2017-02-15

CD73 has both enzymatic and non-enzymatic functions in cells. As a nucleotidase, CD73 plays its enzymatic function by catalyzing the hydrolysis of AMP into adenosine and phosphate. In addition to this, accumulating data have shown that CD73 is a key regulatory molecule involved in cancer growth and metastasis, but this non-enzymatic function of CD73 in cervical cancer cells has not been well studied. CD73 was overexpressed by pcDNA-NT5E expression vector transfection in Hela and SiHa cells. Cell's proliferation and migration were evaluated by MTT and scratch healing assay. The CD73 specific antagonist -APCP was used to inhibit CD73 enzymatic activity. And the effect of APCP on CD73 activity was determined by high performance liquid chromatography (HPLC). Expression level was assessed by qRT-PCR and western blotting. In the present study, we used Hela and SiHa cell lines to evaluate the effects of CD73 on cervical cancer cells proliferation and migration, and further explore the potential regulating mechanisms. Our data showed that CD73 overexpression significantly promoted cervical cancer cells proliferation and migration, and this promotive effect was not reverted by blocking CD73 enzymatic activity, both in Hela and SiHa cells. On the other hand, our data also showed that high concentration of adenosine inhibited Hela and SiHa cells proliferation and migration. These results demonstrated that the promotive effect of CD73 on cervical cancer cells proliferation and migration in vitro was independent from its enzymatic activity (i.e. production of adenosine). Furthermore, the expressions of EGFR, VEGF and Akt were significantly increased in CD73 overexpression Hela and SiHa cells. Our data suggested that CD73 might promote proliferation and migration via potentiating EGFR/Akt and VEGF/Akt pathway, which was independent of CD73 enzyme activity. These data provide a novel insight into the regulating function of CD73 in cancer cells and suggest that CD73 may be promising therapeutic target in cervical cancer.

Beneficial contribution of the arbuscular mycorrhizal fungus, Rhizophagus irregularis, in the protection of Medicago truncatula roots against benzo[a]pyrene toxicity.

PubMed

Lenoir, Ingrid; Fontaine, Joël; Tisserant, Benoît; Laruelle, Frédéric; Lounès-Hadj Sahraoui, Anissa

2017-07-01

Arbuscular mycorrhizal fungi are able to improve plant establishment in polluted soils but little is known about the genes involved in the plant protection against pollutant toxicity by mycorrhization, in particular in the presence of polycyclic aromatic hydrocarbons (PAH). The present work aims at studying in both symbiotic partners, Medicago truncatula and Rhizophagus irregularis: (i) expression of genes putatively involved in PAH tolerance (MtSOD, MtPOX, MtAPX, MtGST, MtTFIIS, and MtTdp1α), (ii) activities of antioxidant (SOD, POX) and detoxification (GST) enzymes, and (iii) H 2 O 2 and the heavy PAH, benzo[a]pyrene (B[a]P) accumulation. In the presence of B[a]P, whereas induction of the enzymatic activities was detected in R. irregularis and non-mycorrhizal roots as well as upregulation of the gene expressions in the non-mycorrhizal roots, downregulation of the gene expressions and decrease of enzyme activities were observed in mycorrhizal roots. Moreover, B[a]P increased H 2 O 2 production in non-mycorrhizal roots and in R. irregularis but not in mycorrhizal roots. In addition, a lower B[a]P bioaccumulation in mycorrhizal roots was measured in comparison with non-mycorrhizal roots. Being less affected by pollutant toxicity, mycorrhizal roots did not activate any defense mechanism either at the gene expression regulation level or at the enzymatic level.

Encapsulation of alpha-amylase into starch-based biomaterials: an enzymatic approach to tailor their degradation rate.

PubMed

Azevedo, Helena S; Reis, Rui L

2009-10-01

This paper reports the effect of alpha-amylase encapsulation on the degradation rate of a starch-based biomaterial. The encapsulation method consisted in mixing a thermostable alpha-amylase with a blend of corn starch and polycaprolactone (SPCL), which were processed by compression moulding to produce circular disks. The presence of water was avoided to keep the water activity low and consequently to minimize the enzyme activity during the encapsulation process. No degradation of the starch matrix occurred during processing and storage (the encapsulated enzyme remained inactive due to the absence of water), since no significant amount of reducing sugars was detected in solution. After the encapsulation process, the released enzyme activity from the SPCL disks after 28days was found to be 40% comparatively to the free enzyme (unprocessed). Degradation studies on SPCL disks, with alpha-amylase encapsulated or free in solution, showed no significant differences on the degradation behaviour between both conditions. This indicates that alpha-amylase enzyme was successfully encapsulated with almost full retention of its enzymatic activity and the encapsulation of alpha-amylase clearly accelerates the degradation rate of the SPCL disks, when compared with the enzyme-free disks. The results obtained in this work show that degradation kinetics of the starch polymer can be controlled by the amount of encapsulated alpha-amylase into the matrix.

Electroactive chitosan nanoparticles for the detection of single-nucleotide polymorphisms using peptide nucleic acids.

PubMed

Kerman, Kagan; Saito, Masato; Tamiya, Eiichi

2008-08-01

Here we report an electrochemical biosensor that would allow for simple and rapid analysis of nucleic acids in combination with nuclease activity on nucleic acids and electroactive bionanoparticles. The detection of single-nucleotide polymorphisms (SNPs) using PNA probes takes advantage of the significant structural and physicochemical differences between the full hybrids and SNPs in PNA/DNA and DNA/DNA duplexes. Ferrocene-conjugated chitosan nanoparticles (Chi-Fc) were used as the electroactive indicator of hybridization. Chi-Fc had no affinity towards the neutral PNA probe immobilized on a gold electrode (AuE) surface. When the PNA probe on the electrode surface hybridized with a full-complementary target DNA, Chi-Fc electrostatically attached to the negatively-charged phosphate backbone of DNA on the surface and gave rise to a high electrochemical oxidation signal from ferrocene at approximately 0.30 V. Exposing the surface to a single-stranded DNA specific nuclease, Nuclease S1, was found to be very effective for removing the nonspecifically adsorbed SNP DNA. An SNP in the target DNA to PNA made it susceptible to the enzymatic digestion. After the enzymatic digestion and subsequent exposure to Chi-Fc, the presence of SNPs was determined by monitoring the changes in the electrical current response of Chi-Fc. The method provided a detection limit of 1 fM (S/N = 3) for the target DNA oligonucleotide. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism (GMO) in standard Roundup Ready soybean samples. PNA-mediated PCR amplification of real DNA samples was performed to detect SNPs related to alcohol dehydrogenase (ALDH). Chitosan nanoparticles are promising biomaterials for various analytical and pharmaceutical applications.

Selective enzymatic cleavage and labeling for sensitive capillary electrophoresis laser-induced fluorescence analysis of oxidized DNA bases.

PubMed

Li, Cuiping; Wang, Hailin

2015-08-07

Oxidatively generated DNA damage is considered to be a significant contributing factor to cancer, aging, and age-related human diseases. It is important to detect oxidatively generated DNA damage to understand and clinically diagnosis diseases caused by oxidative damage. In this study, using selective enzymatic cleavage and quantum dot (QD) labeling, we developed a novel capillary electrophoresis-laser induced fluorescence method for the sensitive detection of oxidized DNA bases. First, oxidized DNA bases are recognized and removed by one DNA base excision repair glycosylase, leaving apurinic and apyrimidinic sites (AP sites) at the oxidized positions. The AP sites are further excised by the AP nicking activity of the chosen glycosylase, generating a nucleotide gap with 5'- and 3'- phosphate groups. After dephosphorylation with one alkaline phosphatase, a biotinylated ddNTP is introduced into the nucleotide space within the DNA strand by DNA polymerase I. The biotin-tagged DNA is further labeled with a QD-streptavidin conjugate via non-covalent interactions. The DNA-bound QD is well-separated from excess DNA-unbound QD by highly efficient capillary electrophoresis and is sensitively detected by online coupled laser-induced fluorescence analysis. Using this method, we can assess the trace levels of oxidized DNA bases induced by the Fenton reaction and UV irradiation. Interestingly, the use of the formamidopyrimidine glycosylase (FPG) protein and endonuclease VIII enables the detection of oxidized purine and pyrimidine bases, respectively. Using the synthesized standard DNA, the approach has low limits of detection of 1.1×10(-19)mol in mass and 2.9pM in concentration. Copyright © 2015 Elsevier B.V. All rights reserved.

Changes on digestive enzymes during initial ontogeny in the three-spot cichlid Cichlasoma trimaculatum.

PubMed

Toledo-Solís, F J; Uscanga-Martínez, A; Guerrero-Zárate, R; Márquez-Couturier, G; Martínez-García, R; Camarillo-Coop, S; Perales-García, N; Rodríguez-Valencia, W; Gómez-Gómez, M A; Álvarez-González, C A

2015-02-01

A study was performed in order to understand the development of digestive enzymes during initial ontogeny of Cichlasoma trimaculatum, for which the activity of acidic and alkaline proteases, lipases, amylases and phosphatases was determined by means of biochemical and electrophoretic analysis. Our results showed that the activity of alkaline proteases, trypsin and chymotrypsin is present from day 6 after hatching (dah) during exogenous feeding with Artemia nauplii. The activities of carboxypeptidase A and leucine aminopeptidase are present from the first days, increasing at 6 dah and reaching their maximum activity at 9 dah while acid protease activity started at 9 dah. Furthermore, the lipase activity is detected on 6 dah and keeps increasing and decreasing on 17 dah. Amylase activity is detected on 3 dah, presenting fluctuations until 45 dah, where it reaches its maximum activity. Acid and alkaline phosphatases are detected from 3 dah and reach a maximum activity between 13 and 19 dah. The SDS-PAGE electrophoresis revealed six types of bands in the alkaline proteases, with molecular weight between 113.4 and 20.4 kDa. First three bands appear on 6 dah, but it is until 11 dah when all isoforms appear. Based on these results, it is considered that this species completes its digestive enzymatic machinery from day 9 after hatching, therefore is recommended to perform the transition from live feed to inert feed at 15 dah.

Enzymatic generation of galactose-rich oligosaccharides/oligomers from potato rhamnogalacturonan I pectic polysaccharides.

PubMed

Khodaei, Nastaran; Karboune, Salwa

2016-04-15

Potato pulp by-product rich in galactan-rich rhamnogalacturonan I (RG I) was investigated as a new source of oligosaccharides with potential prebiotic properties. The efficiency of selected monocomponent enzymes and multi-enzymatic preparations to generate oligosaccharides/oligomers from potato RG I was evaluated. These overall results of yield were dependent on the activity profile of the multi-enzymatic preparations. Highest oligo-RG I yield of 93.9% was achieved using multi-enzymatic preparation (Depol 670L) with higher hydrolytic activity toward side chains of RG I as compared to its backbone. Main oligo-RG I products were oligosaccharides with DP of 2-12 (79.8-100%), while the oligomers with DP of 13-70 comprised smaller proportion (0.0-20.2%). Galactose (58.9-91.2%, w/w) was the main monosaccharide of oligo-RG I, while arabinose represented 0.0-12.1%. An understanding of the relationship between the activity profile of multi-enzymatic preparations and the yield/DP of oligo-RG I was achieved. This is expected to provide the capability to generate galacto- and galacto(arabino) oligosaccharides and their corresponding oligomers from an abundant by-product. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effect of endogenous and exogenous enzymatic treatment of green vanilla beans on extraction of vanillin and main aromatic compounds.

PubMed

Pardío, Violeta T; Flores, Argel; López, Karla M; Martínez, David I; Márquez, Ofelia; Waliszewski, Krzysztof N

2018-06-01

Endogenous and exogenous enzymatic hydrolysis carried out to obtain vanilla extracts with higher concentrations of vanillin using green vanilla beans. Sequences initiated with freezing of green vanilla beans at - 1 °C for 24 h, followed by endogenous hydrolysis under optimal β-glucosidase activity at 4.2 and 35 °C for 96 h, exogenous hydrolysis with Crystalzyme PML-MX at pH 5.0 and 40 °C for 72 h, and ethanol extraction at 40% (v v -1 ) for 30 days. In the proposed method, 200 g of fresh green vanilla beans with 84% moisture (32 g dry base) were used to obtain a liter of single fold vanilla extract. This method allowed the release of 82.57% of the theoretically available vanillin from its precursor glucovanillin with 5.78 g 100 g -1 green vanilla beans (dry base). Vanillic acid, p -hydroxybenzaldehyde and vanillyl alcohol were also released and found in commercial and enzymatic extracts. Glucovanillin was detected in commercial and traditional extracts but was absent in enzymatic extracts, indicating incomplete hydrolysis during the curing process. An in vitro assay was conducted to determine if the presence of peroxidase during hydrolysis might affect overall vanillin concentration. Results showed that POD can use vanillin as a substrate under conditions similar to those in which hydrolysis was conducted (pH 5.0 and 50 °C), possibly explaining why vanillin concentration was not complete at the end of the process.

Determination of glucose and ethanol after enzymatic hydrolysis and fermentation of biomass using Raman spectroscopy.

PubMed

Shih, Chien-Ju; Smith, Emily A

2009-10-27

Raman spectroscopy has been used for the quantitative determination of the conversion efficiency at each step in the production of ethanol from biomass. The method requires little sample preparation; therefore, it is suitable for screening large numbers of biomass samples and reaction conditions in a complex sample matrix. Dilute acid or ammonia-pretreated corn stover was used as a model biomass for these studies. Ammonia pretreatment was suitable for subsequent measurements with Raman spectroscopy, but dilute acid-pretreated corn stover generated a large background signal that surpassed the Raman signal. The background signal is attributed to lignin, which remains in the plant tissue after dilute acid pretreatment. A commercial enzyme mixture was used for the enzymatic hydrolysis of corn stover, and glucose levels were measured with a dispersive 785 nm Raman spectrometer. The glucose detection limit in hydrolysis liquor by Raman spectroscopy was 8 g L(-1). The mean hydrolysis efficiency for three replicate measurements obtained with Raman spectroscopy (86+/-4%) was compared to the result obtained using an enzymatic reaction with UV-vis spectrophotometry detection (78+/-8%). The results indicate good accuracy, as determined using a Student's t-test, and better precision for the Raman spectroscopy measurement relative to the enzymatic detection assay. The detection of glucose in hydrolysis broth by Raman spectroscopy showed no spectral interference, provided the sample was filtered to remove insoluble cellulose prior to analysis. The hydrolysate was further subjected to fermentation to yield ethanol. The detection limit for ethanol in fermentation broth by Raman spectroscopy was found to be 6 g L(-1). Comparison of the fermentation efficiencies measured by Raman spectroscopy (80+/-10%) and gas chromatography-mass spectrometry (87+/-9%) were statistically the same. The work demonstrates the utility of Raman spectroscopy for screening the entire conversion process to generate lignocellulosic ethanol.

Co3O4 based non-enzymatic glucose sensor with high sensitivity and reliable stability derived from hollow hierarchical architecture.

PubMed

Tian, Liangliang; He, Gege; Cai, Yanhua; Wu, Shenping; Su, Yongyao; Yan, Hengqing; Yang, Cong; Chen, Yanling; Li, Lu

2018-02-16

Inspired by kinetics, the design of hollow hierarchical electrocatalysts through large-scale integration of building blocks is recognized as an effective approach to the achievement of superior electrocatalytic performance. In this work, a hollow, hierarchical Co 3 O 4 architecture (Co 3 O 4 HHA) was constructed using a coordinated etching and precipitation (CEP) method followed by calcination. The resulting Co 3 O 4 HHA electrode exhibited excellent electrocatalytic activity in terms of high sensitivity (839.3 μA mM -1 cm -2 ) and reliable stability in glucose detection. The high sensitivity could be attributed to the large specific surface area (SSA), ample unimpeded penetration diffusion paths and high electron transfer rate originating from the unique two-dimensional (2D) sheet-like character and hollow porous architecture. The hollow hierarchical structure also affords sufficient interspace for accommodation of volume change and structural strain, resulting in enhanced stability. The results indicate that Co 3 O 4 HHA could have potential for application in the design of non-enzymatic glucose sensors, and that the construction of hollow hierarchical architecture provides an efficient way to design highly active, stable electrocatalysts.

Co3O4 based non-enzymatic glucose sensor with high sensitivity and reliable stability derived from hollow hierarchical architecture

NASA Astrophysics Data System (ADS)

Tian, Liangliang; He, Gege; Cai, Yanhua; Wu, Shenping; Su, Yongyao; Yan, Hengqing; Yang, Cong; Chen, Yanling; Li, Lu

2018-02-01

Inspired by kinetics, the design of hollow hierarchical electrocatalysts through large-scale integration of building blocks is recognized as an effective approach to the achievement of superior electrocatalytic performance. In this work, a hollow, hierarchical Co3O4 architecture (Co3O4 HHA) was constructed using a coordinated etching and precipitation (CEP) method followed by calcination. The resulting Co3O4 HHA electrode exhibited excellent electrocatalytic activity in terms of high sensitivity (839.3 μA mM-1 cm-2) and reliable stability in glucose detection. The high sensitivity could be attributed to the large specific surface area (SSA), ample unimpeded penetration diffusion paths and high electron transfer rate originating from the unique two-dimensional (2D) sheet-like character and hollow porous architecture. The hollow hierarchical structure also affords sufficient interspace for accommodation of volume change and structural strain, resulting in enhanced stability. The results indicate that Co3O4 HHA could have potential for application in the design of non-enzymatic glucose sensors, and that the construction of hollow hierarchical architecture provides an efficient way to design highly active, stable electrocatalysts.

Characterization of selected cellulolytic activities of multi-enzymatic complex system from Penicillium funiculosum.

PubMed

Karboune, Salwa; Geraert, Pierre-André; Kermasha, Selim

2008-02-13

The presence of endo-1,4-beta-D-glucanase, cellobiohydrolase, and beta-glucosidase activities in a multi-enzymatic complex system from Penicillium funiculosum was investigated. The interesting feature of these enzymes is their synergistic action for the hydrolysis of the native cellulose into glucose units. Both endo-1,4-beta-D-glucanase and cellobiohydrolase showed broader pH activity profiles, with pH optima of 4.0 and 4.0-5.0, respectively. However, beta-glucosidase activity showed a narrow pH-activity profile, with an optimum pH of 4.5. The different cellulolytic activities were stable in the acidic pH range of 2.5-6.0 and showed a similar optimal temperature of 60 degrees C. Although beta-glucosidase has shown a close catalytic efficiency as that of endo-1,4-beta-D-glucanase, its thermal stability was lower. However, the thermal stability profile of cellobiohydrolase was close to that of endo-1,4-beta-D-glucanase. The results also revealed the presence of high levels of endo-1,3-1,4-beta-D-glucanase, endo-1,3-beta- d-glucanase, and pectinase activities in the multi-enzymatic cellulolytic complex system. Moreover, the investigated multi-enzymatic complex system was effective in degrading the nonstarch polysaccharides of soybean meal.

Effect of structural modifications of ganglioside GM2 on intra-molecular carbohydrate-to-carbohydrate interaction and enzymatic susceptibility.

PubMed

Li, Yu-Teh; Li, Su-Chen; Kiso, Makoto; Ishida, Hideharu; Mauri, Laura; Raimondi, Laura; Bernardi, Anna; Sonnino, Sandro

2008-03-01

The effect of inter-molecular carbohydrate-to-carbohydrate interaction on basic cell biological processes has been well documented and appreciated. In contrast, very little is known about the intra-molecular carbohydrate-to-carbohydrate interaction. The presence of an interaction between the GalNAc and the Neu5Ac in GM2 detected by NMR spectroscopy represents a well-defined intra-molecular carbohydrate-to-carbohydrate interaction. This intriguing interaction is responsible for the GM2-epitope, GalNAcbeta1-->4(Neu5Acalpha2-->3)Gal-, to exhibit a rigid and compact conformation. We hypothesized that this compact conformation may be the cause for both the GalNAc and the Neu5Ac in GM2 to be refractory to enzymatic hydrolysis and the GM2 activator protein is able to interact with the compact trisaccharide GM2-epitope, rendering the GalNAc and the Neu5Ac accessible to beta-hexosaminidase A and sialidase. We have used a series of structurally modified GM2 to study the effect of modifications of sugar chains on the conformation and enzymatic susceptibility of this ganglioside. Our hypothesis was borne out by the fact that when the GalNAcbeta1-->4Gal linkage in GM2 was converted to the GalNAcbeta1-->6Gal, both the GalNAc and the Neu5Ac became susceptible to beta-hexosaminidase A and sialidase, respectively, without GM2 activator protein. We hope our work will engender interest in identifying other intra-molecular carbohydrate-to-carbohydrate interactions in glycoconjugates.

Non-Enzymatic Glucose Biosensor Based on CuO-Decorated CeO2 Nanoparticles

PubMed Central

Guan, Panpan; Li, Yongjian; Zhang, Jie; Li, Wei

2016-01-01

Copper oxide (CuO)-decorated cerium oxide (CeO2) nanoparticles were synthesized and used to detect glucose non-enzymatically. The morphological characteristics and structure of the nanoparticles were characterized through transmission electron microscopy, X-ray photoelectron spectroscopy, and X-ray diffraction. The sensor responses of electrodes to glucose were investigated via an electrochemical method. The CuO/CeO2 nanocomposite exhibited a reasonably good sensitivity of 2.77 μA mM−1cm−2, an estimated detection limit of 10 μA, and a good anti-interference ability. The sensor was also fairly stable under ambient conditions. PMID:28335287

Monitoring the enzymatic conversion of urea to ammonium by conventional or microchip capillary electrophoresis with contactless conductivity detection.

PubMed

Schuchert-Shi, Aiping; Hauser, Peter C

2008-05-15

Capillary electrophoresis with contactless conductivity detection was used to directly quantify the ammonium produced in the enzymatic conversion of urea with urease. This allowed the characterization of the reaction without having to use more elaborate indirect optical methods for quantification. The maximum rate of reaction, V(max), was determined as 5.1 mmol x mL(-1) x min(-1), and the Michaelis-Menten constant, K(m), was determined as 16 mM. Furthermore, the method was successfully applied to the determination of urea in clinical samples of human blood by using a conventional capillary and a microchip device.

pH sensing characteristics and biosensing application of solution-gated reduced graphene oxide field-effect transistors.

PubMed

Sohn, Il-Yung; Kim, Duck-Jin; Jung, Jin-Heak; Yoon, Ok Ja; Thanh, Tien Nguyen; Quang, Trung Tran; Lee, Nae-Eung

2013-07-15

Solution-gated reduced graphene oxide field-effect transistors (R-GO FETs) were investigated for pH sensing and biochemical sensing applications. A channel of a networked R-GO film formed by self-assembly was incorporated as a sensing layer into a solution-gated FET structure for pH sensing and the detection of acetylcholine (Ach), which is a neurotransmitter in the nerve system, through enzymatic reactions. The fabricated R-GO FET was sensitive to protons (H(+)) with a pH sensitivity of 29 mV/pH in terms of the shift of the charge neutrality point (CNP), which is attributed to changes in the surface potential caused by the interaction of protons with OH surface functional groups present on the R-GO surface. The R-GO FET immobilized with acetylcholinesterase (AchE) was used to detect Ach in the concentration range of 0.1-10mM by sensing protons generated during the enzymatic reactions. The results indicate that R-GO FETs provide the capability to detect protons, demonstrating their applicability as a biosensing device for enzymatic reactions. Copyright © 2013 Elsevier B.V. All rights reserved.

Quantitative imaging of disease signatures through radioactive decay signal conversion

PubMed Central

Thorek, Daniel LJ; Ogirala, Anuja; Beattie, Bradley J; Grimm, Jan

2013-01-01

In the era of personalized medicine there is an urgent need for in vivo techniques able to sensitively detect and quantify molecular activities. Sensitive imaging of gamma rays is widely used, but radioactive decay is a physical constant and signal is independent of biological interactions. Here we introduce a framework of novel targeted and activatable probes excited by a nuclear decay-derived signal to identify and measure molecular signatures of disease. This was accomplished utilizing Cerenkov luminescence (CL), the light produced by β-emitting radionuclides such as clinical positron emission tomography (PET) tracers. Disease markers were detected using nanoparticles to produce secondary Cerenkov-induced fluorescence. This approach reduces background signal compared to conventional fluorescence imaging. In addition to information from a PET scan, we demonstrate novel medical utility by quantitatively determining prognostically relevant enzymatic activity. This technique can be applied to monitor other markers and facilitates a shift towards activatable nuclear medicine agents. PMID:24013701

9-Hydroxyprostaglandin dehydrogenase activity in the adult rat kidney. Regional distribution and sub-fractionation.

PubMed

Asciak, C P; Domazet, Z

1975-02-20

1. Catabolism of prostaglandin F2alpha in the adult rat kidney takes place by the following sequence of enzymatic steps: (1) 15-hydroxyprostaglandin dehydrogenase; (2) prostaglandin delta13-reductase; and (3) 9-hydroxyprostaglandin dehydrogenase. 2. 9-Hydroxyprostaglandin dehydrogenase activity was highest in the cortex with lesser amounts in the medulla and negligible activity detected in the papilla. A similar distribution was observed for 15-hydroxyprostaglandin dehydrogenase and prostaglandin delta13-reductase. 3. Most of the 9-hydroxyprostaglandin dehydrogenase activity in the homogenate was found in the high-speed supernatant as also observed for 15-hydroxyprostaglandin dehydrogenase and prostaglandin delta13-reductase. 4. These observations indicate that the rat kidney contains an abundance of prostaglandin-catabolising enzymes which favour formation of metabolites of the E-type.

Cobalt oxide nanosheets wrapped onto nickel foam for non-enzymatic detection of glucose

NASA Astrophysics Data System (ADS)

Meng, Shangjun; Wu, Meiyan; Wang, Qian; Dai, Ziyang; Si, Weili; Huang, Wei; Dong, Xiaochen

2016-08-01

Ultra-sensitive and highly selective detection of glucose is essential for the clinical diagnosis of diabetes. In this paper, an ultra-sensitive glucose sensor was successfully fabricated based on cobalt oxide (Co3O4) nanosheets directly grown on nickel foam through a simple hydrothermal method. Characterizations indicated that the Co3O4 nanosheets are completely and uniformly wrapped onto the surface of nickel foam to form a three-dimensional heterostructure. The resulting self-standing electrochemical electrode presents a high performance for the non-enzymatic detection of glucose, including short response time (<10 s), ultra-sensitivity (12.97 mA mM-1 cm-2), excellent selectivity and low detection limit (0.058 μM, S/N = 3). These results indicate that Co3O4 nanosheets wrapped onto nickel foam are a low-cost, practical, and high performance electrochemical electrode for bio sensing.

Purification and characteristics of an inducible by polycyclic aromatic hydrocarbons NADP(+)-dependent naphthalenediol dehydrogenase (NDD) in Mucor circinelloides YR-1.

PubMed

Camacho-Morales, Reyna Lucero; Zazueta-Novoa, Vanesa; Casillas, Juana Lizbeth González; Ballesteros, Elizabeth Aranda; Bote, Juan Antonio Ocampo; Zazueta-Sandoval, Roberto

2014-05-01

We detected NADP(+)-dependent dihydrodiol dehydrogenase (DD) activity in a cell-free extract from Mucor circinelloides YR-1, after high-speed centrifugation. We analyzed the enzymatic activity in the cytosolic fraction by zymograms, as described previously, and eight different DD activity bands were revealed. Five constitutive DD activities (DD1-5) were present when glucose was used as carbon source and three inducible activities (NDD, PDD1 and PDD2) when aromatic hydrocarbon compounds were used. NDD activity was induced all of the aromatic hydrocarbon compounds. The highest DD activity inducer was naphthalene and the lowest was pyrene. One of the enzymes showed higher activity with cis-naphthalene-diol rather than with trans-nahthalenediol as a substrate. We purified this particular enzyme to homogeneity and found that it had an isoelectric point of 4.6. The molecular weight for the native protein was 197.4kDa and 49.03±0.5kDa for the monomer that conforms it, suggesting a homotetrameric structure for the complete enzyme. Polyclonal antibodies were raised against it and obtained. NDD activity was almost totally inhibited when antibodies were used at low concentrations, and in native immunoblots only one band, which corresponds to the activity band detected in the zymograms, could be detected. In denaturing PAGE immunoblots only one band was detected. This band corresponds to the purified protein band of 49kDa detected in SDS-PAGE gels. The other two inducible enzymes PDD1 and PDD2 were present only when phenanthrene was used as sole carbon source in the culture media. Copyright © 2014 Elsevier Inc. All rights reserved.

Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines

PubMed Central

Godlewska, Marlena; Krasuska, Wanda

2018-01-01

Thyroid peroxidase (TPO) is an enzyme and autoantigen expressed in thyroid and breast tissues. Thyroid TPO undergoes a complex maturation process however, nothing is known about post-translational modifications of breast-expressed TPO. In this study, we have investigated the biochemical properties of TPO expressed in normal and cancerous human breast tissues, and the maturation process and antigenicity of TPO present in a panel of human breast tissue-derived cell lines. We found that the molecular weight of breast TPO was slightly lower than that of thyroid TPO due to decreased glycosylation and as suggest results of Western blot also shorter amino acid chain. Breast TPO exhibit enzymatic activity and isoelectric point comparable to that of thyroid TPO. The biochemical properties of TPO expressed in mammary cell lines and normal thyrocytes are similar regarding glycan content, molecular weight and isoelectric point. However, no peroxidase activity and dimer formation was detected in any of these cell lines since the majority of TPO protein was localized in the cytoplasmic compartment, and the TPO expression at the cell surface was too low to detect its enzymatic activity. Lactoperoxidase, a protein highly homologous to TPO expressed also in breast tissues, does not influence the obtained data. TPO expressed in the cell lines was recognized by a broad panel of TPO-specific antibodies. Although some differences in biochemical properties between thyroid and breast TPO were observed, they do not seem to be critical for the overall three-dimensional structure. This conclusion is supported by the fact that TPO expressed in breast tissues and cell lines reacts well with conformation-sensitive antibodies. Taking into account a close resemblance between both proteins, especially high antigenicity, future studies should investigate the potential immunotherapies directed against breast-expressed TPO and its specific epitopes. PMID:29513734

Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines.

PubMed

Godlewska, Marlena; Krasuska, Wanda; Czarnocka, Barbara

2018-01-01

Thyroid peroxidase (TPO) is an enzyme and autoantigen expressed in thyroid and breast tissues. Thyroid TPO undergoes a complex maturation process however, nothing is known about post-translational modifications of breast-expressed TPO. In this study, we have investigated the biochemical properties of TPO expressed in normal and cancerous human breast tissues, and the maturation process and antigenicity of TPO present in a panel of human breast tissue-derived cell lines. We found that the molecular weight of breast TPO was slightly lower than that of thyroid TPO due to decreased glycosylation and as suggest results of Western blot also shorter amino acid chain. Breast TPO exhibit enzymatic activity and isoelectric point comparable to that of thyroid TPO. The biochemical properties of TPO expressed in mammary cell lines and normal thyrocytes are similar regarding glycan content, molecular weight and isoelectric point. However, no peroxidase activity and dimer formation was detected in any of these cell lines since the majority of TPO protein was localized in the cytoplasmic compartment, and the TPO expression at the cell surface was too low to detect its enzymatic activity. Lactoperoxidase, a protein highly homologous to TPO expressed also in breast tissues, does not influence the obtained data. TPO expressed in the cell lines was recognized by a broad panel of TPO-specific antibodies. Although some differences in biochemical properties between thyroid and breast TPO were observed, they do not seem to be critical for the overall three-dimensional structure. This conclusion is supported by the fact that TPO expressed in breast tissues and cell lines reacts well with conformation-sensitive antibodies. Taking into account a close resemblance between both proteins, especially high antigenicity, future studies should investigate the potential immunotherapies directed against breast-expressed TPO and its specific epitopes.

Herbivore species identity and composition affect soil enzymatic activity through altered plant composition in a coastal tallgrass prairie

USDA-ARS?s Scientific Manuscript database

Although single species of herbivores are known to affect soil microbial communities, the effects of herbivore species identity and functional composition on soil microbes is unknown. We tested the effects of single species of orthopterans and multiple species combinations on soil enzymatic activity...

Parasites of the deep-sea fish Mora moro (Risso, 1810) from the NW Mediterranean Sea and relationship with fish diet and enzymatic biomarkers

NASA Astrophysics Data System (ADS)

Dallarés, Sara; Constenla, María; Padrós, Francesc; Cartes, Joan E.; Solé, Montse; Carrassón, Maite

2014-10-01

Specimens of Mora moro were collected in two seasons and three localities of the Balearic Sea (NW Mediterranean Sea) and parasitological, dietary (to prey-species level), enzymatic and histological data were obtained, alongside with environmental information (T, S, O2). The relationships among fish parasite load, condition indices, diet, enzymatic activity of muscular acetylcholinesterase (AChE) and lactate dehydrogenase (LDH), intensity of splenic melano-macrophage centres (MMC) and hepatic granulomas were tested. M. moro showed a rich and abundant parasite fauna, and was a new host record for 17 out of the 18 different endoparasite taxa found. Significant differences were detected among locality-season groups, in turn related to different environmental variables, for Anisakidae gen. sp., Anisakis Type II and Tetraphyllidea fam. gen. sp.; thus, they are proposed as potentially useful as biological tags for geographical discrimination of M. moro in the NW Mediterranean Sea. Detailed relationships were found between parasite taxa and prey ingested (e.g. Anisakidae gen. sp. related with meso-bathypelagic crustaceans; Anisakis Type I with benthopelagic squids). Most parasites were linked to samples with highest levels of near-bottom O2, which is consistent with direct relationships found between near-bottom O2 and zooplankton biomass in the Balearic Basin. Total parasite abundance and the abundance of Tetraphyllidea fam. gen. sp. showed a significant relationship with the activity of AChE and the abundance of Anisakis Type II with LDH. AChE was associated with hepatosomatic index (HSI) and condition factor (K); LDH with gonadosomatic index (GSI), K and fish total length (TL). LDH activity showed differences among sampling groups. Splenic MMC and hepatic granulomas were not associated with fish parasite load. A positive relationship was found between MMC area and fish TL and LDH activity.

[Comparative characteristics of microbial proteases by the level of hydrolysis of protein substrates].

PubMed

Rimareva, L V; Overchenko, M B; Serba, E M; Trifonova, V V

1997-01-01

Screening of enzyme preparations displaying a maximum proteolytic activity at pH 4.0-5.5 and effecting deep proteolysis of plant proteins was performed. Amyloprotooryzin prepared from Aspergillus oryzae 387 containing a complex of proteolytic enzymes was the most effective. The amino acid composition of the hydrolysates obtained was studied. Amyloprotooryzin increased the contents of amino acids by 108-227%, depending on the substrate used. The enzymatic complex of amyloprotooryzin was studied; in addition, proteases, alpha-amylase, exo-beta-glucanase, and xylanase were detected in the complex.

Detection of glutamate and acetylcholine with organic electrochemical transistors based on conducting polymer/platinum nanoparticle composites.

PubMed

Kergoat, Loïg; Piro, Benoît; Simon, Daniel T; Pham, Minh-Chau; Noël, Vincent; Berggren, Magnus

2014-08-27

The aim of the study is to open a new scope for organic electrochemical transistors based on PEDOT:PSS, a material blend known for its stability and reliability. These devices can leverage molecular electrocatalysis by incorporating small amounts of nano-catalyst during the transistor manufacturing (spin coating). This methodology is very simple to implement using the know-how of nanochemistry and results in efficient enzymatic activity transduction, in this case utilizing choline oxidase and glutamate oxidase. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inulin hydrolysis by inulinase immobilized covalently on magnetic nanoparticles prepared with wheat gluten hydrolysates.

PubMed

Torabizadeh, Homa; Mahmoudi, Asieh

2018-03-01

Inulinase can produce a high amount of fructose syrup from inulin in a one-step enzymatic process. Inulinase from Aspergillus niger was immobilized covalently on Fe 3 O 4 magnetic nanoparticles functionalized with wheat gluten hydrolysates (WGHs). Wheat gluten was enzymatically hydrolyzed by two endopeptidases Alcalase and Neutrase and related nanoparticles were prepared by desolvation method. Magnetite nanoparticles were coated with WGHs nanoparticles and then inulinase was immobilized onto it using glutaraldehyde as crosslinking agent. Parallel studies employing differential scanning calorimetry and field emmision scanning electron microscopy were carried out to observe functional and structural variations in free inulinase during immobilization. Optimum temperature of immobilized inulinase was increased, while, pH and K m values were decreased compared to free enzyme. Overall, a 12.3 folds rise was detected in enzyme half-life value after Immobilization at 75 °C and enzyme preserved 70% of its initial activity after 12 cycles of hydrolysis with 75% of enzyme loading.

Manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium is enzymatically active and accumulates to high levels in transgenic maize seed.

PubMed

Clough, Richard C; Pappu, Kameshwari; Thompson, Kevin; Beifuss, Katherine; Lane, Jeff; Delaney, Donna E; Harkey, Robin; Drees, Carol; Howard, John A; Hood, Elizabeth E

2006-01-01

Manganese peroxidase (MnP) has been implicated in lignin degradation and thus has potential applications in pulp and paper bleaching, enzymatic remediation and the textile industry. Transgenic plants are an emerging protein expression platform that offer many advantages over traditional systems, in particular their potential for large-scale industrial enzyme production. Several plant expression vectors were created to evaluate the accumulation of MnP from the wood-rot fungus Phanerochaete chrysosporium in maize seed. We showed that cell wall targeting yielded full-length MnP, whereas cytoplasmic localization resulted in multiple truncated peroxidase polypeptides as detected by immunoblot analysis. In addition, the use of a seed-preferred promoter dramatically increased the expression levels and reduced the negative effects on plant health. Multiple independent transgenic lines were backcrossed with elite inbred corn lines for several generations with the maintenance of high-level expression, indicating genetic stability of the transgene.

A Computational Methodology to Overcome the Challenges Associated With the Search for Specific Enzyme Targets to Develop Drugs Against Leishmania major

PubMed Central

Catharina, Larissa; Lima, Carlyle Ribeiro; Franca, Alexander; Guimarães, Ana Carolina Ramos; Alves-Ferreira, Marcelo; Tuffery, Pierre; Derreumaux, Philippe; Carels, Nicolas

2017-01-01

We present an approach for detecting enzymes that are specific of Leishmania major compared with Homo sapiens and provide targets that may assist research in drug development. This approach is based on traditional techniques of sequence homology comparison by similarity search and Markov modeling; it integrates the characterization of enzymatic functionality, secondary and tertiary protein structures, protein domain architecture, and metabolic environment. From 67 enzymes represented by 42 enzymatic activities classified by AnEnPi (Analogous Enzymes Pipeline) as specific for L major compared with H sapiens, only 40 (23 Enzyme Commission [EC] numbers) could actually be considered as strictly specific of L major and 27 enzymes (19 EC numbers) were disregarded for having ambiguous homologies or analogies with H sapiens. Among the 40 strictly specific enzymes, we identified sterol 24-C-methyltransferase, pyruvate phosphate dikinase, trypanothione synthetase, and RNA-editing ligase as 4 essential enzymes for L major that may serve as targets for drug development. PMID:28638238

A Computational Methodology to Overcome the Challenges Associated With the Search for Specific Enzyme Targets to Develop Drugs Against Leishmania major.

PubMed

Catharina, Larissa; Lima, Carlyle Ribeiro; Franca, Alexander; Guimarães, Ana Carolina Ramos; Alves-Ferreira, Marcelo; Tuffery, Pierre; Derreumaux, Philippe; Carels, Nicolas

2017-01-01

We present an approach for detecting enzymes that are specific of Leishmania major compared with Homo sapiens and provide targets that may assist research in drug development. This approach is based on traditional techniques of sequence homology comparison by similarity search and Markov modeling; it integrates the characterization of enzymatic functionality, secondary and tertiary protein structures, protein domain architecture, and metabolic environment. From 67 enzymes represented by 42 enzymatic activities classified by AnEnPi (Analogous Enzymes Pipeline) as specific for L major compared with H sapiens , only 40 (23 Enzyme Commission [EC] numbers) could actually be considered as strictly specific of L major and 27 enzymes (19 EC numbers) were disregarded for having ambiguous homologies or analogies with H sapiens . Among the 40 strictly specific enzymes, we identified sterol 24-C-methyltransferase, pyruvate phosphate dikinase, trypanothione synthetase, and RNA-editing ligase as 4 essential enzymes for L major that may serve as targets for drug development.

Mediation of donor–acceptor distance in an enzymatic methyl transfer reaction

PubMed Central

Zhang, Jianyu; Kulik, Heather J.; Martinez, Todd J.; Klinman, Judith P.

2015-01-01

Enzymatic methyl transfer, catalyzed by catechol-O-methyltransferase (COMT), is investigated using binding isotope effects (BIEs), time-resolved fluorescence lifetimes, Stokes shifts, and extended graphics processing unit (GPU)-based quantum mechanics/molecular mechanics (QM/MM) approaches. The WT enzyme is compared with mutants at Tyr68, a conserved residue that is located behind the reactive sulfur of cofactor. Small (>1) BIEs are observed for an S-adenosylmethionine (AdoMet)-binary and abortive ternary complex containing 8-hydroxyquinoline, and contrast with previously reported inverse (<1) kinetic isotope effects (KIEs). Extended GPU-based computational studies of a ternary complex containing catecholate show a clear trend in ground state structures, from noncanonical bond lengths for WT toward solution values with mutants. Structural and dynamical differences that are sensitive to Tyr68 have also been detected using time-resolved Stokes shift measurements and molecular dynamics. These experimental and computational results are discussed in the context of active site compaction that requires an ionization of substrate within the enzyme ternary complex. PMID:26080432

Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 degrees C.

PubMed

Faustini, Massimo; Torre, Maria Luisa; Stacchezzini, Simona; Norberti, Roberta; Consiglio, Anna Lange; Porcelli, Franca; Conte, Ubaldo; Munari, Eleonora; Russo, Vincenzo; Vigo, Daniele

2004-01-01

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.

Membrane Phospholipid Augments Cytochrome P4501a Enzymatic Activity by Modulating Structural Conformation during Detoxification of Xenobiotics

PubMed Central

Ghosh, Manik C.; Ray, Arun K.

2013-01-01

Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment. PMID:23469105

Membrane phospholipid augments cytochrome P4501a enzymatic activity by modulating structural conformation during detoxification of xenobiotics.

PubMed

Ghosh, Manik C; Ray, Arun K

2013-01-01

Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment.

Hydrogen sulfide production from cysteine and homocysteine by periodontal and oral bacteria.

PubMed

Yoshida, Akihiro; Yoshimura, Mamiko; Ohara, Naoya; Yoshimura, Shigeru; Nagashima, Shiori; Takehara, Tadamichi; Nakayama, Koji

2009-11-01

Hydrogen sulfide is one of the predominant volatile sulfur compounds (VSCs) produced by oral bacteria. This study developed and evaluated a system for detecting hydrogen sulfide production by oral bacteria. L-methionine-alpha-deamino-gamma-mercaptomethane-lyase (METase) and beta carbon-sulfur (beta C-S) lyase were used to degrade homocysteine and cysteine, respectively, to produce hydrogen sulfide. Enzymatic reactions resulting in hydrogen sulfide production were assayed by reaction with bismuth trichloride, which forms a black precipitate when mixed with hydrogen sulfide. The enzymatic activities of various oral bacteria that result in hydrogen sulfide production and the capacity of bacteria from periodontal sites to form hydrogen sulfide in reaction mixtures containing L-cysteine or DL-homocysteine were assayed. With L-cysteine as the substrate, Streptococcus anginosus FW73 produced the most hydrogen sulfide, whereas Porphyromonas gingivalis American Type Culture Collection (ATCC) 33277 and W83 and Fusobacterium nucleatum ATCC 10953 produced approximately 35% of the amount produced by the P. gingivalis strains. Finally, the hydrogen sulfide found in subgingival plaque was analyzed. Using bismuth trichloride, the hydrogen sulfide produced by oral bacteria was visually detectable as a black precipitate. Hydrogen sulfide production by oral bacteria was easily analyzed using bismuth trichloride. However, further innovation is required for practical use.

Development of an extraction method for the quantification of lignans in espresso coffee by using HPLC-MS/MS triple quadrupole.

PubMed

Angeloni, Simone; Navarini, Luciano; Sagratini, Gianni; Torregiani, Elisabetta; Vittori, Sauro; Caprioli, Giovanni

2018-06-20

Lignans are polyphenolic compounds that are considered phytoestrogens for their plant origins and they possess different biological activities. Three different extraction methods, i.e., 'dilute and shoot', acidic hydrolysis and enzymatic digestion, have been compared for extracting lignans (secoisolariciresinol, SECO, matairesinol, MAT and lariciresinol LARI) from espresso coffee (EC) by using high performance liquid chromatography (HPLC) tandem mass spectrometry (MS/MS). The best recovery values (SECO: 97%, LARI: 98% and MAT: 93%) were obtained by using enzymatic hydrolysis with Clara-Diastase at 10% (w/v), keeping the sample at 37 °C for 3 hours. For this reason, this method has been chosen and then applied to quantify lignans in nine different EC samples from five different geographical origins (Brazil, Colombia, El Salvador, Ethiopia and India). SECO and LARI were found in all EC samples from 27.9 to 52.0 μg l -1 and from 5.3 to 27.8 μg l -1 , respectively, contrary to MAT that it was not possible to detect it in each type of coffee. This method confirms the high specificity and sensitivity of MS/MS system for detecting bioactives in complex matrix such as coffee. This article is protected by copyright. All rights reserved.

Bio-conversion of apple pomace into ethanol and acetic acid: Enzymatic hydrolysis and fermentation.

PubMed

Parmar, Indu; Rupasinghe, H P Vasantha

2013-02-01

Enzymatic hydrolysis of cellulose present in apple pomace was investigated using process variables such as enzyme activity of commercial cellulase, pectinase and β-glucosidase, temperature, pH, time, pre-treatments and end product separation. The interaction of enzyme activity, temperature, pH and time had a significant effect (P<0.05) on release of glucose. Optimal conditions of enzymatic saccharification were: enzyme activity of cellulase, 43units; pectinase, 183units; β-glucosidase, 41units/g dry matter (DM); temperature, 40°C; pH 4.0 and time, 24h. The sugars were fermented using Saccharomyces cerevisae yielding 19.0g ethanol/100g DM. Further bio-conversion using Acetobacter aceti resulted in the production of acetic acid at a concentration of 61.4g/100g DM. The present study demonstrates an improved process of enzymatic hydrolysis of apple pomace to yield sugars and concomitant bioconversion to produce ethanol and acetic acid. Copyright © 2012 Elsevier Ltd. All rights reserved.

PROTEIN ADDUCTS AS BIOMAKERS OF EXPOSURE TO ORGANOPHOSPHORUS COMPOUNDS

PubMed Central

Marsillach, Judit; Costa, Lucio G.; Furlong, Clement E.

2013-01-01

Exposure to organophosphorus (OP) compounds can lead to serious neurological damage or death. Following bioactivation by the liver cytochromes P450, the OP metabolites produced are potent inhibitors of serine active-site enzymes including esterases, proteases and lipases. OPs may form adducts on other cellular proteins. Blood cholinesterases (ChEs) have long served as biomarkers of OP exposure in humans. However, the enzymatic assays used for biomonitoring OP exposures have several drawbacks. A more useful approach will focus on multiple biomarkers and avoid problems with the enzymatic activity assays. OP inhibitory effects result from a covalent bond with the active-site serine of the target enzymes. The serine OP adducts become irreversible following a process referred to as aging where one alkyl group dissociates over variable lengths of time depending on the OP adduct. The OP-adducted enzyme then remains in circulation until it is degraded, allowing for a longer window of detection compared with direct analysis of OPs or their metabolites. Mass spectrometry (MS) provides a very sensitive method for identification of post-translational protein modifications. MS analyses of the percentage adduction of the active-site serine of biomarker proteins such as ChEs will eliminate the need for basal activity levels of the individual and will provide for a more accurate determination of OP exposure. MS analysis of biomarker proteins also provides information about the OP that has caused inhibition. Other useful biomarker proteins include other serine hydrolases, albumin, tubulin and transferrin. PMID:23261756

Molecular cloning, expression and enzymatic characterization of glutathione S-transferase from Antarctic sea-ice bacteria Pseudoalteromonas sp. ANT506.

PubMed

Shi, Yonglei; Wang, Quanfu; Hou, Yanhua; Hong, Yanyan; Han, Xiao; Yi, Jiali; Qu, Junjie; Lu, Yi

2014-01-01

A glutathione S-transferase (GST) gene from Antarctic sea-ice bacteria Pseudoalteromonas sp. ANT506 (namely PsGST), was cloned and expressed in Escherichia coli. The open reading frame of PsGST comprised 654 bp encoding a protein of 217 amino acids with a calculated molecular size of 24.3 kDa. The rPsGST possesses the conserved amino acid defining the binding sites of glutathione (G-site) and substrate binding pocket (H-site) in GST N_3 family. PsGST was expressed in E. coli and the recombinant PsGST (rPsGST) was purified by Ni-affinity chromatography with a high specific activity of 74.21 U/mg. The purified rPsGST showed maximum activity at 40 °C and exhibited 14.2% activity at 0 °C. It was completely inactivated at 50 °C for 40 min. These results indicated that rPsGST was a typical cold active GST with low thermostability. The enzyme was little affected by H2O2 and Triton X-100, and 50.2% of the remaining activity was detected in the presence of high salt concentrations (2M NaCl). The enzymatic Km values for CDNB and GSH was 0.22 mM and 1.01 mM, respectively. These specific enzyme properties may be related to the survival environment of Antarctic sea ice bacteria. Copyright © 2013 Elsevier GmbH. All rights reserved.

Mitochondrial dysfunction is involved in the toxic activity of boric acid against Saprolegnia.

PubMed

Ali, Shimaa E; Thoen, Even; Evensen, Øystein; Wiik-Nielsen, Jannicke; Gamil, Amr A A; Skaar, Ida

2014-01-01

There has been a significant increase in the incidence of Saprolegnia infections over the past decades, especially after the banning of malachite green. Very often these infections are associated with high economic losses in salmonid farms and hatcheries. The use of boric acid to control the disease has been investigated recently both under in vitro and in vivo conditions, however its possible mode of action against fish pathogenic Saprolegnia is not known. In this study, we have explored the transformation in Saprolegnia spores/hyphae after exposure to boric acid (1 g/L) over a period 4-24 h post treatment. Using transmission electron microscopy (TEM), early changes in Saprolegnia spores were detected. Mitochondrial degeneration was the most obvious sign observed following 4 h treatment in about 20% of randomly selected spores. We also investigated the effect of the treatment on nuclear division, mitochondrial activity and function using confocal laser scanning microscopy (CLSM). Fluorescence microscopy was also used to test the effect of treatment on mitochondrial membrane potential and formation of reactive oxygen species. Additionally, the viability and proliferation of treated spores that correlated to mitochondrial enzymatic activity were tested using an MTS assay. All obtained data pointed towards changes in the mitochondrial structure, membrane potential and enzymatic activity following treatment. We have found that boric acid has no effect on the integrity of membranes of Saprolegnia spores at concentrations tested. It is therefore likely that mitochondrial dysfunction is involved in the toxic activity of boric acid against Saprolegnia spp.

A new insight into the physiological role of bile salt hydrolase among intestinal bacteria from the genus Bifidobacterium.

PubMed

Jarocki, Piotr; Podleśny, Marcin; Glibowski, Paweł; Targoński, Zdzisław

2014-01-01

This study analyzes the occurrence of bile salt hydrolase in fourteen strains belonging to the genus Bifidobacterium. Deconjugation activity was detected using a plate test, two-step enzymatic reaction and activity staining on a native polyacrylamide gel. Subsequently, bile salt hydrolases from B. pseudocatenulatum and B. longum subsp. suis were purified using a two-step chromatographic procedure. Biochemical characterization of the bile salt hydrolases showed that the purified enzymes hydrolyzed all of the six major human bile salts under the pH and temperature conditions commonly found in the human gastrointestinal tract. Next, the dynamic rheometry was applied to monitor the gelation process of deoxycholic acid under different conditions. The results showed that bile acids displayed aqueous media gelating properties. Finally, gel-forming abilities of bifidobacteria exhibiting bile salt hydrolase activity were analyzed. Our investigations have demonstrated that the release of deconjugated bile acids led to the gelation phenomenon of the enzymatic reaction solution containing purified BSH. The presented results suggest that bile salt hydrolase activity commonly found among intestinal microbiota increases hydrogel-forming abilities of certain bile salts. To our knowledge, this is the first report showing that bile salt hydrolase activity among Bifidobacterium is directly connected with the gelation process of bile salts. In our opinion, if such a phenomenon occurs in physiological conditions of human gut, it may improve bacterial ability to colonize the gastrointestinal tract and their survival in this specific ecological niche.

Unraveling the Enzymatic Activity of Oxygenated Carbon Nanotubes and Their Application in the Treatment of Bacterial Infections.

PubMed

Wang, Huan; Li, Penghui; Yu, Dongqin; Zhang, Yan; Wang, Zhenzhen; Liu, Chaoqun; Qiu, Hao; Liu, Zhen; Ren, Jinsong; Qu, Xiaogang

2018-05-17

Carbon nanotubes (CNTs) and their derivatives have emerged as a series of efficient biocatalysts to mimic the function of natural enzymes in recent years. However, the unsatisfiable enzymatic efficiency usually limits their practical usage ranging from materials science to biotechnology. Here, for the first time, we present the synthesis of several oxygenated-group-enriched carbon nanotubes (o-CNTs) via a facile but green approach, as well as their usage as high-performance peroxidase mimics for biocatalytic reaction. Exhaustive characterizations of the enzymatic activity of o-CNTs have been provided by exploring the accurate effect of various oxygenated groups on their surface including carbonyl, carboxyl, and hydroxyl groups. Because of the "competitive inhibition" effect among all of these oxygenated groups, the catalytic efficiency of o-CNTs is significantly enhanced by weakening the presence of noncatalytic sites. Furthermore, the admirable enzymatic activity of these o-CNTs has been successfully applied in the treatment of bacterial infections, and the results of both in vitro and in vivo nanozyme-mediated bacterial clearance clearly demonstrate the feasibility of o-CNTs as robust peroxidase mimics to effectively decrease the bacterial viability under physiological conditions. We believe that the present study will not only facilitate the construction of novel efficient nanozymes by rationally adjusting the degree of the "competitive inhibition" effect, but also broaden the biological usage of o-CNT-based nanomaterials via their satisfactory enzymatic activity.

Bio-nanogate controlled enzymatic reaction for virus sensing.

PubMed

Wang, Ronghui; Xu, Lizhou; Li, Yanbin

2015-05-15

The objective of this study was to develop an aptamer-based bifunctional bio-nanogate, which could selectively respond to target molecules, and control enzymatic reaction for electrochemical measurements. It was successfully applied for sensitive, selective, rapid, quantitative, and label-free detection of avian influenza viruses (AIV) H5N1. A nanoporous gold film with pore size of ~20 nm was prepared by a metallic corrosion method, and the purity was checked by energy-dispersive X-ray spectroscopy (EDS) study. To improve the performance of the bio-nanogate biosensor, its main analytical parameters were studied and optimized. We demonstrated that the developed bio-nanogate was capable of controlling enzymatic reaction for AIV H5N1 sensing within 1h with a detection limit of 2(-9)HAU (hemagglutination units). The enzymatic reaction was able to cause significant current change due to the presence of target AIV. A linear relationship was found in the virus titer range of 2(-10)-2(2)HAU. No interference was observed from non-target AIV subtypes such as H1N1, H2N2, H4N8 and H7N2. The developed approach could be adopted for sensing of other viruses. Copyright © 2014 Elsevier B.V. All rights reserved.

Mapping of Functional Domains of the Lipid Kinase Phosphatidylinositol 4-Kinase Type III Alpha Involved in Enzymatic Activity and Hepatitis C Virus Replication

PubMed Central

Harak, Christian; Radujkovic, Danijela; Taveneau, Cyntia; Reiss, Simon; Klein, Rahel; Bressanelli, Stéphane

2014-01-01

ABSTRACT The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an endoplasmic reticulum (ER)-resident enzyme that synthesizes phosphatidylinositol 4-phosphate (PI4P). PI4KIIIα is an essential host factor for hepatitis C virus (HCV) replication. Interaction with HCV nonstructural protein 5A (NS5A) leads to kinase activation and accumulation of PI4P at intracellular membranes. In this study, we investigated the structural requirements of PI4KIIIα in HCV replication and enzymatic activity. Therefore, we analyzed PI4KIIIα mutants for subcellular localization, reconstitution of HCV replication in PI4KIIIα knockdown cell lines, PI4P induction in HCV-positive cells, and lipid kinase activity in vitro. All mutants still interacted with NS5A and localized in a manner similar to that of the full-length enzyme, suggesting multiple regions of PI4KIIIα are involved in NS5A interaction and subcellular localization. Interestingly, the N-terminal 1,152 amino acids were dispensable for HCV replication, PI4P induction, and enzymatic function, whereas further N-terminal or C-terminal deletions were deleterious, thereby defining the minimal PI4KIIIα core enzyme at a size of ca. 108 kDa. Additional deletion of predicted functional motifs within the C-terminal half of PI4KIIIα also were detrimental for enzymatic activity and for the ability of PI4KIIIα to rescue HCV replication, with the exception of a proposed nuclear localization signal, suggesting that the entire C-terminal half of PI4KIIIα is involved in the formation of a minimal enzymatic core. This view was supported by structural modeling of the PI4KIIIα C terminus, suggesting a catalytic center formed by an N- and C-terminal lobe and an armadillo-fold motif, which is preceded by three distinct alpha-helical domains probably involved in regulation of enzymatic activity. IMPORTANCE The lipid kinase PI4KIIIα is of central importance for cellular phosphatidylinositol metabolism and is a key host cell factor of hepatitis C virus replication. However, little is known so far about the structure of this 240-kDa protein and the functional importance of specific subdomains regarding lipid kinase activity and viral replication. This work focuses on the phenotypic analysis of distinct PI4KIIIα mutants in different biochemical and cell-based assays and develops a structural model of the C-terminal enzymatic core. The results shed light on the structural and functional requirements of enzymatic activity and the determinants required for HCV replication. PMID:24920820

Development of conductometric biosensors based on alkaline phosphatases for the water quality control

NASA Astrophysics Data System (ADS)

Berezhetskyy, A.

2008-09-01

Researches are focused on the elaboration of enzymatic microconductometric device for heavy metal ions detection in water solutions. The manuscript includes a general introduction, the first chapter contains bibliographic review, the second chapter described the fundamentals of conductometric transducers, the third chapter examining the possibility to create and to optimize conductometric biosensor based on bovine alkaline phosphatase for heavy metals ions detection, the fourth chapter devoted to creation and optimization of conductometric biosensor based on alkaline phosphatase active microalgae and sol gel technology, the last chapter described application of the proposed algal biosensor for measurements of heavy metal ions toxicity of waste water, general conclusions stating the progresses achieved in the field of environmental monitoring

Identification of Residues That Affect Oligomerization and/or Enzymatic Activity of Influenza Virus H5N1 Neuraminidase Proteins

PubMed Central

Dai, Meiling; Guo, Hongbo; Dortmans, Jos C. F. M.; Dekkers, Jojanneke; Nordholm, Johan; Daniels, Robert; van Kuppeveld, Frank J. M.; de Vries, Erik

2016-01-01

ABSTRACT Influenza A virus (IAV) attachment to and release from sialoside receptors is determined by the balance between hemagglutinin (HA) and neuraminidase (NA). The molecular determinants that mediate the specificity and activity of NA are still poorly understood. In this study, we aimed to design the optimal recombinant soluble NA protein to identify residues that affect NA enzymatic activity. To this end, recombinant soluble versions of four different NA proteins from H5N1 viruses were compared with their full-length counterparts. The soluble NA ectodomains were fused to three commonly used tetramerization domains. Our results indicate that the particular oligomerization domain used does not affect the Km value but may affect the specific enzymatic activity. This particularly holds true when the stalk domain is included and for NA ectodomains that display a low intrinsic ability to oligomerize. NA ectodomains extended with a Tetrabrachion domain, which forms a nearly parallel four-helix bundle, better mimicked the enzymatic properties of full-length proteins than when other coiled-coil tetramerization domains were used, which probably distort the stalk domain. Comparison of different NA proteins and mutagenic analysis of recombinant soluble versions thereof resulted in the identification of several residues that affected oligomerization of the NA head domain (position 95) and therefore the specific activity or sialic acid binding affinity (Km value; positions 252 and 347). This study demonstrates the potential of using recombinant soluble NA proteins to reveal determinants of NA assembly and enzymatic activity. IMPORTANCE The IAV HA and NA glycoproteins are important determinants of host tropism and pathogenicity. However, NA is relatively understudied compared to HA. Analysis of soluble versions of these glycoproteins is an attractive way to study their activities, as they are easily purified from cell culture media and applied in downstream assays. In the present study, we analyzed the enzymatic activity of different NA ectodomains with three commonly used tetramerization domains and compared them with full-length NA proteins. By performing a mutagenic analysis, we identified several residues that affected NA assembly, activity, and/or substrate binding. In addition, our results indicate that the design of the recombinant soluble NA protein, including the particular tetramerization domain, is an important determinant for maintaining the enzymatic properties within the head domain. NA ectodomains extended with a Tetrabrachion domain better mimicked the full-length proteins than when the other tetramerization domains were used. PMID:27512075

Enzymatic hydrolysis of oleuropein from Olea europea (olive) leaf extract and antioxidant activities.

PubMed

Yuan, Jiao-Jiao; Wang, Cheng-Zhang; Ye, Jian-Zhong; Tao, Ran; Zhang, Yu-Si

2015-02-11

Oleuropein (OE), the main polyphenol in olive leaf extract, is likely to decompose into hydroxytyrosol (HT) and elenolic acid under the action of light, acid, base, high temperature. In the enzymatic process, the content of OE in olive leaf extract and enzyme are key factors that affect the yield of HT. A selective enzyme was screened from among 10 enzymes with a high OE degradation rate. A single factor (pH, temperature, time, enzyme quantity) optimization process and a Box-Behnken design were studied for the enzymatic hydrolysis of 81.04% OE olive leaf extract. Additionally, enzymatic hydrolysis results with different substrates (38.6% and 81.04% OE) were compared and the DPPH antioxidant properties were also evaluated. The result showed that the performance of hydrolysis treatments was best using hemicellulase as a bio-catalyst, and the high purity of OE in olive extract was beneficial to biotransform OE into HT. The optimal enzymatic conditions for achieving a maximal yield of HT content obtained by the regression were as follows: pH 5, temperature 55 °C and enzyme quantity 55 mg. The experimental result was 11.31% ± 0.15%, and the degradation rate of OE was 98.54%. From the present investigation of the antioxidant activity determined by the DPPH method, the phenol content and radical scavenging effect were both decreased after enzymatic hydrolysis by hemicellulase. However, a high antioxidant activity of the ethyl acetate extract enzymatic hydrolysate (IC50 = 41.82 μg/mL) was demonstated. The results presented in this work suggested that hemicellulase has promising and attractive properties for industrial production of HT, and indicated that HT might be a valuable biological component for use in pharmaceutical products and functional foods.

Continuous monitoring of enzymatic activity within native electrophoresis gels: Application to mitochondrial oxidative phosphorylation complexes

PubMed Central

Covian, Raul; Chess, David; Balaban, Robert S.

2012-01-01

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction media recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase where catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. PMID:22975200

Continuous monitoring of enzymatic activity within native electrophoresis gels: application to mitochondrial oxidative phosphorylation complexes.

PubMed

Covian, Raul; Chess, David; Balaban, Robert S

2012-12-01

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light-scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze the enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction medium recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high-resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase in which catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. Published by Elsevier Inc.

Rapid Microarray Detection of DNA and Proteins in Microliter Volumes with SPR Imaging Measurements

PubMed Central

Seefeld, Ting Hu; Zhou, Wen-Juan; Corn, Robert M.

2011-01-01

A four chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm × 18 mm biochip consists of four 3 μL microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The twelve element (4 × 3) SPRI microarray consists of gold thin film spots (1 mm2 area; 45 nm thickness) each in individually addressable 0.5 μL volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels; the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (single stranded DNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements were also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss was proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18,000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 seconds in the microliter volume format. PMID:21488682

Pitfalls in the detection of cholesterol in Huntington's disease models.

PubMed

Marullo, Manuela; Valenza, Marta; Leoni, Valerio; Caccia, Claudio; Scarlatti, Chiara; De Mario, Agnese; Zuccato, Chiara; Di Donato, Stefano; Carafoli, Ernesto; Cattaneo, Elena

2012-10-11

Background Abnormalities in brain cholesterol homeostasis have been reported in Huntington's disease (HD), an adult-onset neurodegenerative disorder caused by an expansion in the number of CAG repeats in the huntingtin (HTT) gene. However, the results have been contradictory with respect to whether cholesterol levels increase or decrease in HD models. Biochemical and mass spectrometry methods show reduced levels of cholesterol precursors and cholesterol in HD cells and in the brains of several HD animal models. Abnormal brain cholesterol homeostasis was also inferred from studies in HD patients. In contrast, colorimetric and enzymatic methods indicate cholesterol accumulation in HD cells and tissues. Here we used several methods to investigate cholesterol levels in cultured cells in the presence or absence of mutant HTT protein. Results Colorimetric and enzymatic methods with low sensitivity gave variable results, whereas results from a sensitive analytical method, gas chromatography-mass spectrometry, were more reliable. Sample preparation, high cell density and cell clonality also influenced the detection of intracellular cholesterol. Conclusions Detection of cholesterol in HD samples by colorimetric and enzymatic assays should be supplemented by detection using more sensitive analytical methods. Care must be taken to prepare the sample appropriately. By evaluating lathosterol levels using isotopic dilution mass spectrometry, we confirmed reduced cholesterol biosynthesis in knock-in cells expressing the polyQ mutation in a constitutive or inducible manner. *Correspondence should be addressed to Elena Cattaneo: elena.cattaneo@unimi.it.

Detection of ingested nitromethane and reliable creatinine assessment using multiple common analytical methods.

PubMed

Murphy, Christine M; Devlin, John J; Beuhler, Michael C; Cheifetz, Paul; Maynard, Susan; Schwartz, Michael D; Kacinko, Sherri

2018-04-01

Nitromethane, found in fuels used for short distance racing, model cars, and model airplanes, produces a falsely elevated serum creatinine with standard creatinine analysis via the Jaffé method. Erroneous creatinine elevation often triggers extensive testing, leads to inaccurate diagnoses, and delayed or inappropriate medical interventions. Multiple reports in the literature identify "enzymatic assays" as an alternative method to detect the true value of creatinine, but this ambiguity does not help providers translate what type of enzymatic assay testing can be done in real time to determine if there is indeed false elevation. We report seven cases of ingested nitromethane where creatinine was determined via Beckman Coulter ® analyser using the Jaffé method, Vitros ® analyser, or i-Stat ® point-of-care testing. Nitromethane was detected and semi-quantified using a common clinical toxic alcohol analysis method, and quantified by headspace-gas chromatography-mass spectrometry. When creatinine was determined using i-Stat ® point-of-care testing or a Vitros ® analyser, levels were within the normal range. Comparatively, all initial creatinine levels obtained via the Jaffé method were elevated. Nitromethane concentrations ranged from 42 to 310 μg/mL. These cases demonstrate reliable assessment of creatinine through other enzymatic methods using a Vitros ® analyser or i-STAT ® . Additionally, nitromethane is detectable and quantifiable using routine alcohols gas chromatography analysis and by headspace-gas chromatography-mass spectrometry.

[The effect of enzymatic treatment using proteases on properties of persistent sodium current in CA1 pyramidal neurons of rat hippocampus].

PubMed

Lun'ko, O O; Isaiev, D S; Maxymiuk, O P; Kryshtal', O O; Isaieva, O V

2014-01-01

We investigated the effect of proteases, widely used for neuron isolation in electrophysiological studies, on the amplitude and kinetic characteristics of persistent sodium current (I(NaP)) in hippocampal CA1 pyramidal neurons. Properties of I(NaP) were studied on neurons isolated by mechanical treatment (control group) and by mechanical and enzymatic treatment using pronase E (from Streptomyces griseus) or protease type XXIII (from Aspergillus oryzae). We show that in neurons isolated with pronase E kinetic of activation and density of I(NaP) was unaltered. Enzymatic treatment with protease type XXIII did not alter I(NaP) activation but result in significant decrease in I(NaP) density. Our data indicates that enzymatic treatment using pronase E for neuron isolation is preferable for investigation of I(NaP).

Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway.

PubMed

Keller, Markus A; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V; Griffin, Julian L; Ralser, Markus

2016-01-01

Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks.

Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway

PubMed Central

Keller, Markus A.; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V.; Griffin, Julian L.; Ralser, Markus

2016-01-01

Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks. PMID:26824074

Glucose Oxidase-Mediated Polymerization as a Platform for Dual-Mode Signal Amplification and Biodetection

PubMed Central

Berron, Brad J; Johnson, Leah M; Ba, Xiao; McCall, Joshua D; Alvey, Nicholas J; Anseth, Kristi S; Bowman, Christopher N

2011-01-01

We report the first use of a polymerization-based ELISA substrate solution employing enzymatically mediated radical polymerization as a dual-mode amplification strategy. Enzymes are selectively coupled to surfaces to generate radicals that subsequently lead to polymerization-based amplification (PBA) and biodetection. Sensitivity and amplification of the polymerization-based detection system were optimized in a microwell strip format using a biotinylated microwell surface with a glucose oxidase (GOx)–avidin conjugate. The immobilized GOx is used to initiate polymerization, enabling the detection of the biorecognition event visually or through the use of a plate reader. Assay response is compared to that of an enzymatic substrate utilizing nitroblue tetrazolium in a simplified assay using biotinylated wells. The polymerization substrate exhibits equivalent sensitivity (2 µg/mL of GOx-avidin) and over three times greater signal amplification than this traditional enzymatic substrate since each radical that is enzymatically generated leads to a large number of polymerization events. Enzyme-mediated polymerization proceeds in an ambient atmosphere without the need for external energy sources, which is an improvement upon previous PBA platforms. Substrate formulations are highly sensitive to both glucose and iron concentrations at the lowest enzyme concentrations. Increases in amplification time correspond to higher assay sensitivities with no increase in non-specific signal. Finally, the polymerization substrate generated a signal to noise ratio of 14 at the detection limit (156 ng/mL) in an assay of transforming growth factor-beta. Biotechnol. Bioeng. 2011; 108:1521–1528. © 2011 Wiley Periodicals, Inc. PMID:21337335

Purification and substrate specificities of a fructanase from Kluyveromyces marxianus isolated from the fermentation process of Mezcal.

PubMed

Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre

2011-02-01

A fructanase, produced by a Kluyveromyces marxianus strain isolated during the fermentation step of the elaboration process of "Mezcal de Guerrero" was purified and biochemically characterized. The active protein was a glycosylated dimer with a molecular weight of approximately 250 kDa. The specific enzymatic activity of the protein was determined for different substrates: sucrose, inulin, Agave tequilana fructan, levan and Actilight® and compared with the activity of Fructozyme®. The hydrolysis profile of the different substrates analyzed by HPAEC-PAD showed that the enzyme has different affinities over the substrates tested with a sucrose/inulin enzymatic activity ratio (S/I) of 125. For the hydrolysis of Agave tequilana fructans, the enzyme also showed a higher enzymatic activity and specificity than Fructozyme®, which is important for its potential application in the tequila industry. Copyright © 2010 Elsevier Ltd. All rights reserved.

Evidence of oleuropein degradation by olive leaf protein extract.

PubMed

De Leonardis, Antonella; Macciola, Vincenzo; Cuomo, Francesca; Lopez, Francesco

2015-05-15

The enzymatic activity of raw protein olive leaf extract has been investigated in vivo, on olive leaf homogenate and, in vitro with pure oleuropein and other phenolic substrates. At least two types of enzymes were found to be involved in the degradation of endogenous oleuropein in olive leaves. As for the in vitro experiments, the presence of active polyphenoloxidase and β-glucosidase was determined by HPLC and UV-Visible spectroscopy. Interestingly, both the enzymatic activities were found to change during the storage of olive leaves. Specifically, the protein extracts obtained from fresh leaves showed the presence of both the enzymatic activities, because oleuropein depletion occurred simultaneously with the formation of the oleuropein aglycon, 3,4-DHPEA-EA. In comparison leaves subjected to the drying process showed a polyphenoloxidase activity leading exclusively to the formation of oxidation products responsible for the typical brown coloration of the reaction solution. Copyright © 2014 Elsevier Ltd. All rights reserved.

Proton mediated control of biochemical reactions with bioelectronic pH modulation

NASA Astrophysics Data System (ADS)

Deng, Yingxin; Miyake, Takeo; Keene, Scott; Josberger, Erik E.; Rolandi, Marco

2016-04-01

In Nature, protons (H+) can mediate metabolic process through enzymatic reactions. Examples include glucose oxidation with glucose dehydrogenase to regulate blood glucose level, alcohol dissolution into carboxylic acid through alcohol dehydrogenase, and voltage-regulated H+ channels activating bioluminescence in firefly and jellyfish. Artificial devices that control H+ currents and H+ concentration (pH) are able to actively influence biochemical processes. Here, we demonstrate a biotransducer that monitors and actively regulates pH-responsive enzymatic reactions by monitoring and controlling the flow of H+ between PdHx contacts and solution. The present transducer records bistable pH modulation from an “enzymatic flip-flop” circuit that comprises glucose dehydrogenase and alcohol dehydrogenase. The transducer also controls bioluminescence from firefly luciferase by affecting solution pH.

[Ornithine decarboxylase in mammalian organs and tissues at hibernation and artificial hypobiosis].

PubMed

Logvinovich, O S; Aksenova, G E

2013-01-01

Ornithine decarboxylase (ODC, EC 4.1.1.17.) is a short-lived and dynamically regulated enzyme of polyamines biosynthesis. Regulation of functional, metabolic and proliferative state of organs and tissues involves the modifications of the ODC enzymatic activity. The organ-specific changes in ODC activity were revealed in organs and tissues (liver, spleen, bone marrow, kidney, and intestinal mucosa) of hibernating mammals - squirrels Spermophilus undulates - during the hibernating season. At that, a positive correlation was detected between the decline and recovery of the specialized functions of organs and tissues and the respective modifications of ODC activity during hibernation bouts. Investigation of changes in ODC activity in organs and tissues of non-hibernating mammals under artificial hypobiosis showed that in Wistar rats immediately after exposure to hypothermia-hypoxia-hypercapnia (hypobiosis) the level of ODC activity was low in thymus, spleen, small intestine mucosa, neocortex, and liver. The most marked reduction in enzyme activity was observed in actively proliferating tissues: thymus, spleen, small intestine mucosa. In bone marrow of squirrels, while in a state of torpor, as well as in thymus of rats after exposure to hypothermia-hypoxia-hypercapnia, changes in the ODC activity correlated with changes in the rate of cell proliferation (by the criterion of cells distribution over cell cycle). The results obtained, along with the critical analysis of published data, indicate that the ODC enzyme is involved in biochemical adaptation of mammals to natural and artificial hypobiosis. A decline in the ODC enzymatic activity indicates a decline in proliferative, functional, and metabolic activity of organs and tissues of mammals (bone marrow, mucosa of small intestine, thymus, spleen, neocortex, liver, kidneys) when entering the state of hypobiosis.

A sensitive and specific radiochromatographic assay of fatty acid amide hydrolase activity.

PubMed

Maccarrone, M; Bari, M; Agrò, A F

1999-02-15

A radiochromatographic method has been set up in order to determine fatty acid amide hydrolase (FAAH) activity, based on reversed-phase high-performance liquid chromatography and on-line scintillation counting. The reaction products were separated using a C18 column eluted with methanol-water-acetic acid and quantitated with an external standard. Baseline separation of the acid product from the substrate was completed in less than 4 min, with a detection limit of 2.5 fmol arachidonic acid at a signal to noise ratio of 4:1. The method enabled to determine the kinetic constants (i.e., apparent Km of 2.0 +/- 0.2 microM and Vmax of 800 +/- 75 pmol. min-1. mg protein-1 toward anandamide) and the substrate specificity of human brain FAAH, as well as the extent of enzyme inhibition by some anandamide congeners. The femtomole sensitivity and the accuracy of the method allow detection and characterization of the activity of FAAH in very minute tissue samples or in samples where the enzymatic activity is very low. Copyright 1999 Academic Press.

Alkaline phosphatase labeled SERS active sandwich immunoassay for detection of Escherichia coli

NASA Astrophysics Data System (ADS)

Bozkurt, Akif Goktug; Buyukgoz, Guluzar Gorkem; Soforoglu, Mehmet; Tamer, Ugur; Suludere, Zekiye; Boyaci, Ismail Hakki

2018-04-01

In this study, a sandwich immunoassay method utilizing enzymatic activity of alkaline phosphatase (ALP) on 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for Escherichia coli (E. coli) detection was developed using surface enhanced Raman spectroscopy (SERS). For this purpose, spherical magnetic gold coated core-shell nanoparticles (MNPs-Au) and rod shape gold nanoparticles (Au-NRs) were synthesized and modified for immunomagnetic separation (IMS) of E. coli from the solution. In order to specify the developed method to ALP activity, Au-NRs were labeled with this enzyme. After successful construction of the immunoassay, BCIP substrate was added to produce the SERS-active product; 5-bromo-4-chloro-3-indole (BCI). A good linearity (R2 = 0.992) was established between the specific SERS intensity of BCI at 600 cm- 1 and logarithmic E. coli concentration in the range of 1.7 × 101-1.7 × 106 cfu mL- 1. LOD and LOQ values were also calculated and found to be 10 cfu mL- 1 and 30 cfu mL- 1, respectively.

Enzymatic hydrolysis of carotenoid fatty acid esters of red pepper (Capsicum annuum L.) by a lipase from Candida rugosa.

PubMed

Breithaupt, D E

2000-01-01

Analyses of red pepper extracts which had been pretreated with lipase type VII (EC 3.1.1.3.) from Candida rugosa showed for the first time pepper carotenoid esters to be substrates of this enzyme. However, the extent of enzymatic hydrolysis depends on the respective carotenoid and was not quantitative compared to chemical saponification. After enzymatic cleavage, 67-89% of total capsanthin, 61-65% of total zeaxanthin, 70-81% of total beta-cryptoxanthin and 70-86% of total violaxanthin were detected in free form. Nevertheless, the method described here offers the possibility to cleave in part several carotenoid esters originating from red pepper quickly and under comparatively mild reaction conditions. Replacement of the generally performed alkaline hydrolysis by enzymatic cleavage allows the resulting product to be used in food industry as "natural" coloring agent e.g. to colour cheese and jellies.

Effect of exogenous metal ions and mechanical stress on rice processed in thermal-solid enzymatic reaction system related to further alcoholic fermentation efficiency.

PubMed

Xu, Enbo; Wu, Zhengzong; Jiao, Aiquan; Jin, Zhengyu

2018-02-01

Metal-rich thermal-solid enzymatic processing of rice combined with yeast fermentation was investigated. 8 Metal ions were exogenously supplied at 0.05, 0.5 and 5mmol/100g (MG) rice prior to static high pressure enzymatic cooking (HPEC) and dynamic enzymatic extrusion cooking (EEC). Treated rice and its fermentation efficiency (FE) were characterized by rapid viscosity analyzer (RVA), UV-Vis, FT-IR and atomic absorption spectrophotometer (AAS). The optimum pH range of enzyme in solid system (>4.9) was broader than in a liquid system (>5.5). Cations decreased enzymatic activity in HPEC probably due to metal-induced aggregation of rice matrix with reduced reacting area as well as strengthened structure of starch/polysaccharides modified by metals. While using the EEC with mechanical mixing/shearing, relative activity was activated to 110 and 120% by Mg 2+ (0.05-0.5MG) and Ca 2+ (0.05-5MG), respectively. Furthermore, the effectiveness of residual ions to promote further FE was found to follow the order: Ca 2+ >K + >Zn 2+ >Mg 2+ >Mn 2+ >Na + ≈Control>Fe 2+ >Cu 2+ , individually. Copyright © 2017 Elsevier Ltd. All rights reserved.

Assessment of collagen quality associated with non-enzymatic cross-links in human bone using Fourier-transform infrared imaging.

PubMed

Schmidt, F N; Zimmermann, E A; Campbell, G M; Sroga, G E; Püschel, K; Amling, M; Tang, S Y; Vashishth, D; Busse, B

2017-04-01

Aging and many disease conditions, most notably diabetes, are associated with the accumulation of non-enzymatic cross-links in the bone matrix. The non-enzymatic cross-links, also known as advanced glycation end products (AGEs), occur at the collagen tissue level, where they are associated with reduced plasticity and increased fracture risk. In this study, Fourier-transform infrared (FTIR) imaging was used to detect spectroscopic changes associated with the formation of non-enzymatic cross-links in human bone collagen. Here, the non-enzymatic cross-link profile was investigated in one cohort with an in vitro ribose treatment as well as another cohort with an in vivo bisphosphonate treatment. With FTIR imaging, the two-dimensional (2D) spatial distribution of collagen quality associated with non-enzymatic cross-links was measured through the area ratio of the 1678/1692cm -1 subbands within the amide I peak, termed the non-enzymatic crosslink-ratio (NE-xLR). The NE-xLR increased by 35% in the ribation treatment group in comparison to controls (p<0.005), with interstitial bone tissue being more susceptible to the formation of non-enzymatic cross-links. Ultra high-performance liquid chromatography, fluorescence microscopy, and fluorometric assay confirm a correlation between the non-enzymatic cross-link content and the NE-xLR ratio in the control and ribated groups. High resolution FTIR imaging of the 2D bone microstructure revealed enhanced accumulation of non-enzymatic cross-links in bone regions with higher tissue age (i.e., interstitial bone). This non-enzymatic cross-link ratio (NE-xLR) enables researchers to study not only the overall content of AGEs in the bone but also its spatial distribution, which varies with skeletal aging and diabetes mellitus and provides an additional measure of bone's propensity to fracture. Copyright © 2017 Elsevier Inc. All rights reserved.

Assessment of collagen quality associated with non-enzymatic cross-links in human bone using Fourier-transform infrared imaging

PubMed Central

Schmidt, F.N.; Zimmermann, E.A.; Campbell, G.M.; Sroga, G.E.; Püschel, K.; Amling, M.; Tang, S. Y.; Vashishth, D.; Busse, B.

2017-01-01

Aging and many disease conditions, most notably diabetes, are associated with the accumulation of non-enzymatic cross-links in the bone matrix. The non-enzymatic crosslinks, also known as advanced glycation end products (AGEs), occur at the collagen tissue level, where they are associated with reduced plasticity and increased fracture risk. In this study, Fourier-transform infrared (FTIR) imaging was used to detect spectroscopic changes associated with the formation of non-enzymatic cross-links in human bone collagen. Here, the non-enzymatic cross-link profile was investigated in one cohort with an in vitro ribose treatment as well as another cohort with an in vivo bisphosphonate treatment. With FTIR imaging, the two-dimensional (2D) spatial distribution of collagen quality associated with non-enzymatic cross-links was measured through the area ratio of the 1678/1692 cm−1 subbands within the amide I peak, termed the non-enzymatic crosslink-ratio (NE-xLR). The NE-xLR increased by 35% in the ribation treatment group in comparison to controls (p< 0.005), with interstitial bone tissue being more susceptible to the formation of non-enzymatic cross-links. Ultra high performance liquid chromatography, fluorescence microscopy, and fluorometric assay confirm a correlation between the non-enzymatic cross-link content and the NE-xLR ratio in the control and ribated groups. High resolution FTIR imaging of the 2D bone microstructure revealed enhanced accumulation of non-enzymatic cross-links in bone regions with higher tissue age (i.e., interstitial bone). This non-enzymatic cross-link ratio (NE-xLR) enables researchers to study not only the overall content of AGEs in the bone but also its spatial distribution, which varies with skeletal aging and diabetes mellitus and provides an additional measure of bone's propensity to fracture. PMID:28109917

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gopolan,G.; Thwin, M.; Gopalakrishnakone, P.

Russell's viper (Vipera russelli, also known as Daboia russelli) is one of the major causes of fatal snakebites. To date, five Daboia russelli subspecies have been recognized. Daboiatoxin (DbTx) is the main lethal phospholipase A{sub 2} (PLA{sub 2}) toxin in the venom of D. russelli siamensis (Myanmar viper) and has strong neurotoxic, myotoxic and cytotoxic activities. DbTx and its homologous neurotoxins viperotoxin F from D. russelli formosensis (Taiwan viper) and vipoxin from the Bulgarian sand viper V. ammodytes meridionalis consist of complexes between a nontoxic acidic PLA2 protein and an enzymatically active basic PLA2. DbTx and viperotoxin F are presynapticmore » toxins, while vipoxin is postsynaptic. The two chains of DbTx have been separated and their PLA2 enzymatic activity has been measured using the secretory PLA2 assay kit. The enzymatic activity of DbTx chain B is reduced by 30% of its original activity by chain A in a unimolar ratio, thus indicating that DbTx chain A acts as an inhibitor. The lethal activity of the two chains has also been studied in male albino mice and chain A is less lethal than chain B. The crystal structure of DbTx has also been determined and its structural details are compared with those of the two homologues. Furthermore, an attempt is made to correlate the sequence and structural determinants of these toxins with their enzymatic activities and their pharmacological effects.« less

Recent data concerning heparanase: focus on fibrosis, inflammation and cancer.

PubMed

Secchi, Maria Francesca; Masola, Valentina; Zaza, Gianluigi; Lupo, Antonio; Gambaro, Giovanni; Onisto, Maurizio

2015-12-01

Heparanase (HPSE) is a multitasking protein characterized by enzymatic and non-enzymatic activities. By means of its enzymatic activity, HPSE catalyzes the cutting of the side chains of heparan sulfate (HS) proteoglycans, thereby inducing the remodeling of the extracellular matrix and basement membranes. Thanks to the cleavage of HS, HPSE also promotes the release and diffusion of several HS-linked molecules such as growth factors, cytokines and enzymes. In addition to degrading HS chains, HPSE has non-enzymatic functions that trigger several signaling pathways. This signaling activity is achieved by interacting with transmembrane proteins, activating kinases such as Akt and Src, or modulating the activity of factors such as FGF-2 and TGF-β. Several studies have recently highlighted a possible intracellular activity for HPSE, particularly at nuclear level. While HPSE activity is quite limited in physiological conditions, its demonstrated increasing involvement in various pathological conditions, such as in tumor progression and renal disease, have attracted the attention of a growing number of researchers. The fact that no other molecule is capable of performing the same function as HPSE makes this enzyme an attractive potential target of medical treatment. With this short conceptual overview, we aim to provide an update on current knowledge concerning the HPSE protein in the experimental and clinical settings, paying particular attention to its role in fibrosis, inflammation and cancer.

Vancomycin analogues containing monosaccharides exhibit improved antibiotic activity: a combined one-pot enzymatic glycosylation and chemical diversification strategy.

PubMed

Thayer, Desiree A; Wong, Chi-Huey

2006-09-18

Many natural products contain carbohydrate moieties that contribute to their biological activity. Manipulation of the carbohydrate domain of natural products through multiple glycosylations to identify new derivatives with novel biological activities has been a difficult and impractical process. We report a practical one-pot enzymatic approach with regeneration of cosubstrates to synthesize analogues of vancomycin that contain an N-alkyl glucosamine, which exhibited marked improvement in antibiotic activity against a vancomycin-resistant strain of Enterococcus.

Double enzymatic cascade reactions within FeSe-Pt@SiO2 nanospheres: synthesis and application toward colorimetric biosensing of H2O2 and glucose.

PubMed

Qiao, Fengmin; Wang, Zhenzhen; Xu, Ke; Ai, Shiyun

2015-10-07

A facile process was developed for the synthesis of FeSe-Pt@SiO2 nanospheres based on the hydrothermal treatment of FeCl3·6H2O, selenium and NaBH4 in ethanolamine solvent, followed by reducing HPtCl4 with NaBH4 in the presence of FeSe particles to obtain FeSe coated with Pt NPs (FeSe-Pt), ending with a surfactant assembled sol-gel process to obtain FeSe-Pt@SiO2. The morphology and composition of FeSe-Pt@SiO2 were characterized by transmission electron microscopy, high resolution TEM, X-ray diffraction and Fourier transform infrared spectroscopy. Structural analyses revealed that FeSe-Pt@SiO2 nanospheres were of regular spherical shape with smooth surfaces due to the SiO2 shells, compared with FeSe particles with 150 nm lateral diameter. The prepared FeSe-Pt@SiO2 nanospheres possessed both intrinsic glucose oxidase (GOx-) and peroxidase-mimic activities, and we engineered an artificial enzymatic cascade system with high activity and stability based on this nanostructure. The good catalytic performance of the composites could be attributed to the synergy between the functions of FeSe particles and Pt NPs. Significantly, the FeSe-Pt@SiO2 nanospheres as robust nanoreactors can catalyze a self-organized cascade reaction, which includes oxidation of glucose by oxygen to yield gluconic acid and H2O2, and then oxidation of 3,3,5,5-tetramethylbenzidine (TMB) by H2O2 to produce a colour change. Colorimetric detection of H2O2 and glucose using the FeSe-Pt@SiO2 nanospheres was conducted with high detection sensitivities, 0.227 nM and 1.136 nM, respectively, demonstrating the feasibility of practical sensing applications. It is therefore believed that our findings in this study could open up the possibility of utilizing FeSe-Pt@SiO2 nanospheres as enzymatic mimics in diagnostic and biotechnology fields.

Activity of selected hydrolytic enzymes in Allium sativum L. anthers.

PubMed

Winiarczyk, Krystyna; Gębura, Joanna

2016-05-01

The aim of the study was to determine enzymatic activity in sterile Allium sativum anthers in the final stages of male gametophyte development (the stages of tetrads and free microspores). The analysed enzymes were shown to occur in the form of numerous isoforms. In the tetrad stage, esterase activity was predominant, which was manifested by the greater number of isoforms of the enzyme. In turn, in the microspore stage, higher numbers of isoforms of acid phosphatases and proteases were detected. The development of sterile pollen grains in garlic is associated with a high level of protease and acid phosphatase activity and lower level of esterase activities in the anther locule. Probably this is the first description of the enzymes activity (ACPH, EST, PRO) in the consecutives stages of cell wall formation which is considered to be one of the causes of male sterility in flowering plant. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Application of activity-based protein profiling to study enzyme function in adipocytes.

PubMed

Galmozzi, Andrea; Dominguez, Eduardo; Cravatt, Benjamin F; Saez, Enrique

2014-01-01

Activity-based protein profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways. © 2014 Elsevier Inc. All rights reserved.

Non-enzymatic electrochemical glucose sensor based on NiMoO4 nanorods

NASA Astrophysics Data System (ADS)

Wang, Dandan; Cai, Daoping; Huang, Hui; Liu, Bin; Wang, Lingling; Liu, Yuan; Li, Han; Wang, Yanrong; Li, Qiuhong; Wang, Taihong

2015-04-01

A non-enzymatic glucose sensor based on the NiMoO4 nanorods has been fabricated for the first time. The electrocatalytic performance of the NiMoO4 nanorods’ modified electrode toward glucose oxidation was evaluated by cyclic voltammetry and amperometry. The NiMoO4 nanorods’ modified electrode showed a greatly enhanced electrocatalytic property toward glucose oxidation, as well as an excellent anti-interference and a good stability. Impressively, good accuracy and high precision for detecting glucose concentration in human serum samples were obtained. These excellent sensing properties, combined with good reproducibility and low cost, indicate that NiMoO4 nanorods are a promising candidate for non-enzymatic glucose sensors.

Signal Amplification Technologies for the Detection of Nucleic Acids: from Cell-Free Analysis to Live-Cell Imaging.

PubMed

Fozooni, Tahereh; Ravan, Hadi; Sasan, Hosseinali

2017-12-01

Due to their unique properties, such as programmability, ligand-binding capability, and flexibility, nucleic acids can serve as analytes and/or recognition elements for biosensing. To improve the sensitivity of nucleic acid-based biosensing and hence the detection of a few copies of target molecule, different modern amplification methodologies, namely target-and-signal-based amplification strategies, have already been developed. These recent signal amplification technologies, which are capable of amplifying the signal intensity without changing the targets' copy number, have resulted in fast, reliable, and sensitive methods for nucleic acid detection. Working in cell-free settings, researchers have been able to optimize a variety of complex and quantitative methods suitable for deploying in live-cell conditions. In this study, a comprehensive review of the signal amplification technologies for the detection of nucleic acids is provided. We classify the signal amplification methodologies into enzymatic and non-enzymatic strategies with a primary focus on the methods that enable us to shift away from in vitro detecting to in vivo imaging. Finally, the future challenges and limitations of detection for cellular conditions are discussed.

Detection of DNA Sequences Refractory to PCR Amplification Using a Biophysical SERRS Assay (Surface Enhanced Resonant Raman Spectroscopy)

PubMed Central

Feuillie, Cécile; Merheb, Maxime M.; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2014-01-01

The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction – based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage. PMID:25502338

Detection of DNA sequences refractory to PCR amplification using a biophysical SERRS assay (Surface Enhanced Resonant Raman Spectroscopy).

PubMed

Feuillie, Cécile; Merheb, Maxime M; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2014-01-01

The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction - based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage.

Fabrication of few-layer graphene film based field effect transistor and its application for trace-detection of herbicide atrazine

NASA Astrophysics Data System (ADS)

Thanh Cao, Thi; Chuc Nguyen, Van; Binh Nguyen, Hai; Thang Bui, Hung; Thu Vu, Thi; Phan, Ngoc Hong; Thang Phan, Bach; Hoang, Le; Bayle, Maxime; Paillet, Matthieu; Sauvajol, Jean Louis; Phan, Ngoc Minh; Tran, Dai Lam

2016-09-01

We describe the fabrication of highly sensitive graphene-based field effect transistor (FET) enzymatic biosensor for trace-detection of atrazine. The few-layers graphene films were prepared on polycrystalline copper foils by atmospheric pressure chemical vapor deposition method using an argon/hydrogen/methane mixture. The characteristics of graphene films were investigated by scanning electron microscopy, transmission electron microscopy and Raman spectroscopy. The results indicated low uniformity of graphene layers, which is probably induced by heterogeneous distribution of graphene nucleation sites on the Cu surface. The pesticide detection is accomplished through the measurement of the drain-source current variations of the FET sensor upon the urea enzymatic hydrolysis reaction. The obtained biosensor is able to detect atrazine with a sensitivity of 56 μA/logCATZ in range between 2 × 10-4 and 20 ppb and has a limit of detection as low as 0.05 ppt. The elaboration of such highly sensitive biosensors will provide better biosensing performances for the detection of biochemical targets.

Polyluminol/hydrogel composites as new electrochemiluminescent-active sensing layers.

PubMed

Leca-Bouvier, Béatrice D; Sassolas, Audrey; Blum, Loïc J

2014-09-01

This paper reports on electrochemiluminescent sensors and biosensors based on polyluminol/hydrogel composite sensing layers using chemical or biological membranes as hydrogel matrices. In this work, luminol is electropolymerized under near-neutral conditions onto screen-printed electrode (SPE)-supported hydrogel films. The working electrode coated with a hydrogel film is soaked in a solution containing monomeric luminol units, allowing the monomeric luminol units to diffuse inside the porous matrix to the electrode surface where they are electropolymerized by cyclic voltammetry (CV). Sensors and enzymatic biosensors for H2O2 and choline detection, respectively, have been developed, using choline oxidase (ChOD) as a model enzyme. In this case, hydrogel is used both as the enzymatic immobilization matrix and as a template for the electrosynthesis of polyluminol. The enzyme was immobilized by entrapment in the gel matrix during its formation before electropolymerization of the monomer. Several parameters have been optimized in terms of polymerization conditions, enzyme loading, and average pore size. Using calcium alginate or tetramethoxysilane (TMOS)-based silica as porous matrix, H2O2 and choline detection are reported down to micromolar concentrations with three orders of magnitude wide dynamic ranges starting from 4 × 10(-7) M. Polyluminol/hydrogel composites appear as suitable electrochemiluminescence (ECL)-active sensing layers for the design of new reagentless and disposable easy-to-use optical sensors and biosensors, using conventional TMOS-based silica gel or the more original and easier to handle calcium alginate, reported here for the first time in such a configuration, as the biocompatible hydrogel matrix.

Enzymatic degradation of cyclic 2,3-diphosphoglycerate to 2,3-diphosphoglycerate in Methanobacterium thermoautotrophicum.

PubMed

Sastry, M V; Robertson, D E; Moynihan, J A; Roberts, M F

1992-03-24

2,3-Diphosphoglycerate (2,3-DPG) has been found to be the product of the enzymatic degradation of cyclic 2,3-diphosphoglycerate (cDPG) in the archaebacterium Methanobacterium thermoautotrophicum delta H. Although 2,3-DPG has not previously been detected as a major soluble component of M. thermoautotrophicum, large pools accumulated at an incubation temperature of 50 degrees C (below the optimum growth temperature of 62 degrees C). Under these conditions, cellular activity was significantly decreased; a return of the culture to the optimum growth temperature restored the 2,3-DPG pool back to original low levels and caused steady-state cDPG levels to increase again. While 13CO2-pulse/12CO2-chase experiments at 50 degrees C showed that the cDPG turned over, the appearance of 2,3-DPG at NMR-visible concentrations required at least 10 h. Production of 2,3-DPG in vivo was prevented by exposure of the cells to O2. The enzyme responsible for this hydrolysis of cDPG was purified by affinity chromatography and appears to be a 33-kDa protein. Activity was detected in the presence of oxygen and was enhanced by a solution of 1 M KCl, 25 mM MgCl2, and dithiothreitol. Both Km and Vmax have been determined at 37 degrees C; kinetics also indicate that in vitro the product, 2,3-DPG, is an inhibitor of cDPG hydrolysis. These findings are discussed in view of a proposed role for cDPG in methanogens.

Genes from the medicinal leech (Hirudo medicinalis) coding for unusual enzymes that specifically cleave endo-epsilon (gamma-Glu)-Lys isopeptide bonds and help to dissolve blood clots.

PubMed

Zavalova, L; Lukyanov, S; Baskova, I; Snezhkov, E; Akopov, S; Berezhnoy, S; Bogdanova, E; Barsova, E; Sverdlov, E D

1996-11-27

We previously detected in salivary gland secretions of the medicinal leech (Hirudo medicinalis) a novel enzymatic activity, endo-epsilon(gamma-Glu)-Lys isopeptidase, which cleaves isopeptide bonds formed by transglutaminase (Factor XIIIa) between glutamine gamma-carboxamide and the epsilon-amino group of lysine. Such isopeptide bonds, either within or between protein polypeptide chains are formed in many biological processes. However, before we started our work no enzymes were known to be capable of specifically splitting isopeptide bonds in proteins. The isopeptidase activity we detected was specific for isopeptide bonds. The enzyme was termed destabilase. Here we report the first purification of destabilase, part of its amino acid sequence isolation and sequencing of two related cDNAs derived from the gene family that encodes destabilase proteins, and the detection of isopeptidase activity encoded by one of these cDNAs cloned in a baculovirus expression vector. The deduced mature protein products of these cDNAs contain 115 and 116 amino acid residues, including 14 highly conserved Cys residues, and are formed from precursors containing specific leader peptides. No homologous sequences were found in public databases.

Zinc oxide inverse opal enzymatic biosensor

NASA Astrophysics Data System (ADS)

You, Xueqiu; Pikul, James H.; King, William P.; Pak, James J.

2013-06-01

We report ZnO inverse opal- and nanowire (NW)-based enzymatic glucose biosensors with extended linear detection ranges. The ZnO inverse opal sensors have 0.01-18 mM linear detection range, which is 2.5 times greater than that of ZnO NW sensors and 1.5 times greater than that of other reported ZnO sensors. This larger range is because of reduced glucose diffusivity through the inverse opal geometry. The ZnO inverse opal sensors have an average sensitivity of 22.5 μA/(mM cm2), which diminished by 10% after 35 days, are more stable than ZnO NW sensors whose sensitivity decreased by 10% after 7 days.

Production and characterization of enzymatic cocktail produced by Aspergillus niger using green macroalgae as nitrogen source and its application in the pre-treatment for biogas production from Ulva rigida.

PubMed

Karray, Raida; Hamza, Manel; Sayadi, Sami

2016-09-01

Marine macroalgae are gaining more and more importance as a renewable feedstock for durable bioenergy production, but polysaccharides of this macroalgae are structurally complex in its chemical composition. The use of enzymatic hydrolysis may provide new pathways in the conversion of complex polysaccharides to fermentable sugars. In this study, an enzymatic cocktail with high specificity was first isolated from Aspergillus niger using the green macroalgae Ulva rigida as nitrogen source. The cocktail is rich on β-glucosidase, pectinase and carboxy-methyl-cellulase (CMCase). The highest activity was obtained with β-glucosidase (109IUmL(-1)) and pectinase (76IUmL(-1)), while CMCase present the lowest activity 4.6IUmL(-1). The U. rigida pre-treatment with this enzymatic cocktail showed high rate of reduced sugar release, and could bring promising prospects for enzymatic pre-treatment of the biogas production from U. rigida biomass which reached 1175mLgCODint(-1). Copyright © 2016 Elsevier Ltd. All rights reserved.

Microbial production of metabolites and associated enzymatic reactions under high pressure.

PubMed

Dong, Yongsheng; Jiang, Hua

2016-11-01

High environmental pressure exerts an external stress on the survival of microorganisms that are commonly found under normal pressure. In response, many growth traits alter, including cell morphology and physiology, cellular structure, metabolism, physical and chemical properties, the reproductive process, and defense mechanisms. The high-pressure technology (HP) has been industrially utilized in pressurized sterilization, synthesis of stress-induced products, and microbial/enzymatic transformation of chemicals. This article reviews current research on pressure-induced production of metabolites in normal-pressure microbes and their enzymatic reactions. Factors that affect the production of such metabolites are summarized, as well as the effect of pressure on the performance of microbial fermentation and the yield of flavoring compounds, different categories of induced enzymatic reactions and their characteristics in the supercritical carbon dioxide fluid, effects on enzyme activity, and the selection of desirable bacterial strains. Technological challenges are discussed, and future research directions are proposed. Information presented here will benefit the research, development, and application of the HP technology to improve microbial fermentation and enzymatic production of biologically active substances, thereby help to meet their increasing demand from the ever-expanding market.

Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: BoNT/A2, /F4, and /F5.

PubMed

Kalb, Suzanne R; Baudys, Jakub; Smith, Theresa J; Smith, Leonard A; Barr, John R

2014-04-01

Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum.

The activity of Rhizomuchor miehei lipase as a biocatalyst in enzymatic acylation of cyclic alcohol

NASA Astrophysics Data System (ADS)

Iftitah, Elvina Dhiaul; Srihardyastuti, Arie; Ariefin, Mokhamat

2017-03-01

We report the activity of Rhizomuchor miehei lipase (RML) as a biocatalyst, in particular the investigations concerning the effort of substrate-structure reactivity on the enzymatic acylation. The acylation was studied using acetic anhydride as an acyl donor and performed in n-hexane as a solvent. The selectivity of the enzymatic acylation was revealed by Gas Chromatography-Mass Spectra. We observed that, RML has shown different behavior when catalyzing the acylation of isopulegol and mixture of isopulegol and citronellal (ratio 1:1). The chemoselectivity for the O-acylation was improved when the acyl acceptor included mixture of isopulegol and citronellal

Performance of glucose/O2 enzymatic fuel cell based on supporting electrodes over-coated by polymer-nanogold particle composite with entrapped enzymes

NASA Astrophysics Data System (ADS)

Huo, W. S.; Zeng, H.; Yang, Y.; Zhang, Y. H.

2017-03-01

Enzymatic electrodes over-coated by thin film of nano-composite made up of polymer and functionalized nano-gold particle was prepared. Glucose/O2 membrane-free enzymatic fuel cell based on nano-composite based electrodes with incorporated glucose oxidase and laccase was assembled. This enzymatic fuel cell exhibited high energy out-put density even when applied in human serum. Catalytic cycle involved in enzymatic fuel cell was limited by oxidation of glucose occurred on bioanode resulting from impact of sophisticated interaction between active site in glucose oxidase and nano-gold particle on configuration of redox center of enzyme molecule which crippled catalytic efficiency of redox protein.

Interleukin-1beta and interleukin-6 disturb the antioxidant enzyme system in bovine chondrocytes: a possible explanation for oxidative stress generation.

PubMed

Mathy-Hartert, M; Hogge, L; Sanchez, C; Deby-Dupont, G; Crielaard, J M; Henrotin, Y

2008-07-01

Beside matrix metalloproteinases, reactive oxygen species (ROS) are the main biochemical factors of cartilage degradation. To prevent ROS toxicity, chondrocytes possess a well-coordinated enzymatic antioxidant system formed principally by superoxide dismutases (SODs), catalase (CAT) and glutathione peroxidase (GPX). This work was designed to assess the effects of interleukin (IL)-1beta and IL-6 on the enzymatic activity and gene expression of SODs, CAT and GPX in bovine chondrocytes. Bovine chondrocytes were cultured in monolayer for 4-96 h in the absence or in the presence of IL-1beta (0.018-1.8ng/ml) or IL-6 (10-100 ng/ml). To study signal transduction pathway, inhibitors of mitogen-activated protein kinases (MAPK) (PD98059, SB203580 and SP600125) (5-20 microM) and nuclear factor (NF)-kappaB inhibitors [BAY11-7082 (1-10 microM) and MG132 (0.1-10 microM)] were used. SODs, CAT and GPX enzymatic activities were evaluated in cellular extract by using colorimetric enzymatic assays. Mn SODs, Cu/Zn SOD, extracellular SOD (EC SOD), CAT and GPX gene expressions were quantified by real-time and quantitative polymerase chain reaction (PCR). Mn SOD and GPX activities were dose and time-dependently increased by IL-1beta. In parallel, IL-1beta markedly enhanced Mn SOD and GPX gene expressions, but decreased Cu/Zn SOD, EC SOD and CAT gene expressions. Induction of SOD enzymatic activity and Mn SOD mRNA expression were inhibited by NF-kappaB inhibitors but not by MAPK inhibitors. IL-6 effects were similar but weaker than those of IL-1beta. In conclusion, IL-1beta, and to a lesser extend IL-6, dysregulates enzymatic antioxidant defenses in chondrocyte. These changes could lead to a transient accumulation of H(2)O(2) in mitochondria, and consequently to mitochondria damage. These changes contribute to explain the mitochondrial dysfunction observed in osteoarthritis chondrocytes.

How Mg2+ ions lower the SN2@P barrier in enzymatic triphosphate hydrolysis.

PubMed

van Bochove, Marc A; Roos, Goedele; Fonseca Guerra, Célia; Hamlin, Trevor A; Bickelhaupt, F Matthias

2018-04-03

Our quantum chemical activation strain analyses demonstrate how Mg2+ lowers the barrier of the enzymatic triphosphate hydrolysis through two distinct mechanisms: (a) weakening of the leaving-group bond, thereby decreasing activation strain; and (b) transition state (TS) stabilization through enhanced electrophilicity of the triphosphate PPP substrate, thereby strengthening the interaction with the nucleophile.

Bioremediation of cooking oil waste using lipases from wastes

PubMed Central

do Prado, Débora Zanoni; Facanali, Roselaine; Marques, Márcia Mayo Ortiz; Nascimento, Augusto Santana; Fernandes, Célio Junior da Costa; Zambuzzi, William Fernando

2017-01-01

Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases. PMID:29073166

Bioremediation of cooking oil waste using lipases from wastes.

PubMed

Okino-Delgado, Clarissa Hamaio; Prado, Débora Zanoni do; Facanali, Roselaine; Marques, Márcia Mayo Ortiz; Nascimento, Augusto Santana; Fernandes, Célio Junior da Costa; Zambuzzi, William Fernando; Fleuri, Luciana Francisco

2017-01-01

Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases.

Show Yourself, Asparaginase: An Enzymatic Reaction Explained through a Hands-On Interactive Activity

PubMed Central

2017-01-01

Determining the catalytic activity of an enzyme can be the perfect method for its identification, for example during purification procedures or for isolation purposes. Herein, we used a pharmaceutically relevant protein to bring the concept of enzymatic activity to the classroom. We designed a hands-on interactive activity in which a medically relevant enzyme, asparaginase, was distinguished from a nonenzymatic protein based on its specific enzymatic activity. The experiment was carried out in the classroom, designed to impact different educational levels from elementary to high school. Our main purposes were to promote the emerging field of protein-based drugs as a source of scientific careers in bionanotechnology and to show the students an image of a “scientist” as that of a common and educated person working in an exciting profession. In addition of being inexpensive, this activity proved to be adaptable for various educational levels and can be easily implemented in different scenarios, for example, scientific fairs, some schools, and so forth. PMID:29599566

The Oxidative Metabolism of Fossil Hydrocarbons and Sulfide Minerals by the Lithobiontic Microbial Community Inhabiting Deep Subterrestrial Kupferschiefer Black Shale.

PubMed

Włodarczyk, Agnieszka; Lirski, Maciej; Fogtman, Anna; Koblowska, Marta; Bidziński, Grzegorz; Matlakowska, Renata

2018-01-01

Black shales are one of the largest reservoirs of fossil organic carbon and inorganic reduced sulfur on Earth. It is assumed that microorganisms play an important role in the transformations of these sedimentary rocks and contribute to the return of organic carbon and inorganic sulfur to the global geochemical cycles. An outcrop of deep subterrestrial ~256-million-year-old Kupferschiefer black shale was studied to define the metabolic processes of the deep biosphere important in transformations of organic carbon and inorganic reduced sulfur compounds. This outcrop was created during mining activity 12 years ago and since then it has been exposed to the activity of oxygen and microorganisms. The microbial processes were described based on metagenome and metaproteome studies as well as on the geochemistry of the rock. The microorganisms inhabiting the subterrestrial black shale were dominated by bacterial genera such as Pseudomonas, Limnobacter, Yonghaparkia, Thiobacillus, Bradyrhizobium , and Sulfuricaulis . This study on black shale was the first to detect archaea and fungi, represented by Nitrososphaera and Aspergillus genera, respectively. The enzymatic oxidation of fossil aliphatic and aromatic hydrocarbons was mediated mostly by chemoorganotrophic bacteria, but also by archaea and fungi. The dissimilative enzymatic oxidation of primary reduced sulfur compounds was performed by chemolithotrophic bacteria. The geochemical consequences of microbial activity were the oxidation and dehydrogenation of kerogen, as well as oxidation of sulfide minerals.

The hidden impact of forest management on the decomposition of soil organic matter

NASA Astrophysics Data System (ADS)

Schöning, Ingo; Schrumpf, Marion

2017-04-01

Decomposition in soils is a key ecosystem function. Extracellular enzymes mediate the decomposition of soil organic matter and the mineralization of carbon (C), nitrogen (N), sulfur (S) and phosphorus (P). Forest management is assumed to affect decomposition processes through tree species selection, thinning and harvesting. In this study, we assessed the impact of forest management on the magnitude of soil enzymatic activities and soil respiration using the silvicultural management intensity indicator (SMI) introduced by Schall & Ammer (2013). We collected mineral soil samples (0-10 cm) from 150 forest plots in three different German regions (Schorfheide-Chorin, Hainich-Dün, Schwäbische Alb) and determined basic properties such as pH, soil texture, soil C and N contents. An aliquot of each soil sample was used to determine potential activities of enzymes involved in the C, N, P and S cycle (ß-glucosidase, N-actyl-glucosaminidase, phosphatase, sulfatase). Another aliquot was incubated (20 ̊C, 60% WHC) for 14 days and the evolving CO2 was determined. The main drivers of potential enzymatic activities and soil respiration were the site conditions such as clay contents and pH values. The effects of forest management were much lower but still significant. This shows that forest management has an impact on decomposition which is only detectable with high number of replicates.

Purification and properties of beta-galactosidase from Aspergillus nidulans.

PubMed

Díaz, M; Pedregosa, A M; de Lucas, J R; Torralba, S; Monistrol, I F; Laborda, F

1996-12-01

Beta-Galactosidase from mycelial extract of Aspergillus nidulans has been purified by substrate affinity chromatography and used to obtain anti-beta-galactosidase polyclonal antibodies. A. nidulans growing in lactose as carbon source synthesizes one active form of beta-galactosidase which seems to be a multimeric enzyme of 450 kDa composed of monomers with 120 and 97 kDa. Although the enzyme was not released to the culture medium, some enzymatic activity was detected in a cell-wall extract, thus suggesting that it can be an extracellular enzyme. Beta-Galactosidase of A. nidulans is a very unstable enzyme with an optimum pH value of 7.5 and an optimum temperature of 30 degrees C. It was only active against beta-galactoside substrates like lactose and p-nitrophenyl-beta-D-galactoside (PNPG).

Ureide metabolism during seedling development in French bean (Phaseolus vulgaris).

PubMed

Quiles, Francisco Antonio; Raso, María José; Pineda, Manuel; Piedras, Pedro

2009-01-01

French bean (Phaseolus vulgaris) is a legume that transports most of the atmospheric nitrogen fixed in its nodules to the aerial parts of the plant as ureides. Changes in ureide content and in enzymatic activities involved in their metabolism were identified in the cotyledons and embryonic axes during germination and early seedling development. Accumulation of ureides (ca. 1300 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds. Throughout germination, the total amount of ureides slightly decreased to about 1200 nmol, but increased both in cotyledons and in embryonic axes after radicle emergence. In the axes, the ureides were almost equally distributed in roots, hypocotyls and epicotyls. The pattern of ureide distribution was not affected by the presence of nitrate or sucrose in the media up to 6 days after imbibition. Ureides are synthesized from purines because allopurinol (a xanthine dehydrogenase inhibitor) blocks the increase of ureides. Allantoin and allantoate-degrading activities were detected in French bean dried seeds, whereas no ureidoglycolate-degrading activity was detected. During germination, the levels of the three activities remain unchanged in cotyledons. After radicle emergence, the levels of activities in cotyledons changed. Allantoin-degrading activity increased, allantoate-degrading activity decreased and ureidoglycolate-degrading activity remained undetectable in cotyledons. In developing embryonic axes, the three activities were detected throughout germination and early seedling development. The embryonic axes are able to synthesize ureides, because those compounds accumulated in axes without cotyledons.

Monoamine oxidase B layer-by-layer film fabrication and characterization toward dopamine detection.

PubMed

Miyazaki, Celina Massumi; Pereira, Tamyris Paschoal; Mascagni, Daniela Branco Tavares; de Moraes, Marli Leite; Ferreira, Marystela

2016-01-01

In this work nanostructured film composites of the monoamine oxidase B (MAO-B) enzyme, free or encapsulated in liposomes, were fabricated by the layer-by-layer (LbL) self-assembly technique, employing polyethylene imine (PEI) as polycation. Initially, the MAO-B enzyme was incorporated into liposomes in order to preserve its enzymatic structure ensuring their activity and catalytic stability. The LbL film growth was monitored by surface plasmon resonance (SPR) by gold resonance angle shift analysis after each bilayer deposition. Subsequently, the films were applied as amperometric biosensors for dopamine detection using Prussian Blue (PB) as the electron mediator. The biosensor fabricated by MAO-B incorporated into liposomes composed of DPPG:POPG in the ratio (1:4) (w/w) showed the best performance with a sensitivity of 0.86 (μA cm(-2))/(mmol L(-1)) and a detection limit of 0.33 mmol L(-1).

Detection of damaged DNA bases by DNA glycosylase enzymes.

PubMed

Friedman, Joshua I; Stivers, James T

2010-06-22

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we call the search complex (SC). Sliding is frequently punctuated by the formation of a transient "interrogation" complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.

Mycoplasma agalactiae MAG_5040 is a Mg2+-Dependent, Sugar-Nonspecific SNase Recognised by the Host Humoral Response during Natural Infection

PubMed Central

Cacciotto, Carla; Addis, Maria Filippa; Coradduzza, Elisabetta; Carcangiu, Laura; Nuvoli, Anna Maria; Tore, Gessica; Dore, Gian Mario; Pagnozzi, Daniela; Uzzau, Sergio; Chessa, Bernardo; Pittau, Marco; Alberti, Alberto

2013-01-01

In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants. PMID:23469065

Long-term effects of nickel oxide nanoparticles on performance, microbial enzymatic activity, and microbial community of a sequencing batch reactor.

PubMed

Wang, Sen; Li, Zhiwei; Gao, Mengchun; She, Zonglian; Guo, Liang; Zheng, Dong; Zhao, Yangguo; Ma, Bingrui; Gao, Feng; Wang, Xuejiao

2017-02-01

The nitrogen and phosphorus removal, microbial enzymatic activity, and microbial community of a sequencing batch reactor (SBR) were evaluated under long-term exposure to nickel oxide nanoparticles (NiO NPs). High NiO NP concentration (over 5 mg L -1 ) affected the removal of chemical oxygen demand, nitrogen, and phosphorus. The presence of NiO NP inhibited the microbial enzymatic activities and reduced the nitrogen and phosphorus removal rates of activated sludge. The microbial enzymatic activities of the activated sludge showed a similar variation trend to the nitrogen and phosphorus removal rates with the increase in NiO NP concentration from 0 to 60 mg L -1 . The Ni content in the effluent and activated sludge showed an increasing trend with the increase in NiO NP concentration. Some NiO NPs were absorbed on the sludge surface or penetrate the cell membrane into the interior of microbial cells in the activated sludge. NiO NP facilitated the increase in reactive oxygen species by disturbing the balance between the oxidation and anti-oxidation processes, and the variation in lactate dehydrogenase demonstrated that NiO NP could destroy the cytomembrane and cause variations in the microbial morphology and physiological function. High-throughput sequencing demonstrated that the microbial community of SBR had some obvious changes at 0-60 mg L -1 NiO NPs at the phyla, class and genus levels. Copyright © 2016 Elsevier Ltd. All rights reserved.

D-Amino acid oxidase bio-functionalized platforms: Toward an enhanced enzymatic bio-activity

NASA Astrophysics Data System (ADS)

Herrera, Elisa; Valdez Taubas, Javier; Giacomelli, Carla E.

2015-11-01

The purpose of this work is to study the adsorption process and surface bio-activity of His-tagged D-amino acid oxidase (DAAO) from Rhodotorula gracilis (His6-RgDAAO) as the first step for the development of an electrochemical bio-functionalized platform. With such a purpose this work comprises: (a) the His6-RgDAAO bio-activity in solution determined by amperometry, (b) the adsorption mechanism of His6-RgDAAO on bare gold and carboxylated modified substrates in the absence (substrate/COO-) and presence of Ni(II) (substrate/COO- + Ni(II)) determined by reflectometry, and (c) the bio-activity of the His6-RgDAAO bio-functionalized platforms determined by amperometry. Comparing the adsorption behavior and bio-activity of His6-RgDAAO on these different solid substrates allows understanding the contribution of the diverse interactions responsible for the platform performance. His6-RgDAAO enzymatic performance in solution is highly improved when compared to the previously used pig kidney (pk) DAAO. His6-RgDAAO exhibits an amperometrically detectable bio-activity at concentrations as low as those expected on a bio-functional platform; hence, it is a viable bio-recognition element of D-amino acids to be coupled to electrochemical platforms. Moreover, His6-RgDAAO bio-functionalized platforms exhibit a higher surface activity than pkDAAO physically adsorbed on gold. The platform built on Ni(II) modified substrates present enhanced bio-activity because the surface complexes histidine-Ni(II) provide with site-oriented, native-like enzymes. The adsorption mechanism responsible of the excellent performance of the bio-functionalized platform takes place in two steps involving electrostatic and bio-affinity interactions whose prevalence depends on the degree of surface coverage.

Effect of structural modifications of ganglioside GM2 on intra-molecular carbohydrate-to-carbohydrate interaction and enzymatic susceptibility

PubMed Central

Li, Yu-Teh; Li, Su-Chen; Kiso, Makoto; Ishida, Hideharu; Mauri, Laura; Raimondi, Laura; Bernardi, Anna; Sonnino, Sandro

2008-01-01

Summary The effect of inter-molecular carbohydrate-to-carbohydrate interaction on basic cell biological processes has been well documented and appreciated. In contrast, very little is known about the intra-molecular carbohydrate-to-carbohydrate interaction. The presence of an interaction between the GalNAc and the Neu5Ac in GM2 detected by NMR spectroscopy represents a well-defined intra-molecular carbohydrate-to-carbohydrate interaction. This intriguing interaction is responsible for the GM2-epitope, GalNAcβ1Π4(Neu5Acα2Π3)Gal-, to exhibit a rigid and compact conformation. We hypothesized that this compact conformation may be the cause for both the GalNAc and the Neu5Ac in GM2 to be refractory to enzymatic hydrolysis and the GM2 activator protein is able to interact with the compact trisaccharide GM2-epitope, rendering the GalNAc and the Neu5Ac accessible to β-hexosaminidase A and sialidase. We have used a series of structurally modified GM2 to study the effect of modifications of sugar chains on the conformation and enzymatic susceptibility of this ganglioside. Our hypothesis was borne out by the fact that when the GalNAcβ1Π4Gal linkage in GM2 was converted to the GalNAcβ1Π6Gal, both the GalNAc and the Neu5Ac became susceptible to β-hexosaminidase A and sialidase, respectively, without GM2 activator protein. We hope our work will engender interest in identifying other intra-molecular carbohydrate-to-carbohydrate interactions in glycoconjugates. PMID:17967427

Real-time investigation of mannosyltransferase function of a Xylella fastidiosa recombinant GumH protein using QCM-D.

PubMed

Alves, Claudia A; Pedroso, Mariele M; de Moraes, Marcela C; Souza, Dulce H F; Cass, Quezia B; Faria, Ronaldo C

2011-05-20

Xylella fastidiosa is a gram-negative bacterium that causes serious diseases in economically important crops, including grapevine, coffee, and citrus fruits. X. fastidiosa colonizes the xylem vessels of the infected plants, thereby blocking water and nutrient transport. The genome sequence of X. fastidiosa has revealed an operon containing nine genes possibly involved in the synthesis of an exopolisaccharide (EPS) named fastidian gum that can be related with the pathogenicity of this bacterium. The α-1,3-mannosyltransferase (GumH) enzyme from X. fastidiosa is involved in fastidian gum production. GumH is responsible for the transfer of mannose from guanosine diphosphate mannose (GDP-man) to the cellobiose-pyrophosphate-polyprenol carrier lipid (CPP-Lip) during the assembly and biosynthesis of EPS. In this work, a method for real-time detection of recombinant GumH enzymatic activity was successfully developed using a Quartz Crystal Microbalance with dissipation monitoring (QCM-D). The QCM-D transducer was strategically modified with CPP-Lip by using a solid-supported lipid bilayer that makes use of a self-assembled monolayer of 1-undecanethiol. Monitoring the real-time CPP-Lip QCM-D transducer in the presence of GDP-man and GumH enzyme shows a mass increase, indicating the transfer of mannose. The real-time QCM-D determination of mannosyltransferase function was validated by a High Performance Liquid Chromatography (LC) method developed for determination of GDP produced by enzymatic reaction. LC results confirmed the activity of recombinant GumH protein, which is the first enzyme involved in the biosynthesis of the EPS from X. fastidiosa enzymatically characterized. Copyright © 2011 Elsevier Inc. All rights reserved.

Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines.

PubMed

Hanavan, Paul D; Borges, Chad R; Katchman, Benjamin A; Faigel, Douglas O; Ho, Thai H; Ma, Chen-Ting; Sergienko, Eduard A; Meurice, Nathalie; Petit, Joachim L; Lake, Douglas F

2015-07-30

Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a "proof-of-principle" that enzymatic inhibition of QSOX1 may have clinical relevancy.

Binding of Nickel to Testicular Glutamate–Ammonia Ligase Inhibits Its Enzymatic Activity

PubMed Central

SUN, YINGBIAO; OU, YOUNG; CHENG, MIN; RUAN, YIBING; VAN DER HOORN, FRANS A.

2016-01-01

SUMMARY Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate–ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure. PMID:21254280

Signal-on electrochemical assay for label-free detection of TdT and BamHI activity based on grown DNA nanowire-templated copper nanoclusters.

PubMed

Hu, Yufang; Zhang, Qingqing; Xu, Lihua; Wang, Jiao; Rao, Jiajia; Guo, Zhiyong; Wang, Sui

2017-11-01

Electrochemical methods allow fast and inexpensive analysis of enzymatic activity. Here, a simple and yet efficient "signal-on" electrochemical assay for sensitive, label-free detection of DNA-related enzyme activity was established on the basis of terminal deoxynucleotidyl transferase (TdT)-mediated extension strategy. TdT, which is a template-independent DNA polymerase, can catalyze the sequential addition of deoxythymidine triphosphate (dTTP) at the 3'-OH terminus of single-stranded DNA (ssDNA); then, the TdT-yield T-rich DNA nanowires can be employed as the synthetic template of copper nanoclusters (CuNCs). Grown DNA nanowires-templated CuNCs (noted as DNA-CuNCs) were attached onto graphene oxide (GO) surface and exhibited unique electrocatalytic activity to H 2 O 2 reduction. Under optimal conditions, the proposed biosensor was utilized for quantitatively monitoring TdT activity, with the observed LOD of 0.1 U/mL. It also displayed high selectivity to TdT with excellent stability, and offered a facile, convenient electrochemical method for TdT-relevant inhibitors screening. Moreover, the proposed sensor was successfully used for BamHI activity detection, in which a new 3'-OH terminal was exposed by the digestion of a phosphate group. Ultimately, it has good prospects in DNA-related enzyme-based biochemical studies, disease diagnosis, and drug discovery. Graphical Abstract Extraordinary TdT-generated DNA-CuNCs are synthesized and act as a novel electrochemical sensing platform for sensitive detection of TdT and BamHI activity in biological environments.

Enzymatic activity in the surface microlayer and subsurface water in the harbour channel

NASA Astrophysics Data System (ADS)

Perliński, Piotr; Mudryk, Zbigniew J.; Antonowicz, Józef

2017-09-01

Hydrolytic activity of eight extracellular enzymes was determined spectrofluorimetric method in the surface microlayer and subsurface water in the harbour channel in Ustka. The ranking order of the potential enzyme activity rates in the studied water layers was as follows: lipase > phosphatase > aminopeptidase > β-glucosidase > α-glucosidase > xylanase > cellulase > chitinase. The level of activity of all studied hydrolases was higher in the surface microlayer than subsurface water. No clear gradients in the level of enzymatic activity were determined along the horizontal profile of the studied channel. Activity of extracellular enzymes was strongly influenced by the season.

Development of a Novel Separase Inhibitor, Sepin 1, for Breast Cancer Therapy

DTIC Science & Technology

2016-06-01

Wolinowska,  R.;  Tyski,  S.,  Synthesis  and  in  vitro  Antibacterial   Activity   of  5‐Halogenomethylsulfonyl‐  Benzimidazole and Benzotriazole...incidence of relapse, metastasis, and a lower 5-year overall survival rate. We hypothesize that modulation of Separase enzymatic activity constitutes a...mammary tumorigenesis, and intratumoral heterogeneity in mice.4,6 We hypothesize that modulation of Separase enzymatic activity constitutes a new

Effects of Pulsed Electric Field (PEF) Treatment on Enhancing Activity and Conformation of α-Amylase.

PubMed

Tian, Mei-ling; Fang, Ting; Du, Mu-ying; Zhang, Fu-sheng

2016-04-01

To explore an efficient, safe, and speedy application of pulsed electric field (PEF) technology for enzymatic modification, effects of PEF treatment on the enzymatic activity, property and kinetic parameters of α-amylase were investigated. Conformational transitions were also studied with the aid of circular dichroism (CD) and fluorescence spectra. The maximum enzymatic activity of α-amylase was obtained under 15 kV/cm electric field intensity and 100 mL/min flow velocity PEF treatment, in which the enzymatic activity increased by 22.13 ± 1.14% compared with control. The activation effect could last for 18 h at 4 °C. PEF treatment could widen the range of optimum temperature for α-amylase, however, it barely exerted any effect on the optimum pH. On the other hand, α-amylase treated by PEF showed an increase of Vmax, t1/2 and ΔG, whereas a decrease of Km and k were observed. Furthermore, it can be observed from fluorescence and CD spectra that PEF treatment had increased the number of amino acid residues, especially that of tryptophan, on α-amylase surface with enhanced α-helices by 34.76% and decreased random coil by 12.04% on α-amylase when compared with that of untreated. These changes in structure had positive effect on enhancing α-amylase activity and property.

Enzymatically Active Microgels from Self-Assembling Protein Nanofibrils for Microflow Chemistry.

PubMed

Zhou, Xiao-Ming; Shimanovich, Ulyana; Herling, Therese W; Wu, Si; Dobson, Christopher M; Knowles, Tuomas P J; Perrett, Sarah

2015-06-23

Amyloid fibrils represent a generic class of protein structure associated with both pathological states and with naturally occurring functional materials. This class of protein nanostructure has recently also emerged as an excellent foundation for sophisticated functional biocompatible materials including scaffolds and carriers for biologically active molecules. Protein-based materials offer the potential advantage that additional functions can be directly incorporated via gene fusion producing a single chimeric polypeptide that will both self-assemble and display the desired activity. To succeed, a chimeric protein system must self-assemble without the need for harsh triggering conditions which would damage the appended functional protein molecule. However, the micrometer to nanoscale patterning and morphological control of protein-based nanomaterials has remained challenging. This study demonstrates a general approach for overcoming these limitations through the microfluidic generation of enzymatically active microgels that are stabilized by amyloid nanofibrils. The use of scaffolds formed from biomaterials that self-assemble under mild conditions enables the formation of catalytic microgels while maintaining the integrity of the encapsulated enzyme. The enzymatically active microgel particles show robust material properties and their porous architecture allows diffusion in and out of reactants and products. In combination with microfluidic droplet trapping approaches, enzymatically active microgels illustrate the potential of self-assembling materials for enzyme immobilization and recycling, and for biological flow-chemistry. These design principles can be adopted to create countless other bioactive amyloid-based materials with diverse functions.

Understanding fibroblast activation protein (FAP): substrates, activities, expression and targeting for cancer therapy.

PubMed

Hamson, Elizabeth J; Keane, Fiona M; Tholen, Stefan; Schilling, Oliver; Gorrell, Mark D

2014-06-01

Fibroblast activation protein (FAP) is best known for its heightened expression in tumour stroma. This atypical serine protease has both dipeptidyl peptidase and endopeptidase activities, cleaving substrates at a post-proline bond. FAP expression is difficult to detect in non-diseased adult organs, but is greatly upregulated in sites of tissue remodelling, which include liver fibrosis, lung fibrosis, atherosclerosis, arthritis, tumours and embryonic tissues. Due to its restricted expression pattern and dual enzymatic activities, FAP is emerging as a unique therapeutic target. However, methods to exploit and target this protease are advancing more rapidly than knowledge of the fundamental biology of FAP. This review highlights this imbalance, emphasising the need to better define the substrate repertoire and expression patterns of FAP to elucidate its role in biological and pathological processes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ultrathin NiCo2O4 nanowalls supported on a 3D nanoporous gold coated needle for non-enzymatic amperometric sensing of glucose.

PubMed

Li, Weiwei; Qi, Hui; Wang, Baogang; Wang, Qiyu; Wei, Shuting; Zhang, Xiaolin; Wang, Ying; Zhang, Lei; Cui, Xiaoqiang

2018-01-24

A disposable needle-type of hybrid electrode was prepared from a core of stainless steel needle whose surface was modified with a 3D nanoporous gold/NiCo 2 O 4 nanowall hybrid structure for electrochemical non-enzymatic glucose detection. This hybrid electrode, best operated at 0.45 V (vs. SCE) in solutions of pH 13 has a linear response in the 0.01 to 21 mM glucose concentration range, a response time of <1 s, and a 1 μM detection limit (at an S/N ratio of 3). The remarkable enhancement compared to the solid gold/NiCo 2 O 4 and stainless steel/NiCo 2 O 4 hybrid electrodes in electrochemical performance is assumed to originate from the good electrical conductivity and large surface area of the hybrid electrode, which enhance the transport of mass and charge during electrochemical reactions. This biosensor was also applied to real sample analysis with little interferences. The electrode is disposable and considered to be a promising tool for non-enzymatic sensing of glucose in a variety of practical situations. Graphical abstract Ultrathin NiCo 2 O 4 nanowalls supported on nanoporous gold that is coated on a stainless steel needle was fabricated for sensitive non-enzymatic amperometric sensing of glucose.

Impact of potato cultivation and cattle farming on physicochemical parameters and enzymatic activities of Neotropical high Andean Páramo ecosystem soils.

PubMed

Avellaneda-Torres, Lizeth Manuela; León Sicard, Tomás Enrique; Torres Rojas, Esperanza

2018-08-01

The Andean Páramos are high mountain ecosystems whose soils are essential for the management of South American water resources, but research on anthropic impacts to these soils is currently minimal and insufficient. The objective of this study was to evaluate the impacts of potato (Solanum tuberosum) cultivation and livestock on the physicochemical parameters and enzymatic activities that determine the soil quality of the Neotropical high Andean Páramo ecosystem in the Nevados National Natural Park (Nevados NNP) in Colombia. It was hypothesised that sites with potato crops and livestock farming would exhibit significant changes in soil physicochemical parameters and enzymatic activities compared with Páramo sites that have been conserved without agriculture. Samples were collected from soils under potato cultivation, livestock and Páramo (subject to the lowest degree of human intervention possible), on three farms in the El Bosque District at three different altitudes (Buenos Aires, El Edén and La Secreta) during two seasons (dry and rainy). The results showed that none of the physical parameters under study presented statistically significant differences due to the type of use (livestock, potato crop or Páramo), season of sampling (dry or rainy season) or altitude (different farms). The chemical parameters that statistically significantly differed due to land use were organic carbon, cation exchange capacity, calcium, potassium, and ammonium and those that showed statistically significant differences associated with the sampling timing were organic carbon, nitrogen, cation exchange capacity, total carbon, C/N and nitrate. Additionally, there were differences in organic carbon due to the altitude of the farms. With respect to enzymatic activities, those of β-glucosidase, phosphodiesterase and urease significantly decreased in soils under potato cultivation and livestock relative to those of Páramo, but those of acid phosphatase and protease increased significantly under potato cropping and livestock. The activities of β-glucosidase, acid phosphatase, alkaline phosphatase, phosphodiesterase and protease were higher during the dry season than the rainy season, and the activities of β-glucosidase, acid phosphatase and urease decreased statistically in the lower-altitude farm (La Secreta). These decreases in enzymatic activities are attributable to changes in the organic carbon of the soil. This study provides a novel insight on the relationships between land use and the physicochemical parameters and enzymatic activities of Páramo soils (which have been minimally studied to date) at different altitudes and during different seasons. The results suggest that changes in agricultural practices should be implemented to maintain the organic carbon of soil and, therefore, its enzymatic activities. Copyright © 2018 Elsevier B.V. All rights reserved.

Ultrasensitive dual-channel detection of matrix metalloproteinase-2 in human serum using gold-quantum dot core-satellite nanoprobes.

PubMed

Zheng, Tingting; Zhang, Rui; Zhang, Qingfeng; Tan, Tingting; Zhang, Kui; Zhu, Jun-Jie; Wang, Hui

2013-09-18

We have developed a robust enzymatic peptide cleavage-based assay for the ultrasensitive dual-channel detection of matrix metalloproteinase-2 (MMP-2) in human serum using gold-quantum dot (Au-QD) core-satellite nanoprobes.

Facile synthesis of a silver nanoparticles/polypyrrole nanocomposite for non-enzymatic glucose determination.

PubMed

Poletti Papi, Maurício A; Caetano, Fabio R; Bergamini, Márcio F; Marcolino-Junior, Luiz H

2017-06-01

The present work describes the synthesis of a new conductive nanocomposite based on polypyrrole (PPy) and silver nanoparticles (PPy-AgNP) based on a facile reverse microemulsion method and its application as a non-enzymatic electrochemical sensor for glucose detection. Focusing on the best sensor performance, all experimental parameters used in the synthesis of nanocomposite were optimized based on its electrochemical response for glucose. Characterization of the optimized material by FT-IR, cyclic voltammetry, and DRX measurements and TEM images showed good monodispersion of semispherical Ag nanoparticles capped by PPy structure, with size average of 12±5nm. Under the best analytical conditions, the proposed sensor exhibited glucose response in linear dynamic range of 25 to 2500μmolL -1 , with limit of detection of 3.6μmolL -1 . Recovery studies with human saliva samples varying from 99 to 105% revealed the accuracy and feasibility of a non-enzymatic electrochemical sensor for glucose determination by easy construction and low-cost. Copyright © 2017 Elsevier B.V. All rights reserved.

[Detection of UGT1A1*28 Polymorphism Using Fragment Analysis].

PubMed

Huang, Ying; Su, Jian; Huang, Xiaosui; Lu, Danxia; Xie, Zhi; Yang, Suqing; Guo, Weibang; Lv, Zhiyi; Wu, Hongsui; Zhang, Xuchao

2017-12-20

Uridine-diphosphoglucuronosyl transferase 1A1 (UGT1A1), UGT1A1*28 polymorphism can reduce UGT1A1 enzymatic activity, which may lead to severe toxicities in patients who receive irinotecan. This study tries to build a fragment analysis method to detect UGT1A1*28 polymorphism. A total of 286 blood specimens from the lung cancer patients who were hospitalized in Guangdong General Hospital between April 2014 to May 2015 were detected UGT1A1*28 polymorphism by fragment analysis method. Comparing with Sanger sequencing, precision and accuracy of the fragment analysis method were 100%. Of the 286 patients, 236 (82.5% harbored TA6/6 genotype, 48 (16.8%) TA 6/7 genotype and 2 (0.7%) TA7/7 genotype. Our data suggest hat the fragment analysis method is robust for detecting UGT1A1*28 polymorphism in clinical practice. It's simple, time-saving, and easy-to-carry.

Nanoplasmonic Biosensor Using Localized Surface Plasmon Resonance Spectroscopy for Biochemical Detection.

PubMed

Zhang, Diming; Zhang, Qian; Lu, Yanli; Yao, Yao; Li, Shuang; Liu, Qingjun

2017-01-01

Localized surface plasmon resonance (LSPR) associated with metal nanostructures has developed into a highly useful sensor technique. Optical LSPR spectroscopy of nanostructures often shows sharp absorption and scattering peaks, which can be used to probe several bio-molecular interactions. Here, we report nanoplasmonic biosensors using LSPR on nanocup arrays (nanoCA) to recognize bio-molecular binding for biochemical detection. These sensors can be modified to quantify binding of small molecules to proteins for odorant and explosive detections. Electrochemical LSPR biosensors can also be designed by coupling electrochemistry and LSPR spectroscopy measurements. Multiple sensing information can be obtained and electrochemical LSPR property can be investigated for biosensors. In some applications, the electrochemical LSPR biosensor can be used to quantify immunoreactions and enzymatic activity. The biosensors exhibit better performance than those of conventional optical LSPR measurements. With multi-transducers, the nanoplasmonic biosensor can provide a promising approach for bio-detection in environmental monitoring, healthcare diagnostics, and food quality control.

Pitfalls in the detection of cholesterol in Huntington’s disease models

PubMed Central

Marullo, Manuela; Valenza, Marta; Leoni, Valerio; Caccia, Claudio; Scarlatti, Chiara; De Mario, Agnese; Zuccato, Chiara; Di Donato, Stefano; Carafoli, Ernesto; Cattaneo, Elena

2012-01-01

Background Abnormalities in brain cholesterol homeostasis have been reported in Huntington’s disease (HD), an adult-onset neurodegenerative disorder caused by an expansion in the number of CAG repeats in the huntingtin (HTT) gene. However, the results have been contradictory with respect to whether cholesterol levels increase or decrease in HD models. Biochemical and mass spectrometry methods show reduced levels of cholesterol precursors and cholesterol in HD cells and in the brains of several HD animal models. Abnormal brain cholesterol homeostasis was also inferred from studies in HD patients. In contrast, colorimetric and enzymatic methods indicate cholesterol accumulation in HD cells and tissues. Here we used several methods to investigate cholesterol levels in cultured cells in the presence or absence of mutant HTT protein. Results Colorimetric and enzymatic methods with low sensitivity gave variable results, whereas results from a sensitive analytical method, gas chromatography-mass spectrometry, were more reliable. Sample preparation, high cell density and cell clonality also influenced the detection of intracellular cholesterol. Conclusions Detection of cholesterol in HD samples by colorimetric and enzymatic assays should be supplemented by detection using more sensitive analytical methods. Care must be taken to prepare the sample appropriately. By evaluating lathosterol levels using isotopic dilution mass spectrometry, we confirmed reduced cholesterol biosynthesis in knock-in cells expressing the polyQ mutation in a constitutive or inducible manner. *Correspondence should be addressed to Elena Cattaneo: elena.cattaneo@unimi.it PMID:23145355

Detection of benzo[a]pyrene diol epoxide-DNA adducts in peripheral blood lymphocytes and antibodies to the adducts in serum from coke oven workers.

PubMed Central

Harris, C C; Vahakangas, K; Newman, M J; Trivers, G E; Shamsuddin, A; Sinopoli, N; Mann, D L; Wright, W E

1985-01-01

Coke oven workers are exposed to high levels of carcinogenic polycyclic aromatic hydrocarbons, including benzo[a]pyrene (B[a]P), and are at increased risk of lung cancer. Since B[a]P is enzymatically activated to 7 beta,8 alpha-dihydroxy(9 alpha, 10 alpha)epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE) that forms adducts with DNA, the presence of these adducts was measured in DNA from peripheral blood lymphocytes by synchronous fluorescence spectrophotometry and enzyme radioimmunoassay. Approximately two-thirds of the workers had detectable levels of B[a]PDE-DNA adducts. Antibodies to the DNA adducts were also found in the serum of 27% of the workers. B[a]PDE-DNA adducts were not detectable in lymphocytes and antibodies to the adducts were not detected in sera from a control group of nonsmoking laboratory workers. DNA adducts and/or antibodies to the adducts indicate exposure to B[a]P and its metabolic activation to the carcinogenic metabolite that covalently binds to and damages DNA. Detection of adducts and antibodies to them may also be useful as internal dosimeters of the pathobiological effective doses of chemical carcinogens. PMID:2413443

Chitosan-based biocatalytic nanoparticles for pollutant removal from wastewater.

PubMed

Alarcón-Payán, Dulce A; Koyani, Rina D; Vazquez-Duhalt, Rafael

2017-05-01

Chitosan, a renewable biopolymer has the prospective applications in different fields due to its gelation capacity. Nanoconfiguration of chitosan through ionotropic gelation to encapsulate enzymatic activity offers numerous potential applications. In the present study, the preparation and characterization of chitosan nanoparticles loaded with versatile peroxidase are reported. Their performance in bioremediation process and the resistance enhancement against natural microbial biodegradation were studied. The average diameter of enzymatic nanoparticles was 120nm and showed a high enzyme loading capacity. The kinetic parameters of nanoparticles exhibited a slightly lower catalytic activity (k cat ), similar affinity constant (Km) for hydrogen peroxide and higher Km value for the phenolic compound when compared with the free enzyme. The enzymatic nanoparticles showed higher thermostability and the same pH activity profile than those from free enzyme. Ten phenolic compounds, including pesticides, halogenated compounds, endocrine disruptors and antibacterials were transformed by the enzymatic nanoparticles. The transformation rate was lower than those obtained with free enzyme suggesting mass transfer limitations. But very importantly, the enzymatic nanoparticles showed a significant increase of the operational stability in real conditions of wastewater treatment process. Moreover, chemical modification of nanoparticles with different aldehydes still enhanced the operational stability of nanoparticulated enzymes. This enhancement of stability in real conditions and the potential use of biocatalytic nanoparticles in bioremediation processes are discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

HO-1 and CO decrease platelet-derived growth factor-induced vascular smooth muscle cell migration via inhibition of Nox1

PubMed Central

Rodriguez, Andres I.; Gangopadhyay, Archana; Kelley, Eric E.; Pagano, Patrick J.; Zuckerbraun, Brian S.; Bauer, Philip M.

2009-01-01

Objective Heme oxygenase-1 (HO-1), via its enzymatic degradation products, exhibits cell and tissue protective effects in models of vascular injury and disease. The migration of vascular smooth muscle cells (VSMC) from the medial to the intimal layer of blood vessels plays an integral role in the development of a neointima in these models. Despite this, there are no studies addressing the effect of increased HO-1 expression on VSMC migration. Results and Methods The effects of increased HO-1 expression as well as biliverdin, bilirubin, and carbon monoxide (CO), were studied in in vitro models of VSMC migration. Induction of HO-1 or CO, but not biliverdin or bilirubin, inhibited VSMC migration. This effect was mediated by the inhibition of Nox1 as determined by a range of approaches including detection of intracellular superoxide, NADPH oxidase activity measurements, and siRNA experiments. Furthermore, CO decreased PDGF-stimulated, redox-sensitive signaling pathways. Conclusion Herein we demonstrate that increased HO-1 expression and CO decreases PDGF-stimulated VSMC migration via inhibition of Nox1 enzymatic activity. These studies reveal a novel mechanism by which HO-1 and CO may mediate their beneficial effects in arterial inflammation and injury. PMID:19875720

Method for the enzymatic production of hydrogen

DOEpatents

Woodward, Jonathan; Mattingly, Susan M.

1999-01-01

The present invention is an enzymatic method for producing hydrogen comprising the steps of: a) forming a reaction mixture within a reaction vessel comprising a substrate capable of undergoing oxidation within a catabolic reaction, such as glucose, galactose, xylose, mannose, sucrose, lactose, cellulose, xylan and starch. The reaction mixture further comprises an amount of glucose dehydrogenase in an amount sufficient to catalyze the oxidation of the substrate, an amount of hydrogenase sufficient to catalyze an electron-requiring reaction wherein a stoichiometric yield of hydrogen is produced, an amount of pH buffer in an amount sufficient to provide an environment that allows the hydrogenase and the glucose dehydrogenase to retain sufficient activity for the production of hydrogen to occur and also comprising an amount of nicotinamide adenine dinucleotide phosphate sufficient to transfer electrons from the catabolic reaction to the electron-requiring reaction; b) heating the reaction mixture at a temperature sufficient for glucose dehydrogenase and the hydrogenase to retain sufficient activity and sufficient for the production of hydrogen to occur, and heating for a period of time that continues until the hydrogen is no longer produced by the reaction mixture, wherein the catabolic reaction and the electron-requiring reactions have rates of reaction dependent upon the temperature; and c) detecting the hydrogen produced from the reaction mixture.

Method for the enzymatic production of hydrogen

DOEpatents

Woodward, J.; Mattingly, S.M.

1999-08-24

The present invention is an enzymatic method for producing hydrogen comprising the steps of: (a) forming a reaction mixture within a reaction vessel comprising a substrate capable of undergoing oxidation within a catabolic reaction, such as glucose, galactose, xylose, mannose, sucrose, lactose, cellulose, xylan and starch; the reaction mixture also comprising an amount of glucose dehydrogenase in an amount sufficient to catalyze the oxidation of the substrate, an amount of hydrogenase sufficient to catalyze an electron-requiring reaction wherein a stoichiometric yield of hydrogen is produced, an amount of pH buffer in an amount sufficient to provide an environment that allows the hydrogenase and the glucose dehydrogenase to retain sufficient activity for the production of hydrogen to occur and also comprising an amount of nicotinamide adenine dinucleotide phosphate sufficient to transfer electrons from the catabolic reaction to the electron-requiring reaction; (b) heating the reaction mixture at a temperature sufficient for glucose dehydrogenase and the hydrogenase to retain sufficient activity and sufficient for the production of hydrogen to occur, and heating for a period of time that continues until the hydrogen is no longer produced by the reaction mixture, wherein the catabolic reaction and the electron-requiring reactions have rates of reaction dependent upon the temperature; and (c) detecting the hydrogen produced from the reaction mixture. 8 figs.

Comparative analysis of lysyl oxidase (like) family members in pulmonary fibrosis.

PubMed

Aumiller, Verena; Strobel, Benjamin; Romeike, Merrit; Schuler, Michael; Stierstorfer, Birgit E; Kreuz, Sebastian

2017-03-10

Extracellular matrix (ECM) composition and stiffness are major driving forces for the development and persistence of fibrotic diseases. Lysyl oxidase (LOX) and LOX-like (LOXL) proteins play crucial roles in ECM remodeling due to their collagen crosslinking and intracellular functions. Here, we systematically investigated LOX/L expression in primary fibroblasts and epithelial cells under fibrotic conditions, Bleomycin (BLM) induced lung fibrosis and in human IPF tissue. Basal expression of all LOX/L family members was detected in epithelial cells and at higher levels in fibroblasts. Various pro-fibrotic stimuli broadly induced LOX/L expression in fibroblasts, whereas specific induction of LOXL2 and partially LOX was observed in epithelial cells. Immunohistochemical analysis of lung tissue from 14 IPF patients and healthy donors revealed strong induction of LOX and LOXL2 in bronchial and alveolar epithelium as well as fibroblastic foci. Using siRNA experiments we observed that LOXL2 and LOXL3 were crucial for fibroblast-to-myofibroblast transition (FMT). As FMT could only be reconstituted with an enzymatically active LOXL2 variant, we conclude that LOXL2 enzymatic function is crucial for fibroblast transdifferentiation. In summary, our study provides a comprehensive analysis of the LOX/L family in fibrotic lung disease and indicates prominent roles for LOXL2/3 in fibroblast activation and LOX/LOXL2 in IPF.

Mitochondrial Dysfunction Is Involved in the Toxic Activity of Boric Acid against Saprolegnia

PubMed Central

Ali, Shimaa E.; Thoen, Even; Evensen, Øystein; Wiik-Nielsen, Jannicke; Gamil, Amr A. A.; Skaar, Ida

2014-01-01

There has been a significant increase in the incidence of Saprolegnia infections over the past decades, especially after the banning of malachite green. Very often these infections are associated with high economic losses in salmonid farms and hatcheries. The use of boric acid to control the disease has been investigated recently both under in vitro and in vivo conditions, however its possible mode of action against fish pathogenic Saprolegnia is not known. In this study, we have explored the transformation in Saprolegnia spores/hyphae after exposure to boric acid (1 g/L) over a period 4–24 h post treatment. Using transmission electron microscopy (TEM), early changes in Saprolegnia spores were detected. Mitochondrial degeneration was the most obvious sign observed following 4 h treatment in about 20% of randomly selected spores. We also investigated the effect of the treatment on nuclear division, mitochondrial activity and function using confocal laser scanning microscopy (CLSM). Fluorescence microscopy was also used to test the effect of treatment on mitochondrial membrane potential and formation of reactive oxygen species. Additionally, the viability and proliferation of treated spores that correlated to mitochondrial enzymatic activity were tested using an MTS assay. All obtained data pointed towards changes in the mitochondrial structure, membrane potential and enzymatic activity following treatment. We have found that boric acid has no effect on the integrity of membranes of Saprolegnia spores at concentrations tested. It is therefore likely that mitochondrial dysfunction is involved in the toxic activity of boric acid against Saprolegnia spp. PMID:25354209

Potentiometric sensing of nuclease activities and oxidative damage of single-stranded DNA using a polycation-sensitive membrane electrode.

PubMed

Ding, Jiawang; Qin, Wei

2013-09-15

A simple, general and label-free potentiometric method to measure nuclease activities and oxidative DNA damage in a homogeneous solution using a polycation-sensitive membrane electrode is reported. Protamine, a linear polyionic species, is used as an indicator to report the cleavage of DNA by nucleases such as restriction and nonspecific nucleases, and the damage of DNA induced by hydroxyl radicals. Measurements can be done with a titration mode or a direct detection mode. For the potentiometric titration mode, the enzymatic cleavage dramatically affects the electrostatical interaction between DNA and protamine and thus shifts the response curve for the potentiometric titration of the DNA with protamine. Under the optimized conditions, the enzyme activities can be sensed potentiometrically with detection limits of 2.7×10(-4)U/µL for S1 nuclease, and of 3.9×10(-4)U/µL for DNase I. For the direct detection mode, a biocomplex between protamine and DNA is used as a substrate. The nuclease of interest cleaves the DNA from the protamine/DNA complex into smaller fragments, so that free protamine is generated and can be detected potentiometrically via the polycation-sensitive membrane electrode. Using a direct measurement, the nuclease activities could be rapidly detected with detection limits of 3.2×10(-4)U/µL for S1 nuclease, and of 4.5×10(-4)U/µL for DNase I. Moreover, the proposed potentiometric assays demonstrate the potential applications in the detection of hydroxyl radicals. It is anticipated that the present potentiometric strategy will provide a promising platform for high-throughput screening of nucleases, reactive oxygen species and the drugs with potential inhibition abilities. Copyright © 2013 Elsevier B.V. All rights reserved.

Correlation of Biological Activity and Reactor Performance in Biofiltration of Toluene with the Fungus Paecilomyces variotii CBS115145

PubMed Central

García-Peña, Inés; Hernández, Sergio; Auria, Richard; Revah, Sergio

2005-01-01

A biofiltration system inoculated with the mold Paecilomyces variotii CBS115145 showed a toluene elimination capacity (EC) of around 250 g/m3 of biofilter/h, which was higher than the values usually reported for bacteria. P. variotii assimilated m- and p-cresols but not the o isomer. Initial toluene hydroxylation occurred both on the methyl group and through the p-cresol pathway. These results were corroborated by detecting benzyl alcohol, benzaldehyde, and p-cresol as volatile intermediates. In liquid cultures with toluene as a substrate, the activity of toluene oxygenase (TO) was 5.6 nmol of O2/min/mg of biomass, and that of benzyl alcohol dehydrogenase was 16.2 nmol of NADH/min/mg of protein. Toluene biodegradation determined from the TO activity in the biofilter depended on the biomass distribution and the substrate concentration. The specific enzymatic activity decreased from 6.3 to 1.9 nmol of O2/min/mg of biomass along the reactor. Good agreement was found between the EC calculated from the TO activity and the EC measured on the biofilter. The results were confirmed by short-time biofiltration experiments. Average EC measured in different biofiltration experiments and EC calculated from the TO activity showed a linear relation, suggesting that in the biofilters, EC was limited by biological reaction. As the enzymatic activities of P. variotii were similar to those reported for bacteria, the high performance of the fungal biofilters can possibly be explained by the increased transfer of the hydrophobic compounds, including oxygen, from the gas phase to the mycelia, overcoming the transfer problems associated with the flat bacterial biofilms. PMID:16085815

Correlation of biological activity and reactor performance in biofiltration of toluene with the fungus Paecilomyces variotii CBS115145.

PubMed

García-Peña, Inés; Hernández, Sergio; Auria, Richard; Revah, Sergio

2005-08-01

A biofiltration system inoculated with the mold Paecilomyces variotii CBS115145 showed a toluene elimination capacity (EC) of around 250 g/m3 of biofilter/h, which was higher than the values usually reported for bacteria. P. variotii assimilated m- and p-cresols but not the o isomer. Initial toluene hydroxylation occurred both on the methyl group and through the p-cresol pathway. These results were corroborated by detecting benzyl alcohol, benzaldehyde, and p-cresol as volatile intermediates. In liquid cultures with toluene as a substrate, the activity of toluene oxygenase (TO) was 5.6 nmol of O2/min/mg of biomass, and that of benzyl alcohol dehydrogenase was 16.2 nmol of NADH/min/mg of protein. Toluene biodegradation determined from the TO activity in the biofilter depended on the biomass distribution and the substrate concentration. The specific enzymatic activity decreased from 6.3 to 1.9 nmol of O2/min/mg of biomass along the reactor. Good agreement was found between the EC calculated from the TO activity and the EC measured on the biofilter. The results were confirmed by short-time biofiltration experiments. Average EC measured in different biofiltration experiments and EC calculated from the TO activity showed a linear relation, suggesting that in the biofilters, EC was limited by biological reaction. As the enzymatic activities of P. variotii were similar to those reported for bacteria, the high performance of the fungal biofilters can possibly be explained by the increased transfer of the hydrophobic compounds, including oxygen, from the gas phase to the mycelia, overcoming the transfer problems associated with the flat bacterial biofilms.

Enzymatic properties of the glycine D-alanine [corrected] aminopeptidase of Aspergillus oryzae and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji).

PubMed

Marui, Junichiro; Matsushita-Morita, Mayumi; Tada, Sawaki; Hattori, Ryota; Suzuki, Satoshi; Amano, Hitoshi; Ishida, Hiroki; Yamagata, Youhei; Takeuchi, Michio; Kusumoto, Ken-Ichi

2012-01-01

The gdaA gene encoding S12 family glycine-D-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the D-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high D-stereospecificity and efficiently released N-terminal glycine and D-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and D-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and D-alanine aminopeptidase activity detected at a pH range of 6 to 9.

Coronary vasospasm induced in transgenic mouse with increased phospholipase C-δ1 activity.

PubMed

Shibutani, Shuji; Osanai, Tomohiro; Ashitate, Toshihiro; Sagara, Shigeki; Izumiyama, Kei; Yamamoto, Yuko; Hanada, Kenji; Echizen, Takashi; Tomita, Hirofumi; Fujita, Takeshi; Miwa, Takeshi; Matsubara, Hiroaki; Homma, Yoshimi; Okumura, Ken

2012-02-28

We reported that phospholipase C (PLC)-δ1 activity was enhanced 3-fold in patients with coronary spastic angina. We detected variant PLC-δ1 with replacement of arginine 257 by histidine (R257H) showing increased enzymatic activity. We tested the hypothesis that increased PLC-δ1 activity causes enhanced coronary vasomotility. We generated transgenic (TG) mice with human R257H variant PLC-δ1 in vascular smooth muscle cells. PLC enzymatic activity in the coronary artery was increased by 2.57 and 1.89 times, respectively, in homozygous and heterozygous TG compared with wild-type (WT) mice. ST elevation after ergometrine occurred in 17 of 18 homozygous TG, 6 of 20 heterozygous TG, and 3 of 22 WT mice (P<0.01, homozygous TG versus WT; P<0.05, homozygous TG versus heterozygous TG; P=NS, heterozygous TG versus WT). ST elevation was associated with bradyarrhythmias in homozygous TG mice. Focal coronary artery narrowing was documented with the microvascular filling technique in 3 of 5 homozygous TG mice after ergometrine but not in any of 7 WT mice (P<0.05). In the isolated Langendorff hearts, coronary perfusion pressure was increased after ergometrine in homozygous TG mice (P<0.01) but not in heterozygous TG or WT mice. Coronary perfusion pressure increase after prostaglandin F2α was similar among homozygous TG, heterozygous TG, and WT mice. Cultured rat aortic smooth muscle cells transfected with variant PLC-δ1 showed a higher PLC activity than those with WT PLC-δ1 (P<0.05) and furthermore showed greater intracellular Ca2+ response to acetylcholine in variant than in WT PLC-δ1 (P<0.05). Increased PLC-δ1 activity enhances coronary vasomotility such as that seen in patients with coronary spastic angina.

Functional Characterization of the Vitamin K2 Biosynthetic Enzyme UBIAD1

PubMed Central

Hirota, Yoshihisa; Nakagawa, Kimie; Sawada, Natsumi; Okuda, Naoko; Suhara, Yoshitomo; Uchino, Yuri; Kimoto, Takashi; Funahashi, Nobuaki; Kamao, Maya; Tsugawa, Naoko; Okano, Toshio

2015-01-01

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) plays a significant role in vitamin K2 (MK-4) synthesis. We investigated the enzymological properties of UBIAD1 using microsomal fractions from Sf9 cells expressing UBIAD1 by analysing MK-4 biosynthetic activity. With regard to UBIAD1 enzyme reaction conditions, highest MK-4 synthetic activity was demonstrated under basic conditions at a pH between 8.5 and 9.0, with a DTT ≥0.1 mM. In addition, we found that geranyl pyrophosphate and farnesyl pyrophosphate were also recognized as a side-chain source and served as a substrate for prenylation. Furthermore, lipophilic statins were found to directly inhibit the enzymatic activity of UBIAD1. We analysed the aminoacid sequences homologies across the menA and UbiA families to identify conserved structural features of UBIAD1 proteins and focused on four highly conserved domains. We prepared protein mutants deficient in the four conserved domains to evaluate enzyme activity. Because no enzyme activity was detected in the mutants deficient in the UBIAD1 conserved domains, these four domains were considered to play an essential role in enzymatic activity. We also measured enzyme activities using point mutants of the highly conserved aminoacids in these domains to elucidate their respective functions. We found that the conserved domain I is a substrate recognition site that undergoes a structural change after substrate binding. The conserved domain II is a redox domain site containing a CxxC motif. The conserved domain III is a hinge region important as a catalytic site for the UBIAD1 enzyme. The conserved domain IV is a binding site for Mg2+/isoprenyl side-chain. In this study, we provide a molecular mapping of the enzymological properties of UBIAD1. PMID:25874989

Production of xylooligosaccharide from wheat bran by microwave assisted enzymatic hydrolysis.

PubMed

Wang, Tseng-Hsing; Lu, Shin

2013-06-01

The effective production of xylooligosaccharides (XOS) from wheat bran was investigated. Wheat bran contains rich hemicellulose which can be hydrolyzed by enzyme; the XOS were obtained by microwave assisted enzymatic hydrolysis. To improve the productivity of XOS, repeated microwave assisted enzymatic hydrolysis and activated carbon adsorption method was chosen to eliminate macromolecules in the XOS. On the basis of experimental data, an industrial XOS production process consisting of pretreatment, repeated microwave assisted enzymatic treatment and purification was designed. Using the designed process, 3.2g dry of purified XOS was produced from 50 g dry wheat bran powder. Copyright © 2012 Elsevier Ltd. All rights reserved.

Biphasic photoelectrochemical sensing strategy based on in situ formation of CdS quantum dots for highly sensitive detection of acetylcholinesterase activity and inhibition.

PubMed

Hou, Ting; Zhang, Lianfang; Sun, Xinzhi; Li, Feng

2016-01-15

Herein, we reported a facile and highly sensitive biphasic photoelectrochemical (PEC) sensing strategy based on enzymatic product-mediated in situ formation of CdS quantum dots (QDs), and assayed the activity and inhibition of acetylcholinesterase (AChE) in its optimal state. Upon the hydrolysis of acetylthiocholine catalyzed by AChE, the product thiocholine stabilizes the in situ formation of CdS QDs in homogenous solution. Due to the electrostatic attraction, the resulting tertiary amino group-functionalized CdS QDs are attached to the surface of the negatively charged indium tin oxide (ITO) electrode, generating significant PEC response upon illumination in the presence of electron donors. By taking full advantage of the in situ formation of CdS QDs in homogenous solution, this strategy is capable of detecting AChE activity and inhibition in its optimal state. A directly measured detection limit of 0.01mU/mL for AChE activity is obtained, which is superior to those obtained by some fluorescence methods. The inhibition of AChE activity by aldicarb is successfully detected, and the corresponding IC50 is determined to be 13μg/L. In addition to high sensitivity and good selectivity, this strategy also exhibits additional advantages of simplicity, low cost and easy operation. To the best of our knowledge, the as-proposed strategy is the first example demonstrating the application of CdS QDs formed in situ for biphasic PEC detection of enzyme activity and inhibition. More significantly, it opens up a new horizon for the development of homogenous PEC sensing platforms, and has great potential in probing many other analytes. Copyright © 2015 Elsevier B.V. All rights reserved.

Fluorescein as a Visible-Light-Induced Oxidase Mimic for Signal-Amplified Colorimetric Assay of Carboxylesterase by an Enzymatic Cascade Reaction.

PubMed

Liu, Li; Sun, Chaoqun; Yang, Juan; Shi, Ying; Long, Yijuan; Zheng, Huzhi

2018-04-20

We have found that fluorescein possesses high visible-light-induced oxidase mimetic activity and could transform colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB) without unstable and destructive H 2 O 2 under visible-light illumination. Instead, fluorescein uses oxygen as a mild and green electron acceptor, and its activity can be easily controlled by the switching "on/off" of visible light. In addition, the visible-light-induced catalytic mechanism was elucidated in detail and, as the main reactive species h + and O 2 .- accounted for TMB oxidation. Based on the fact that fluorescein diacetate (FDA) possessed no activity and generated active fluorescein in situ in the presence of carboxylesterase (CaE), a signal-amplified sensing platform through a cascade reaction for CaE detection was constructed. Our proposed sensing system displayed excellent analytical performance for the detection of CaE in a wide linear range from 0.040 to 20 U L -1 with a low detection limit of 0.013 U L -1 . This work not only changes the conventional concept that fluorescein is generally considered to be photocatalytically inert, but also provides a novel sensing strategy by tailoring the enzyme mimetic activity of fluorescein derivatives with analyte. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A facile low-cost enzymatic paper-based assay for the determination of urine creatinine.

PubMed

Talalak, Kwanrutai; Noiphung, Julaluk; Songjaroen, Temsiri; Chailapakul, Orawon; Laiwattanapaisal, Wanida

2015-11-01

Creatinine is one of many markers used to investigate kidney function. This paper describes a low-cost enzymatic paper-based analytical device (enz-PAD) for determining urine creatinine. The disposable dead volumes of creatinine enzyme reagents from an automatic analyser cassette were utilised. Whatman No. 3 paper was cut into long rectangular shapes (4×40 mm(2)) on which the enzyme reagents, R1 and R2, were adsorbed in two consecutive regions. The assay was performed by immersing test strips into urine samples contained in microwells to allow creatinine in the sample to react with immobilised active ingredients and, then, traverse via capillary action to the detection area where chromogen products accumulated. The method is based on hydrogen peroxide (H2O2) formation via creatinine conversion using creatininase, creatinase, and sarcosine oxidase. The liberated H2O2 reacts with 4-aminophenazone and 2,4,6-triiodo-3-hydroxybenzoic acid to form quinoneimine with a pink-red colour at the detection zone. The linear range of the creatinine assay was 2.5-25 mg dL(-1) (r(2)=0.983), and the detection limit was 2.0 mg dL(-1). The colorimetric enz-PAD for the creatinine assay was highly correlated with a conventional alkaline picrate method when real urine samples were evaluated (r(2)=0.977; n=40). This simple and nearly zero-cost paper-based device provides a novel alternative method for screening urinary creatinine and will be highly beneficial for developing countries. Copyright © 2015 Elsevier B.V. All rights reserved.

Label-free fluorescent enzymatic assay of citrate synthase by CoA-Au(I) co-ordination polymer and its application in a multi-enzyme logic gate cascade.

PubMed

Li, Yong; Wang, Huixia; Dai, Futao; Li, Pei; Jin, Xin; Huang, Yan; Nie, Zhou; Yao, Shouzhuo

2016-12-15

Citrate synthase (CS) is one of the key metabolic enzymes in the Krebs tricarboxylic acid (TCA) cycle. It regulates energy generation in mitochondrial respiration by catalysing the reaction between oxaloacetic acid (OAA) and acetyl coenzyme A (Ac-CoA) to generate citrate and coenzyme A (CoA). CS has been shown to be a biomarker of neurological diseases and various kinds of cancers. Here, a label-free fluorescent assay has been developed for homogeneously detecting CS and its inhibitor based on the in situ generation of CoA-Au(I) co-ordination polymer (CP) and the fluorescence signal-on by SYBR Green II-stained CoA-Au(I) CP. Because of the unique property of the CoA-Au(I) CP, this CS activity assay method could achieve excellent selectivity and sensitivity, with a linear range from 0.0033 U/μL to 0.264 U/μL and a limit of detection to be 0.00165 U/μL. Meanwhile, this assay method has advantages of being facile and cost effective with quick detection. Moreover, based on this method, a biomimetic logic system was established by rationally exploiting the cascade enzymatic interactions in TCA cycle for chemical information processing. In the TCA cycle-derived logic system, an AND-AND-AND-cascaded gate was rigorously operated step by step in one pot, and is outputted by a label-free fluorescent signal with visualized readout. Copyright © 2016 Elsevier B.V. All rights reserved.

Electrochemical sensing of modified ABO3 perovskite: LaFe0.8 R0.2O3(R= Cr, Co, Al)

NASA Astrophysics Data System (ADS)

Vidya Rajan, N.; Alexander, L. K.

2017-06-01

Perovskite LaFeO3 with orthorhombic structure has been synthesized by citric acid mediated solution method. The effectiveness of ionic radii and Oxidation state of the doping material on ionic conductivity of the host matrix was evaluated by B-site (Fe) doping on LaFeO3 with Cr, Co and Al, resulting LaFe0.8 R0.2O3 (R = Cr, Co, Al). XRD with Rietveld refinement and Raman spectroscopic analysis demonstrate successful synthesis. The effect of the 20% B site doping on electrochemical activity is reported. The doped materials exhibit a decrease in sensing activity towards the non enzymatic detection of H2O2.

Inhibition of trypsin by condensed tannins and wine.

PubMed

Gonçalves, Rui; Soares, Susana; Mateus, Nuno; de Freitas, Victor

2007-09-05

Phenolic compounds are abundant vegetable secondary metabolites in the human diet. The ability of procyanidin oligomers and wine polyphenols to inhibit trypsin activity was studied using a versatile and reliable in vitro method. The hydrolysis of the chromogenic substrate N-benzoyl-d,l-arginine-p-nitroanilide (BApNA) by trypsin was followed by spectrophotometry in the presence and absence of condensed tannins and wine. A clear relationship between the degree of polymerization of procyanidins and enzymatic inhibition was observed. Trypsin activity inhibition was also detected in several types of wine. In general, the inhibition increased with the concentration of phenolic compounds in wines. These results may be relevant when considering these compounds as antinutritional factors, thereby contributing to a reduced absorption of nutrients.

Peripheral blood lymphocytes express recombination-activating genes 1 and 2 during Epstein-Barr virus-induced infectious mononucleosis.

PubMed

Wagner, Hans-Joachim; Scott, Rona S; Buchwald, Dedra; Sixbey, John W

2004-09-01

Implicit in the persistence of Epstein-Barr virus (EBV) in B lymphocytes is the successful circumvention of ongoing cell selection for competence of B cell receptors (BCRs). Because the EBV infection of B cells in vitro induces enzymatic machinery that is responsible for secondary immunoglobulin gene rearrangement, we examined the expression of the recombination-activating genes (RAGs) in peripheral blood mononuclear cells (PBMCs) from 26 patients with infectious mononucleosis (IM). RAG1 and/or RAG2 RNA was detected in PBMCs from 42% of patients with IM but not from healthy control subjects. EBV may usurp the cellular mechanism that diversifies the BCR, to guarantee a level of survival signaling sufficient for its own persistence.

Production of Cellulolytic and Hemicellulolytic Enzymes From Aureobasidium pulluans on Solid State Fermentation

NASA Astrophysics Data System (ADS)

Leite, Rodrigo Simões Ribeiro; Bocchini, Daniela Alonso; da Silva Martins, Eduardo; Silva, Dênis; Gomes, Eleni; da Silva, Roberto

This article investigates a strain of the yeast Aureobasidium pullulans for cellulase and hemicellulase production in solid state fermentation. Among the substrates analyzed, the wheat bran culture presented the highest enzymatic production (1.05 U/mL endoglucanase, 1.3 U/mL β-glucosidase, and 5.0 U/mL xylanase). Avicelase activity was not detected. The optimum pH and temperature for xylanase, endoglucanase and β-glucosidase were 5.0 and 50, 4.5 and 60, 4.0 and 75°C, respectively. These enzymes remained stable between a wide range of pH. The β-glucosidase was the most thermostable enzyme remaining 100% active when incubated at 75°C for 1 h.

Ni-Co bimetal nanowires filled multiwalled carbon nanotubes for the highly sensitive and selective non-enzymatic glucose sensor applications

PubMed Central

Ramachandran, K.; Raj kumar, T.; Babu, K. Justice; Gnana kumar, G.

2016-01-01

The facile, time and cost efficient and environmental benign approach has been developed for the preparation of Nickel (Ni)-Cobalt (Co) alloy nanowires filled multiwalled carbon nanotubes (MWCNTs) with the aid of mesoporous silica nanoparticles (MSN)/Ni-Co catalyst. The controlled incorporation of Ni-Co nanostructures in the three dimensional (3D) pore structures of MSN yielded the catalytically active system for the MWCNT growth. The inner surface of MWCNTs was quasi-continuously filled with face-centered cubic (fcc) structured Ni-Co nanowires. The as-prepared nanostructures were exploited as non-enzymatic electrochemical sensor probes for the reliable detection of glucose. The electrochemical measurements illustrated that the fabricated sensor exhibited an excellent electrochemical performance toward glucose oxidation with a high sensitivity of 0.695 mA mM−1 cm−2, low detection limit of 1.2 μM, a wide linear range from 5 μM–10 mM and good selectivity. The unprecedented electrochemical performances obtained for the prepared nanocomposite are purely attributed to the synergistic effects of Ni-Co nanowires and MWCNTs. The constructed facile, selective and sensitive glucose sensor has also endowed its reliability in analyzing the human serum samples, which wide opened the new findings for exploring the novel nanostructures based glucose sensor devices with affordable cost and good stability. PMID:27833123

Conjugation of glucose oxidase onto Mn-doped ZnS quantum dots for phosphorescent sensing of glucose in biological fluids.

PubMed

Wu, Peng; He, Yu; Wang, He-Fang; Yan, Xiu-Ping

2010-02-15

Integrating various enzymes with nanomaterials provides various nanohybrids with new possibilities in biosensor applications. Furthermore, the enzymatic activity and stability are also improved due to the large surface area of nanomaterials. Here we report the conjugation of glucose oxidase (GOD) onto phosphorescent Mn-doped ZnS quantum dots (QDs) using 1-ethyl-3-(3-dimethylaminopropy)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) as coupling reagents for glucose biosensing based on the effective quenching of the room temperature phosphorescence (RTP) of Mn-doped ZnS QDs by the H(2)O(2) generated from GOD-catalyzed oxidation of glucose. The obtained bioconjugate not only provided improved enzymatic performance with Michaelis-Menten constant of 0.70 mM but also favored biological applications because the phosphorescent detection mode avoided the interference from autofluorescence and scattering light from the biological matrix. In addition, the GOD-conjugated Mn-doped ZnS QDs showed better thermal stability in the temperature range of 20-80 degrees C. The GOD-Mn-doped ZnS QDs based RTP sensor for glucose gave a detection limit of 3 microM and two linear ranges from 10 microM to 0.1 mM and from 0.1 to 1 mM. The developed biosensor was successfully applied to the determination of glucose in real serum samples without the need for any complicated sample pretreatments.

Ni-Co bimetal nanowires filled multiwalled carbon nanotubes for the highly sensitive and selective non-enzymatic glucose sensor applications

NASA Astrophysics Data System (ADS)

Ramachandran, K.; Raj Kumar, T.; Babu, K. Justice; Gnana Kumar, G.

2016-11-01

The facile, time and cost efficient and environmental benign approach has been developed for the preparation of Nickel (Ni)-Cobalt (Co) alloy nanowires filled multiwalled carbon nanotubes (MWCNTs) with the aid of mesoporous silica nanoparticles (MSN)/Ni-Co catalyst. The controlled incorporation of Ni-Co nanostructures in the three dimensional (3D) pore structures of MSN yielded the catalytically active system for the MWCNT growth. The inner surface of MWCNTs was quasi-continuously filled with face-centered cubic (fcc) structured Ni-Co nanowires. The as-prepared nanostructures were exploited as non-enzymatic electrochemical sensor probes for the reliable detection of glucose. The electrochemical measurements illustrated that the fabricated sensor exhibited an excellent electrochemical performance toward glucose oxidation with a high sensitivity of 0.695 mA mM-1 cm-2, low detection limit of 1.2 μM, a wide linear range from 5 μM-10 mM and good selectivity. The unprecedented electrochemical performances obtained for the prepared nanocomposite are purely attributed to the synergistic effects of Ni-Co nanowires and MWCNTs. The constructed facile, selective and sensitive glucose sensor has also endowed its reliability in analyzing the human serum samples, which wide opened the new findings for exploring the novel nanostructures based glucose sensor devices with affordable cost and good stability.

Association between enzymatic and non-enzymatic antioxidant defense mechanism with apolipoprotein E genotypes in Alzheimer disease.

PubMed

Kharrazi, Hadi; Vaisi-Raygani, Asad; Rahimi, Zohreh; Tavilani, Haidar; Aminian, Mahdi; Pourmotabbed, Tayebeh

2008-08-01

There are evidence suggesting that APOE-varepsilon4 allele play an important role in the pathogenesis of Alzheimer's disease (AD) by reducing peripheral levels and activities of a broad spectrum of nonenzymatic and enzymatic antioxidants systems. However, the link between APOE genotype, oxidative stress, and AD has yet to be established. In this study we examined whether antioxidant defense mechanism exacerbates the risk of AD in individual carrying APOE-varepsilon4 allele in a population from Tehran, Iran. We determined the enzymatic activities of the erythrocyte Cu-Zn superoxide dismutase (Cu-Zn SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and serum level of total antioxidant status(TAS) in various APOE genotypes in 91 patients with AD and 91 healthy subjects as control group (age and sex-matched). The results showed that the TAS level and the activities of enzymatic antioxidants CAT and GSH-Px were significantly lower and the SOD activity was significantly higher in AD patients compared to controls. The AD patients with APOE-varepsilon4 allele genotype had significantly lower serum TAS concentration and lower erythrocytes GSH-Px and CAT activities (p=0.001) but significantly higher erythrocytes Cu-Zn SOD activity (p=0.001) than the non-APOE-varepsilon4 carrier AD and the control group. In addition, the association observed between the factors involved in an antioxidant defense mechanism and APOE-varepsilon4 allele in AD increased with age of the subjects. These data indicate that the reduced serum level of TAS and activity of CAT, GSH-Px and increased SOD exacerbate the risk of AD in individuals carrying APOE-varepsilon4 allele. The reduced antioxidants defense in APOE-varepsilon4 allele carrier may contribute to beta-amyloidosis. This effect, however, is more pronounced in the AD patients older than 75 years of age. This suggests that a therapeutic modality should be considered for these subjects.

A real-time fluorescence assay for protease activity and inhibitor screening based on the aggregation-caused quenching of a perylene probe.

PubMed

Wang, Yan; Zhang, Zhifang; Zhang, Ya; Yu, Cong

2018-06-01

We have established a real-time and label-free fluorescence turn-on strategy for protease activity detection and inhibitor screening via peptide-induced aggregation-caused quenching of a perylene probe. Because of electrostatic interactions and high hydrophilicity, poly-l-glutamic acid sodium salt (PGA; a negatively charged peptide) could induce aggregation of a positively charged perylene probe (probe 1) and the monomer fluorescence of probe 1 was effectively quenched. After a protease was added, PGA was enzymatically hydrolyzed into small fragments and probe 1 disaggregated. The fluorescence recovery of probe 1 was found to be proportional to the concentration of protease in the range from 0 to 1 mU/ml. The detection limit was down to 0.1 mU/ml. In the presence of a protease inhibitor, protease activity was inhibited and fluorescence recovery reduced. Moreover, we demonstrated the potential application of our method in a complex mixture sample including 1% human serum. Our method is simple, fast and cost effective. Copyright © 2018 John Wiley & Sons, Ltd.

Enzyme-linked electrochemical DNA ligation assay using magnetic beads.

PubMed

Stejskalová, Eva; Horáková, Petra; Vacek, Jan; Bowater, Richard P; Fojta, Miroslav

2014-07-01

DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5'-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

Effect of wheat and Miscanthus straw biochars on soil enzymatic activity, ecotoxicity, and plant yield

NASA Astrophysics Data System (ADS)

Mierzwa-Hersztek, Monika; Gondek, Krzysztof; Klimkowicz-Pawlas, Agnieszka; Baran, Agnieszka

2017-07-01

The variety of technological conditions and raw materials from which biochar is produced is the reason why its soil application may have different effects on soil properties and plant growth. The aim of this study was to evaluate the effect of the addition of wheat straw and Miscanthus giganteus straw (5 t DM ha-1) and biochar obtained from this materials in doses of 2.25 and 5 t DM ha-1 on soil enzymatic activity, soil ecotoxicity, and plant yield (perennial grass mixture with red clover). The research was carried out under field conditions on soil with the granulometric composition of loamy sand. No significant effect of biochar amendment on soil enzymatic activity was observed. The biochar-amended soil was toxic to Vibrio fischeri and exhibited low toxicity to Heterocypris incongruens. Application of wheat straw biochar and M. giganteus straw biochar in a dose of 5 t DM ha-1 contributed to an increase in plant biomass production by 2 and 14%, respectively, compared to the soil with mineral fertilisation. Biochars had a more adverse effect on soil enzymatic activity and soil ecotoxicity to H. incongruens and V. fischeri than non-converted wheat straw and M. giganteus straw, but significantly increased the grass crop yield.

Conformational change results in loss of enzymatic activity of jack bean urease on its interaction with silver nanoparticle.

PubMed

Ponnuvel, Shobana; Subramanian, Balakumar; Ponnuraj, Karthe

2015-10-01

Urease is an enzyme produced by microbes such as bacteria, yeast and fungi. Plants also produce this enzyme. Urease action splits urea into ammonia and carbamate. This action is having important implications in agro-chemical, medicinal and environment. Therefore there is always a constant search for new and novel compounds which could inhibit this enzyme. Here we have studied the interaction of jack bean urease (JBU) with silver nanoparticle to analyze the influence of the resultant protein corona formation on the catalytic property of JBU. Several techniques like UV-Vis, gel shift assay and CD spectroscopy have been used to characterize this interaction. Urease activity assay suggests that the protein corona formation inhibits the enzymatic action of JBU. The loss of enzymatic action could be either due to the nanoparticle blocking the active site of JBU or a conformational change in the protein. The CD spectra of JBU-AgNP complexes clearly revealed significant changes in the secondary structural composition of the JBU and this could be the reason for the loss of enzymatic activity of JBU. This study revealed an interesting observation, where the interaction of AgNP with JBU resulted destabilization of hexameric nature of JBU which is otherwise highly stable. The results of the present study could be useful in the development of nanoparticle based material for inhibiting the ureolytic activity of ureases in different fields.

Single molecule thermodynamics in biological motors.

PubMed

Taniguchi, Yuichi; Karagiannis, Peter; Nishiyama, Masayoshi; Ishii, Yoshiharu; Yanagida, Toshio

2007-04-01

Biological molecular machines use thermal activation energy to carry out various functions. The process of thermal activation has the stochastic nature of output events that can be described according to the laws of thermodynamics. Recently developed single molecule detection techniques have allowed each distinct enzymatic event of single biological machines to be characterized providing clues to the underlying thermodynamics. In this study, the thermodynamic properties in the stepping movement of a biological molecular motor have been examined. A single molecule detection technique was used to measure the stepping movements at various loads and temperatures and a range of thermodynamic parameters associated with the production of each forward and backward step including free energy, enthalpy, entropy and characteristic distance were obtained. The results show that an asymmetry in entropy is a primary factor that controls the direction in which the motor will step. The investigation on single molecule thermodynamics has the potential to reveal dynamic properties underlying the mechanisms of how biological molecular machines work.

Enzymatic detection of As(III) in aqueous solution using alginate immobilized pumpkin urease: optimization of process variables by response surface methodology.

PubMed

Talat, Mahe; Prakash, Om; Hasan, S H

2009-10-01

Urease immobilized on alginate was utilized to detect and quantify As(3+) in aqueous solution. Urease from the seeds of pumpkin (vegetable waste) was purified to apparent homogeneity by heat treatment and gel filtration (Sephadex G-200). Further enzyme was entrapped in 3.5% alginate beads. Urea hydrolysis by enzyme revealed a clear dependence on the concentration and interaction time of As(3+). The process variables effecting the quantitation of As(3+) was investigated using central composite design with Minitab 15 software. The predicted results were found in good agreement (R(2)=96.71%) with experimental results indicating the applicability of proposed model. The multiple regression analysis and ANOVA showed that enzyme activity decreased with increase of As(3+) concentration and interaction time. 3D plot and contour plot between As(3+) concentration and interaction time was helpful to predict residual activity of enzyme for a particular As(3+) at a particular time.

Comparison of analytical methods for the determination of histamine in reference canned fish samples

NASA Astrophysics Data System (ADS)

Jakšić, S.; Baloš, M. Ž.; Mihaljev, Ž.; Prodanov Radulović, J.; Nešić, K.

2017-09-01

Two screening methods for histamine in canned fish, an enzymatic test and a competitive direct enzyme-linked immunosorbent assay (CD-ELISA), were compared with the reversed-phase liquid chromatography (RP-HPLC) standard method. For enzymatic and CD-ELISA methods, determination was conducted according to producers’ manuals. For RP-HPLC, histamine was derivatized with dansyl-chloride, followed by RP-HPLC and diode array detection. Results of analysis of canned fish, supplied as reference samples for proficiency testing, showed good agreement when histamine was present at higher concentrations (above 100 mg kg-1). At a lower level (16.95 mg kg-1), the enzymatic test produced some higher results. Generally, analysis of four reference samples according to CD-ELISA and RP-HPLC showed good agreement for histamine determination (r=0.977 in concentration range 16.95-216 mg kg-1) The results show that the applied enzymatic test and CD-ELISA appeared to be suitable screening methods for the determination of histamine in canned fish.

A simple and sensitive enzymatic method for cholesterol quantification in macrophages and foam cells

PubMed Central

Robinet, Peggy; Wang, Zeneng; Hazen, Stanley L.; Smith, Jonathan D.

2010-01-01

A precise and sensitive method for measuring cellular free and esterified cholesterol is required in order to perform studies of macrophage cholesterol loading, metabolism, storage, and efflux. Until now, the use of an enzymatic cholesterol assay, commonly used for aqueous phase plasma cholesterol assays, has not been optimized for use with solid phase samples such as cells, due to inefficient solubilization of total cholesterol in enzyme compatible solvents. We present an efficient solubilization protocol compatible with an enzymatic cholesterol assay that does not require chemical saponification or chromatographic separation. Another issue with enzyme compatible solvents is the presence of endogenous peroxides that interfere with the enzymatic cholesterol assay. We overcame this obstacle by pretreatment of the reaction solution with the enzyme catalase, which consumed endogenous peroxides resulting in reduced background and increased sensitivity in our method. Finally, we demonstrated that this method for cholesterol quantification in macrophages yields results that are comparable to those measured by stable isotope dilution gas chromatography with mass spectrometry detection. In conclusion, we describe a sensitive, simple, and high-throughput enzymatic method to quantify cholesterol in complex matrices such as cells. PMID:20688754

Extracellular enzymatic activities and physiological profiles of yeasts colonizing fruit trees.

PubMed

Molnárová, Jana; Vadkertiová, Renáta; Stratilová, Eva

2014-07-01

Yeasts form a significant and diverse part of the phyllosphere microbiota. Some yeasts that inhabit plants have been found to exhibit extracellular enzymatic activities. The aim of the present study was to investigate the ability of yeasts isolated from leaves, fruits, and blossoms of fruit trees cultivated in Southwest Slovakia to produce extracellular enzymes, and to discover whether the yeasts originating from these plant organs differ from each other in their physiological properties. In total, 92 strains belonging to 29 different species were tested for: extracellular protease, β-glucosidase, lipase, and polygalacturonase activities; fermentation abilities; the assimilation of xylose, saccharose and alcohols (methanol, ethanol, glycerol); and for growth in a medium with 33% glucose. The black yeast Aureobasidium pullulans showed the largest spectrum of activities of all the species tested. Almost 70% of the strains tested demonstrated some enzymatic activity, and more than 90% utilized one of the carbon compounds tested. Intraspecies variations were found for the species of the genera Cryptococcus and Pseudozyma. Interspecies differences of strains exhibiting some enzymatic activities and utilizing alcohols were also noted. The largest proportion of the yeasts exhibited β-glucosidase activity and assimilated alcohols independently of their origin. The highest number of strains positive for all activities tested was found among the yeasts associated with leaves. Yeasts isolated from blossoms assimilated saccharose and D-xylose the most frequently of all the yeasts tested. The majority of the fruit-inhabiting yeasts grew in the medium with higher osmotic pressure. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation and comparison of Abbott Jaffe and enzymatic creatinine methods: Could the old method meet the new requirements?

PubMed

Küme, Tuncay; Sağlam, Barıs; Ergon, Cem; Sisman, Ali Rıza

2018-01-01

The aim of this study is to evaluate and compare the analytical performance characteristics of the two creatinine methods based on the Jaffe and enzymatic methods. Two original creatinine methods, Jaffe and enzymatic, were evaluated on Architect c16000 automated analyzer via limit of detection (LOD) and limit of quantitation (LOQ), linearity, intra-assay and inter-assay precision, and comparability in serum and urine samples. The method comparison and bias estimation using patient samples according to CLSI guideline were performed on 230 serum and 141 urine samples by analyzing on the same auto-analyzer. The LODs were determined as 0.1 mg/dL for both serum methods and as 0.25 and 0.07 mg/dL for the Jaffe and the enzymatic urine method respectively. The LOQs were similar with 0.05 mg/dL value for both serum methods, and enzymatic urine method had a lower LOQ than Jaffe urine method, values at 0.5 and 2 mg/dL respectively. Both methods were linear up to 65 mg/dL for serum and 260 mg/dL for urine. The intra-assay and inter-assay precision data were under desirable levels in both methods. The higher correlations were determined between two methods in serum and urine (r=.9994, r=.9998 respectively). On the other hand, Jaffe method gave the higher creatinine results than enzymatic method, especially at the low concentrations in both serum and urine. Both Jaffe and enzymatic methods were found to meet the analytical performance requirements in routine use. However, enzymatic method was found to have better performance in low creatinine levels. © 2017 Wiley Periodicals, Inc.

Xylan extraction from pretreated sugarcane bagasse using alkaline and enzymatic approaches.

PubMed

Sporck, Daniele; Reinoso, Felipe A M; Rencoret, Jorge; Gutiérrez, Ana; Del Rio, José C; Ferraz, André; Milagres, Adriane M F

2017-01-01

New biorefinery concepts are necessary to drive industrial use of lignocellulose biomass components. Xylan recovery before enzymatic hydrolysis of the glucan component is a way to add value to the hemicellulose fraction, which can be used in papermaking, pharmaceutical, and food industries. Hemicellulose removal can also facilitate subsequent cellulolytic glucan hydrolysis. Sugarcane bagasse was pretreated with an alkaline-sulfite chemithermomechanical process to facilitate subsequent extraction of xylan by enzymatic or alkaline procedures. Alkaline extraction methods yielded 53% (w/w) xylan recovery. The enzymatic approach provided a limited yield of 22% (w/w) but produced the xylan with the lowest contamination with lignin and glucan components. All extracted xylans presented arabinosyl side groups and absence of acetylation. 2D-NMR data suggested the presence of O -methyl-glucuronic acid and p -coumarates only in enzymatically extracted xylan. Xylans isolated using the enzymatic approach resulted in products with molecular weights (Mw) lower than 6 kDa. Higher Mw values were detected in the alkali-isolated xylans. Alkaline extraction of xylan provided a glucan-enriched solid readily hydrolysable with low cellulase loads, generating hydrolysates with a high glucose/xylose ratio. Hemicellulose removal before enzymatic hydrolysis of the cellulosic fraction proved to be an efficient manner to add value to sugarcane bagasse biorefining. Xylans with varied yield, purity, and structure can be obtained according to the extraction method. Enzymatic extraction procedures produce high-purity xylans at low yield, whereas alkaline extraction methods provided higher xylan yields with more lignin and glucan contamination. When xylan extraction is performed with alkaline methods, the residual glucan-enriched solid seems suitable for glucose production employing low cellulase loadings.

Biochemical conversions of lignocellulosic biomass for sustainable fuel-ethanol production in the upper Midwest

NASA Astrophysics Data System (ADS)

Brodeur-Campbell, Michael J.

Biofuels are an increasingly important component of worldwide energy supply. This research aims to understand the pathways and impacts of biofuels production, and to improve these processes to make them more efficient. In Chapter 2, a life cycle assessment (LCA) is presented for cellulosic ethanol production from five potential feedstocks of regional importance to the upper Midwest — hybrid poplar, hybrid willow, switchgrass, diverse prairie grasses, and logging residues — according to the requirements of Renewable Fuel Standard (RFS). Direct land use change emissions are included for the conversion of abandoned agricultural land to feedstock production, and computer models of the conversion process are used in order to determine the effect of varying biomass composition on overall life cycle impacts. All scenarios analyzed here result in greater than 60% reduction in greenhouse gas emissions relative to petroleum gasoline. Land use change effects were found to contribute significantly to the overall emissions for the first 20 years after plantation establishment. Chapter 3 is an investigation of the effects of biomass mixtures on overall sugar recovery from the combined processes of dilute acid pretreatment and enzymatic hydrolysis. Biomass mixtures studied were aspen, a hardwood species well suited to biochemical processing; balsam, a high-lignin softwood species, and switchgrass, an herbaceous energy crop with high ash content. A matrix of three different dilute acid pretreatment severities and three different enzyme loading levels was used to characterize interactions between pretreatment and enzymatic hydrolysis. Maximum glucose yield for any species was 70% of theoretical for switchgrass, and maximum xylose yield was 99.7% of theoretical for aspen. Supplemental β-glucosidase increased glucose yield from enzymatic hydrolysis by an average of 15%, and total sugar recoveries for mixtures could be predicted to within 4% by linear interpolation of the pure species results. Chapter 4 is an evaluation of the potential for producing Trichoderma reesei cellulose hydrolases in the Kluyveromyces lactis yeast expression system. The exoglucanases Cel6A and Cel7A, and the endoglucanase Cel7B were inserted separately into the K. lactis and the enzymes were analyzed for activity on various substrates. Recombinant Cel7B was found to be active on carboxymethyl cellulose and Avicel powdered cellulose substrates. Recombinant Cel6A was also found to be active on Avicel. Recombinant Cel7A was produced, but no enzymatic activity was detected on any substrate. Chapter 5 presents a new method for enzyme improvement studies using enzyme co-expression and yeast growth rate measurements as a potential high-throughput expression and screening system in K. lactis yeast. Two different K. lactis strains were evaluated for their usefulness in growth screening studies, one wild-type strain and one strain which has had the main galactose metabolic pathway disabled. Sequential transformation and co-expression of the exoglucanase Cel6A and endoglucanase Cel7B was performed, and improved hydrolysis rates on Avicel were detectable in the cell culture supernatant. Future work should focus on hydrolysis of natural substrates, developing the growth screening method, and utilizing the K. lactis expression system for directed evolution of enzymes.

[Activity of antioxidative enzymes of the myocardium during ischemia].

PubMed

Gutkin, D V; Petrovich, Iu A

1982-01-01

Activation of lipid peroxidation during myocardial ischemia may be determined by the reduction of the enzymatic antioxidant cell protection. Such a conclusion has been drawn on the basis of an analysis of variation in the activity of superoxide dismutase, glutathion peroxidase and catalase in experimental myocardial ischemia in rats, induced by ligation of the left descending artery of the heart. In the early period of ischemia (1-3 h) the activity of superoxide dismutase and glutation peroxidase markedly decreases. In the periischemic zone, the fall in the enzymatic activity is not so pronounced. The activity of the enzymes does not reach the basic level 5 days after the operation.

Non-enzymatic browning reaction of glucosamine at mild conditions: Relationship between colour formation, radical scavenging activity and α-dicarbonyl compounds production.

PubMed

Hong, Pui Khoon; Betti, Mirko

2016-12-01

Glucosamine (GlcN, 5% w/v) was incubated in either phosphate buffer or ammonium hydroxide solutions at 40 and 60°C for up to 48h in order to yield caramel solutions. Non-enzymatic browning was monitored via changes in absorption at 280, 320 and 420nm and the physico-chemical properties as well as the generation of short chain α-dicarbonyl compounds were evaluated. Accumulation of GlcN autocondensation products (280nm) proceeded in parallel with the development of pre-melanoidins (320nm) and melanoidins (420nm). The reactive α-dicarbonyls were detected at temperature as low as 40°C within 3h with a maximum level of diacetyl recorded at 6h. The caramel solutions showed a high efficacy in scavenging DPPH and ABTS radicals in accordance with the increasing browning intensity. The results suggest that GlcN browning can be modulated according to the specific desired properties to produce a multi-functional food ingredient that has health-promoting effects. Copyright © 2016 Elsevier Ltd. All rights reserved.

Novel synthesis and characterization of Ag@TiO2 core shell nanostructure for non-enzymatic glucose sensor

NASA Astrophysics Data System (ADS)

T, Dayakar; Venkateswara Rao, K.; Vinodkumar, M.; Bikshalu, K.; Chakradhar, B.; Ramachandra Rao, K.

2018-03-01

Ag@TiO2 core-shell nano composite (ATCSNC) were synthesized by using Ocimum tenuiflorum leaves extract through a simple one-step hydrothermal route for Non-enzymatic glucose sensing material. The prepared NCs were characterized and found high crystallinity, red shift absorbance, interface-bonding parameters, rough surface and network like microstructure through XRD, Uv-vis, FTIR, SEM, and TEM. The prepared ATCSNC have been used for fabrication of glassy carbon electrode (GCE) and the same was applied to test its electro catalytic activity of glucose in 0.1 M NaOH. The promising results were recorded for ATCSNC/GCE with a high sensitivity (1968.72 μAm M-1cm-2), wide linear range (1 μM-8.1 mM), good response time (3 s), and excellent low detection limit (0.19 μM, S/N = 3). Furthermore, the designed sensor exhibits admirable stability and reproducibility, as well as attractive achievability for real sample analysis. As such, the proposed ATCSNC could be highly beneficial in the development of sustainable and eco-friendly glucose sensing devices.

An update on anticancer drug development and delivery targeting carbonic anhydrase IX

PubMed Central

Parkkila, Seppo

2017-01-01

The expression of carbonic anhydrase (CA) IX is up-regulated in many types of solid tumors in humans under hypoxic and acidic microenvironment. Inhibition of CA IX enzymatic activity with selective inhibitors, antibodies or labeled probes has been shown to reverse the acidic environment of solid tumors and reduce the tumor growth establishing the significant role of CA IX in tumorigenesis. Thus, the development of potent antitumor drugs targeting CA IX with minimal toxic effects is important for the target-specific tumor therapy. Recently, several promising antitumor agents against CA IX have been developed to treat certain types of cancers in combination with radiation and chemotherapy. Here we review the inhibition of CA IX by small molecule compounds and monoclonal antibodies. The methods of enzymatic assays, biophysical methods, animal models including zebrafish and Xenopus oocytes, and techniques of diagnostic imaging to detect hypoxic tumors using CA IX-targeted conjugates are discussed with the aim to overview the recent progress related to novel therapeutic agents that target CA IX in hypoxic tumors. PMID:29181278

Microbial characteristics of purple paddy soil in response to Pb pollution.

PubMed

Jiang, Qiu-Ju; Zhang, Yue-Qiang; Zhang, La-Mei; Zhou, Xin-Bin; Shi, Xiao-Jun

2014-05-01

The study focused on the change of microbial characteristics affected by Plumbum pollution with purple paddy soil in an incubation experiment. The results showed that low concentration of Plumbum had little effect on most of microbial amounts, biological activity and enzymatic activity. However, denitrifying activity was inhibited severely, and inhibition rate was up to 98%. Medium and high concentration of Plumbum significantly reduced the amounts and activity of all microorganisms and enzymatic activity, which increased with incubation time. Negative correlations were found between Plumbum concentrations and microbial amounts, biological activity and enzymatic activities except fungi and actinomyces. Thus they can be used to indicate the Plumbum pollution levels to some extent. LD(50) of denitrifying bacteria (DB) and ED50 of denitrifying activity were 852mg/kg and 33.5mg/kg. Across all test soil microbes, denitrifying bacteria was most sensitive to Plumbum pollution in purple paddy soil. Value of early warning showed that anaerobic cellulose-decomposing bacteria (ACDB) and actinomyces were also sensitive to Plumbum pollution. We concluded that denitrifying activity, actinomyces, ACDB or DB can be chosen as predictor of Plumbum contamination in purple paddy soil.

Mechanistic investigation in ultrasound induced enhancement of enzymatic hydrolysis of invasive biomass species.

PubMed

Borah, Arup Jyoti; Agarwal, Mayank; Poudyal, Manisha; Goyal, Arun; Moholkar, Vijayanand S

2016-08-01

This study has assessed four invasive weeds, viz. Saccharum spontaneum (SS), Mikania micrantha (MM), Lantana camara (LC) and Eichhornia crassipes (EC) for enzymatic hydrolysis prior to bioalcohol fermentation. Enzymatic hydrolysis of pretreated biomasses of weeds has been conducted with mechanical agitation and sonication under constant (non-optimum) conditions. Profiles of total reducible sugar release have been fitted to HCH-1 model of enzymatic hydrolysis using Genetic Algorithm. Trends in parameters of this model reveal physical mechanism of ultrasound-induced enhancement of enzymatic hydrolysis. Sonication accelerates hydrolysis kinetics by ∼10-fold. This effect is contributed by several causes, attributed to intense micro-convection generated during sonication: (1) increase in reaction velocity, (2) increase in enzyme-substrate affinity, (3) reduction in product inhibition, and (4) enhancement of enzyme activity due to conformational changes in its secondary structure. Enhancement effect of sonication is revealed to be independent of conditions of enzymatic hydrolysis - whether optimum or non-optimum. Copyright © 2016 Elsevier Ltd. All rights reserved.

Chemotactic Activity of Cyclophilin A in the Skin Mucus of Yellow Catfish (Pelteobagrus fulvidraco) and Its Active Site for Chemotaxis

PubMed Central

Dawar, Farman Ullah; Tu, Jiagang; Xiong, Yang; Lan, Jiangfeng; Dong, Xing Xing; Liu, Xiaoling; Khattak, Muhammad Nasir Khan; Mei, Jie; Lin, Li

2016-01-01

Fish skin mucus is a dynamic barrier for invading pathogens with a variety of anti-microbial enzymes, including cyclophilin A (CypA), a multi-functional protein with peptidyl-prolyl cis/trans isomerase (PPIase) activity. Beside various other immunological functions, CypA induces leucocytes migration in vitro in teleost. In the current study, we have discovered several novel immune-relevant proteins in yellow catfish skin mucus by mass spectrometry (MS). The CypA present among them was further detected by Western blot. Moreover, the CypA present in the skin mucus displayed strong chemotactic activity for yellow catfish leucocytes. Interestingly, asparagine (like arginine in mammals) at position 69 was the critical site in yellow catfish CypA involved in leucocyte attraction. These novel efforts do not only highlight the enzymatic texture of skin mucus, but signify CypA to be targeted for anti-inflammatory therapeutics. PMID:27589721

Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

PubMed Central

Mofford, David M.; Reddy, Gadarla Randheer; Miller, Stephen C.

2014-01-01

Beetle luciferases are thought to have evolved from fatty acyl-CoA synthetases present in all insects. Both classes of enzymes activate fatty acids with ATP to form acyl-adenylate intermediates, but only luciferases can activate and oxidize d-luciferin to emit light. Here we show that the Drosophila fatty acyl-CoA synthetase CG6178, which cannot use d-luciferin as a substrate, is able to catalyze light emission from the synthetic luciferin analog CycLuc2. Bioluminescence can be detected from the purified protein, live Drosophila Schneider 2 cells, and from mammalian cells transfected with CG6178. Thus, the nonluminescent fruit fly possesses an inherent capacity for bioluminescence that is only revealed upon treatment with a xenobiotic molecule. This result expands the scope of bioluminescence and demonstrates that the introduction of a new substrate can unmask latent enzymatic activity that differs significantly from an enzyme’s normal function without requiring mutation. PMID:24616520

Intrinsic Peroxidase-like Activity of Ficin

NASA Astrophysics Data System (ADS)

Yang, Yufang; Shen, Dongjun; Long, Yijuan; Xie, Zhixiong; Zheng, Huzhi

2017-02-01

Ficin is classified as a sulfhydryl protease isolated from the latex of fig trees. In most cases, a particular enzyme fits a few types of substrate and catalyzes one type of reaction. In this investigation, we found sufficient proofs for the intrinsic peroxidase-like activity of ficin and designed experiments to examine its effectiveness in a variety of scenarios. Ficin can transform peroxidase substrates to colored products in the existence of H2O2. Our results also indicate that the active sites of peroxidase-like activity of ficin are different from that of protease, which reveals that one enzyme may catalyze more than one kind of substrate to perform different types of reactions. On the basis of these findings, H2O2 releasing from MCF-7 cells was detected successfully. Our findings support a wider application of ficin in biochemistry and open up the possibility of utilizing ficin as enzymatic mimics in biotechnology and environmental monitoring.

Oxidative stress markers in aqueous humor of patients with senile cataracts.

PubMed

Sawada, Hideko; Fukuchi, Takeo; Abe, Haruki

2009-01-01

To investigate the levels of oxidative stress markers in human eyes with senile cataracts. We conducted a retrospective, case-controlled study of 57 patients with senile cataracts. To assess oxidative stress markers in the eye, we measured the enzymatic activities of superoxide dismutase (SOD) and catalase (CAT) as well as the total protein levels in aqueous humor. In aqueous humor, SOD and CAT activity levels were 0.133 +/- 0.020 and 1.223 +/- 0.081 U/ml, respectively; protein levels were 2.372 +/- 0.166 mg/ml (means +/- SEM). We observed a significant increase in SOD activity and the protein level in progressed nuclear cataracts. No significant age-associated difference in antioxidant enzyme levels was detected. Significant increases in the levels of SOD activity and total protein correlated with the severity of the cataract but not with patient age, suggesting that progressed cataract is associated with molecules leaking from the lens capsule.

Substitutions in PBP2b from β-Lactam-resistant Streptococcus pneumoniae Have Different Effects on Enzymatic Activity and Drug Reactivity*

PubMed Central

Calvez, Philippe; Breukink, Eefjan; Roper, David I.; Dib, Mélanie; Contreras-Martel, Carlos; Zapun, André

2017-01-01

Pneumococcus resists β-lactams by expressing variants of its target enzymes, the penicillin-binding proteins (PBPs), with many amino acid substitutions. Up to 10% of the sequence can be modified. These altered PBPs have a much reduced reactivity with the drugs but retain their physiological activity of cross-linking the peptidoglycan, the major constituent of the bacterial cell wall. However, because β-lactams are chemical and structural mimics of the natural substrate, resistance mediated by altered PBPs raises the following paradox: how PBPs that react poorly with the drugs maintain a sufficient level of activity with the physiological substrate. This question is addressed for the first time in this study, which compares the peptidoglycan cross-linking activity of PBP2b from susceptible and resistant strains with their inhibition by different β-lactams. Unexpectedly, the enzymatic activity of the variants did not correlate with their antibiotic reactivity. This finding indicates that some of the numerous amino acid substitutions were selected to restore a viable level of enzymatic activity by a compensatory molecular mechanism. PMID:28062575

Enzymatic reaction paths as determined by transition path sampling

NASA Astrophysics Data System (ADS)

Masterson, Jean Emily

Enzymes are biological catalysts capable of enhancing the rates of chemical reactions by many orders of magnitude as compared to solution chemistry. Since the catalytic power of enzymes routinely exceeds that of the best artificial catalysts available, there is much interest in understanding the complete nature of chemical barrier crossing in enzymatic reactions. Two specific questions pertaining to the source of enzymatic rate enhancements are investigated in this work. The first is the issue of how fast protein motions of an enzyme contribute to chemical barrier crossing. Our group has previously identified sub-picosecond protein motions, termed promoting vibrations (PVs), that dynamically modulate chemical transformation in several enzymes. In the case of human heart lactate dehydrogenase (hhLDH), prior studies have shown that a specific axis of residues undergoes a compressional fluctuation towards the active site, decreasing a hydride and a proton donor--acceptor distance on a sub-picosecond timescale to promote particle transfer. To more thoroughly understand the contribution of this dynamic motion to the enzymatic reaction coordinate of hhLDH, we conducted transition path sampling (TPS) using four versions of the enzymatic system: a wild type enzyme with natural isotopic abundance; a heavy enzyme where all the carbons, nitrogens, and non-exchangeable hydrogens were replaced with heavy isotopes; and two versions of the enzyme with mutations in the axis of PV residues. We generated four separate ensembles of reaction paths and analyzed each in terms of the reaction mechanism, time of barrier crossing, dynamics of the PV, and residues involved in the enzymatic reaction coordinate. We found that heavy isotopic substitution of hhLDH altered the sub-picosecond dynamics of the PV, changed the favored reaction mechanism, dramatically increased the time of barrier crossing, but did not have an effect on the specific residues involved in the PV. In the mutant systems, we observed changes in the reaction mechanism and altered contributions of the mutated residues to the enzymatic reaction coordinate, but we did not detect a substantial change in the time of barrier crossing. These results confirm the importance of maintaining the dynamics and structural scaffolding of the hhLDH PV in order to facilitate facile barrier passage. We also utilized TPS to investigate the possible role of fast protein dynamics in the enzymatic reaction coordinate of human dihydrofolate reductase (hsDHFR). We found that sub-picosecond dynamics of hsDHFR do contribute to the reaction coordinate, whereas this is not the case in the E. coli version of the enzyme. This result indicates a shift in the DHFR family to a more dynamic version of catalysis. The second inquiry we addressed in this thesis regarding enzymatic barrier passage concerns the variability of paths through reactive phase space for a given enzymatic reaction. We further investigated the hhLDH-catalyzed reaction using a high-perturbation TPS algorithm. Though we saw that alternate reaction paths were possible, the dominant reaction path we observed corresponded to that previously elucidated in prior hhLDH TPS studies. Since the additional reaction paths we observed were likely high-energy, these results indicate that only the dominant reaction path contributes significantly to the overall reaction rate. In conclusion, we show that the enzymes hhLDH and hsDHFR exhibit paths through reactive phase space where fast protein motions are involved in the enzymatic reaction coordinate and exhibit a non-negligible contribution to chemical barrier crossing.

Phospholipase C produced by Clostridium botulinum types C and D: comparison of gene, enzymatic, and biological activities with those of Clostridium perfringens alpha-toxin.

PubMed

Fatmawati, Ni Nengah Dwi; Sakaguchi, Yoshihiko; Suzuki, Tomonori; Oda, Masataka; Shimizu, Kenta; Yamamoto, Yumiko; Sakurai, Jun; Matsushita, Osamu; Oguma, Keiji

2013-01-01

Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

Modulation of the Activity of Mycobacterium tuberculosis LipY by Its PE Domain

PubMed Central

Garrett, Christopher K.; Broadwell, Lindsey J.; Hayne, Cassandra K.; Neher, Saskia B.

2015-01-01

Mycobacterium tuberculosis harbors over 160 genes encoding PE/PPE proteins, several of which have roles in the pathogen’s virulence. A number of PE/PPE proteins are secreted via Type VII secretion systems known as the ESX secretion systems. One PE protein, LipY, has a triglyceride lipase domain in addition to its PE domain. LipY can regulate intracellular triglyceride levels and is also exported to the cell wall by one of the ESX family members, ESX-5. Upon export, LipY’s PE domain is removed by proteolytic cleavage. Studies using cells and crude extracts suggest that LipY’s PE domain not only directs its secretion by ESX-5, but also functions to inhibit its enzymatic activity. Here, we attempt to further elucidate the role of LipY’s PE domain in the regulation of its enzymatic activity. First, we established an improved purification method for several LipY variants using detergent micelles. We then used enzymatic assays to confirm that the PE domain down-regulates LipY activity. The PE domain must be attached to LipY in order to effectively inhibit it. Finally, we determined that full length LipY and the mature lipase lacking the PE domain (LipYΔPE) have similar melting temperatures. Based on our improved purification strategy and activity-based approach, we concluded that LipY’s PE domain down-regulates its enzymatic activity but does not impact the thermal stability of the enzyme. PMID:26270534

Enzymatically Active Microgels from Self-Assembling Protein Nanofibrils for Microflow Chemistry

PubMed Central

2015-01-01

Amyloid fibrils represent a generic class of protein structure associated with both pathological states and with naturally occurring functional materials. This class of protein nanostructure has recently also emerged as an excellent foundation for sophisticated functional biocompatible materials including scaffolds and carriers for biologically active molecules. Protein-based materials offer the potential advantage that additional functions can be directly incorporated via gene fusion producing a single chimeric polypeptide that will both self-assemble and display the desired activity. To succeed, a chimeric protein system must self-assemble without the need for harsh triggering conditions which would damage the appended functional protein molecule. However, the micrometer to nanoscale patterning and morphological control of protein-based nanomaterials has remained challenging. This study demonstrates a general approach for overcoming these limitations through the microfluidic generation of enzymatically active microgels that are stabilized by amyloid nanofibrils. The use of scaffolds formed from biomaterials that self-assemble under mild conditions enables the formation of catalytic microgels while maintaining the integrity of the encapsulated enzyme. The enzymatically active microgel particles show robust material properties and their porous architecture allows diffusion in and out of reactants and products. In combination with microfluidic droplet trapping approaches, enzymatically active microgels illustrate the potential of self-assembling materials for enzyme immobilization and recycling, and for biological flow-chemistry. These design principles can be adopted to create countless other bioactive amyloid-based materials with diverse functions. PMID:26030507

Fatty acid biosynthesis revisited: Structure elucidation and metabolic engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beld, Joris; Lee, D. John; Burkart, Michael D.

Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understandingmore » of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases' many intricate structural and regulatory elements. Lastly, in this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field.« less

Effect of alkali lignins with different molecular weights from alkali pretreated rice straw hydrolyzate on enzymatic hydrolysis.

PubMed

Li, Yun; Qi, Benkun; Luo, Jianquan; Wan, Yinhua

2016-01-01

This study investigated the effect of alkali lignins with different molecular weights on enzymatic hydrolysis of lignocellulose. Different alkali lignins fractions, which were obtained from cascade ultrafiltration, were added into the dilute acid pretreated (DAP) and alkali pretreated (AP) rice straws respectively during enzymatic hydrolysis. The results showed that the addition of alkali lignins enhanced the hydrolysis and the enhancement for hydrolysis increased with increasing molecular weights of alkali lignins, with maximum enhancement being 28.69% for DAP and 20.05% for AP, respectively. The enhancement was partly attributed to the improved cellulase activity, and filter paper activity increased by 18.03% when adding lignin with highest molecular weight. It was found that the enhancement of enzymatic hydrolysis was correlated with the adsorption affinity of cellulase on alkali lignins, and the difference in surface charge and hydrophobicity of alkali lignins were responsible for the difference in affinity between cellulase and lignins. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fatty acid biosynthesis revisited: Structure elucidation and metabolic engineering

DOE PAGES

Beld, Joris; Lee, D. John; Burkart, Michael D.

2014-10-20

Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understandingmore » of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases' many intricate structural and regulatory elements. Lastly, in this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field.« less

Cellular localization of Na(+), K(+)-ATPase in the mammalian vestibular system

NASA Technical Reports Server (NTRS)

Kerr, T. P.

1984-01-01

Two different, but complementary, procedures for cellular localization of Na+, K+-ATPase in the guinea pig vestibular system were employed. One of these techniques, devised by Stirling, depends upon the well documented ability of the specific inhibitor ouabain to bind selectively to Na+,K+-ATPase, blocking catalytic activity. Microdisected vestibular tissues are incubated with tritium-labelled (3H-) ouabain, and regions with a high concentration of Na+,K+-ATPase are subsequently identified by light microscope autoradiography. A second method, originated by Ernst, detects inorganic phosphate released from an artificial substrate (nitrophenyl phosphate) by catalytic activity of the enzyme. In the presence of strontium ion, phosphate is precipitated near regions of high activity, then converted to a product which may finally be visualized in the electron microscope. This cytochemical enzymatic reaction is inhibited by ouabain.

Magnetic beads-based DNAzyme recognition and AuNPs-based enzymatic catalysis amplification for visual detection of trace uranyl ion in aqueous environment.

PubMed

Zhang, Hongyan; Lin, Ling; Zeng, Xiaoxue; Ruan, Yajuan; Wu, Yongning; Lin, Minggui; He, Ye; Fu, FengFu

2016-04-15

We herein developed a novel biosensor for the visual detection of trace uranyl ion (UO2(2+)) in aqueous environment with high sensitivity and specificity by using DNAzyme-functionalized magnetic beads (MBs) for UO2(2+) recognition and gold nano-particles (AuNPs)-based enzymatic catalysis oxidation of TMB (3,3',5,5'-tetramethylbenzidine sulfate) for signal generation. The utilization of MBs facilitates the magnetic separation and collection of sensing system from complex sample solution, which leads to more convenient experimental operation and more strong resistibility of the biosensor to the matrix of sample, and the utilization of AuNPs-based enzymatic catalysis amplification greatly improved the sensitivity of the biosensor. Compared with the previous DNAzyme-based UO2(2+) sensors, the proposed biosensor has outstanding advantages such as relative high sensitivity and specificity, operation convenience, low cost and more strong resistibility to the matrix of sample. It can be used to detect as low as 0.02 ppb (74 pM) of UO2(2+) in aqueous environment by only naked-eye observation and 1.89 ppt (7.0 pM) of UO2(2+) by UV-visible spectrophotometer with a recovery of 93-99% and a RSD ≤ 5.0% (n=6) within 3h. Especially, the visual detection limit of 0.02 ppb (74 pM) is much lower than the maximum allowable level of UO2(2+) (130 nM) in the drinking water defined by the U.S. Environmental Protection Agency (EPA), indicating that our method meets the requirement of rapid and on-site detection of UO2(2+) in the aqueous environment by only naked-eye observation. Copyright © 2015 Elsevier B.V. All rights reserved.

Enzymatic Degradation of Aromatic and Aliphatic Polyesters by P. pastoris Expressed Cutinase 1 from Thermobifida cellulosilytica

PubMed Central

Gamerith, Caroline; Vastano, Marco; Ghorbanpour, Sahar M.; Zitzenbacher, Sabine; Ribitsch, Doris; Zumstein, Michael T.; Sander, Michael; Herrero Acero, Enrique; Pellis, Alessandro; Guebitz, Georg M.

2017-01-01

To study hydrolysis of aromatic and aliphatic polyesters cutinase 1 from Thermobifida cellulosilytica (Thc_Cut1) was expressed in P. pastoris. No significant differences between the expression of native Thc_Cut1 and of two glycosylation site knock out mutants (Thc_Cut1_koAsn and Thc_Cut1_koST) concerning the total extracellular protein concentration and volumetric activity were observed. Hydrolysis of poly(ethylene terephthalate) (PET) was shown for all three enzymes based on quantification of released products by HPLC and similar concentrations of released terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) were detected for all enzymes. Both tested aliphatic polyesters poly(butylene succinate) (PBS) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were hydrolyzed by Thc_Cut1 and Thc_Cut1_koST, although PBS was hydrolyzed to significantly higher extent than PHBV. These findings were also confirmed via quartz crystal microbalance (QCM) analysis; for PHBV only a small mass change was observed while the mass of PBS thin films decreased by 93% upon enzymatic hydrolysis with Thc_Cut1. Although both enzymes led to similar concentrations of released products upon hydrolysis of PET and PHBV, Thc_Cut1_koST was found to be significantly more active on PBS than the native Thc_Cut1. Hydrolysis of PBS films by Thc_Cut1 and Thc_Cut1_koST was followed by weight loss and scanning electron microscopy (SEM). Within 96 h of hydrolysis up to 92 and 41% of weight loss were detected with Thc_Cut1_koST and Thc_Cut1, respectively. Furthermore, SEM characterization of PBS films clearly showed that enzyme tretment resulted in morphological changes of the film surface. PMID:28596765

Electrochemical branched-DNA assay for polymerase chain reaction-free detection and quantification of oncogenes in messenger RNA.

PubMed

Lee, Ai-Cheng; Dai, Ziyu; Chen, Baowei; Wu, Hong; Wang, Jun; Zhang, Aiguo; Zhang, Lurong; Lim, Tit-Meng; Lin, Yuehe

2008-12-15

We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcripts in the population of messenger ribonucleic acid (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify the target signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-naphthyl phosphate. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were systematically optimized. A detection limit of 1 fM (1 x 10(-19) mol of target transcripts in 100 microL) and a 3-order-wide dynamic range of target concentration were achieved by the electrochemical bDNA assay. Such limit corresponded to approximately 17 fg of the p185 BCR-ABL fusion transcripts. The specificity and sensitivity of assay enabled direct detection of target transcripts in as little as 4.6 ng of mRNA population without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcripts in mRNA population. A mean transcript copy number of 62,900/ng of mRNA was determined, which was at least 50-fold higher than that of real-time quantitative PCR (qPCR). The finding was consistent with the underestimation of targets by qPCR reported earlier. In addition, the unique design based on bDNA technology increases the assay specificity as only the p185 BCR-ABL fusion transcripts will respond to the detection. The approach thus provides a simple, sensitive, accurate, and quantitative tool alternative to the qPCR for early disease diagnosis.

A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay

PubMed Central

Guo, Hou-Fu; Cho, Eun Jeong; Devkota, Ashwini K.; Chen, Yulong; Russell, William; Phillips, George N.; Yamauchi, Mitsuo; Dalby, Kevin; Kurie, Jonathan M.

2017-01-01

Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated previously using E coli– or insect-based expression systems was either insoluble or enzymatically unstable, and LH2 enzymatic activity assays have measured radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems. PMID:28216326

A non-enzymatic function of 17β-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival

PubMed Central

Rauschenberger, Katharina; Schöler, Katja; Sass, Jörn Oliver; Sauer, Sven; Djuric, Zdenka; Rumig, Cordula; Wolf, Nicole I; Okun, Jürgen G; Kölker, Stefan; Schwarz, Heinz; Fischer, Christine; Grziwa, Beate; Runz, Heiko; Nümann, Astrid; Shafqat, Naeem; Kavanagh, Kathryn L; Hämmerling, Günter; Wanders, Ronald J A; Shield, Julian P H; Wendel, Udo; Stern, David; Nawroth, Peter; Hoffmann, Georg F; Bartram, Claus R; Arnold, Bernd; Bierhaus, Angelika; Oppermann, Udo; Steinbeisser, Herbert; Zschocke, Johannes

2010-01-01

Deficiency of the mitochondrial enzyme 2-methyl-3-hydroxybutyryl-CoA dehydrogenase involved in isoleucine metabolism causes an organic aciduria with atypical neurodegenerative course. The disease-causing gene is HSD17B10 and encodes 17β-hydroxysteroid dehydrogenase type 10 (HSD10), a protein also implicated in the pathogenesis of Alzheimer's disease. Here we show that clinical symptoms in patients are not correlated with residual enzymatic activity of mutated HSD10. Loss-of-function and rescue experiments in Xenopus embryos and cells derived from conditional Hsd17b10−/− mice demonstrate that a property of HSD10 independent of its enzymatic activity is essential for structural and functional integrity of mitochondria. Impairment of this function in neural cells causes apoptotic cell death whilst the enzymatic activity of HSD10 is not required for cell survival. This finding indicates that the symptoms in patients with mutations in the HSD17B10 gene are unrelated to accumulation of toxic metabolites in the isoleucine pathway and, rather, related to defects in general mitochondrial function. Therefore alternative therapeutic approaches to an isoleucine-restricted diet are required. PMID:20077426

A dual enzymatic-biosensor for simultaneous determination of glucose and cholesterol in serum and peritoneal macrophages of diabetic mice: evaluation of the diabetes-accelerated atherosclerosis risk.

PubMed

Huang, Qilin; An, Yarui; Tang, Linlin; Jiang, Xiaoli; Chen, Hua; Bi, Wenji; Wang, Zhongchuan; Zhang, Wen

2011-11-30

In this paper, a novel dual enzymatic-biosensor is described for simultaneous determination of glucose and cholesterol in serum and peritoneal macrophages (PMs) of diabetic mice to evaluate the risk of diabetes-accelerated atherosclerosis. The biosensor was constructed by a three-step method. First, a poly-thionine (PTH) film was assembled on the surface of glassy carbon electrode by cyclic voltammetric electropolymerization of thionine, which serves as an electron transfer mediator (ETM). Second, gold nanoparticles (GNPs) were covered on the surface of PTH facilitating the electron transfer between glucose oxidase (GOx), cholesterol oxidase (ChOx) and electrode. Finally, the enzymes, GOx, cholesterol esterase (ChE), and ChOx, were covalently attached to the PTH layer through a chitosan (CH) linker. The PTH coupled with GNPs provides good selectivity, high sensitivity and little crosstalk for the dual enzymatic-biosensor. The developed biosensor had good electrocatalytic activity toward the oxidations of glucose and cholesterol, exhibiting a linear range from 0.008 mM to 6.0 mM for glucose with a detection limit of 2.0 μM, and a linear range from 0.002 mM to 1.0 mM for cholesterol with a detection limit of 0.6 μM. The results of the diabetic mice demonstrated that the cholesterol level did not change obviously with the increase of glucose level in serum, while the cholesterol level was induced with the increase of the glucose level in PMs. Previous studies have shown that the large accumulation of cholesterol in macrophage could lead to macrophage foam cell formation, which is the hallmark of early atherosclerosis. This study provides useful further evidences for the development of diabetes-accelerated atherosclerosis. Copyright © 2011 Elsevier B.V. All rights reserved.

An association between Schistosoma mansoni worms and an enzymatically-active protease/peptidase in mouse blood.

PubMed

Darani, H Y; Doenhoff, M J

2008-04-01

An enzyme found previously in extracts of adult Schistosoma mansoni worms, that hydrolysed the chromogenic substrate N-acetyl-DL-phenylalanine beta-naphthyl-ester, has here been further investigated and characterized. Evidence that the molecule found in the parasite was antigenically and enzymatically homologous with a constituent of normal mouse plasma has been consolidated using a monospecific serum in immunoelectrophoresis and Western immunoblotting. The molecular size of the enzyme was found to be approximately 70 kDa and it was inhibited by a serine protease inhibitor, but not by inhibitors of other classes of protease. The enzymatic activity found in normal mouse serum was also found in normal rat serum, but not in sera from several other mammalian species.

Assessment of pollution impact on biological activity and structure of seabed bacterial communities in the Port of Livorno (Italy).

PubMed

Iannelli, Renato; Bianchi, Veronica; Macci, Cristina; Peruzzi, Eleonora; Chiellini, Carolina; Petroni, Giulio; Masciandaro, Grazia

2012-06-01

The main objective of this study was to assess the impact of pollution on seabed bacterial diversity, structure and activity in the Port of Livorno. Samples of seabed sediments taken from five selected sites within the port were subjected to chemical analyses, enzymatic activity detection, bacterial count and biomolecular analysis. Five different statistics were used to correlate the level of contamination with the detected biological indicators. The results showed that the port is mainly contaminated by variable levels of petroleum hydrocarbons and heavy metals, which affect the structure and activity of the bacterial population. Irrespective of pollution levels, the bacterial diversity did not diverge significantly among the assessed sites and samples, and no dominance was observed. The type of impact of hydrocarbons and heavy metals was controversial, thus enforcing the supposition that the structure of the bacterial community is mainly driven by the levels of nutrients. The combined use of chemical and biological essays resulted in an in-depth observation and analysis of the existing links between pollution macro-indicators and biological response of seabed bacterial communities. Copyright © 2012 Elsevier B.V. All rights reserved.

Roles of histidine residues in plant vacuolar H(+)-pyrophosphatase.

PubMed

Hsiao, Yi Y; Van, Ru C; Hung, Shu H; Lin, Hsin H; Pan, Rong L

2004-02-15

Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP(i) hydrolysis. Alignment analysis on amino acid sequence demonstrates that vacuolar H(+)-PPase of mung bean contains six highly conserved histidine residues. Previous evidence indicated possible involvement of histidine residue(s) in enzymatic activity and H(+)-translocation of vacuolar H(+)-PPase as determined by using histidine specific modifier, diethylpyrocarbonate [J. Protein Chem. 21 (2002) 51]. In this study, we further attempted to identify the roles of histidine residues in mung bean vacuolar H(+)-PPase by site-directed mutagenesis. A line of mutants with histidine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Among the mutants scrutinized, only the mutation of H716 significantly decreased the enzymatic activity, the proton transport, and the coupling ratio of vacuolar H(+)-PPase. The enzymatic activity of H716A is relatively resistant to inhibition by diethylpyrocarbonate as compared to wild-type and other mutants, indicating that H716 is probably the target residue for the attack by this modifier. The mutation at H716 of V-PPase shifted the optimum pH value but not the T(1/2) (pretreatment temperature at which half enzymatic activity is observed) for PP(i) hydrolytic activity. Mutation of histidine residues obviously induced conformational changes of vacuolar H(+)-PPase as determined by immunoblotting analysis after limited trypsin digestion. Furthermore, mutation of these histidine residues modified the inhibitory effects of F(-) and Na(+), but not that of Ca(2+). Single substitution of H704, H716 and H758 by alanine partially released the effect of K(+) stimulation, indicating possible location of K(+) binding in the vicinity of domains surrounding these residues.

Retinoic Acid 4-Hydroxylase Inducibility and Clinical Response to Isotretinoin in Acne Patients

PubMed Central

Wang, Frank; Kwak, Heh Shin R.; Elbuluk, Nada; Kaczmarek, Anya L.; Hamilton, Ted; Voorhees, John J.; Fisher, Gary J.; Kang, Sewon

2011-01-01

Background The cytochrome P450 enzyme CYP26 (retinoic acid 4-hydroxylase) initiates the catabolism of all-trans retinoic acid (tRA) and limits the effects of tRA. The CYP26 enzyme acts on tRA, but not 13-cis RA (isotretinoin), a retinoid used to treat severe acne. However, 13-cis RA can isomerize to tRA, which can then be metabolized by CYP26. Objective In healthy subjects, we assessed the variability of CYP26 enzymatic activity. We then investigated whether response to oral 13-cis RA among acne patients correlates with variability in CYP26 expression. Methods In healthy subjects, we isolated microsomal fractions from the epidermis of keratome biopsies and measured CYP26 enzymatic activity in untreated skin and skin treated with tRA. Enzymatic activity was determined based on rate of formation of 4-hydroxy RA (pg/min) per mg microsomal protein. Using real-time PCR we quantified CYP26 mRNA induction after tRA application in acne patients who responded or did not respond to one course of 13-cis RA. Results In normal skin (N=118), CYP26 enzymatic activity was widely variable (1–180 pg/min per mg microsomal fraction; mean 42.7 ± 3.5). Furthermore, CYP26 enzymatic activity was inducible in a dose-dependent manner in normal skin following tRA application, but not correlated with age or sex (N=29). In acne patients, CYP26 mRNA induction following 0.1% tRA application did not differ (P>0.05) between subjects who responded (N=8, 587±325 fold) or did not respond (N=8, 657±227 fold) to one course of 13-cis RA. Limitations The small number of acne patients treated with 13-cis RA was a major limitation. Conclusion Factors other than CYP26 activity may determine response to isotretinoin in acne. PMID:19525031

Metabolite secretion, Fe(3+)-reducing activity and wood degradation by the white-rot fungus Trametes versicolor ATCC 20869.

PubMed

Aguiar, André; Gavioli, Daniela; Ferraz, André

2014-11-01

Trametes versicolor is a promising white-rot fungus for the biological pretreatment of lignocellulosic biomass. In the present work, T. versicolor ATCC 20869 was grown on Pinus taeda wood chips under solid-state fermentation conditions to examine the wood-degrading mechanisms employed by this fungus. Samples that were subjected to fungal pretreatment for one-, two- and four-week periods were investigated. The average mass loss ranged from 5 % to 8 % (m m(-)(1)). The polysaccharides were preferentially degraded: hemicellulose and glucan losses reached 13.4 % and 6.9 % (m m(-)(1)) after four weeks of cultivation, respectively. Crude enzyme extracts were obtained and assayed using specific substrates and their enzymatic activities were measured. Xylanases were the predominant enzymes, while cellobiohydrolase activities were marginally detected. Endoglucanase activity, β-glucosidase activity, and wood glucan losses increased up to the second week of biodegradation and remained constant after that time. Although no lignin-degrading enzyme activity was detected, the lignin loss reached 7.5 % (m m(-)(1)). Soluble oxalic acid was detected in trace quantities. After the first week of biodegradation, the Fe(3+)-reducing activity steadily increased with time, but the activity levels were always lower than those observed in the undecayed wood. The progressive wood polymer degradation appeared related to the secretion of hydrolytic enzymes, as well as to Fe(3+)-reducing activity, which was restored in the cultures after the first week of biodegradation. Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Effect of ionic liquid on activity, stability, and structure of enzymes: a review.

PubMed

Naushad, Mu; Alothman, Zied Abdullah; Khan, Abbul Bashar; Ali, Maroof

2012-11-01

Ionic liquids have shown their potential as a solvent media for many enzymatic reactions as well as protein preservation, because of their unusual characteristics. It is also observed that change in cation or anion alters the physiochemical properties of the ionic liquids, which in turn influence the enzymatic reactions by altering the structure, activity, enatioselectivity, and stability of the enzymes. Thus, it is utmost need of the researchers to have full understanding of these influences created by ionic liquids before choosing or developing an ionic liquid to serve as solvent media for enzymatic reaction or protein preservation. So, in the present review, we try to shed light on effects of ionic liquids chemistry on structure, stability, and activity of enzymes, which will be helpful for the researchers in various biocatalytic applications. Copyright © 2012. Published by Elsevier B.V.