Modification of chemical reactivity of enzymatic hydrolysis lignin by ultrasound treatment in dilute alkaline solutions.

PubMed

Ma, Zhuoming; Li, Shujun; Fang, Guizhen; Patil, Nikhil; Yan, Ning

2016-12-01

In this study, we have explored various ultrasound treatment conditions for structural modification of enzymatic hydrolysis lignin (EHL) for enhanced chemical reactivity. The key structural modifications were characterized by using a combination of analytical methods, including, Fourier Transform-Infrared spectroscopy (FTIR), Proton Nuclear Magnetic Resonance ( 1 H NMR), Gel permeation chromatography (GPC), X-ray photoelectron spectroscopy (XPS), and Folin-Ciocalteu (F-C) method. Chemical reactivity of the modified EHL samples was determined by both 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity and their reactivity towards formaldehyde. It was observed that the modified EHL had a higher phenolic hydroxyl group content, a lower molecular weight, a higher reactivity towards formaldehyde, and a greater antioxidant property. The higher reactivity demonstrated by the samples after treatment suggesting that ultrasound is a promising method for modifying enzymatic hydrolysis lignin for value-added applications. Copyright © 2016 Elsevier B.V. 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.

Simultaneous Determination of Four Compounds, Campesterol, Emodin8-O-β-D-Glucopyranoside, Quercetin, and Isoquercitrin in Reynoutria sachalinensis by High-performance Liquid Chromatography-Diode Array Detector

PubMed Central

Eom, Min Rye; Weon, Jin Bae; Jung, Youn Sik; Ryu, Ga Hee; Yang, Woo Seung; Ma, Choong Je

2017-01-01

Background: Reynoutria sachalinensis is a well-known and used herbal medicine to treatment of arthralgia, jaundice, amenorrhea, coughs, carbuncles, and sores. Objective: We have developed high-performance liquid chromatography analysis method for simultaneous determination of isolated four compounds, campesterol, emodin8-O-β-D-glucopyranoside, quercetin, and isoquercitrin from R. sachalinensis is. Materials and Methods: The four compounds were separated on Shiseido C18 column (S-5 μm, 4.6 mm I.D. ×250 mm) at a column temperature of 25°C. The mobile phase composed of water and methanol with gradient elution system, and flow rate is 1.0 ml/min. The detection wavelength was set at 205 nm. Results: Validation of this analytical method was evaluated by linearity, precision, and accuracy test. This established method had good linearity (R2 > 0.997). The relative standard deviation values of intra- and inter-day testing were indicated that <2%, and accuracy is 91.66%–103.31% at intraday and 91.69%–103.31% at intraday. The results of recovery test were 92.60%–108.99%. Conclusion: In these results, developed method was accurate and reliable to the quality evaluation of campesterol, emodin 8-O-β-D-glucopyranoside, quercetin, and isoquercitrin isolated from R. sachalinensis. SUMMARY We have developed high-performance liquid analysis method for simultaneous determination of 4 compounds of Reynoutria sachalinensis. Abbreviations used: HPLC: High-performance liquid chromatography, DAD: Diode array detector, LOD: Limit of detection, LOQ: Limit of quantitation, ICH: International Conference on Harmonisation. PMID:28808389

Identification and antioxidant properties of polyphenols in lotus seed epicarp at different ripening stages.

PubMed

Liu, Ying; Ma, Shuang-shuang; Ibrahim, S A; Li, Er-hu; Yang, Hong; Huang, Wen

2015-10-15

In this study, polyphenols from lotus seed epicarp (PLSE) at three different ripening stages were purified by column chromatography and identified by RP-HPLC and HPLC-ESI-MS(2). The antioxidant activities of PLSE were also investigated. We found that the contents of PLSE at the green ripening stage, half ripening stage and full ripening stage are 13.08%, 10.95% and 6.73% respectively. The levels of catechin, epicatechin, hyperoside, and isoquercitrin in PLSE at the three different ripening stages were different. Moreover, the amounts of catechin and epicatechin decreased, while the contents of hyperoside and isoquercitrin increased as the seed ripened. We found that PLSE at three different ripening stages had good scavenging abilities on DPPH and ABTS(+) radicals. However, the scavenging ability decreased with maturation. Our results may be valuable with regard to the utilization of lotus seed epicarp as a functional food material. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enhanced enzymatic saccharification of corn stover by in situ modification of lignin with poly (ethylene glycol) ether during low temperature alkali pretreatment.

PubMed

Lai, Chenhuan; Tang, Shuo; Yang, Bo; Gao, Ziqi; Li, Xin; Yong, Qiang

2017-11-01

A novel pretreatment process of corn stover was established in this study by in situ modification of lignin with poly (ethylene glycol) diglycidyl ether (PEGDE) during low temperature alkali pretreatment. The addition of PEGDE obviously improved the enzymatic hydrolysis by covalently modifying the residual lignins in substrates. Under the optimized conditions (pretreated with 10% (w/w) NaOH and 10% (w/w) PEGDE at 70°C for 2.5h), the total fermentable sugar yield was increased by 46.4%, from 23.7g to 34.7g per 100g raw materials. Additionally, the remaining activities of exo-glucanase and β-glucosidase in supernatant were increased by 58.6% and 40.6% respectively, demonstrating that the enhancement of enzymatic hydrolysis was mainly due to the alleviation of enzyme non-productive binding. Although the isolated lignin modified with PEGDE enhanced the enzymatic hydrolysis of substrates as well, this in situ lignin modification provided an efficient but simple way to improve enzymatic saccharification. Copyright © 2017. Published by Elsevier Ltd.

Corneal Protection for Burn Patients

DTIC Science & Technology

2011-07-01

enzymatic degradation The methods used to harvest AM, to treat the amnion with proteolytic enzymes and to measure the extent of enzymatic...Multiple enzymatic cleavages of the membrane proteins degrade the AM and release peptide fragments containing free amino groups. We measured the level...appropriate modified AM for the goals of this study. Although these measurements were not part of the original proposal, we will soon be able quantify

Photoelectrochemical NADH Regeneration using Pt-Modified p -GaAs Semiconductor Electrodes

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

Stufano, Paolo; Paris, Aubrey R.; Bocarsly, Andrew

Cofactor regeneration in enzymatic reductions is crucial for the application of enzymes to both biological and energy-related catalysis. Specifically, regenerating NADH from NAD + is of great interest, and using electrochemistry to achieve this end is considered a promising option. Here in this paper, we report the first example of photoelectrochemical NADH regeneration at the illuminated (λ >600 nm), metal-modified p-type semiconductor electrode Pt/p-GaAs. Although bare p-GaAs electrodes produce only enzymatically inactive NAD 2, NADH was produced at the illuminated Pt-modified p-GaAs surface. At low overpotential (–0.75 V vs. Ag/AgCl), Pt/p-GaAs exhibited a seven-fold greater Faradaic efficiency for the formationmore » of NADH than Pt alone, with reduced competition from the hydrogen evolution reaction. Improved Faradaic efficiency and low overpotential suggest the possible utility of Pt/p-GaAs in energy-related NADH-dependent enzymatic processes.« less

Photoelectrochemical NADH Regeneration using Pt-Modified p -GaAs Semiconductor Electrodes

DOE PAGES

Stufano, Paolo; Paris, Aubrey R.; Bocarsly, Andrew

2017-02-22

Cofactor regeneration in enzymatic reductions is crucial for the application of enzymes to both biological and energy-related catalysis. Specifically, regenerating NADH from NAD + is of great interest, and using electrochemistry to achieve this end is considered a promising option. Here in this paper, we report the first example of photoelectrochemical NADH regeneration at the illuminated (λ >600 nm), metal-modified p-type semiconductor electrode Pt/p-GaAs. Although bare p-GaAs electrodes produce only enzymatically inactive NAD 2, NADH was produced at the illuminated Pt-modified p-GaAs surface. At low overpotential (–0.75 V vs. Ag/AgCl), Pt/p-GaAs exhibited a seven-fold greater Faradaic efficiency for the formationmore » of NADH than Pt alone, with reduced competition from the hydrogen evolution reaction. Improved Faradaic efficiency and low overpotential suggest the possible utility of Pt/p-GaAs in energy-related NADH-dependent enzymatic processes.« less

Modified Low Density Lipoprotein and Lipoprotein-Containing Circulating Immune Complexes as Diagnostic and Prognostic Biomarkers of Atherosclerosis and Type 1 Diabetes Macrovascular Disease

PubMed Central

Orekhov, Alexander N.; Bobryshev, Yuri V.; Sobenin, Igor A.; Melnichenko, Alexandra A.; Chistiakov, Dimitry A.

2014-01-01

In atherosclerosis; blood low-density lipoproteins (LDL) are subjected to multiple enzymatic and non-enzymatic modifications that increase their atherogenicity and induce immunogenicity. Modified LDL are capable of inducing vascular inflammation through activation of innate immunity; thus, contributing to the progression of atherogenesis. The immunogenicity of modified LDL results in induction of self-antibodies specific to a certain type of modified LDL. The antibodies react with modified LDL forming circulating immune complexes. Circulating immune complexes exhibit prominent immunomodulatory properties that influence atherosclerotic inflammation. Compared to freely circulating modified LDL; modified LDL associated with the immune complexes have a more robust atherogenic and proinflammatory potential. Various lipid components of the immune complexes may serve not only as diagnostic but also as essential predictive markers of cardiovascular events in atherosclerosis. Accumulating evidence indicates that LDL-containing immune complexes can also serve as biomarker for macrovascular disease in type 1 diabetes. PMID:25050779

Recent Research Trends on the Enzymatic Synthesis of Structured Lipids.

PubMed

Kim, Byung Hee; Akoh, Casimir C

2015-08-01

Structured lipids (SLs) are lipids that have been chemically or enzymatically modified from their natural biosynthetic form. Because SLs are made to possess desired nutritional, physicochemical, or textural properties for various applications in the food industry, many research activities have been aimed at their commercialization. The production of SLs by enzymatic procedures has a great potential in the future market because of the specificity of lipases and phospholipases used as the biocatalysts. The aim of this review is to provide concise information on the recent research trends on the enzymatic synthesis of SLs of commercial interest, such as medium- and long-chain triacylglycerols, human milk fat substitutes, cocoa butter equivalents, trans-free or low-trans plastic fats (such as margarines and shortenings), low-calorie fats/oils, health-beneficial fatty acid-rich fats/oils, mono- or diacylglycerols, and structurally modified phospholipids. This limited review covers 108 research articles published between 2010 and 2014 which were searched in Web of Science. © 2015 Institute of Food Technologists®

Biomonitoring of carcinogenic substances: enzymatic digestion of globin for detecting alkylated amino acids

NASA Astrophysics Data System (ADS)

Bader, Michael; Rauscher, Dankwart; Geibel, Kurt; Angerer, Juergen

1993-03-01

We report the application of proteases for the total hydrolysis of globin with subsequent determination of amino acids. Optimization of the proteolysis was made with respect to enzyme concentration, time of incubation and type of protease. Ethylene oxide modified globin was used to compare the results of the analysis of the N-terminal amino acid valine after enzymatic cleavage to those obtained from the widely used modified Edman procedure. It is shown that the cleavage is of good reproducibility and yields more alkylated amino acid than the Edman procedure.

A new phenylpropanoid glycoside from Jasminum subtriplinerve Blume.

PubMed

Huong, Nguyen Thi Hong; Cu, Nguyen Khac Quynh; Quy, Trinh Van; Zidorn, Christian; Ganzera, Markus; Stuppner, Hermann

2008-01-01

From the ethyl acetate extract of the aerial parts of Jasminum subtriplinerve Blume (Oleaceae), 6'-O-menthiafoloylverbascoside (1), rutin (2), isoverbascoside (4), isooleoverbascoside (6), apiosylverbascoside (7), astragalin (9), isoquercitrin (10), and verbascoside (11) were isolated. Their structures were elucidated by extensive MS and NMR spectroscopy. Amongst 6'-O-menthiafoloylverbascoside (1) is a new phenylpropanoid glycoside.

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.

Antisecretory activity from the flowers of Chiranthodendron pentadactylon and its flavonoids on intestinal fluid accumulation induced by Vibrio cholerae toxin in rats.

PubMed

Velázquez, Claudia; Calzada, Fernando; Esquivel, Baldomero; Barbosa, Elizabeth; Calzada, Samuel

2009-12-10

The flowers of Chiranthodendron pentadactylon Larreat. (Sterculiaceae) has been traditionally used as folk medicine in Mexico for the treatment of gastrointestinal disorders such as diarrhea and dysentery. This study aimed to assess the antisecretory activity which supports the therapeutic use of Chiranthodendron pentadactylon and its flavonoids to treat diarrhea. The methanol extract of Chiranthodendron pentadactylon, subsequent fractions, and flavonoids were evaluated on cholera toxin-induced intestinal secretion in rat jejunal loops model. Three antisecretory flavonoids were isolated by bioassay-guided purification, namely, isoquercitrin 3, (+)-catechin 4 and (-)-epicatechin 5. Among them, epicatechin exhibited the most potent antisecretory activity with ID(50) of 8.3 microM/kg. Its potency was close that of to loperamide (ID(50) 6.1 microM/kg), drug used as control. Isoquercitrin (ID(50) 19.2 microM/kg) and catechin (ID(50) 51.7 microM/kg) showed moderate and weak activity, respectively. The results of the present study lend some support to the anecdotal report for the traditional use of the flowers of Chiranthodendron pentadactylon in the control of dysentery.

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.

Use of natural and modified cyclodextrins as inhibiting agents of peach juice enzymatic browning.

PubMed

López-Nicolas, José M; Pérez-López, Antonio J; Carbonell-Barrachina, Angel; García-Carmona, Francisco

2007-06-27

Although cyclodextrins (CDs) have been successfully used as antibrowning agents in different fruit juices, no research has studied the effect of these compounds on enzymatic browning in peach juice. In this paper, the color of fresh peach juice was evaluated in the presence of two types of natural (alpha-CD and beta-CD) and a modified (maltosyl-beta-CD) CD, and the effectiveness of these compounds as browning inhibitors was determined using the color space CIELAB system. Moreover, to clarify the mechanism by which CDs inhibit peach juice enzymatic browning, the process was kinetically modeled in the absence and presence of CDs using a colorimetric method; the apparent complexation constants between the mixtures of diphenols present in peach juice and some types of CD were calculated. The results show that the highest affinity constant was presented by alpha-CD (Kc = 18.31 mM-1) followed by maltosyl-beta-CD (Kc = 11.17 mM-1), whereas beta-CD was incapable of inhibiting peach juice enzymatic browning. Cyclodextrin; browning; peach; juice; color; polyphenol oxidase.

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.

Rheology and microstructure of gels based on wheat arabinoxylans enzymatically modified in arabinose and xylose

USDA-ARS?s Scientific Manuscript database

Atomic force microscopy (AFM) was used to investigate the microstructure of laccase-induced arabinoxylan (AX) gels for the first time. The effect of the degree of substitution (DS) of AX on gel microstructure was investigated by AFM. AX with three DS values (0.68, 0.61 and 0.51) were enzymatically t...

α-l-rhamnosidase selective for rutin to isoquercitrin transformation from Penicillium griseoroseum MTCC-9224.

PubMed

Yadav, Sarita; Yadava, Sudha; Yadav, Kapil D S

2017-02-01

An α-l-rhamnosidase secreting fungal strain has been isolated from the decaying goose berry (Emblica officinalis) fruit peel. The fungal strain has been identified as Penicillium greoroseum MTCC-9224. The α-l-rhamnosidase of this fungal strain has been purified to homogeneity using a simple procedure involving concentration by ultra filtration and an anion exchange chromatography on DEAE-cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 97kDa in SDS-PAGE analysis. The native-PAGE analysis also gave a single protein band confirming the purity of the enzyme. Using p-nitrophenyl-α-l-rhamnopyranoside as the substrate, K m and k cat values of the enzyme were 0.65mM and 43.65s -1 , respectively. The pH and temperature optima of the enzyme were 6.5 and 57°C, respectively. The activation energy for the thermal denaturation of the enzyme was 27.9kJ/mol. The purified α-l-rhamnosidase hydrolyzed rutin to isoquercitrin and l-rhamnose but has no effect on naringin and hesperidin. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

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.

Electroreduction-based electrochemical-enzymatic redox cycling for the detection of cancer antigen 15-3 using graphene oxide-modified indium-tin oxide electrodes.

PubMed

Park, Seonhwa; Singh, Amardeep; Kim, Sinyoung; Yang, Haesik

2014-02-04

We compare herein biosensing performance of two electroreduction-based electrochemical-enzymatic (EN) redox-cycling schemes [the redox cycling combined with simultaneous enzymatic amplification (one-enzyme scheme) and the redox cycling combined with preceding enzymatic amplification (two-enzyme scheme)]. To minimize unwanted side reactions in the two-enzyme scheme, β-galactosidase (Gal) and tyrosinase (Tyr) are selected as an enzyme label and a redox enzyme, respectively, and Tyr is selected as a redox enzyme label in the one-enzyme scheme. The signal amplification in the one-enzyme scheme consists of (i) enzymatic oxidation of catechol into o-benzoquinone by Tyr and (ii) electroreduction-based EN redox cycling of o-benzoquinone. The signal amplification in the two-enzyme scheme consists of (i) enzymatic conversion of phenyl β-d-galactopyranoside into phenol by Gal, (ii) enzymatic oxidation of phenol into catechol by Tyr, and (iii) electroreduction-based EN redox cycling of o-benzoquinone including further enzymatic oxidation of catechol to o-benzoquinone by Tyr. Graphene oxide-modified indium-tin oxide (GO/ITO) electrodes, simply prepared by immersing ITO electrodes in a GO-dispersed aqueous solution, are used to obtain better electrocatalytic activities toward o-benzoquinone reduction than bare ITO electrodes. The detection limits for mouse IgG, measured with GO/ITO electrodes, are lower than when measured with bare ITO electrodes. Importantly, the detection of mouse IgG using the two-enzyme scheme allows lower detection limits than that using the one-enzyme scheme, because the former gives higher signal levels at low target concentrations although the former gives lower signal levels at high concentrations. The detection limit for cancer antigen (CA) 15-3, a biomarker of breast cancer, measured using the two-enzyme scheme and GO/ITO electrodes is ca. 0.1 U/mL, indicating that the immunosensor is highly sensitive.

Heterogeneous glycation of cancellous bone and its association with bone quality and fragility.

PubMed

Karim, Lamya; Vashishth, Deepak

2012-01-01

Non-enzymatic glycation (NEG) and enzymatic biochemical processes create crosslinks that modify the extracellular matrix (ECM) and affect the turnover of bone tissue. Because NEG affects turnover and turnover at the local level affects microarchitecture and formation and removal of microdamage, we hypothesized that NEG in cancellous bone is heterogeneous and accounts partly for the contribution of microarchitecture and microdamage on bone fragility. Human trabecular bone cores from 23 donors were subjected to compression tests. Mechanically tested cores as well as an additional 19 cores were stained with lead-uranyl acetate and imaged to determine microarchitecture and measure microdamage. Post-yield mechanical properties were measured and damaged trabeculae were extracted from a subset of specimens and characterized for the morphology of induced microdamage. Tested specimens and extracted trabeculae were quantified for enzymatic and non-enzymatic crosslink content using a colorimetric assay and Ultra-high Performance Liquid Chromatography (UPLC). Results show that an increase in enzymatic crosslinks was beneficial for bone where they were associated with increased toughness and decreased microdamage. Conversely, bone with increased NEG required less strain to reach failure and were less tough. NEG heterogeneously modified trabecular microarchitecture where high amounts of NEG crosslinks were found in trabecular rods and with the mechanically deleterious form of microdamage (linear microcracks). The extent of NEG in tibial cancellous bone was the dominant predictor of bone fragility and was associated with changes in microarchitecture and microdamage.

Heterogeneous Glycation of Cancellous Bone and Its Association with Bone Quality and Fragility

PubMed Central

Karim, Lamya; Vashishth, Deepak

2012-01-01

Non-enzymatic glycation (NEG) and enzymatic biochemical processes create crosslinks that modify the extracellular matrix (ECM) and affect the turnover of bone tissue. Because NEG affects turnover and turnover at the local level affects microarchitecture and formation and removal of microdamage, we hypothesized that NEG in cancellous bone is heterogeneous and accounts partly for the contribution of microarchitecture and microdamage on bone fragility. Human trabecular bone cores from 23 donors were subjected to compression tests. Mechanically tested cores as well as an additional 19 cores were stained with lead-uranyl acetate and imaged to determine microarchitecture and measure microdamage. Post-yield mechanical properties were measured and damaged trabeculae were extracted from a subset of specimens and characterized for the morphology of induced microdamage. Tested specimens and extracted trabeculae were quantified for enzymatic and non-enzymatic crosslink content using a colorimetric assay and Ultra-high Performance Liquid Chromatography (UPLC). Results show that an increase in enzymatic crosslinks was beneficial for bone where they were associated with increased toughness and decreased microdamage. Conversely, bone with increased NEG required less strain to reach failure and were less tough. NEG heterogeneously modified trabecular microarchitecture where high amounts of NEG crosslinks were found in trabecular rods and with the mechanically deleterious form of microdamage (linear microcracks). The extent of NEG in tibial cancellous bone was the dominant predictor of bone fragility and was associated with changes in microarchitecture and microdamage. PMID:22514706

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.

In vitro evaluation of digestive and endolysosomal enzymes to cleave CML-modified Ara h 1 peptides

USDA-ARS?s Scientific Manuscript database

The sensory, biological, chemical, and immunological characteristics of foods can be modified non-enzymatically during processing. Notably, these modifications may modulate the allergenic potency of food allergens, such as the Ara h 1 peanut allergen. Carboxymethyl-lysine (CML) modification is a p...

Simultaneous determination and pharmacokinetic study of three flavonoid glycosides in rat plasma by LC-MS/MS after oral administration of Rubus chingii Hu extract.

PubMed

Zan, Tao; Piao, Li; Wei, Yuntao; Gu, Yue; Liu, Baohua; Jiang, Daqing

2018-03-01

A simple and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of isoquercitrin, kaempferol-3-O-rutinoside and tiliroside in rat plasma. Plasma samples were deproteinized with methanol and separated on a Hypersil Gold C 18 column (2.1 × 50 mm, i.d., 3.0 μm) using gradient elution with the mobile phase of water and methanol at a flow rate of 0.4 mL/min. Mass spectrometric detection was performed with negative ion electrospray ionization in selected reaction monitoring mode. All analytes showed good linearity over their investigated concentration ranges (r 2  > 0.99). The lower limit of quantification was 1.0 ng/mL for isoquercitrin and 2.0 ng/mL for kaempferol-3-O-rutinoside and tiliroside, respectively. Intra- and inter-day precisions were <8.2% and accuracy ranged from -11.5 to 9.7%. The mean extraction recoveries of analytes and IS from rat plasma were >80.4%. The assay was successfully applied to investigate the pharmacokinetic study of the three ingredients after oral administration of Rubus chingii Hu to rats. Copyright © 2017 John Wiley & Sons, Ltd.

HDAC Inhibition Modulates Hippocampus-Dependent Long-Term Memory for Object Location in a CBP-Dependent Manner

ERIC Educational Resources Information Center

Haettig, Jakob; Stefanko, Daniel P.; Multani, Monica L.; Figueroa, Dario X.; McQuown, Susan C.; Wood, Marcelo A.

2011-01-01

Transcription of genes required for long-term memory not only involves transcription factors, but also enzymatic protein complexes that modify chromatin structure. Chromatin-modifying enzymes, such as the histone acetyltransferase (HAT) CREB (cyclic-AMP response element binding) binding protein (CBP), are pivotal for the transcriptional regulation…

Effects of cyclodextrin glycosiltransferase modified starch and cyclodextrins on plasma glucose and lipids metabolism in mice

USDA-ARS?s Scientific Manuscript database

The potential functional and nutritional benefits of granular starch treated with cyclodextrin glycosyltransferase (CGTase) and the released cyclodextrins (CDs) were explored in in vivo studies. The metabolic effects of diets in the C57BL/6J mouse containing native and enzymatically modified corn st...

Slow glucose release property of enzyme-synthesized highly branched maltodextrins differs among starch sources.

PubMed

Kittisuban, Phatcharee; Lee, Byung-Hoo; Suphantharika, Manop; Hamaker, Bruce R

2014-07-17

Seven types of starch (waxy corn, normal corn, waxy rice, normal rice, waxy potato, normal potato, and tapioca) were selected to produce slowly digestible maltodextrins by enzymatic modification using a previously developed procedure. Branching enzyme (BE) alone and in combination with β-amylase (BA) were used to increase the amount of α-1,6 branching points, which are slowly hydrolyzed by mucosal α-glucosidases in the small intestine. The enzymatic treatments of all starches resulted in a reduction of the debranched linear chain length distribution and weight-average molecular weight. After α-amylolysis of the enzymatically synthesized-maltodextrins, the proportion of branched α-limit dextrins increased, and consequently a reduction in rate of glucose release by rat intestinal α-glucosidases in vitro. Among the samples, enzyme-modified waxy starches had a more pronounced effect on an increase in the slow digestion property than normal starches. These enzyme-modified maltodextrins show potential as novel functional foods by slowing digestion rate to attain extended glucose release. Copyright © 2014 Elsevier Ltd. All rights reserved.

Investigation of ischemia modified albumin, oxidant and antioxidant markers in acute myocardial infarction

PubMed Central

Hazini, Ahmet; Işıldak, İbrahim; Alpdağtaş, Saadet; Önül, Abdullah; Şenel, Ünal; Kocaman, Tuba; Dur, Ali; Iraz, Mustafa; Uyarel, Hüseyin

2015-01-01

Introduction Acute myocardial infarction (AMI) is still one of the most common causes of death worldwide. In recent years, for diagnosis of myocardial ischemia, a new parameter, called ischemia modified albumin (IMA), which is thought to be more advantageous than common methods, has been researched. Aim In this study, systematic analysis of parameters considered to be related to myocardial ischemia has been performed, comparing between control and myocardial ischemia groups. Material and methods We selected 40 patients with AMI and 25 healthy controls for this study. Ischemia modified albumin levels, glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) antioxidant enzyme activities and non-enzymatic antioxidants such as retinol, α-tocopherol, β-carotene and ascorbic acid levels were investigated in both groups. Glutathione (GSH) and malondialdehyde (MDA) levels, which are indicators of oxidative stress, were compared between patient and control groups. Results Ischemia modified albumin levels were found significantly higher in the AMI diagnosed group when compared with controls. The MDA level was elevated in the patient group, whereas the GSH level was decreased. SOD, GPx and CAT enzyme levels were decreased in the patient group, where it could be presumed that oxidative stress causes the cardiovascular diseases. Conclusions Due to the increased oxidative stress, non-enzymatic and enzymatic antioxidant capacity was affected. Systematic investigation of parameters related to myocardial infarction has been performed, and it is believed that such parameters can contribute to protection and early diagnosis of AMI and understanding the mechanism of development of the disease. PMID:26677379

Carborane-linked 2'-deoxyuridine 5'-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA.

PubMed

Balintová, Jana; Simonova, Anna; Białek-Pietras, Magdalena; Olejniczak, Agnieszka; Lesnikowski, Zbigniew J; Hocek, Michal

2017-11-01

5-[(p-Carborane-2-yl)ethynyl]-2'-deoxyuridine 5'-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

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

Thompson, David N.; Apel, William A.; Thompson, Vicki S.

A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extractsmore » are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.« less

Thermophilic acetylxylan esterase genes and enzymes from alicyclobacillus acidocaldarius and related organisms and methods

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

Thompson, Vicki S.; Thompson, David N.; Reed, David W.

A genetically modified organism comprising at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharide, lignocellulose, hemicellulose, lignin, chitin, heteroxylan, and/or xylan-decorating group; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methodsmore » of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.« less

Phage display and selection of lanthipeptides on the carboxy-terminus of the gene-3 minor coat protein.

PubMed

Urban, Johannes H; Moosmeier, Markus A; Aumüller, Tobias; Thein, Marcus; Bosma, Tjibbe; Rink, Rick; Groth, Katharina; Zulley, Moritz; Siegers, Katja; Tissot, Kathrin; Moll, Gert N; Prassler, Josef

2017-11-15

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products with drug-like properties. To fully exploit the potential of RiPPs as peptide drug candidates, tools for their systematic engineering are required. Here we report the engineering of lanthipeptides, a subclass of RiPPs characterized by multiple thioether cycles that are enzymatically introduced in a regio- and stereospecific manner, by phage display. This was achieved by heterologous co-expression of linear lanthipeptide precursors fused to the widely neglected C-terminus of the bacteriophage M13 minor coat protein pIII, rather than the conventionally used N-terminus, along with the modifying enzymes from distantly related bacteria. We observe that C-terminal precursor peptide fusions to pIII are enzymatically modified in the cytoplasm of the producing cell and subsequently displayed as mature cyclic peptides on the phage surface. Biopanning of large C-terminal display libraries readily identifies artificial lanthipeptide ligands specific to urokinase plasminogen activator (uPA) and streptavidin.

Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

DOEpatents

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Ward, Thomas E.

2016-03-22

A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

DOEpatents

Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

2013-07-23

A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

DOEpatents

Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

2014-04-08

A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

Antioxidant and Anti-Fatigue Constituents of Okra

PubMed Central

Xia, Fangbo; Zhong, Yu; Li, Mengqiu; Chang, Qi; Liao, Yonghong; Liu, Xinmin; Pan, Ruile

2015-01-01

Okra (Abelmoschus esculentus (L.) Moench), a healthy vegetable, is widely spread in tropical and subtropical areas. Previous studies have proven that okra pods possess anti-fatigue activity, and the aim of this research is to clarify the anti-fatigue constituents. To achieve this, we divided okra pods (OPD) into seeds (OSD) and skins (OSK), and compared the contents of total polysaccharides, total polyphenols, total flavonoids, isoquercitrin, and quercetin-3-O-gentiobiose and the antioxidant activity in vitro and anti-fatigue activity in vivo between OSD and OSK. The contents of total polyphenols and total polysaccharides were 29.5% and 14.8% in OSD and 1.25% and 43.1% in OSK, respectively. Total flavonoids, isoquercitrin and quercetin-3-O-gentiobiose (5.35%, 2.067% and 2.741%, respectively) were only detected in OSD. Antioxidant assays, including 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging, ferric reducing antioxidant power (FRAP) and reducing power test, and weight-loaded swimming test showed OSD possessed significant antioxidant and anti-fatigue effects. Moreover, biochemical determination revealed that that anti-fatigue activity of OSD is caused by reducing the levels of blood lactic acid (BLA) and urea nitrogen (BUN), enhancing hepatic glycogen storage and promoting antioxidant ability by lowering malondialdehyde (MDA) level and increasing superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) levels. These results proved okra seeds were the anti-fatigue part of okra pods and polyphenols and flavonoids were active constituents. PMID:26516905

Chlorogenic Acid and Rutin Play a Major Role in the In Vivo Anti-Diabetic Activity of Morus alba Leaf Extract on Type II Diabetic Rats

PubMed Central

Hunyadi, Attila; Martins, Ana; Hsieh, Tusty-Jiuan; Seres, Adrienn; Zupkó, István

2012-01-01

The leaves of the white mulberry tree (Morus alba L.) are used worldwide in traditional medicine as anti-diabetics. Various constituents of mulberry leaves, such as iminosugars (i.e. 1-deoxynojirimicin), flavonoids and related compounds, polysaccharides, glycopeptides and ecdysteroids, have been reported to exert anti-diabetic activity, but knowledge about their contribution to the overall activity is limited. The objective of the present work was to determine the in vivo anti-diabetic activity of an extract of mulberry leaves (MA), and to examine to what extent three major constituents, chlorogenic acid, rutin and isoquercitrin, might contribute to the observed activity. Quantities of the three constituents of interest in the extract were determined by using HPLC-DAD. Activity was determined by using a type II diabetic rat model. After 11 days of per os administration of 250 or 750 mg/kg of MA or the corresponding amounts of each individual compound, a dose dependent decrease of non-fasting blood glucose levels were found for MA, chlorogenic acid and rutin, but not for isoquercitrin. Based on our results, chlorogenic acid and rutin might account for as much as half the observed anti-diabetic activity of MA, hence they can be considered as excellent markers for the quality control of mulberry products. PMID:23185641

Apoptosis triggered by isoquercitrin in bladder cancer cells by activating the AMPK-activated protein kinase pathway.

PubMed

Wu, Ping; Liu, Siyuan; Su, Jianyu; Chen, Jianping; Li, Lin; Zhang, Runguang; Chen, Tianfeng

2017-10-18

Cancer cells are well known to require a constant supply of protein, lipid, RNA, and DNA via altered metabolism for accelerated cell proliferation. Targeting metabolic pathways is, therefore, a promising therapeutic strategy for cancers. Isoquercitrin (ISO) is widely distributed in dietary and medicinal plants and displays selective cytotoxicity to cancer cells, primarily by inducing apoptosis and cell cycle arrest. The aims of this study were to find out whether ISO could stabilize in a bladder-like acidic environment and inhibit bladder cancer cell proliferation by affecting their metabolism, and to investigate its molecular mechanism. In this study, the exposure of T24 bladder cancer cells to ISO (20-80 μM) decreased cell viability by causing ROS overproduction. This ROS change regulated the AMPK signaling pathway, and caused Caspase-dependent apoptosis as well as metabolism dysfunction. Metabolic alterations elevated metabolic pathway variation, which in turn destabilized lipid synthesis and altered anaerobic glycolysis. This linkage was proved by immunoblotting assay, and metabolomics as identified by UHPLC-QTOF-MS. Our findings provide comprehensive evidence that ISO influenced T24 bladder cancer cell metabolism, and that this process was mainly involved in activating the AMPK pathway. This study could lead to an understanding of how ISO suppresses bladder cancer cell growth, and whether the affected cancer metabolism is a common mechanism by which nutritional compounds suppress cancers.

Effects of Bidens pilosa L. var. radiata SCHERFF treated with enzyme on histamine-induced contraction of guinea pig ileum and on histamine release from mast cells.

PubMed

Matsumoto, Takayuki; Horiuchi, Masako; Kamata, Katsuo; Seyama, Yoshiyuki

2009-06-01

The medical mechanism against type I allergies is to block the release or production of chemical mediators from mast cells or to block the H(1)-receptor signaling. We previously reported that the anti-allergic action of the dry powder from Bidens pilosa L. var. radiata SCHERFF treated with the enzyme cellulosine (eMMBP) was dependent on the inhibition of histamine release from mast cells. Here, we investigate that the effect of fractions in eMMBP on the histamine-induced contraction in guinea pig ileum and on the release of histamine in rat peritoneal mast cells. The histamine-induced contraction in guinea pig ileum is dose-dependently inhibited by ketotifen, an antagonist of H(1)-receptor. Fractions contained caffeic acid, caffeoylquinic acid and fractions contained flavonoids such as hyperin and isoquercitrin in eMMBP inhibit histamine release from mast cells, but only flavonoids such as hyperin, isoquercitrin and rutin suppress the histamine-induced contraction in guinea pig ileum. Moreover, the histamine-induced contraction was not affected by caffeic acid, however, such contraction was significantly inhibited by rutin. These results suggest that the primary antagonists of H(1)- receptor are different from the components in eMMBP that inhibit histamine release, and that these components participate in the anti-allergic activity of eMMBP.

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.

Enantioselective Syntheses of (−)-Alloyohimbane and (−)-Yohimbane by an Efficient Enzymatic Desymmetrization Process

PubMed Central

Ghosh, Arun K.; Sarkar, Anindya

2016-01-01

Enantioselective syntheses of (−)-alloyohimbane and (−)-yohimbane was accomplished in a convergent manner. The key step involved a modified mild protocol for the enantioselective enzymatic desymmetrization of meso-diacetate. The protocol provided convenient access to an optically active monoacetate in multi-gram scale in high enantiomeric purity. This monoacetate was converted to (−)-alloyohimbane. Reductive amination of the derived aldehyde causes the isomerization leading to the trans-product and allows the synthesis of (−)-yohimbane. PMID:28757804

A Multiplex Enzymatic Machinery for Cellular Protein S-nitrosylation.

PubMed

Seth, Divya; Hess, Douglas T; Hausladen, Alfred; Wang, Liwen; Wang, Ya-Juan; Stamler, Jonathan S

2018-02-01

S-nitrosylation, the oxidative modification of Cys residues by nitric oxide (NO) to form S-nitrosothiols (SNOs), modifies all main classes of proteins and provides a fundamental redox-based cellular signaling mechanism. However, in contrast to other post-translational protein modifications, S-nitrosylation is generally considered to be non-enzymatic, involving multiple chemical routes. We report here that endogenous protein S-nitrosylation in the model organism E. coli depends principally upon the enzymatic activity of the hybrid cluster protein Hcp, employing NO produced by nitrate reductase. Anaerobiosis on nitrate induces both Hcp and nitrate reductase, thereby resulting in the S-nitrosylation-dependent assembly of a large interactome including enzymes that generate NO (NO synthase), synthesize SNO-proteins (SNO synthase), and propagate SNO-based signaling (trans-nitrosylases) to regulate cell motility and metabolism. Thus, protein S-nitrosylation by NO in E. coli is essentially enzymatic, and the potential generality of the multiplex enzymatic mechanism that we describe may support a re-conceptualization of NO-based cellular signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

[Studies on chemical constituents of Taxillus sutchuenenisis].

PubMed

Chen, Jiang-tao; Feng, Feng

2007-11-01

To study the chemical constituents of Taxillus sutchuenenisis (Lecomte) Danser. Chromatography and spectrum analysis were employed to isolated and elucidate the chemical constituents in the plant. 9 compounds were isolated and identified as quercetin (I), quervetin 3-O-beta-D-galactoside (II), isoquercitrin (III), quercitrin (IV), rutin (V), gallic acid (VI), ferulic acid (VII), beta-sitosterol (VIII), daucosterol (IX), respectively. Compounds III-IX are isolated from this plant for the first time. The work provide evidence for the exploitation and utilization of this plant resouce.

Physico-Chemical Properties and Biodegradability of Genetically Modified Populus trichocarpa and Pinus taeda

NASA Astrophysics Data System (ADS)

Edmunds, Charles Warren

Increasing concerns over greenhouse gas emissions and the finite supply of fossil fuels lead to the goal of utilizing lignocellulosic feedstocks for biofuels, platform chemicals, and biocomposites. Lignin is responsible for the recalcitrance of lignocellulosic biomass and is a major barrier to its deconstruction. Great progress has been made in mapping and modifying the lignin biosynthetic pathway. However, the link between the genetic modification, resulting chemical and physical properties of the wood, and how these properties influence the thermomechanical and recalcitrance to biological and chemical degradation needs further investigation. In this dissertation, the study of modified Populus trichocarpa and Pinus taeda were utilized to accomplish this goal. Thermo-mechanical properties of genetically modified P. trichocarpa with altered lignin content and/or lignin structure were measured with a series of tools including; dynamic mechanical analysis, nuclear magnetic resonance, and wet chemistry techniques. Results demonstrated lignin content and lignin structure likely influence the glass transition temperature (Tg), and that decreased lignin content and the corresponding higher proportion of cell wall carbohydrates may contribute to increased molecular mobility in the wood polymer structure. The effect of lignin biosynthetic pathway modification on biological degradation of these transgenic wood specimens was of interest. However, experimental methods for fungal treatment on small young greenhouse-grown wood specimens are not well established. Therefore, a project was undertaken to develop a method for fungal inoculation and incubation for these unique specimens. Several parameters were tested, and a fungal treatment method was identified with sufficient weight loss after decay and significant reduction in variation of weight loss between replicates compared to previous experiments by direct inoculation of wood with liquid malt extract fungal culture. Utilizing the fungal treatment method which was developed, fungal pretreatment as a potential low-input and environmentally-friendly alternative to conventional pretreatment methods was tested using the white-rot fungus, Ceriporiopsis subvermispora, on wildtype and transgenic P. trichocarpa. In addition to fungal treatment, hot water and dilute acid treatments followed by enzymatic hydrolysis was tested. Results showed no clear relationship between the initial lignin content or syringyl/guaiacyl lignin monomer ratio and weight loss due to fungal treatment. P-hydroxyphenyl lignin monomer degradation of up to 60% during the fungal treatment were observed in cinnamate 3-hydroxylase down-regulated genetic lines. It was demonstrated that fungal treatment in wildtype and several transgenic lines resulted in substantial improvements in sugar yields, up to 2.4-fold increase in glucose yield and 6.7-fold increase in xylose yield after enzymatic hydrolysis. However, some genetic lines showed little benefit from fungal pretreatment, and in general hot water and dilute acid pretreatments showed similar or increased glucose yield compared to fungal treatment. The goal of the last project was to characterize P. taeda which was genetically modified for S lignin production or decreased lignin content. In addition, the amenability to pretreatment and enzymatic hydrolysis were analyzed using hot water and dilute acid pretreatments followed by enzymatic hydrolysis. In the transgenic lines modified for production of syringyl lignin, Maule staining demonstrated the intermittent deposition of syringyl lignin in the secondary xylem, while thioacidolysis showed 13% concentration of S lignin, and solid state NMR demonstrated the occurrence of beta-O-4 linkages in S lignin units. In transgenic lines modified for reduced lignin content, lignin reduction up to 33% was observed, and pretreatment and enzymatic hydrolysis demonstrated increased cellulose conversion in lowlignin samples. These results highlight the potential of softwood to be a viable bioenergy/biochemical feedstock and opens up exciting new avenue of research.

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.

A reagentless enzymatic fluorescent biosensor for glucose based on upconverting glasses, as excitation source, and chemically modified glucose oxidase.

PubMed

Del Barrio, Melisa; Cases, Rafael; Cebolla, Vicente; Hirsch, Thomas; de Marcos, Susana; Wilhelm, Stefan; Galbán, Javier

2016-11-01

Upon near-infrared excitation Tm(3+)+Yb(3+) doped fluorohafnate glasses present upconversion properties and emit visible light. This property permits to use these glasses (UCG) as excitation sources for fluorescent optical biosensors. Taking this into account, in this work a fluorescent biosensor for glucose determination is designed and evaluated. The biosensor combines the UCG and the fluorescence of the enzyme glucose oxidase chemically modified with a fluorescein derivative (GOx-FS), whose intensity is modified during the enzymatic reaction with glucose. Optical parameters have been optimized and a mathematical model describing the behavior of the analytical signal is suggested. Working in FIA mode, the biosensor responds to glucose concentrations up to, at least, 15mM with a limit of detection of 1.9mM. The biosensor has a minimum lifetime of 9 days and has been applied to glucose determination in drinks. The applicability of the sensor was tested by glucose determination in two fruit juices. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Pseudouridine: Still mysterious, but never a fake (uridine)!

PubMed Central

Spenkuch, Felix; Motorin, Yuri; Helm, Mark

2014-01-01

Pseudouridine (Ψ) is the most abundant of >150 nucleoside modifications in RNA. Although Ψ was discovered as the first modified nucleoside more than half a century ago, neither the enzymatic mechanism of its formation, nor the function of this modification are fully elucidated. We present the consistent picture of Ψ synthases, their substrates and their substrate positions in model organisms of all domains of life as it has emerged to date and point out the challenges that remain concerning higher eukaryotes and the elucidation of the enzymatic mechanism. PMID:25616362

The in-capillary DPPH-capillary electrophoresis-the diode array detector combined with reversed-electrode polarity stacking mode for screening and quantifying major antioxidants in Cuscuta chinensis Lam.

PubMed

Liu, Jiao; Tian, Ji; Li, Jin; Azietaku, John Teye; Zhang, Bo-Li; Gao, Xiu-Mei; Chang, Yan-Xu

2016-07-01

An in-capillary 2, 2-diphenyl-1-picrylhydrazyl (DPPH)-CE-the DAD (in-capillary DPPH-CE-DAD) combined with reversed-electrode polarity stacking mode has been developed to screen and quantify the active antioxidant components of Cuscuta chinensis Lam. The operation parameters were optimized with regard to the pH and concentration of buffer solution, SDS, β-CDs, organic modifier, as well as separation voltage and temperature. Six antioxidants including chlorogenic acid, p-coumaric acid, rutin, hyperin, isoquercitrin, and astragalin were screened and the total antioxidant activity of the complex matrix was successfully evaluated based on the decreased peak area of DPPH by the established DPPH-CE-DAD method. Sensitivity was enhanced under reversed-electrode polarity stacking mode and 10- to 31-fold of magnitude improvement in detection sensitivity for each analyte was attained. The results demonstrated that the newly established in-capillary DPPH-CE-DAD method combined with reversed-electrode polarity stacking mode could integrate sample concentration, the oxidizing reaction, separation, and detection into one capillary to fully automate the system. It was considered a suitable technique for the separation, screening, and determination of trace antioxidants in natural products. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Process development of starch hydrolysis using mixing characteristics of Taylor vortices.

PubMed

Masuda, Hayato; Horie, Takafumi; Hubacz, Robert; Ohmura, Naoto; Shimoyamada, Makoto

2017-04-01

In food industries, enzymatic starch hydrolysis is an important process that consists of two steps: gelatinization and saccharification. One of the major difficulties in designing the starch hydrolysis process is the sharp change in its rheological properties. In this study, Taylor-Couette flow reactor was applied to continuous starch hydrolysis process. The concentration of reducing sugar produced via enzymatic hydrolysis was evaluated by varying operational variables: rotational speed of the inner cylinder, axial velocity (reaction time), amount of enzyme, and initial starch content in the slurry. When Taylor vortices were formed in the annular space, efficient hydrolysis occurred because Taylor vortices improved the mixing of gelatinized starch with enzyme. Furthermore, a modified inner cylinder was proposed, and its mixing performance was numerically investigated. The modified inner cylinder showed higher potential for enhanced mixing of gelatinized starch and the enzyme than the conventional cylinder.

Modification of enzymes by use of high-pressure homogenization.

PubMed

Dos Santos Aguilar, Jessika Gonçalves; Cristianini, Marcelo; Sato, Helia Harumi

2018-07-01

High-pressure is an emerging and relatively new technology that can modify various molecules. High-pressure homogenization (HPH) has been used in several studies on protein modification, especially in enzymes used or found in food, from animal, plant or microbial resources. According to the literature, the enzymatic activity can be modulated under pressure causing inactivation, stabilization or activation of the enzymes, which, depending on the point of view could be very useful. Homogenization can generate changes in the structure of the enzyme modifying various chemical bonds (mainly weak bonds) causing different denaturation levels and, consequently, affecting the catalytic activity. This review aims to describe the various alterations due to HPH treatment in enzymes, to show the influence of high-pressure on proteins and to report the HPH effects on the enzymatic activity of different enzymes employed in the food industry and research. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

Chemical Fingerprint and Quantitative Analysis for the Quality Evaluation of Docynia dcne Leaves by High-Performance Liquid Chromatography Coupled with Chemometrics Analysis.

PubMed

Zhang, Xiaoyu; Mei, Xueran; Wang, Zhanguo; Wu, Jing; Liu, Gang; Hu, Huiling; Li, Qijuan

2018-05-24

Docynia dcne leaf from the genus of Docynia Dcne (including three species of Docynia delavayi, Docynia indica and Docynia longiunguis.) is an important raw material of local ethnic minority tea, ethnomedicines and food supplements in southwestern areas of China. However, D. dcne leaves from these three species are usually used confusingly, which could influence the therapeutic effect of it. A rapid and effective method for the chemical fingerprint and quantitative analysis to evaluate the quality of D. dcne leaves was established. The chemometric methods, including similarity analysis, hierarchical cluster analysis and partial least-squares discrimination analysis, were applied to distinguish 30 batches of D. dcne leaf samples from these three species. The above results could validate each other and successfully group these samples into three categories which were closely related to the species of D. dcne leaves. Moreover, isoquercitrin and phlorizin were screened as the chemical markers to evaluate the quality of D. dcne leaves from different species. And the contents of isoquercitrin and phlorizin varied remarkably in these samples, with ranges of 6.41-38.84 and 95.73-217.76 mg/g, respectively. All the results indicated that an integration method of chemical fingerprint couple with chemometrics analysis and quantitative assessment was a powerful and beneficial tool for quality control of D. dcne leaves, and could be applied also for differentiation and quality control of other herbal preparations.

Spondias tuberosa (Anacardiaceae) leaves: profiling phenolic compounds by HPLC-DAD and LC-MS/MS and in vivo anti-inflammatory activity.

PubMed

da Silva Siqueira, Emerson Michell; Félix-Silva, Juliana; de Araújo, Lorena Maria Lima; Fernandes, Julia Morais; Cabral, Bárbara; Gomes, Jacyra Antunes Dos Santos; de Araújo Roque, Alan; Tomaz, José Carlos; Lopes, Norberto Peporine; de Freitas Fernandes-Pedrosa, Matheus; Giordani, Raquel Brandt; Zucolotto, Silvana Maria

2016-10-01

Spondias tuberosa is a medicinal plant used by several local communities in northeast Brazil to treat infections, digestive disorders and inflammatory conditions. The study aimed to identify and quantify the major phenolic in hydroethanolic extract of leaves from S. tuberosa and to evaluate its anti-inflammatory potential. The chemical profile of extract was analyzed by HPLC-DAD and HPLC-MS. The in vivo anti-inflammatory activity was investigated in carrageenan-induced hind paw edema and peritonitis models in mice. Identified and quantified through HPLC-DAD or HPLC-MS analyses of S. tuberosa extract were the following compounds: chlorogenic acid, caffeic acid, rutin and isoquercitrin. The inflammatory response to carrageenan was significantly reduced in both models by S. tuberosa extract. In hind paw edema, the edematogenic response was reduced by up to 63.6% and the myeloperoxidase activity was completely inhibited. In the peritonitis model, the total cell migration into the peritoneal cavity was reduced by up to 65%. The results obtained give evidence of the anti-inflammatory action of S. tuberosa and suggest the potential therapeutic benefit of this plant on inflammatory conditions. The chlorogenic acid, caffeic acid, rutin and isoquercitrin identified and quantified in S. tuberosa leaves enable us to suggest that these compounds could be used as chemical markers for quality control of derivative products from this species. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Some Aspects of Enzymatic Browning in Apples

ERIC Educational Resources Information Center

Liffen, C. L.; Cleeve, H. N.

1975-01-01

Describes material modified from the Nuffield advanced chemistry course to make it meaningful and relevant to pupils in the middle school. Discusses a series of simple experiments on apple browning and summarizes the browning process and its control. (Author/GS)

Anti-complementary constituents of Houttuynia cordata and their targets in complement activation cascade.

PubMed

Jiang, Yun; Lu, Yan; Zhang, Yun-Yi; Chen, Dao-Feng

2014-01-01

Activity-guided fractionation for complement inhibitors led to the isolation of 23 known compounds from Houttuynia cordata Thunb. Seven flavonoids, two alkaloids, one coumarin and two phenols showed anti-complementary activity. Preliminary inhibitory mechanism of four flavonoids, including quercitrin, afzelin, isoquercitrin and quercetin in the complement activation cascade were examined for the first time. The results indicated that the target components of flavonols are different from those of flavonosides, and the glycoside moieties may be necessary to block C3 and C4 components.

Non-enzymatic glucose detection based on phenylboronic acid modified optical fibers

NASA Astrophysics Data System (ADS)

Sun, Xiaolan; Li, Nana; Zhou, Bin; Zhao, Wei; Liu, Liyuan; Huang, Chao; Ma, Longfei; Kost, Alan R.

2018-06-01

A non-enzymatic, sensitive glucose sensor was fabricated based on an evanescent wave absorbing optical fiber probe. The optical fiber sensor was functionalized by fixing a poly (phenylboronic acid) (polyPBA) film onto the conical region of the single mode fiber. The reflected light intensity of the polyPBA-functionalized fiber sensor increased proportionally with glucose concentration in the range of 0-60 mM, and the sensor showed good reproducibility and stability. The developed sensor possessed a high sensitivity of 0.1787%/mM and good linearity. The measurement of glucose concentration in human serum was also demonstrated.

Ferrocene-Modified Linear Poly(ethylenimine) for Enzymatic Immobilization and Electron Mediation.

PubMed

Hickey, David P

2017-01-01

Enzymatic glucose biosensors and biofuel cells make use of the electrochemical transduction between an oxidoreductase enzyme, such as glucose oxidase (GOx), and an electrode to either quantify the amount of glucose in a solution or generate electrical energy. However, many enzymes including GOx are not able to electrochemically interact with an electrode surface directly, but require an external electrochemical relay to shuttle electrons to the electrode. Ferrocene-modified linear poly(ethylenimine) (Fc-LPEI) redox polymers have been designed to simultaneously immobilize glucose oxidase (GOx) at an electrode and mediate electron transfer from their flavin adenine dinucleotide (FAD) active site to the electrode surface. Cross-linked films of Fc-LPEI create hydrogel networks that allow for rapid transport of glucose, while the covalently bound ferrocene moieties are able to facilitate rapid electron transfer due to the ability of ferrocene to exchange electrons between adjacent ferrocene residues. For these reasons, Fc-LPEI films have been widely used in the development of high current density bioanode materials. This chapter describes the synthesis of a commonly used dimethylferrocene-modified linear poly(ethylenimine), as well as the subsequent preparation and electrochemical characterization of a GOx bioanode film utilizing the synthesized polymer.

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.

Hydrophobic modification of jute fiber used for composite reinforcement via laccase-mediated grafting

NASA Astrophysics Data System (ADS)

Dong, Aixue; Yu, Yuanyuan; Yuan, Jiugang; Wang, Qiang; Fan, Xuerong

2014-05-01

Jute fiber is a lignocellulosic material which could be utilized for reinforcement of composites. To improve the compatibility of hydrophilic jute fiber with hydrophobic resin, surface hydrophobization of the fiber is often needed. In this study, the feasibility of laccase-mediated grafting dodecyl gallate (DG) on the jute fiber was investigated. First, the grafting products were characterized by FT-IR, XPS, SEM and AFM. And then the grafting percentage (Gp) and the DG content of the modified jute were determined in terms of weighting and saponification, respectively. The parameters of the enzymatic grafting process were optimized to the target application. Lastly, the hydrophobicity of the jute fabrics was estimated by means of contact angle and wetting time. The mechanical properties and the fracture section of the jute fabric/polypropylene (PP) composites were studied. The results revealed covalently coupling of DG to the jute substrates mediated by laccase. The enzymatic process reached the maximum grafting rate of 4.16% when the jute fabric was incubated in the 80/20 (v/v, %) pH 3 0.2 M acetate buffer/ethanol medium with 1.0 U/mL laccase and 5 mM DG at 50 °C for 4 h. The jute fabric modified with laccase and DG showed increased contact angle of 111.49° and wetting time of at least 30 min, indicating that the surface hydrophobicity of the jute fabric was increased after the enzymatic graft modification with hydrophobic DG. The breaking strength of the modified jute fiber/PP composite was also increased and the fracture section became neat and regular due to the laccase-assisted grafting with DG.

Effect of high oxygen modified atmosphere packaging on microbial growth and sensorial qualities of fresh-cut produce.

PubMed

Jacxsens, L; Devlieghere, F; Van der Steen, C; Debevere, J

2001-12-30

The application of High Oxygen Atmospheres (HOA) (i.e. > 70% O2) for packaging ready-to-eat vegetables was evaluated as an alternative technique for low O2 Equilibrium Modified Atmosphere (EMA) packaging (3% O2-5% CO2-balance N2) for respiring products. Comparative experiments between both techniques were performed in-vitro and in-vivo. Typical spoilage causing microorganisms (Pseudomonas fluorescens, Candida lambica), the moulds Botrytis cinerea, Aspergillus flavus and the opportunistic psychrotrophic human pathogenic microorganism associated with refrigerated minimally processed vegetables. Aeromonas caviae (HG4), showed a retarded growth during the conducted in-vitro studies at 4 degrees C in 70%, 80% and 95% O2 as examples of HOA compared to the in-vitro experiments in 5% O2 (as example of EMA packaging) and the effect was more pronounced in 95% O2. The effect of the high O2-concentrations on the human pathogen Listeria monocytogenes resulted in an extended lag phase (95% O2). The plant pathogen Erwinia carotovora was increasingly stimulated by increasing high O2-concentrations. During a storage experiment of three types of ready-to-eat vegetables (mushroom slices, grated celeriac and shredded chicory endive), which are sensitive to enzymatic browning and microbial spoilage, the effect of EMA and HOA (95% O2-5% N2) on their quality and shelf life was compared. High O2 atmospheres were found to be particularly effective in inhibiting enzymatic browning of the tested vegetables. Also, the microbial quality was better as a reduction in yeast growth was observed. The HOA can be applied as an alternative for low O2 modified atmospheres for some specific types of ready-to-eat vegetables, sensitive to enzymatic browning and spoilage by yeasts.

Rapeseed-straw enzymatic digestibility enhancement by sodium hydroxide treatment under ultrasound irradiation.

PubMed

Kang, Kyeong Eop; Jeong, Gwi-Taek; Park, Don-Hee

2013-08-01

In this study, we carried out sodium hydroxide and sonication pretreatments of rapeseed straw (Brassica napus) to obtain monosugar suitable for production of biofuels. To optimize the pretreatment conditions, we applied a statistical response-surface methodology. The optimal pretreatment conditions using sodium hydroxide under sonication irradiation were determined to be 75.0 °C, 7.0 % sodium hydroxide, and 6.8 h. For these conditions, we predicted 97.3 % enzymatic digestibility. In repeated experiments to validate the predicted value, 98.9 ± 0.3 % enzymatic digestibility was obtained, which was well within the range of the predicted model. Moreover, sonication irradiation was found to have a good effect on pretreatment in the lower temperature range and at all concentrations of sodium hydroxide. According to scanning electron microscopy images, the surface area and pore size of the pretreated rapeseed straw were modified by the sodium hydroxide pretreatment under sonication irradiation.

Responses of enzymatically isolated mammalian vascular smooth muscle cells to pharmacological and electrical stimuli.

PubMed

DeFeo, T T; Morgan, K G

1985-05-01

A modified method for enzymatically isolating mammalian vascular smooth muscle cells has been developed and tested for ferret portal vein smooth muscle. This method produces a high proportion of fully relaxed cells and these cells appear to have normal pharmacological responsiveness. The ED50 values for both alpha stimulation and potassium depolarization are not significantly different in the isolated cells from those obtained from intact strips of ferret portal vein, suggesting that the enzymatic treatment does not destroy receptors or alter the electrical responsiveness of the cells. It was also possible to demonstrate a vasodilatory action of papaverine, nitroprusside and adenosine directly on the isolated cells indicating that the pathways involved are intact in the isolated cells. This method should be of considerable usefulness, particularly in combination with the new fluorescent indicators and cell sorter techniques which require isolated cells.

Application of microscopy technique and high-performance liquid chromatography for quality assessment of the flower bud of Tussilago farfara L. (Kuandonghua)

PubMed Central

Li, Da; Liang, Li; Zhang, Jing; Kang, Tingguo

2015-01-01

Background: Quality control is one of the bottleneck problems limiting the application and development of traditional Chinese medicine (TCM). In recent years, microscopy and high-performance liquid chromatography (HPLC) techniques have been frequently applied in the quality control of TCM. However, studies combining conventional microscopy and HPLC techniques for the quality control of the flower bud of Tussilago farfara L. (Kuandonghua) have not been reported. Objective: This study was undertaken to evaluate the quality of the flower bud of T. farfara L. and to establish the relationships between the quantity of pollen grains and four main bioactive constituents: tussilagone, chlorogenic acid, rutin and isoquercitrin. Materials and Methods: In this study, microscopic examination was used to quantify microscopic characteristics of the flower bud of T. farfara L., and the chemical components were determined by HPLC. The data were analyzed by Statistical Package for the Social Sciences statistics software. Results: The results of the analysis showed that tussilagone, chlorogenic acid, rutin and isoquercitrin were significantly correlated with the quantity of pollen grains in the flower bud of T. farfara L. There is a positive correlation between them. From these results, it can be deduced that the flower bud of T. farfara L. with a greater quantity of pollen grains should be of better quality. Conclusion: The study showed that the established method can be helpful for evaluating the quality of the flower bud of T. farfara L. based on microscopic characteristic constants and chemical quantitation. PMID:26246737

Quantitative analysis of phenolic compounds in Chinese hawthorn (Crataegus spp.) fruits by high performance liquid chromatography-electrospray ionisation mass spectrometry.

PubMed

Liu, Pengzhan; Kallio, Heikki; Lü, Deguo; Zhou, Chuansheng; Yang, Baoru

2011-08-01

Eleven major phenolic compounds (hyperoside, isoquercitrin, chlorogenic acid, ideain, epicatechin, two procyanidin (PA) dimers, three PA trimers and a PA dimer-hexoside) were quantified in the fruits of 22 cultivars/origins of three species of the Chinese hawthorn (Crataegus spp.) by HPLC-ESI-MS-SIR. Hyperoside (0.1-0.8mg/g dry mass [DM]), isoquercitrin (0.1-0.3mg/g DM), chlorogenic acid (0.2-1.6mg/g DM), epicatechin (0.9-11.7mg/g DM), PA B2 (0.7-12.4mg/g DM), PA dimer II (0.1-1.5mg/g DM), PA trimer I (0.1-2.7mg/g DM), PA trimer II (0.7-6.9mg/g DM), PA trimer III (0.01-1.2mg/g DM) and a PA dimer-hexoside (trace-1.1mg/g DM) were detected in all the samples. Ideain (0.0-0.7mg/g DM) was found in all the samples except Crataegus scabrifolia. Significant correlations between the contents of individual PA aglycons were observed (r>0.9, P<0.01). A strong correlation between flavonols was also shown (r=0.71, P<0.01). Fruits of Crataegus pinnatifida var. major had higher contents of PAs but lower contents of flavonols compared with Crataegus brettschneideri. The fruits of C. scabrifolia contained the highest level of PA dimer-hexoside, which was present in trace amounts in the fruits of C. pinnatifida. Copyright © 2011 Elsevier Ltd. All rights reserved.

Chemo-enzymatic synthesis of physiologically modified avenanthramides

USDA-ARS?s Scientific Manuscript database

Avenanthramides are a group of phenolic alkaloids produced, among food crops, uniquely by oats. These metabolites function as phytoalexins in vegetative tissue and they are produced in the grain where their function is unknown. In vitro the avenanthramides inhibit the activation of nuclear factor ka...

Versatile RNA tetra-U helix linking motif as a toolkit for nucleic acid nanotechnology.

PubMed

Bui, My N; Brittany Johnson, M; Viard, Mathias; Satterwhite, Emily; Martins, Angelica N; Li, Zhihai; Marriott, Ian; Afonin, Kirill A; Khisamutdinov, Emil F

2017-04-01

RNA nanotechnology employs synthetically modified ribonucleic acid (RNA) to engineer highly stable nanostructures in one, two, and three dimensions for medical applications. Despite the tremendous advantages in RNA nanotechnology, unmodified RNA itself is fragile and prone to enzymatic degradation. In contrast to use traditionally modified RNA strands e.g. 2'-fluorine, 2'-amine, 2'-methyl, we studied the effect of RNA/DNA hybrid approach utilizing a computer-assisted RNA tetra-uracil (tetra-U) motif as a toolkit to address questions related to assembly efficiency, versatility, stability, and the production costs of hybrid RNA/DNA nanoparticles. The tetra-U RNA motif was implemented to construct four functional triangles using RNA, DNA and RNA/DNA mixtures, resulting in fine-tunable enzymatic and thermodynamic stabilities, immunostimulatory activity and RNAi capability. Moreover, the tetra-U toolkit has great potential in the fabrication of rectangular, pentagonal, and hexagonal NPs, representing the power of simplicity of RNA/DNA approach for RNA nanotechnology and nanomedicine community. Copyright © 2017 Elsevier Inc. All rights reserved.

New AdoMet Analogues as Tools for Enzymatic Transfer of Photo-Cross-Linkers and Capturing RNA-Protein Interactions.

PubMed

Muttach, Fabian; Mäsing, Florian; Studer, Armido; Rentmeister, Andrea

2017-05-02

Elucidation of biomolecular interactions is of utmost importance in biochemistry. Photo-cross-linking offers the possibility to precisely determine RNA-protein interactions. However, despite the inherent specificity of enzymes, approaches for site-specific introduction of photo-cross-linking moieties into nucleic acids are scarce. Methyltransferases in combination with synthetic analogues of their natural cosubstrate S-adenosyl-l-methionine (AdoMet) allow for the post-synthetic site-specific modification of biomolecules. We report on three novel AdoMet analogues bearing the most widespread photo-cross-linking moieties (aryl azide, diazirine, and benzophenone). We show that these photo-cross-linkers can be enzymatically transferred to the methyltransferase target, that is, the mRNA cap, with high efficiency. Photo-cross-linking of the resulting modified mRNAs with the cap interacting protein eIF4E was successful with aryl azide and diazirine but not benzophenone, reflecting the affinity of the modified 5' caps. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

An enzymatic biosensor based on three-dimensional ZnO nanotetrapods spatial net modified AlGaAs/GaAs high electron mobility transistors

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

Song, Yu; Bioengineering Program, Lehigh University, Bethlehem, Pennsylvania 18015; Zhang, Xiaohui

2014-11-24

We designed and constructed three dimensional (3D) zinc oxide Nanotetrapods (T-ZnOs) modified AlGaAs/GaAs high electron mobility transistors (HEMTs) for enzymatic uric acid (UA) detection. The chemical vapor deposition synthesized T-ZnOs was distributed on the gate areas of HEMTs in order to immobilize uricase and improve the sensitivity of the HEMTs. Combining with the high efficiency of enzyme immobilization by T-ZnOs and high sensitivity from HEMT, the as-constructed uricase/T-ZnOs/HEMTs biosensor showed fast response towards UA at ∼1 s, wide linear range from 0.2 nM to 0.2 mM and the low detect limit at 0.2 nM. The results point out an avenue to design electronic devicemore » as miniaturized lab-on-chip device for high sensitive and specific in biomedical and clinical diagnosis applications.« less

Impacts on the metabolome of down-regulating polyphenol oxidase in transgenic potato tubers

USDA-ARS?s Scientific Manuscript database

Tubers of potato (Solanum tuberosum L. cv. Estima) genetically modified (GM) to reduce polyphenol oxidase (PPO) activity and enzymatic discolouration were assessed for changes in the metabolome using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography (GC)-MS. Metabolome changes ...

Identification of Maillard reaction induced chemical modifications on Ara h 1

USDA-ARS?s Scientific Manuscript database

The Maillard reaction is a non-enzymatic glycation reaction between proteins and reducing sugars that can modify nut allergens during thermal processing. These modifications can alter the structural and immunological properties of these allergens, and may result in increased IgE binding. Here, we ...

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.

Sodium hydroxide pretreatment of genetically modified switchgrass for improved enzymatic release of sugars

USDA-ARS?s Scientific Manuscript database

Overcoming biomass recalcitrance to biological conversion has been the focus of enormous research efforts in the cellulosic biofuel area in the past decades. In this study, Alamo switchgrass was genetically transformed to suppress the expression of 4-coumarate-CoA ligase (4CL). The transgenic plants...

NMR and enzymology of modified DNA/protein interactions

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

Kennedy, M.A.

1994-12-31

We have found distinct DNA structure and base dynamics precisely at the TpA cleavage site in the TTTAAA AHA III endonuclease restriction sequence. Hence, the unusual base stacking and mobility found in this sequence may be important to the mechanism of enzymatic cleavage of the phophodiester bond.

Optimization of Process Conditions for Enzymatic Modification of Alternan using Dextranase from Chaetomium erraticum

USDA-ARS?s Scientific Manuscript database

Alternan is a unique branched glucan with alternating a-(1 ' 6) and a-(1 ' 3) backbone linkages. We previously described the modification of alternan to a reduced molecular weight form using dextranase from Penicillium sp. The solution viscosity properties of this modified alternan resemble those ...

Recruiting the Students to Fight Cancer: Total Synthesis of Goniothalamin

ERIC Educational Resources Information Center

Nahra, Fady; Riant, Olivier

2015-01-01

A modified total synthesis of (S)-goniothalamin is described for an advanced course in organic chemistry. This experiment gives students an opportunity to handle organometallic reagents and perform an enzymatic kinetic resolution and a metathesis reaction, all in the same synthesis. Furthermore, students learn flame-drying techniques for the…

Development of Microbial and Enzymatic Fuel Cells for Bio-Inspired Power Sources

DTIC Science & Technology

2009-03-01

that of the oxidation of NADH as possible.[30] A variety of organic mediators have been studied for the anode, including phenazines ,[38] dyes,[39,40...glucose-6-phosphate dehydrogenase on the rotating graphite disc electrode modified with phenazine methosulfate. Enzyme Microb. Technol. 1993, 15 (6), 525

Plasmid-mediated fosfomycin resistance is due to enzymatic modification of the antibiotic.

PubMed Central

Llaneza, J; Villar, C J; Salas, J A; Suarez, J E; Mendoza, M C; Hardisson, C

1985-01-01

The molecular mechanism of plasmid-mediated resistance to fosfomycin is described. The antibiotic was inactivated intracellularly and remained inside the cells. Modification was also obtained from cell extracts and was not energy dependent. The modifying enzyme seems to have sulfhydryl groups in its active center. PMID:3899003

Arg354 in the catalytic centre of bovine liver catalase is protected from methylglyoxal-mediated glycation.

PubMed

Scheckhuber, Christian Q

2015-12-30

In addition to controlled post-translational modifications proteins can be modified with highly reactive compounds. Usually this leads to a compromised functionality of the protein. Methylglyoxal is one of the most common agents that attack arginine residues. Methylglyoxal is also regarded as a pro-oxidant that affects cellular redox homeostasis by contributing to the formation of reactive oxygen species. Antioxidant enzymes like catalase are required to protect the cell from oxidative damage. These enzymes are also targets for methylglyoxal-mediated modification which could severely affect their catalytic activity in breaking down reactive oxygen species to less reactive or inert compounds. Here, bovine liver catalase was incubated with high levels of methylglyoxal to induce its glycation. This treatment did not lead to a pronounced reduction of enzymatic activity. Subsequently methylglyoxal-mediated arginine modifications (hydroimidazolone and dihydroxyimidazolidine) were quantitatively analysed by sensitive nano high performance liquid chromatography/electron spray ionisation/tandem mass spectrometry. Whereas several arginine residues displayed low to moderate levels of glycation (e.g., Arg93, Arg365, Arg444) Arg354 in the active centre of catalase was never found to be modified. Bovine liver catalase is able to tolerate very high levels of the modifying α-oxoaldehyde methylglyoxal so that its essential enzymatic function is not impaired.

A non-enzymatic amperometric hydrogen peroxide sensor based on iron nanoparticles decorated reduced graphene oxide nanocomposite.

PubMed

Amanulla, Baishnisha; Palanisamy, Selvakumar; Chen, Shen-Ming; Velusamy, Vijayalakshmi; Chiu, Te-Wei; Chen, Tse-Wei; Ramaraj, Sayee Kannan

2017-02-01

A simple and facile green process was used for the synthesis of iron nanoparticles (FeNPs) decorated reduced graphene oxide (rGO) nanocomposite by using Ipomoea pes-tigridis leaf extract as a reducing and stabilizing agent. The as-prepared rGO/FeNPs nanocomposite was characterized by transmission electron microscopy, X-ray spectroscopy and Fourier transform infrared spectroscopy. The nanocomposite was further modified on the glassy carbon electrode and used for non-enzymatic sensing of hydrogen peroxide (H 2 O 2 ). Cyclic voltammetry results reveal that rGO/FeNPs nanocomposite has excellent electro-reduction behavior to H 2 O 2 when compared to the response of FeNPs and rGO modified electrodes. Furthermore, the nanocomposite modified electrode shows 9 and 6 folds enhanced reduction current response to H 2 O 2 than that of rGO and FeNPs modified electrodes. Amperometric method was further used to quantify the H 2 O 2 using rGO/FeNPs nanocomposite, and the response was linear over the concentration ranging from 0.1μM to 2.15mM. The detection limit and sensitivity of the sensor were estimated as 0.056μM and 0.2085μAμM -1 cm -2 , respectively. The fabricated sensor also utilized for detection of H 2 O 2 in the presence of potentially active interfering species, and found high selectivity towards H 2 O 2 . Copyright © 2016 Elsevier Inc. All rights reserved.

Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

NASA Astrophysics Data System (ADS)

Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

2015-04-01

On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC bacteria. Therefore the applicability of on-site enzymatic activity determination as a direct surrogate or proxy parameter for microbiological standard assays and quantification of fecal indicator bacteria (FIB) concentration could not be approved and further research in this field is necessary. Presently we conclude that rapid on-site detection of enzymatic activity is applicable for surface water monitoring and that it constitutes a complementary on-site monitoring parameter with high potential. Selection of the type of measured enzymatic activities has to be done on a catchment-specific basis and further work is needed to learn more about its detailed information characteristics in different habitats. The accomplishment of this method detecting continuous data of enzymatic activity in high temporal resolution caused by a target bacterial member is on the way of becoming a powerful tool for water quality monitoring, health related water quality- and early warning requirements.

Quantitative analysis of immobilized penicillinase using enzyme-modified AlGaN/GaN field-effect transistors.

PubMed

Müntze, Gesche Mareike; Baur, Barbara; Schäfer, Wladimir; Sasse, Alexander; Howgate, John; Röth, Kai; Eickhoff, Martin

2015-02-15

Penicillinase-modified AlGaN/GaN field-effect transistors (PenFETs) are utilized to systematically investigate the covalently immobilized enzyme penicillinase under different experimental conditions. We demonstrate quantitative evaluation of covalently immobilized penicillinase layers on pH-sensitive field-effect transistors (FETs) using an analytical kinetic PenFET model. This kinetic model is explicitly suited for devices with thin enzyme layers that are not diffusion-limited, as it is the case for the PenFETs discussed here. By means of the kinetic model it was possible to extract the Michaelis constant of covalently immobilized penicillinase as well as relative transport coefficients of the different species associated with the enzymatic reaction which, exempli gratia, give information about the permeability of the enzymatic layer. Based on this analysis we quantify the reproducibility and the stability of the analyzed PenFETs over the course of 33 days as well as the influence of pH and buffer concentration on the properties of the enzymatic layer. Thereby the stability measurements reveal a Michalis constant KM of (67 ± 13)μM while the chronological development of the relative transport coefficients suggests a detachment of physisorbed penicillinase during the first two weeks since production. Our results show that AlGaN/GaN PenFETs prepared by covalent immobilization of a penicillinase enzyme layer present a powerful tool for quantitative analysis of enzyme functionality. Copyright © 2014 Elsevier B.V. All rights reserved.

Influence of nanosecond pulsed plasma on the non-enzymatic pathway for the generation of nitric oxide from L-arginine and the modification of graphite oxide to increase the solar cell efficiency.

PubMed

Attri, Pankaj; Park, Ji Hoon; Gaur, Jitender; Kumar, Naresh; Park, Dae Hoon; Jeon, Su Nam; Park, Bong Sang; Chand, Suresh; Uhm, Han Sup; Choi, Eun Ha

2014-09-14

In this work, we demonstrated the action of nanosecond pulsed plasma (NPP) on the generation of nitric oxide (NO) from the non-enzymatic pathway and on the modification of graphite oxide (GO) sheets to increase polymer solar cells (PSCs) efficiency. NO is an important signal and an effector molecule in animals, which is generated from the enzyme-catalyzed oxidation of L-arginine to NO and L-citrulline. Hence, L-arginine is an important biological precursor for NO formation. Therefore, we developed a new non-enzymatic pathway for the formation of NO and L-citrulline using NPP and characterized the pathway using NO detection kit, NMR, liquid chromatography/capillary electrophoresis-mass spectrometry (LC/CE-MS) for both quantitative and qualitative bioanalysis. We then synthesized and modified the functional groups of GO using NPP, and it was characterised by X-ray photoelectron spectroscopy (XPS), confocal Raman spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) imaging, cathodoluminescence (CL) and work function using γ-FIB. Further, we also tested the power conversion efficiency of the PSCs devices with modified GO that is similar to the one obtained with poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) as HTL. This work is perceived to have great implications for inexpensive and efficient methodology for NO generation and modification of GO, which are applicable in materials from nanomaterials to biomolecules.

Stimulatory effects of advanced glycation endproducts (AGEs) on fibronectin matrix assembly.

PubMed

Pastino, Alexandra K; Greco, Todd M; Mathias, Rommel A; Cristea, Ileana M; Schwarzbauer, Jean E

2017-05-01

Advanced glycation endproducts (AGEs) are a heterogeneous group of compounds that form via non-enzymatic glycation of proteins throughout our lifespan and at a higher rate in certain chronic diseases such as diabetes. AGEs contribute to the progression of fibrosis, in part by stimulating cellular pathways that affect gene expression. Long-lived ECM proteins are targets for non-enzymatic glycation but the question of whether the AGE-modified ECM leads to excess ECM accumulation and fibrosis remains unanswered. In this study, cellular changes due to AGE accretion in the ECM were investigated. Non-enzymatic glycation of proteins in a decellularized fibroblast ECM was achieved by incubating the ECM in a solution of methylglyoxal (MGO). Mass spectrometry of fibronectin (FN) isolated from the glycated matrix identified twenty-eight previously unidentified MGO-derived AGE modification sites including functional sites such as the RGD integrin-binding sequence. Mesangial cells grown on the glycated, decellularized matrix assembled increased amounts of FN matrix. Soluble AGE-modified bovine serum albumin (BSA) also stimulated FN matrix assembly and this effect was reduced by function-blocking antibodies against the receptor for AGE (RAGE). These results indicate that cells respond to AGEs by increasing matrix assembly and that RAGE is involved in this response. This raises the possibility that the accumulation of ECM during the progression of fibrosis may be enhanced by cell interactions with AGEs on a glycated ECM. Copyright © 2016 Elsevier B.V. All rights reserved.

Quercetin and quercetin 3-O-glycosides from Bauhinia longifolia (Bong.) Steud. show anti-Mayaro virus activity.

PubMed

dos Santos, Alda E; Kuster, Ricardo M; Yamamoto, Kristie A; Salles, Tiago S; Campos, Renata; de Meneses, Marcelo D F; Soares, Márcia R; Ferreira, Davis

2014-03-28

The arthropod-borne Mayaro virus (MAYV) causes 'Mayaro fever', a disease of medical significance, primarily affecting individuals in permanent contact with forested areas in tropical South America. Recently, MAYV has attracted attention due to its likely urbanization. Currently, there are no licensed drugs against most mosquito-transmitted viruses. Here, we investigated the in vitro anti-MAYV activity of the flavonoids quercetin and its derivatives from the Brazilian shrub Bauhinia longifolia (Bong.) Steud. Flavonoids were purified by chromatographic fractionation from leaf extracts of B. longifolia and chemically identified as quercetin and quercetin glycosides using spectroscopic techniques. Cytotoxicity of purified flavonoids and of EtOAc- and n-BuOH-containing flavonoid mixtures was measured by the dye-uptake assay while their antiviral activity was evaluated by a virus yield inhibition assay. The following flavonoids were purified from B. longifolia leaves: non-glycosylated quercetin and its glycosides guaijaverin, quercitrin, isoquercitrin, and hyperin. EtOAc and n-BuOH fractions containing these flavonoids demonstrated the highest antiviral activity of all tested substances, while quercetin had the highest antiviral activity amongst purified flavonoids. Quercetin, EtOAc, or n-BuOH fractions inhibited MAYV production by more than 90% at 25 μg/mL, displaying a stronger antiviral effect than the licensed antiviral ribavirin. A mixture of the isomers isoquercitrin and hyperin had a modest antiviral effect (IC90 = 104.9), while guaijaverin and quercitrin did not show significant antiviral activity. B. longifolia is a good source of flavonoids with anti-Mayaro virus activity. This is the first report of the activity of quercetin and its derivatives against an alphavirus.

Quercetin and quercetin 3-O-glycosides from Bauhinia longifolia (Bong.) Steud. show anti-Mayaro virus activity

PubMed Central

2014-01-01

Background The arthropod-borne Mayaro virus (MAYV) causes ‘Mayaro fever’, a disease of medical significance, primarily affecting individuals in permanent contact with forested areas in tropical South America. Recently, MAYV has attracted attention due to its likely urbanization. Currently, there are no licensed drugs against most mosquito-transmitted viruses. Here, we investigated the in vitro anti-MAYV activity of the flavonoids quercetin and its derivatives from the Brazilian shrub Bauhinia longifolia (Bong.) Steud. Methods Flavonoids were purified by chromatographic fractionation from leaf extracts of B. longifolia and chemically identified as quercetin and quercetin glycosides using spectroscopic techniques. Cytotoxicity of purified flavonoids and of EtOAc- and n-BuOH-containing flavonoid mixtures was measured by the dye-uptake assay while their antiviral activity was evaluated by a virus yield inhibition assay. Results The following flavonoids were purified from B. longifolia leaves: non-glycosylated quercetin and its glycosides guaijaverin, quercitrin, isoquercitrin, and hyperin. EtOAc and n-BuOH fractions containing these flavonoids demonstrated the highest antiviral activity of all tested substances, while quercetin had the highest antiviral activity amongst purified flavonoids. Quercetin, EtOAc, or n-BuOH fractions inhibited MAYV production by more than 90% at 25 μg/mL, displaying a stronger antiviral effect than the licensed antiviral ribavirin. A mixture of the isomers isoquercitrin and hyperin had a modest antiviral effect (IC90 = 104.9), while guaijaverin and quercitrin did not show significant antiviral activity. Conclusions B. longifolia is a good source of flavonoids with anti-Mayaro virus activity. This is the first report of the activity of quercetin and its derivatives against an alphavirus. PMID:24678592

Viral Mimicry to Usurp Ubiquitin and SUMO Host Pathways

PubMed Central

Wimmer, Peter; Schreiner, Sabrina

2015-01-01

Posttranslational modifications (PTMs) of proteins include enzymatic changes by covalent addition of cellular regulatory determinants such as ubiquitin (Ub) and small ubiquitin-like modifier (SUMO) moieties. These modifications are widely used by eukaryotic cells to control the functional repertoire of proteins. Over the last decade, it became apparent that the repertoire of ubiquitiylation and SUMOylation regulating various biological functions is not restricted to eukaryotic cells, but is also a feature of human virus families, used to extensively exploit complex host-cell networks and homeostasis. Intriguingly, besides binding to host SUMO/Ub control proteins and interfering with the respective enzymatic cascade, many viral proteins mimic key regulatory factors to usurp this host machinery and promote efficient viral outcomes. Advanced detection methods and functional studies of ubiquitiylation and SUMOylation during virus-host interplay have revealed that human viruses have evolved a large arsenal of strategies to exploit these specific PTM processes. In this review, we highlight the known viral analogs orchestrating ubiquitin and SUMO conjugation events to subvert and utilize basic enzymatic pathways. PMID:26343706

Liquid nitrogen pretreatment of eucalyptus sawdust and rice hull for enhanced enzymatic saccharification.

PubMed

Castoldi, Rafael; Correa, Vanesa G; de Morais, Gutierrez Rodrigues; de Souza, Cristina G M; Bracht, Adelar; Peralta, Rosely A; Peralta-Muniz Moreira, Regina F; Peralta, Rosane M

2017-01-01

In this work, liquid nitrogen was used for the first time in the pretreatment of plant biomasses for purposes of enzymatic saccharification. After treatment (cryocrushing), the initial rates of the enzymatic hydrolysis of eucalyptus sawdust and rice hull were increased more than ten-fold. Cryocrushing did not modify significantly the contents of cellulose, hemicellulose and lignin in both eucalyptus sawdust and rice hulls. However, substantial disorganization of the lignocellulosic materials in consequence of the pretreatment could be observed by electron microscopy. Cryocrushing was highly efficient in improving the saccharification of the holocellulose component of the plant biomasses (from 4.3% to 54.1% for eucalyptus sawdust and from 3.9% to 40.6% for rice hull). It is important to emphasize that it consists in a simple operation with low requirements of water and chemicals, no corrosion, no release of products such as soluble phenolics, furfural and hydroxymethylfurfural and no waste generation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Modelling the Effects of Ageing Time of Starch on the Enzymatic Activity of Three Amylolytic Enzymes

PubMed Central

Guerra, Nelson P.; Pastrana Castro, Lorenzo

2012-01-01

The effect of increasing ageing time (t) of starch on the activity of three amylolytic enzymes (Termamyl, San Super, and BAN) was investigated. Although all the enzymatic reactions follow michaelian kinetics, v max decreased significantly (P < 0.05) and K M increased (although not always significantly) with the increase in t. The conformational changes produced in the starch chains as a consequence of the ageing seemed to affect negatively the diffusivity of the starch to the active site of the enzymes and the release of the reaction products to the medium. A similar effect was observed when the enzymatic reactions were carried out with unaged starches supplemented with different concentrations of gelatine [G]. The inhibition in the amylolytic activities was best mathematically described by using three modified forms of the Michaelis-Menten model, which included a term to consider, respectively, the linear, exponential, and hyperbolic inhibitory effects of t and [G]. PMID:22666116

Amaranth addition to enzymatically modified wheat flour improves dough functionality, bread immunoreactivity and quality.

PubMed

Heredia-Sandoval, N G; Calderón de la Barca, A M; Carvajal-Millán, E; Islas-Rubio, A R

2018-01-24

Consumers with gluten-related disorders require gluten-free (GF) foods to avoid an immune response. Alternative to the use of non-gluten containing grains to prepare GF bread, the gluten reactivity has been greatly reduced using a proline specific cleavage enzyme, however, the gluten functionality was lost. The aim of this study was to evaluate the effect of adding an amaranth flour blend (AFB) to enzymatically modified wheat-flour proteins on dough functionality and to evaluate the immunoreactivity and acceptability of the prepared bread. First, wheat flour (20% w/v, substrate) was hydrolyzed using 8.4 U mg -1 protein Aspergillus niger prolyl-endopeptidase (AnPEP) for 8 h at 40 °C under constant agitation. Four types of breads were prepared with the same formulation except for the type of flour (14% w.b.): wheat flour (WF), WF-AFB unmodified not incubated, WF-AFB unmodified incubated and WF-AFB modified. The protein composition and free thiols were analyzed before and after amaranth addition, and the flour and bread proteins were run using SDS-PAGE and immune-detected in blots with IgA from celiac disease patients. The immunoreactive gluten content, specific volume and bread acceptability were evaluated. The polymeric proteins and free thiol groups of WF decreased after AnPEP treatment. The electrophoretic patterns of the modified flour and bread proteins were different and the IgA-immunodetection in blots was highly reduced, particularly for the higher molecular weight subunits. The addition of AFB to the modified wheat flour prepared using AnPEP improved the dough functionality by increasing the thiol groups and allowed the preparation of a sensorially acceptable bread with only 60 mg kg -1 immunoreactive gluten.

Enzymatic modification of phospholipids for functional applications and human nutrition.

PubMed

Guo, Zheng; Vikbjerg, Anders F; Xu, Xuebing

2005-05-01

Rapid progress in biochemistry of phospholipids and evolution of modern bioengineering has brought forth a number of novel concepts and technical advancements in the modification of phospholipids for industrial applications and human nutrition. Highlights cover preparation of novel phospholipid analogs based on the latest understanding of pivotal role of phospholipids in manifold biological processes, exploration of remarkable application potentials of phospholipids in meliorating human health, as well as development of new chemical and biotechnological approaches applied to the modification of phospholipids. This work reviews the natural occurrence and structural characteristics of phospholipids, their updated knowledge on manifold biological and nutritional functions, traditional and novel physical and chemical approaches to modify phospholipids as well as their applications to obtain novel phospholipids, and brief introduction of the efforts focusing on de novo syntheses of phospholipids. Special attention is given to the summary of molecular structural characteristics and catalytic properties of multiple phospholipases, which helps to interpret experimental phenomena and to improve reaction design. This will of course provide fundamental bases also for the development of enzymatic technology to produce structured or modified phospholipids.

A Class of Reactive Acyl-CoA Species Reveals the Non-Enzymatic Origins of Protein Acylation

PubMed Central

Wagner, Gregory R.; Bhatt, Dhaval P.; O’Connell, Thomas M.; Thompson, J. Will; Dubois, Laura G.; Backos, Donald S.; Yang, Hao; Mitchell, Grant A.; Ilkayeva, Olga R.; Stevens, Robert D.; Grimsrud, Paul A.; Hirschey, Matthew D.

2017-01-01

SUMMARY The mechanisms underlying the formation of acyl protein modifications remain poorly understood. By investigating the reactivity of endogenous acyl-CoA metabolites, we found a class of acyl-CoAs that undergoes intramolecular catalysis to form reactive intermediates which non-enzymatically modify proteins. Based on this mechanism, we predicted, validated, and characterized a protein modification: 3-hydroxy-3-methylglutaryl(HMG)-lysine. In a model of altered HMG-CoA metabolism, we found evidence of two additional protein modifications: 3-methylglutaconyl(MGc)-lysine and 3-methylglutaryl(MG)-lysine. Using quantitative proteomics, we compared the ‘acylomes’ of two reactive acyl-CoA species, namely HMG-CoA and glutaryl-CoA, which are generated in different pathways. We found proteins that are uniquely modified by each reactive metabolite, as well as common proteins and pathways. We identified the tricarboxylic acid cycle as a pathway commonly regulated by acylation, and validated malate dehydrogenase as a key target. These data uncover a fundamental relationship between reactive acyl-CoA species and proteins, and define a new regulatory paradigm in metabolism. PMID:28380375

Electrochemiluminescence of luminol at the titanate nanotubes modified glassy carbon electrode.

PubMed

Xu, Guifang; Zeng, Xiaoxue; Lu, Shuangyan; Dai, Hong; Gong, Lingshan; Lin, Yanyu; Wang, Qingping; Tong, Yuejin; Chen, Guonan

2013-01-01

A new strategy for the construction of a sensitive and stable electrochemiluminescent platform based on titanate nanotubes (TNTs) and Nafion composite modified electrode for luminol is described, TNTs contained composite modified electrodes that showed some photocatalytic activity toward luminol electrochemiluminescence emission, and thus could dramatically enhance luminol light emission. This extremely sensitive and stable platform allowed a decrease of the experiment electrochemiluminescence luminol reagent. In addition, in luminol solution at low concentrations, we compared the capabilities of a bare glassy carbon electrode with the TNT composite modified electrode for hydrogen peroxide detection. The results indicated that compared with glassy carbon electrode this platform was extraordinarily sensitive to hydrogen peroxide. Therefore, by combining with an appropriate enzymatic reaction, this platform would be a sensitive matrix for many biomolecules.

Mapping Protein Surface Accessibility via an Electron Transfer Dissociation Selectively Cleavable Hydrazone Probe*

PubMed Central

Vasicek, Lisa; O'Brien, John P.; Browning, Karen S.; Tao, Zhihua; Liu, Hung-Wen; Brodbelt, Jennifer S.

2012-01-01

A protein's surface influences its role in protein-protein interactions and protein-ligand binding. Mass spectrometry can be used to give low resolution structural information about protein surfaces and conformations when used in combination with derivatization methods that target surface accessible amino acid residues. However, pinpointing the resulting modified peptides upon enzymatic digestion of the surface-modified protein is challenging because of the complexity of the peptide mixture and low abundance of modified peptides. Here a novel hydrazone reagent (NN) is presented that allows facile identification of all modified surface residues through a preferential cleavage upon activation by electron transfer dissociation coupled with a collision activation scan to pinpoint the modified residue in the peptide sequence. Using this approach, the correlation between percent reactivity and surface accessibility is demonstrated for two biologically active proteins, wheat eIF4E and PARP-1 Domain C. PMID:22393264

Change in the Chemical Profile of Mangifera indica Leaves after their Metabolism in the Tropidacris collaris Grasshopper.

PubMed

da Silva, Rodolfo R; Moraes, Marcilio M; Camara, Claudio A G; Ramos, Clécio S

2015-11-01

This present work addresses research on the discovery of new compounds from natural sources. It is based on a study of Mangifera indica leaf metabolism by the Tropidacris collaris grasshopper. We found that the grasshopper hydrolyzed the flavonoid isoquercitrin to quercetin when the O-glycosidic bond was broken and sugar released as a probable energy source for the insect. There was not, however, hydrolysis of the major compound in the leaves, mangiferin, which contains the C-glycosidic bond. All compounds were isolated and their chemical structure determined by UV, IR, MS, 1H and 13C NMR.

Componential profile and amylase inhibiting activity of phenolic compounds from Calendula officinalis L. leaves.

PubMed

Olennikov, Daniil N; Kashchenko, Nina I

2014-01-01

An ethanolic extract and its ethyl acetate-soluble fraction from leaves of Calendula officinalis L. (Asteraceae) were found to show an inhibitory effect on amylase. From the crude extract fractions, one new phenolic acid glucoside, 6'-O-vanilloyl-β-D-glucopyranose, was isolated, together with twenty-four known compounds including five phenolic acid glucosides, five phenylpropanoids, five coumarins, and nine flavonoids. Their structures were elucidated based on chemical and spectral data. The main components, isoquercitrin, isorhamnetin-3-O-β-D-glucopyranoside, 3,5-di-O-caffeoylquinic acid, and quercetin-3-O-(6''-acetyl)-β-D-glucopyranoside, exhibited potent inhibitory effects on amylase.

Flavonoids in Helichrysum pamphylicum inhibit mammalian type I DNA topoisomerase.

PubMed

Topcu, Zeki; Ozturk, Bintug; Kucukoglu, Ozlem; Kilinc, Emrah

2008-01-01

DNA topoisomerases are important targets for cancer chemotherapy. We investigated the effects of a methanolic extract of Helichrysum pamphylicum on mammalian DNA topoisomerase I via in vitro plasmid supercoil relaxation assays. The extracts manifested a considerable inhibition of the enzyme's activity in a dose-dependent manner. We also performed a HPLC analysis to identify the flavonoid content of the H. pamphylicum extract and tested the identified flavonoids; luteolin, luteolin-4-glucoside, naringenin, helichrysinA and isoquercitrin, on DNA topoisomerase I activity. The measurement of the total antioxidant capacity of the flavonoid standards suggested that the topoisomerase inhibition might be correlated with the antioxidant capacity of the plant.

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.

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.

Paper-based enzymatic microfluidic fuel cell: From a two-stream flow device to a single-stream lateral flow strip

NASA Astrophysics Data System (ADS)

González-Guerrero, Maria José; del Campo, F. Javier; Esquivel, Juan Pablo; Giroud, Fabien; Minteer, Shelley D.; Sabaté, Neus

2016-09-01

This work presents a first approach towards the development of a cost-effective enzymatic paper-based glucose/O2 microfluidic fuel cell in which fluid transport is based on capillary action. A first fuel cell configuration consists of a Y-shaped paper device with the fuel and the oxidant flowing in parallel over carbon paper electrodes modified with bioelectrocatalytic enzymes. The anode consists of a ferrocenium-based polyethyleneimine polymer linked to glucose oxidase (GOx/Fc-C6-LPEI), while the cathode contains a mixture of laccase, anthracene-modified multiwall carbon nanotubes, and tetrabutylammonium bromide-modified Nafion (MWCNTs/laccase/TBAB-Nafion). Subsequently, the Y-shaped configuration is improved to use a single solution containing both, the anolyte and the catholyte. Thus, the electrolytes pHs of the fuel and the oxidant solutions are adapted to an intermediate pH of 5.5. Finally, the fuel cell is run with this single solution obtaining a maximum open circuit of 0.55 ± 0.04 V and a maximum current and power density of 225 ± 17 μA cm-2 and 24 ± 5 μW cm-2, respectively. Hence, a power source closer to a commercial application (similar to conventional lateral flow test strips) is developed and successfully operated. This system can be used to supply the energy required to power microelectronics demanding low power consumption.

Enzymatic biosensor based on entrapment of d-amino acid oxidase on gold nanofilm/MWCNTs nanocomposite modified glassy carbon electrode by sol-gel network: Analytical applications for d-alanine in human serum.

PubMed

Shoja, Yalda; Rafati, Amir Abbas; Ghodsi, Javad

2017-05-01

Sensing and determination of d-alanine is studied by using an enzymatic biosensor which was constructed on the basis of d-amino acid oxidase (DAAO) immobilization by sol-gel film onto glassy carbon electrode surface modified with nanocomposite of gold nanofilm (Au-NF) and multiwalled carbon nanotubes (MWCNTs). The Au-NF/MWCNT nanocomposite was prepared by applying the potentiostatic technique for electrodeposition of Au-NF on the MWCNT immobilized on glassy carbon electrode surface. The modified electrode is investigated by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), linear sweep voltammetry (LSV) and cyclic voltammetry(CV) techniques. The linear sweep voltammetry was used for determination of d-alanine and the results showed an excellent linear relationship between biosensor response and d-alanine concentration ranging from 0.25μM to 4.5μM with correction coefficient of 0.999 (n=20). Detection limit for the fabricated sensor was calculated about 20nM (for S/N=3) and sensitivity was about 56.1μAμM -1 cm -2 . The developed biosensor exhibited rapid and accurate response to d-alanine, a good stability (4 weeks) and an average recovery of 98.9% in human serum samples. Copyright © 2017 Elsevier Inc. All rights reserved.

Improvement on sugar cane bagasse hydrolysis using enzymatic mixture designed cocktail.

PubMed

Bussamra, Bianca Consorti; Freitas, Sindelia; Costa, Aline Carvalho da

2015-01-01

The aim of this work was to study cocktail supplementation for sugar cane bagasse hydrolysis, where the enzymes were provided from both commercial source and microorganism cultivation (Trichoderma reesei and genetically modified Escherichia coli), followed by purification. Experimental simplex lattice mixture design was performed to optimize the enzymatic proportion. The response was evaluated through hydrolysis microassays validated here. The optimized enzyme mixture, comprised of T. reesei fraction (80%), endoglucanase (10%) and β-glucosidase (10%), converted, theoretically, 72% of cellulose present in hydrothermally pretreated bagasse, whereas commercial Celluclast 1.5L converts 49.11%±0.49. Thus, a rational enzyme mixture designed by using synergism concept and statistical analysis was capable of improving biomass saccharification. Copyright © 2015 Elsevier Ltd. All rights reserved.

Biorecognition of Escherichia coli K88 adhesin for glycated porcine albumin.

PubMed

Sarabia-Sainz, Andre-i; Ramos-Clamont, Gabriela; Candia-Plata, Ma María del Carmen; Vázquez-Moreno, Luz

2009-03-01

Escherichia coli (E. coli) that expresses galactose-reactive lectins, like K88 adhesin, causes high mortality among piglets. Carbohydrates that compete for adhesion could serve as an alternative for disease prevention. Porcine serum albumin (PSA) was modified by non-enzymatic glycation with lactose to produce PSA-Lac or PSA-Glc beta (1-4) Gal, as confirmed by reduction of available free amino groups, increased molecular mass and by Ricinus communis lectin recognition. E. coli K88 binds to PSA-Lac treatments containing three and four lactoses, respectively. In addition, PSA-Lac partially inhibited K88 strain adherence to mucins. These results suggest that neoglycoconjugates obtained by non-enzymatic glycation of proteins may serve in the prophylaxis of piglets' diarrhea.

Surface modification of lignocellulosic fibers using high-frequency ultrasound

Treesearch

Jayant B. Gadhe; Ram B. Gupta; Thomas Elder

2005-01-01

Enzymatic and chemical oxidation of fiber surfaces has been reported in the literature as a method for producing medium density fiberboards without using synthetic adhesives. This work focuses on modifying the surface properties of wood fibers by the generation of free radicals using high-frequency ultrasound. A sonochemical reactor operating at 610 kHz is used to...

Effects of chemical versus enzymatic processing of kenaf fibers on poly(hydroxybutyrate-co-valerate)/poly(butylene adipate-co-terephthalate) composite properties

USDA-ARS?s Scientific Manuscript database

The effect of fiber retting on crystallization and mechanical performance was investigated. A poly(hydroxybutyrate-co-valerate) (PHBV) and poly(butylene adipate-co-terephthalate) (PBAT) blend in a 80/20 ratio was modified using 5% by weight kenaf (Hibiscus cannabinus L.) fiber. Fibers were retted us...

Use of modified atmosphere packaging to preserve mushroom quality during storage.

PubMed

Palacios, Irene; Moro, Carlos; Lozano, Miguel; D'Arrigo, Matilde; Guillamón, Eva; García-Lafuente, Ana; Villares, Ana

2011-09-01

Mushrooms have attracted much attention due to their excellent nutritional and sensory properties. However, they are highly perishable and rapidly lose their organoleptic characteristics. Many methods have been employed for mushroom storage, such as packaging, blanching, canning, or freeze drying. Among them, modified atmosphere packaging (MAP) has been widely employed for preserving fresh mushrooms. MAP provides an affordable packaging system that partly avoids enzymatic browning, fermentation and other biochemical processes by maintaining a controlled gas atmosphere. Several factors, including optimum CO2 and O2 partial pressures, permeability, package material, thickness, or product weight, must be considered in order to design a suitable modified atmosphere package for mushrooms. Thus, different strategies are available to preserve mushroom quality after harvest. The article presents some promising patents on use of modified atmosphere packaging to preserve mushroom quality during storage.

Nanosized insulin-complexes based on biodegradable amine-modified graft polyesters poly[vinyl-3-(diethylamino)-propylcarbamate-co-(vinyl acetate)-co-(vinyl alcohol)]-graft-poly(L-lactic acid): protection against enzymatic degradation, interaction with Caco-2 cell monolayers, peptide transport and cytotoxicity.

PubMed

Simon, Michael; Behrens, Isabel; Dailey, Lea Anne; Wittmar, Matthias; Kissel, Thomas

2007-05-01

Non-parenteral insulin delivery by the oral route is limited by epithelial barriers and enzymatic degradation. Nanosized insulin-complexes based on amine modified comb-like polyesters were designed to overcome these barriers. Protection of insulin in nanocomplexes (NC) from enzymatic degradation was investigated. The interaction with enterocyte-like Caco-2 cells in terms of cytotoxicity, transport through and uptake in the cell layers was evaluated by measuring transepithelial electrical resistance (TEER), release of lactate dehydrogenase (LDH) and insulin transport. The protection capacity of NC and their interaction with Caco-2 cells varied strongly as a function of lactide-grafting (hydrophobicity). With increasing lactide-grafting (P(26) < or = P(26)-1(LL) < or = P(26)-2(LL)) NC protected up to 95% of the insulin against degradation by trypsin. Transport and uptake into cell monolayers increased with higher l-lactid grafting. About 25% of a 1.25mg/ml TRITC-insulin NC dispersion with P(26)-2(LL) was recovered in Caco-2 cells after 90 min. A concentration dependent cytotoxicity profile was observed showing elevated LDH release and decreased TEER values. The cytotoxicity correlates with the surfactant like character of the polymers, decreasing the surface tension to 46 mN/m for the amphiphilic P(26)-2(LL). The observed TEER decrease was reversible after 20 h, suggesting that the biodegradable comb-polyesters offer a promising approach to overcome mucosal barriers.

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.

A Comparative evaluation of Graphene oxide based materials for Electrochemical non-enzymatic sensing of Curcumin

NASA Astrophysics Data System (ADS)

Dey, Nibedita; Devasena, T.; Sivalingam, Tamilarasu

2018-02-01

This work reports a comparative study on the development of a sensitive voltammetric method for the assay of diferuloylmethane which is fabricated using cost-effective sensing material graphene oxide (GO modified electrode) and reduced graphene oxide (rGO modified electrode) modified on glassy carbon electrode respectively. The prepared materials were characterized using SEM, XRD, FTIR, and Raman techniques to understand the formation. Between the both modified electrodes, rGO modified electrode demonstrated a lower limit detection of 0.9 pM and good signal quality. But, the better linear dynamic range for detection was found to be 1 nm to 100 nM for GO and 0.1 nM to 10 nM for rGO modified electrodes respectively. The repeatability is checked for seven cycles and interference studies were also performed for checking the sensors’ selectivity to curcumin. rGO modified electrode and GO modified electrode both shows specific signals for Diferuloylmethane under conditions similar to physiology. But, with better properties over GO modified electrode, rGO modified electrode is suggested a better candidate for real-time usability in sensing. The detection limit reported is the lowest till date for the given plant drug using any sensing assay.

Electrochemical biosensor based on glucose oxidase encapsulated within enzymatically synthesized poly(1,10-phenanthroline-5,6-dione).

PubMed

Ciftci, Hakan; Oztekin, Yasemin; Tamer, Ugur; Ramanaviciene, Almira; Ramanavicius, Arunas

2014-11-01

This study is focused on the investigation of electrocatalytic effect of glucose oxidase (GOx) immobilized on the graphite rod (GR) electrode. The enzyme modified electrode was prepared by encapsulation of immobilized GOx within enzymatically formed poly(1,10-phenanthroline-5,6-dione) (pPD) film. The electrochemical responses of such enzymatic electrode (pPD/GOx/GR) vs. different glucose concentrations were examined chronoamperometrically in acetate-phosphate buffer solution (A-PBS), pH 6.0, under aerobic or anaerobic conditions. Amperometric signals of the pPD/GOx/GR electrode exhibited well-defined hyperbolic dependence upon glucose concentration. Amperometric signals at 100mM of glucose were 41.17 and 32.27 μA under aerobic and anaerobic conditions, respectively. Amperometric signals of the pPD/GOx/GR electrode decreased by 6% within seven days. The pPD/GOx/GR electrode showed excellent selectivity in the presence of dopamine and uric acid. Furthermore it had a good reproducibility and repeatability with standard deviation of 9.4% and 8.0%, respectively. Copyright © 2014 Elsevier B.V. All rights reserved.

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

Preparation of catalytically active, covalent α-polylysine-enzyme conjugates via UV/vis-quantifiable bis-aryl hydrazone bond formation.

PubMed

Grotzky, Andrea; Manaka, Yuichi; Kojima, Taisuke; Walde, Peter

2011-01-10

Covalent UV/vis-quantifiable bis-aryl hydrazone bond formation was investigated for the preparation of conjugates between α-poly-d-lysine (PDL) and either α-chymotrypsin (α-CT) or horseradish peroxidase (HRP). PDL and the enzymes were first modified via free amino groups with the linking reagents succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic, at pH 7.6) and succinimidyl 4-formylbenzoate (S-4FB, at pH 7.2), respectively. The modified PDL and enzymes were then conjugated at pH 4.7, whereby polymer chains carrying several enzymes were obtained. Kinetics of the bis-aryl hydrazone bond formation was investigated spectrophotometrically at 354 nm. Retention of the enzymatic activity after conjugate formation was confirmed by using the substrates N-succinimidyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide (for α-CT) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, for HRP). Thus, not only a mild and efficient preparation and convenient quantification of a conjugate between the polycationic α-polylysine and enzymes could be shown, but also the complete preservation of the enzymatic activity.

Miniaturized flow system based on enzyme modified PMMA microreactor for amperometric determination of glucose.

PubMed

Cerdeira Ferreira, Luís Marcos; da Costa, Eric Tavares; do Lago, Claudimir Lucio; Angnes, Lúcio

2013-09-15

This paper describes the development of a microfluidic system having as main component an enzymatic reactor constituted by a microchannel assembled in poly(methyl methacrylate) (PMMA) substrate connected to an amperometric detector. A CO2 laser engraving machine was used to make the channels, which in sequence were thermally sealed. The internal surfaces of the microchannels were chemically modified with polyethyleneimine (PEI), which showed good effectiveness for the immobilization of the glucose oxidase enzyme using glutaraldehyde as crosslinking agent, producing a very effective microreactor for the detection of glucose. The hydrogen peroxide generated by the enzymatic reaction was detected in an electrochemical flow cell localized outside of the reactor using a platinum disk as the working electrode. The proposed system was applied to the differential amperometric determination of glucose content in soft drinks showing good repeatability (DPR=1.72%, n=50), low detection limit (1.40×10(-6)molL(-1)), high sampling frequency (calculated as 345 samples h(-1)), and relatively good stability for long-term use. The results were in close agreement with those obtained by the classical spectrophotometric method utilized to quantify glucose in biological fluids. Copyright © 2013 Elsevier B.V. All rights reserved.

Genetic engineering including superseding microinjection: new ways to make GM pigs.

PubMed

Galli, Cesare; Perota, Andrea; Brunetti, Dario; Lagutina, Irina; Lazzari, Giovanna; Lucchini, Franco

2010-01-01

Techniques for genetic engineering of swine are providing genetically modified animals of importance for the field of xenotransplantation, animal models for human diseases and for a variety of research applications. Many of these modifications have been directed toward avoiding naturally existing cellular and antibody responses to species-specific antigens. A number of techniques are today available to engineering the genome of mammals, these range from the well established less efficient method of DNA microinjection into the zygote, the use of viral vectors, to the more recent use of somatic cell nuclear transfer. The use of enzymatic engineering that are being developed now will refine the precision of the genetic modification combined with the use of new vectors like transposons. The use of somatic cell nuclear transfer is currently the most efficient way to generate genetically modified pigs. The development of enzymatic engineering with zinc-finger nucleases, recombinases and transposons will revolutionize the field. Nevertheless, genetic engineering in large domesticated animals will remain a challenging task. Recent improvements in several fields of cell and molecular biology offer new promises and opportunities toward an easier, cost-effective and efficient generation of transgenic pigs. © 2010 John Wiley & Sons A/S.

Chocolate HILIC phases: development and characterization of novel saccharide-based stationary phases by applying non-enzymatic browning (Maillard reaction) on amino-modified silica surfaces.

PubMed

Schuster, Georg; Lindner, Wolfgang

2011-06-01

Novel saccharide-based stationary phases were developed by applying non-enzymatic browning (Maillard Reaction) on aminopropyl silica material. During this process, the reducing sugars glucose, lactose, maltose, and cellobiose served as "ligand primers". The reaction cascade using cellobiose resulted in an efficient chromatographic material which further served as our model Chocolate HILIC column. (Chocolate refers to the fact that these phases are brownish.) In this way, an amine backbone was introduced to facilitate convenient manipulation of selectivity by additional attractive or repulsive ionic solute-ligand interactions in addition to the typical HILIC retention mechanism. In total, six different test sets and five different mobile phase compositions were investigated, allowing a comprehensive evaluation of the new polar column. It became evident that, besides the so-called HILIC retention mechanism based on partition phenomena, additional adsorption mechanisms, including ionic interactions, take place. Thus, the new column is another example of a HILIC-type column characterized by mixed-modal retention increments. The glucose-modified materials exhibited the relative highest overall hydrophobicity of all grafted Chocolate HILIC columns which enabled retention of lipophilic analytes with high water content mobile phases.

Enzymatically Modified Starch Favorably Modulated Intestinal Transit Time and Hindgut Fermentation in Growing Pigs

PubMed Central

Newman, M. A.; Zebeli, Q.; Velde, K.; Grüll, D.; Molnar, T.; Kandler, W.; Metzler-Zebeli, B. U.

2016-01-01

Aside from being used as stabilizing agents in many processed foods, chemically modified starches may act as functional dietary ingredients. Therefore, development of chemically modified starches that are less digestible in the upper intestinal segments and promote fermentation in the hindgut receives considerable attention. This study aimed to investigate the impact of an enzymatically modified starch (EMS) on nutrient flow, passage rate, and bacterial activity at ileal and post-ileal level. Eight ileal-cannulated growing pigs were fed 2 diets containing 72% purified starch (EMS or waxy cornstarch as control) in a cross-over design for 10 d, followed by a 4-d collection of feces and 2-d collection of ileal digesta. On d 17, solid and liquid phase markers were added to the diet to determine ileal digesta flow for 8 h after feeding. Reduced small intestinal digestion after the consumption of the EMS diet was indicated by a 10%-increase in ileal flow and fecal excretion of dry matter and energy compared to the control diet (P<0.05). Moreover, EMS feeding reduced ileal transit time of both liquid and solid fractions compared to the control diet (P<0.05). The greater substrate flow to the large intestine with the EMS diet increased the concentrations of total and individual short-chain fatty acids (SCFA) in feces (P<0.05). Total bacterial 16S rRNA gene abundance was not affected by diet, whereas the relative abundance of the Lactobacillus group decreased (P<0.01) by 50% and of Enterobacteriaceae tended (P<0.1) to increase by 20% in ileal digesta with the EMS diet compared to the control diet. In conclusion, EMS appears to resemble a slowly digestible starch by reducing intestinal transit and increasing SCFA in the distal large intestine. PMID:27936165

Rice straw as a feedstock for biofuels: Availability, recalcitrance, and chemical properties: Rice straw as a feedstock for biofuels

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

Satlewal, Alok; Agrawal, Ruchi; Bhagia, Samarthya

The surplus availability of rice straw, its limited usage and environment pollution caused by inefficient burning has fostered research for its valorization to biofuels. This review elucidates the current status of rice straw potential around the globe along with recent advances in revealing the critical factors responsible for its recalcitrance and chemical properties. The role and accumulation of high silica content in rice straw has been elucidated with its impact on enzymatic hydrolysis in a biorefinery environment. The correlation of different pretreatment approaches in modifying the physiochemical properties of rice straw and improving the enzymatic accessibility has also been discussed.more » This study highlights new challenges, resolutions and opportunities for rice straw based biorefineries.« less

A novel computational method to simulate non-enzymatic self-replication. [Abstract only

NASA Technical Reports Server (NTRS)

Navarro-Gonzalez, Rafael; Reggia, James A.; Wu, Jayoung; Chou, Hui-Hsien

1994-01-01

Non-enzymatic, template-directed synthesis of oligonucleotides has been extensively studied in the laboratory as a model to understand the kind of chemical processes that might have contributed to the origin of life on Earth. Several oligonucleotides have been shown to catalyze the synthesis of their complements from activated mononucleotides; however, a restricted number of them have been found to self-replicate. Recently we developed an efficient modified cellular automata method that supports the study of self-replicating oligonucleotides. With this method the oligonucleotide molecules are represented as active cells imbedded in a two-dimensional array of inactive cells symbolizing the environment. Random movements and probability-governed chemical reactions occurring in a cellular space can effectively simulate the experimental behavior observed in self-directed replication of oligonucleotides.

Componential Profile and Amylase Inhibiting Activity of Phenolic Compounds from Calendula officinalis L. Leaves

PubMed Central

Olennikov, Daniil N.; Kashchenko, Nina I.

2014-01-01

An ethanolic extract and its ethyl acetate-soluble fraction from leaves of Calendula officinalis L. (Asteraceae) were found to show an inhibitory effect on amylase. From the crude extract fractions, one new phenolic acid glucoside, 6′-O-vanilloyl-β-D-glucopyranose, was isolated, together with twenty-four known compounds including five phenolic acid glucosides, five phenylpropanoids, five coumarins, and nine flavonoids. Their structures were elucidated based on chemical and spectral data. The main components, isoquercitrin, isorhamnetin-3-O-β-D-glucopyranoside, 3,5-di-O-caffeoylquinic acid, and quercetin-3-O-(6′′-acetyl)-β-D-glucopyranoside, exhibited potent inhibitory effects on amylase. PMID:24683352

Total chemical synthesis of modified histones

NASA Astrophysics Data System (ADS)

Qi, Yun-Kun; Ai, Hua-Song; Li, Yi-Ming; Yan, Baihui

2018-02-01

In the post-genome era, epigenetics has received increasing attentions in recent years. The post-translational modifications (PTMs) of four core histones play central roles in epigenetic regulation of eukaryotic genome by either directly altering the biophysical properties of nucleosomes or by recruiting other effector proteins. In order to study the biological functions and structural mechanisms of these histone PTMs, an obligatory step is to prepare a sufficient amount of homogeneously modified histones. This task cannot be fully accomplished either by recombinant technology or enzymatic modification. In this context, synthetic chemists have developed novel protein synthetic tools and state-of-the-art chemical ligation strategies for the preparation of homologous modified histones. In this review, we summarize the recent advances in the preparation of modified histones, focusing on the total chemical synthesis strategies. The importance and potential of synthetic chemistry for the study of histone code will be also discussed.

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.

Secretion profiles of fungi as potential tools for metal ecotoxicity assessment: a study of enzymatic system in Trametes versicolor.

PubMed

Lebrun, Jérémie D; Demont-Caulet, Nathalie; Cheviron, Nathalie; Laval, Karine; Trinsoutrot-Gattin, Isabelle; Mougin, Christian

2011-01-01

The relationship between the expression of extracellular enzymatic system and a metal stress is scarce in fungi, hence limiting the possible use of secretion profiles as tools for metal ecotoxicity assessment. In the present study, we investigated the effect of Zn, Cu, Pb and Cd, tested alone or in equimolar cocktail, on the secretion profiles at enzymatic and protein levels in Trametesversicolor. For that purpose, extracellular hydrolases (acid phosphatase, β-glucosidase, β-galactosidase and N-acetyl-β-glucosaminidase) and ligninolytic oxidases (laccase, Mn-peroxidase) were monitored in liquid cultures. Fungal secretome was analyzed by electrophoresis and laccase secretion was characterized by western-blot and mass spectrometry analyses. Our results showed that all hydrolase activities were inhibited by the metals tested alone or in cocktail, whereas oxidase activities were specifically stimulated by Cu, Cd and metal cocktail. At protein level, metal exposure modified the electrophoretic profiles of fungal secretome and affected the diversity of secreted proteins. Two laccase isoenzymes, LacA and LacB, identified by mass spectrometry were differentially glycosylated according to the metal exposure. The amount of secreted LacA and LacB was strongly correlated with the stimulation of laccase activity by Cu, Cd and metal cocktail. These modifications of extracellular enzymatic system suggest that fungal oxidases could be used as biomarkers of metal exposure. Copyright © 2010 Elsevier Ltd. All rights reserved.

Role for phospholipid acyl chains and cholesterol in pulmonary infections and inflammation

PubMed Central

Shaikh, Saame Raza; Fessler, Michael B.

2016-01-01

Bacterial and viral respiratory tract infections result in millions of deaths worldwide and are currently the leading cause of death from infection. Acute inflammation is an essential element of host defense against infection, but can be damaging to the host when left unchecked. Effective host defense requires multiple lipid mediators, which collectively have proinflammatory and/or proresolving effects on the lung. During pulmonary infections, phospholipid acyl chains and cholesterol can be chemically and enzymatically oxidized, as well as truncated and modified, producing complex mixtures of bioactive lipids. We review recent evidence that phospholipids and cholesterol and their derivatives regulate pulmonary innate and adaptive immunity during infection. We first highlight data that oxidized phospholipids generated in the lung during infection stimulate pattern recognition receptors, such as TLRs and scavenger receptors, thereby amplifying the pulmonary inflammatory response. Next, we discuss evidence that oxidation of endogenous pools of cholesterol during pulmonary infections produces oxysterols that also modify the function of both innate and adaptive immune cells. Last, we conclude with data that n-3 polyunsaturated fatty acids, both in the form of phospholipid acyl chains and through enzymatic processing into endogenous proresolving lipid mediators, aid in the resolution of lung inflammation through distinct mechanisms. Unraveling the complex mechanisms of induction and function of distinct classes of bioactive lipids, both native and modified, may hold promise for developing new therapeutic strategies for improving pulmonary outcomes in response to infection. PMID:27286794

A novel rapid synthesis of Fe{sub 2}O{sub 3}/graphene nanocomposite using ferrate(VI) and its application as a new kind of nanocomposite modified electrode as electrochemical sensor

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

Karimi, Mohammad Ali, E-mail: ma_karimi43@yahoo.com; Department of Chemistry & Nanoscience and Nanotechnology Research Laboratory; Banifatemeh, Fatemeh

2015-10-15

Highlights: • A novel rapid synthesis of rGO–Fe{sub 2}O{sub 3} nanocomposite was developed using Fe(VI). • Fe(VI) as an environmentally friendly oxidant was introduced for GO synthesis. • Synthesized rGO–Fe{sub 2}O{sub 3} nanocomposite was applied as electrochemical sensor. • A non-enzymatic sensor was developed for H{sub 2}O{sub 2}. - Abstract: In this study, a novel, simple and sensitive non-enzymatic hydrogen peroxide electrochemical sensor was developed using reduced graphene oxide/Fe{sub 2}O{sub 3} nanocomposite modified glassy carbon electrode. This nanocomposite was synthesized by reaction of sodium ferrate with graphene in alkaline media. This reaction completed in 5 min and the products weremore » stable and its deposition on the surface of electrode is investigated. It has been found the apparent charge transfer rate constant (ks) is 0.52 and transfer coefficient (α) is 0.61 for electron transfer between the modifier and glassy carbon electrode. Electrochemical behavior of this electrode and its ability to catalyze the electro-reduction of H{sub 2}O{sub 2} has been studied by cyclic voltammetry and chronoamperometry at different experimental conditions. The analytical parameters showed the good ability of electrode as a sensor for H{sub 2}O{sub 2} amperometric reduction.« less

Analytical criteria for performance characteristics of IgE binding methods for evaluating safety of biotech food products.

PubMed

Holzhauser, Thomas; Ree, Ronald van; Poulsen, Lars K; Bannon, Gary A

2008-10-01

There is detailed guidance on how to perform bioinformatic analyses and enzymatic degradation studies for genetically modified crops under consideration for approval by regulatory agencies; however, there is no consensus in the scientific community on the details of how to perform IgE serum studies. IgE serum studies are an important safety component to acceptance of genetically modified crops when the introduced protein is novel, the introduced protein is similar to known allergens, or the crop is allergenic. In this manuscript, we describe the characteristics of the reagents, validation of assay performance, and data analysis necessary to optimize the information obtained from serum testing of novel proteins and genetically modified (GM) crops and to make results more accurate and comparable between different investigations.

Direct growth of metal-organic frameworks thin film arrays on glassy carbon electrode based on rapid conversion step mediated by copper clusters and hydroxide nanotubes for fabrication of a high performance non-enzymatic glucose sensing platform.

PubMed

Shahrokhian, Saeed; Khaki Sanati, Elnaz; Hosseini, Hadi

2018-07-30

The direct growth of self-supported metal-organic frameworks (MOFs) thin film can be considered as an effective strategy for fabrication of the advanced modified electrodes in sensors and biosensor applications. However, most of the fabricated MOFs-based sensors suffer from some drawbacks such as time consuming for synthesis of MOF and electrode making, need of a binder or an additive layer, need of expensive equipment and use of hazardous solvents. Here, a novel free-standing MOFs-based modified electrode was fabricated by the rapid direct growth of MOFs on the surface of the glassy carbon electrode (GCE). In this method, direct growth of MOFs was occurred by the formation of vertically aligned arrays of Cu clusters and Cu(OH) 2 nanotubes, which can act as both mediator and positioning fixing factor for the rapid formation of self-supported MOFs on GCE surface. The effect of both chemically and electrochemically formed Cu(OH) 2 nanotubes on the morphological and electrochemical performance of the prepared MOFs were investigated. Due to the unique properties of the prepared MOFs thin film electrode such as uniform and vertically aligned structure, excellent stability, high electroactive surface area, and good availability to analyte and electrolyte diffusion, it was directly used as the electrode material for non-enzymatic electrocatalytic oxidation of glucose. Moreover, the potential utility of this sensing platform for the analytical determination of glucose concentration was evaluated by the amperometry technique. The results proved that the self-supported MOFs thin film on GCE is a promising electrode material for fabricating and designing non-enzymatic glucose sensors. Copyright © 2018 Elsevier B.V. All rights reserved.

Genetic Determinants for Enzymatic Digestion of Lignocellulosic Biomass Are Independent of Those for Lignin Abundance in a Maize Recombinant Inbred Population

DOE PAGES

Penning, Bryan W.; Sykes, Robert W.; Babcock, Nicholas C.; ...

2014-06-27

Biotechnological approaches to reduce or modify lignin in biomass crops are predicated on the assumption that it is the principal determinant of the recalcitrance of biomass to enzymatic digestion for biofuels production. We defined quantitative trait loci (QTL) in the Intermated B73 x 3 Mo17 recombinant inbred maize (Zea mays) population using pyrolysis molecular-beam mass spectrometry to establish stem lignin content and an enzymatic hydrolysis assay to measure glucose and xylose yield. Among five multiyear QTL for lignin abundance, two for 4-vinylphenol abundance, and four for glucose and/or xylose yield, not a single QTL for aromatic abundance and sugar yieldmore » was shared. A genome-wide association study for lignin abundance and sugar yield of the 282- member maize association panel provided candidate genes in the 11 QTL of the B73 and Mo17 parents but showed that many other alleles impacting these traits exist among this broader pool of maize genetic diversity. B73 and Mo17 genotypes exhibited large differences in gene expression in developing stem tissues independent of allelic variation. Combining these complementary genetic approaches provides a narrowed list of candidate genes. A cluster of SCARECROW-LIKE9 and SCARECROW-LIKE14 transcription factor genes provides exceptionally strong candidate genes emerging from the genome-wide association study. In addition to these and genes associated with cell wall metabolism, candidates include several other transcription factors associated with vascularization and fiber formation and components of cellular signaling pathways. Finally, these results provide new insights and strategies beyond the modification of lignin to enhance yields of biofuels from genetically modified biomass.« less

Genetic Determinants for Enzymatic Digestion of Lignocellulosic Biomass Are Independent of Those for Lignin Abundance in a Maize Recombinant Inbred Population1[W][OPEN

PubMed Central

Penning, Bryan W.; Sykes, Robert W.; Babcock, Nicholas C.; Dugard, Christopher K.; Held, Michael A.; Klimek, John F.; Shreve, Jacob T.; Fowler, Matthew; Ziebell, Angela; Davis, Mark F.; Decker, Stephen R.; Turner, Geoffrey B.; Mosier, Nathan S.; Springer, Nathan M.; Thimmapuram, Jyothi; Weil, Clifford F.; McCann, Maureen C.; Carpita, Nicholas C.

2014-01-01

Biotechnological approaches to reduce or modify lignin in biomass crops are predicated on the assumption that it is the principal determinant of the recalcitrance of biomass to enzymatic digestion for biofuels production. We defined quantitative trait loci (QTL) in the Intermated B73 × Mo17 recombinant inbred maize (Zea mays) population using pyrolysis molecular-beam mass spectrometry to establish stem lignin content and an enzymatic hydrolysis assay to measure glucose and xylose yield. Among five multiyear QTL for lignin abundance, two for 4-vinylphenol abundance, and four for glucose and/or xylose yield, not a single QTL for aromatic abundance and sugar yield was shared. A genome-wide association study for lignin abundance and sugar yield of the 282-member maize association panel provided candidate genes in the 11 QTL of the B73 and Mo17 parents but showed that many other alleles impacting these traits exist among this broader pool of maize genetic diversity. B73 and Mo17 genotypes exhibited large differences in gene expression in developing stem tissues independent of allelic variation. Combining these complementary genetic approaches provides a narrowed list of candidate genes. A cluster of SCARECROW-LIKE9 and SCARECROW-LIKE14 transcription factor genes provides exceptionally strong candidate genes emerging from the genome-wide association study. In addition to these and genes associated with cell wall metabolism, candidates include several other transcription factors associated with vascularization and fiber formation and components of cellular signaling pathways. These results provide new insights and strategies beyond the modification of lignin to enhance yields of biofuels from genetically modified biomass. PMID:24972714

Genetic Determinants for Enzymatic Digestion of Lignocellulosic Biomass Are Independent of Those for Lignin Abundance in a Maize Recombinant Inbred Population.

PubMed

Penning, Bryan W; Sykes, Robert W; Babcock, Nicholas C; Dugard, Christopher K; Held, Michael A; Klimek, John F; Shreve, Jacob T; Fowler, Matthew; Ziebell, Angela; Davis, Mark F; Decker, Stephen R; Turner, Geoffrey B; Mosier, Nathan S; Springer, Nathan M; Thimmapuram, Jyothi; Weil, Clifford F; McCann, Maureen C; Carpita, Nicholas C

2014-08-01

Biotechnological approaches to reduce or modify lignin in biomass crops are predicated on the assumption that it is the principal determinant of the recalcitrance of biomass to enzymatic digestion for biofuels production. We defined quantitative trait loci (QTL) in the Intermated B73 × Mo17 recombinant inbred maize (Zea mays) population using pyrolysis molecular-beam mass spectrometry to establish stem lignin content and an enzymatic hydrolysis assay to measure glucose and xylose yield. Among five multiyear QTL for lignin abundance, two for 4-vinylphenol abundance, and four for glucose and/or xylose yield, not a single QTL for aromatic abundance and sugar yield was shared. A genome-wide association study for lignin abundance and sugar yield of the 282-member maize association panel provided candidate genes in the 11 QTL of the B73 and Mo17 parents but showed that many other alleles impacting these traits exist among this broader pool of maize genetic diversity. B73 and Mo17 genotypes exhibited large differences in gene expression in developing stem tissues independent of allelic variation. Combining these complementary genetic approaches provides a narrowed list of candidate genes. A cluster of SCARECROW-LIKE9 and SCARECROW-LIKE14 transcription factor genes provides exceptionally strong candidate genes emerging from the genome-wide association study. In addition to these and genes associated with cell wall metabolism, candidates include several other transcription factors associated with vascularization and fiber formation and components of cellular signaling pathways. These results provide new insights and strategies beyond the modification of lignin to enhance yields of biofuels from genetically modified biomass. © 2014 American Society of Plant Biologists. All Rights Reserved.

Enzymatically interesterified fats based on mutton tallow and walnut oil suitable for cosmetic emulsions.

PubMed

Kowalska, M; Mendrycka, M; Zbikowska, A; Stawarz, S

2015-02-01

Formation of emulsion systems based on interesterified fats was the objective of the study. Enzymatic interesterification was carried out between enzymatic mutton tallow and walnut oil in the proportions 2 : 3 (w/w) to produce fats not available in nature. At the beginning of the interesterification process, the balance between the interesterification and fat hydrolysis was intentionally disturbed by adding more water to the catalyst (Lipozyme IR MR) of the reaction to produce more of the polar fraction monoacylglycerols [MAGs] and diacylglycerols [DAGs]. To obtain a greater quantity of MAGs and DAGs in the reaction environment via hydrolysis, water was added (11, 13, 14, 16 w-%) to the enzymatic preparation. The obtained fats were used to form emulsions. The emulsions were evaluated with respect to sensory and skin moisturizing properties by 83 respondents. Determination of emulsion stability using temperature and centrifugal tests was carried out. Morphology and the type of emulsions were determined. The respondents described the skin to which the emulsions in testing were applied as smooth, pleasant to touch and adequately moisturized. The work has demonstrated that interesterification of a mutton tallow and walnut oil blend resulted in new fats with very interesting characteristics of triacylglycerols that are not present in the environment. The results of the present work indicate the possibility of application of fats with the largest quantity of MAGs and DAGs as a fat base of emulsions in the cosmetic industries. The hypothesis assumed in this work of producing additional quantities of MAGs and DAGs (in the process of enzymatic interesterification) responsible for the stability of the system was confirmed. It should be pointed out that the emulsions based on interesterified fats exhibited a greater level of moisturization of the skin than the emulsions containing non-interesterified fat. Also, in the respondents' opinion, the emulsion containing fat, which was modified during enzymatic interesterification when 13% of water was added to the enzymatic preparation, exhibited the best sensory profile. © 2014 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Pretreatment and enzymatic hydrolysis of lignocellulosic biomass

NASA Astrophysics Data System (ADS)

Corredor, Deisy Y.

The performance of soybean hulls and forage sorghum as feedstocks for ethanol production was studied. The main goal of this research was to increase fermentable sugars' yield through high-efficiency pretreatment technology. Soybean hulls are a potential feedstock for production of bio-ethanol due to their high carbohydrate content (≈50%) of nearly 37% cellulose. Soybean hulls could be the ideal feedstock for fuel ethanol production, because they are abundant and require no special harvesting and additional transportation costs as they are already in the plant. Dilute acid and modified steam-explosion were used as pretreatment technologies to increase fermentable sugars yields. Effects of reaction time, temperature, acid concentration and type of acid on hydrolysis of hemicellulose in soybean hulls and total sugar yields were studied. Optimum pretreatment parameters and enzymatic hydrolysis conditions for converting soybean hulls into fermentable sugars were identified. The combination of acid (H2SO4, 2% w/v) and steam (140°C, 30 min) efficiently solubilized the hemicellulose, giving a pentose yield of 96%. Sorghum is a tropical grass grown primarily in semiarid and dry parts of the world, especially in areas too dry for corn. The production of sorghum results in about 30 million tons of byproducts mainly composed of cellulose, hemicellulose, and lignin. Forage sorghum such as brown midrib (BMR) sorghum for ethanol production has generated much interest since this trait is characterized genetically by lower lignin concentrations in the plant compared with conventional types. Three varieties of forage sorghum and one variety of regular sorghum were characterized and evaluated as feedstock for fermentable sugar production. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM) and X-Ray diffraction were used to determine changes in structure and chemical composition of forage sorghum before and after pretreatment and enzymatic hydrolysis process. Up to 72% of hexose yield and 94% of pentose yield were obtained using "modified" steam explosion with 2% sulfuric acid at 140°C for 30 min and enzymatic hydrolysis with cellulase (15 FPU/g cellulose) and beta-glucosidase (50 CBU/g cellulose).

In vitro non-enzymatic ribation reduces post-yield strain accommodation in cortical bone.

PubMed

Willett, Thomas L; Sutty, Sibi; Gaspar, Anne; Avery, Nick; Grynpas, Marc

2013-02-01

Non-enzymatic glycation (NEG) and advanced glycation endproducts (AGEs) may contribute to bone fragility in various diseases, ageing, and other conditions by modifying bone collagen and causing degraded mechanical properties. In this study, we sought to further understand how collagen modification in an in vitro non-enzymatic ribation model leads to loss of cortical bone toughness. Previous in vitro studies using non-enzymatic ribation reported loss of ductility in the cortical bone. Increased crosslinking is most commonly blamed for these changes; however, some studies report positive correlations between measures of total collagen crosslinking and work-to-fracture/toughness measurements whilst correlations between general NEG and measures of ductility are often negative. Fifteen bone beam triplets were cut from bovine metatarsi. Each provided one native non-incubated control, one incubated control and one ribated specimen. Incubation involved simulated body fluid±ribose for fourteen days at 37°C. Pentosidine and pyridinoline crosslinks were measured using HPLC. Three-point bending tests quantified mechanical properties. Fracture surfaces were examined using scanning electron microscopy. The effects of ribation on bone collagen molecular stability and intermolecular connectivity were investigated using differential scanning calorimetry and hydrothermal isometric tension testing. Ribation caused increased non-enzymatic collagen modification and pentosidine content (16mmol/mol collagen) and inferior post-yield mechanical behaviour, especially post-yield strain and flexural toughness. Fracture surfaces were smoother with less collagen fibril deformation or tearing than observed in controls. In the ribated group only, pentosidine content and thermomechanical measures of crosslinking were positively correlated with measures of strain accommodation and energy absorption before failure. Non-enzymatic ribation and the resulting modifications reduce cortical bone pseudo-plasticity through a reduced capacity for post-yield strain accommodation. However, the positive correlations we have found suggest that increased crosslinking may not provide a complete explanation for this embrittlement. Copyright © 2012 Elsevier Inc. All rights reserved.

Alteration and modulation of protein activity by varying post-translational modification

DOEpatents

Thompson, David N; Reed, David W; Thompson, Vicki S; Lacey, Jeffrey A; Apel, William A

2015-03-03

Embodiments of the invention include methods of altering the enzymatic activity or solubility of an extremophilic enzyme or post-translationally modifying a protein of interest via using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.

Alteration and modulation of protein activity by varying post-translational modification

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

Thompson, David N.; Reed, David W.; Thompson, Vicki S.

Embodiments of the invention include methods of altering the enzymatic activity or solubility of an extremophilic enzyme or post-translationally modifying a protein of interest via using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.

Megalin-Mediated Oligonucleotide Trafficking for Breast Cancer Chemosensitization

DTIC Science & Technology

2010-08-01

role of apoprotein J (clusterin) in atherogenesis: binding to enzymatically modified low- density lipoprotein reduces fatty acid-mediated cytotoxicity...therapeutics trafficking [1]. High megalin expression may reflect the level of chemoresistance of the cancer cells [2], while clusterin is known to cause...targeting moieties such as apolipoprotein E (Apo-E) [5] will enhance siRNA delivery into breast cancer cells which reportedly express high megalin level

Resistant Starch but Not Enzymatically Modified Waxy Maize Delays Development of Diabetes in Zucker Diabetic Fatty Rats.

PubMed

Hedemann, Mette Skou; Hermansen, Kjeld; Pedersen, Sven; Bach Knudsen, Knud Erik

2017-05-01

Background: The incidence of type 2 diabetes (T2D) is increasing worldwide, and nutritional management of circulating glucose may be a strategic tool in the prevention of T2D. Objective: We studied whether enzymatically modified waxy maize with an increased degree of branching delayed the onset of diabetes in male Zucker diabetic fatty (ZDF) rats. Methods: Forty-eight male ZDF rats, aged 5 wk, were divided into 4 groups and fed experimental diets for 9 wk that contained 52.95% starch: gelatinized corn starch (S), glucidex (GLU), resistant starch (RS), or enzymatically modified starch (EMS). Blood glucose after feed deprivation was assessed every second week; blood samples taken at run-in and at the end of the experiment were analyzed for glycated hemoglobin (HbA1c) and plasma glucose, insulin, and lipids. During weeks 2 and 8, urine was collected for metabolomic analysis. Results: Based on blood glucose concentrations in feed-deprived rats, none of the groups developed diabetes. However, in week 9, plasma glucose after feed deprivation was significantly lower in rats fed the S and RS diets (13.5 mmol/L) than in rats fed the GLU and EMS diets (17.0-18.9 mmol/L), and rats fed RS had lower HbA1c (4.9%) than rats fed the S, GLU, and EMS (5.6-6.1%) diets. The homeostasis model assessment of insulin resistance was significantly lower in rats fed RS than in rats fed the other diets (185 compared with 311-360), indicating that rats fed the S, GLU, and EMS diets were diabetic, and a 100% higher urine excretion during week 8 in rats fed the GLU and EMS diets than that of rats fed S and RS showed that they were diabetic. Urinary nontargeted metabolomics revealed that the diabetic state of rats fed S, GLU, and EMS diets influenced microbial metabolism, as well as amino acid, lipid, and vitamin metabolism. Conclusions: EMS did not delay the onset of diabetes in ZDF rats, whereas rats fed RS showed no signs of diabetes. © 2017 American Society for Nutrition.

Improved ethanol yield and reduced Minimum Ethanol Selling Price (MESP) by modifying low severity dilute acid pretreatment with deacetylation and mechanical refining: 1) Experimental

PubMed Central

2012-01-01

Background Historically, acid pretreatment technology for the production of bio-ethanol from corn stover has required severe conditions to overcome biomass recalcitrance. However, the high usage of acid and steam at severe pretreatment conditions hinders the economic feasibility of the ethanol production from biomass. In addition, the amount of acetate and furfural produced during harsh pretreatment is in the range that strongly inhibits cell growth and impedes ethanol fermentation. The current work addresses these issues through pretreatment with lower acid concentrations and temperatures incorporated with deacetylation and mechanical refining. Results The results showed that deacetylation with 0.1 M NaOH before acid pretreatment improved the monomeric xylose yield in pretreatment by up to 20% while keeping the furfural yield under 2%. Deacetylation also improved the glucose yield by 10% and the xylose yield by 20% during low solids enzymatic hydrolysis. Mechanical refining using a PFI mill further improved sugar yields during both low- and high-solids enzymatic hydrolysis. Mechanical refining also allowed enzyme loadings to be reduced while maintaining high yields. Deacetylation and mechanical refining are shown to assist in achieving 90% cellulose yield in high-solids (20%) enzymatic hydrolysis. When fermentations were performed under pH control to evaluate the effect of deacetylation and mechanical refining on the ethanol yields, glucose and xylose utilizations over 90% and ethanol yields over 90% were achieved. Overall ethanol yields were calculated based on experimental results for the base case and modified cases. One modified case that integrated deacetylation, mechanical refining, and washing was estimated to produce 88 gallons of ethanol per ton of biomass. Conclusion The current work developed a novel bio-ethanol process that features pretreatment with lower acid concentrations and temperatures incorporated with deacetylation and mechanical refining. The new process shows improved overall ethanol yields compared to traditional dilute acid pretreatment. The experimental results from this work support the techno-economic analysis and calculation of Minimum Ethanol Selling Price (MESP) detailed in our companion paper. PMID:22888758

Enzymatically modified starch up-regulates expression of incretins and sodium-coupled monocarboxylate transporter in jejunum of growing pigs.

PubMed

Metzler-Zebeli, B U; Ertl, R; Grüll, D; Molnar, T; Zebeli, Q

2017-07-01

Dietary effects on the host are mediated via modulation of the intestinal mucosal responses. The present study investigated the effect of an enzymatically modified starch (EMS) product on the mucosal expression of genes related to starch digestion, sugar and short-chain fatty acid (SCFA) absorption and incretins in the jejunum and cecum in growing pigs. Moreover, the impact of the EMS on hepatic expression of genes related to glucose and lipid metabolism, and postprandial serum metabolites were assessed. Barrows (n=12/diet; initial BW 29 kg) were individually fed three times daily with free access to a diet containing either EMS or waxy corn starch as control (CON) for 10 days. The enzymatic modification led to twice as many α-1,6-glycosidic bonds (~8%) in the amylopectin fraction in the EMS in comparison with the non-modified native waxy corn starch (4% α-1,6-glycosidic bonds). Linear discriminant analysis revealed distinct clustering of mucosal gene expression for EMS and CON diets in jejunum. Compared with the CON diet, the EMS intake up-regulated jejunal expression of sodium-coupled monocarboxylate transporter (SMCT), glucagon-like peptide-1 (GLP1) and gastric inhibitory polypeptide (GIP) (P<0.05) and intestinal alkaline phosphatase (ALPI) (P=0.08), which may be related to greater luminal SCFA availability, whereas cecal gene expression was unaffected by diet. Hepatic peroxisome proliferator-activated receptor γ (PPARγ) expression tended (P=0.07) to be down-regulated in pigs fed the EMS diet compared with pigs fed the CON diet, which may explain the trend (P=0.08) of 30% decrease in serum triglycerides in pigs fed the EMS diet. Furthermore, pigs fed the EMS diet had a 50% higher (P=0.03) serum urea concentration than pigs fed the CON diet potentially indicating an increased use of glucogenic amino acids for energy acquisition in these pigs. Present findings suggested the jejunum as the target site to influence the intestinal epithelium and altered lipid and carbohydrate metabolism by EMS feeding.

Chemical Modification of Polysaccharides

PubMed Central

Cumpstey, Ian

2013-01-01

This review covers methods for modifying the structures of polysaccharides. The introduction of hydrophobic, acidic, basic, or other functionality into polysaccharide structures can alter the properties of materials based on these substances. The development of chemical methods to achieve this aim is an ongoing area of research that is expected to become more important as the emphasis on using renewable starting materials and sustainable processes increases in the future. The methods covered in this review include ester and ether formation using saccharide oxygen nucleophiles, including enzymatic reactions and aspects of regioselectivity; the introduction of heteroatomic nucleophiles into polysaccharide chains; the oxidation of polysaccharides, including oxidative glycol cleavage, chemical oxidation of primary alcohols to carboxylic acids, and enzymatic oxidation of primary alcohols to aldehydes; reactions of uronic-acid-based polysaccharides; nucleophilic reactions of the amines of chitosan; and the formation of unsaturated polysaccharide derivatives. PMID:24151557

Synergic effect studies of the bi-enzymatic system laccase-peroxidase in a voltammetric biosensor for catecholamines.

PubMed

Leite, Oldair D; Lupetti, Karina O; Fatibello-Filho, Orlando; Vieira, Iolanda C; Barbosa, Aneli de M

2003-04-10

Several bi-enzymatic carbon paste biosensors modified with enzymes laccase from Pleurotus ostreatus fungi and peroxidase from zucchini (Cucurbita pepo) were constructed for evaluating the synergic effect of the two enzymes on the voltammetric biosensor response for various catecholamines. Initially was investigated the effect of pH from 5.0 to 7.5, temperature from 25 to 50 degrees C, initial stirring time from 30 to 150 s, scan rate from 10 to 60 mVs(-1) and potential pulse amplitude from 10 to 60 mV on the biosensor response for several catecholamines such as dopamine, adrenaline, isoprenaline and l-dopa. It was observed a biosensor signal increase employing both enzymes, indicating thus there is a synergic effect between laccase and peroxidase, verified also in spectrophotometric studies, in the determination of these catecholamines.

APPLICATION OF DRY HAWTHORN (CRATAEGUS OXYACANTHA L.) EXTRACT IN NATURAL TOPICAL FORMULATIONS.

PubMed

Stelmakiene, Ada; Ramanauskiene, Kristina; Petrikaite, Vilma; Jakstas, Valdas; Briedis, Vitalis

2016-07-01

There is a great potential for a semi-solid preparation for topical application to the skin that would use materials of natural origin not only as an active substance but also as its base. The aim of this research was to model semisolid preparations containing hawthorn extract and to determine the effect of their bases (carriers) on the release of active components from experimental dosage forms, based on the results of the in vitro studies of the bioactivity of hawthorn active components and ex vivo skin penetration studies. The active compounds of hawthorn were indentified and quantified by validated HPLC method. The antimicrobial and anti-radical activity of dry hawthorn extract were evaluated by methods in vitro. The penetration of active substances into the full undamaged human skin was evaluated by method ex vivo. Natural topical composition was chosen according to the results of release of active compounds. Release experiments were performed with modified Franz type diffusion cells. B.ceieus was the most sensitive bacteria for the hawthorn extract. Extract showed antiradical activity, however the penetration was limited. Only traces of hyperoside and isoquercitrin were founded in epidermis. Protective topical preparation with shea butter released 41.4-42.4% of active substances. Four major compounds of dry hawthorn extract were identified. The research showed that extract had antimicrobial and antiradical activity, however compounds of hawthorn stay on the surface of the undamaged human skin. Topical preparation containing beeswax did not release active compounds. Beeswax was identified as suspending agent. Topical preparations released active compounds when shea butter was used instead of beeswax.

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

The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity*

PubMed Central

Peters, Diane E.; Szabo, Roman; Friis, Stine; Shylo, Natalia A.; Uzzun Sales, Katiuchia; Holmbeck, Kenn; Bugge, Thomas H.

2014-01-01

The membrane-anchored serine protease prostasin (CAP1/PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. Prostasin null (Prss8−/−) mice lack barrier formation and display fatal postnatal dehydration. In sharp contrast, mice homozygous for a point mutation in the Prss8 gene, which causes the substitution of the active site serine within the catalytic histidine-aspartate-serine triad with alanine and renders prostasin catalytically inactive (Prss8Cat−/Cat− mice), develop barrier function and are healthy when followed for up to 20 weeks. This striking difference could not be explained by genetic modifiers or by maternal effects, as these divergent phenotypes were displayed by Prss8−/− and Prss8Cat−/Cat− mice born within the same litter. Furthermore, Prss8Cat−/Cat− mice were able to regenerate epidermal covering following cutaneous wounding. This study provides the first demonstration that essential in vivo functions of prostasin are executed by a non-enzymatic activity of this unique membrane-anchored serine protease. PMID:24706745

Extraction of green labeled pectins and pectic oligosaccharides from plant byproducts.

PubMed

Zykwinska, Agata; Boiffard, Marie-Hélène; Kontkanen, Hanna; Buchert, Johanna; Thibault, Jean-François; Bonnin, Estelle

2008-10-08

Green labeled pectins were extracted by an environmentally friendly way using proteases and cellulases being able to act on proteins and cellulose present in cell walls. Pectins were isolated from different plant byproducts, i.e., chicory roots, citrus peel, cauliflower florets and leaves, endive, and sugar beet pulps. Enzymatic extraction was performed at 50 degrees C for 4 h, in order to fulfill the conditions required for microbiological safety of extracted products. High methoxy (HM) pectins of high molar mass were extracted with three different enzyme mixtures. These pectins were subsequently demethylated with two pectin methyl esterases (PMEs), either the fungal PME from Aspergillus aculeatus or the orange PME. It was further demonstrated that high molar mass low methoxy (LM) pectins could also be extracted directly from cell walls by adding the fungal PME to the mixture of protease and cellulase. Moreover, health benefit pectic oligosaccharides, the so-called modified hairy regions, were obtained after enzymatic treatment of the residue recovered after pectin extraction. The enzymatic method demonstrates that it is possible to convert vegetable byproducts into high-added value compounds, such as pectins and pectic oligosaccharides, and thus considerably reduce the amount of these residues generated by food industries.

Novel synthesis and characterization of pristine Cu nanoparticles for the non-enzymatic glucose biosensor.

PubMed

Dayakar, T; Rao, K Venkateswara; Bikshalu, K; Rajendar, V; Park, Si-Hyun

2017-07-01

Non enzymatic electrochemical glucose sensing was developed based on pristine Cu Nanopartilces (NPs)/Glassy Carbon Electrode (GCE) which can be accomplished by simple green method via ocimum tenuiflorum leaf extract. Then, the affect of leaf extract addition on improving Structural, Optical and electrochemical properties of pristine cu NPs was investigated. The synthesized Cu NPs were characterized with X-ray diffraction (X-ray), Uv-Visible spectroscopy (Uv-Vis), Fourier transformation infrared spectroscopy (FTIR), Particle size distribution (PSA), Scanning electron microscopy (SEM), Energy dispersive X-ray spectroscopy (EDS), Transmission electron microscopy (TEM) for structural optical and morphological studies respectively. The synthesized Cu NPs were coated over glassy carbon electrode (GCE) to study the electrochemical response of glucose by cyclic voltammetry and ampherometer. The results indicates that the modified biosensor shows a remarkable sensitivity (1065.21 μA mM -1  cm -2 ), rapid response time (<3s), wide linear range (1 to 7.2 mM), low detection limit (0.038 μM at S/N = 3). Therefore, the prepared Cu NPs by the Novel Bio-mediated route were exploited to construct a non-enzymatic glucose biosensor for sustainable clinical field applications.

Effect of enzymatic degradation of chitosan in polyhydroxybutyrate/chitosan/calcium phosphate composites on in vitro osteoblast response.

PubMed

Giretova, Maria; Medvecky, Lubomir; Stulajterova, Radoslava; Sopcak, Tibor; Briancin, Jaroslav; Tatarkova, Monika

2016-12-01

Polyhydroxybutyrate/chitosan/calcium phosphate composites are interesting biomaterials for utilization in regenerative medicine and they may by applied in reconstruction of deeper subchondral defects. Insufficient informations were found in recent papers about the influence of lysozyme degradation of chitosan in calcium phosphate/chitosan based composites on in vitro cytotoxicity and proliferation activity of osteoblasts. The effect of enzymatic chitosan degradation on osteoblasts proliferation was studied on composite films in which the porosity of origin 3D scaffolds was eliminated and the surface texture was modified. The significantly enhanced proliferation activity with faster population growth of osteoblasts were found on enzymatically degraded biopolymer composite films with α-tricalcium phosphate and nanohydroxyapatite. No cytotoxicity of composite films prepared from lysozyme degraded scaffolds containing a large fraction of low molecular weight chitosans (LMWC), was revealed after 10 days of cultivation. Contrary to above in the higher cytotoxicity origin untreated nanohydroxyapatite films and porous composite scaffolds. The results showed that the synergistic effect of surface distribution, morphology of nanohydroxyapatite particles, microtopography and the presence of LMWC due to chitosan degradation in composite films were responsible for compensation of the cytotoxicity of nanohydroxyapatite composite films or porous composite scaffolds.

STUDIES ON THE MECHANISM OF EXPERIMENTAL PROTEINURIA INDUCED BY RENIN

PubMed Central

Deodhar, Sharad D.; Cuppage, Francis E.; Gableman, E.

1964-01-01

Renin-induced proteinuria in the rat was investigated, with special emphasis on the relationship between the enzymatic activity and the proteinuric effect of renin. The dependence of the proteinuric effect on the enzymatic activity was shown by using (a) renin preparations of widely varying purity and (b) chemically modified "active" and "inactive" renin derivatives. Angiotensin II, the pressor product of the enzymatic action of renin, also produced significant proteinuria. Adrenalectomy abolished the proteinuria induced by renin. Proteinuria, however, occurred as a result of pretreatment with DOCA, or aldosterone, or without treatment, 7 to 8 weeks after adrenalectomy. Electron microscopic studies of the kidney at the time of maximal proteinuria showed focal flattening and fusion of epithelial foot processes, as well as swelling and vesicle formation in endothelial and epithelial cells of the glomeruli. Studies with intravenously injected saccharated iron oxide showed increased permeability of the glomerular capillary basement membrane to these particles. These changes were transient and were not seen 24 hours after renin injection. Adrenalectomy prevented these changes. It is concluded that renin, acting through angiotensin, causes glomerular capillary damage with increased permeability of these structures to protein and resultant proteinuria. The adrenal glands participate in a permissive role in this phenomenon. PMID:14212126

Incorporation of single-walled carbon nanotubes into ferrocene-modified linear polyethylenimine redox polymer films.

PubMed

Tran, Tu O; Lammert, Emily G; Chen, Jie; Merchant, Stephen A; Brunski, Daniel B; Keay, Joel C; Johnson, Matthew B; Glatzhofer, Daniel T; Schmidtke, David W

2011-05-17

In this study, we describe the effects of incorporating single-walled carbon nanotubes (SWNTs) into redox polymer-enzyme hydrogels. The hydrogels were constructed by combining the enzyme glucose oxidase with a redox polymer (Fc-C(6)-LPEI) in which ferrocene was attached to linear poly(ethylenimine) by a six-carbon spacer. Incorporation of SWNTs into these films changed their morphology and resulted in a significant increase in the enzymatic response at saturating glucose concentrations (3 mA/cm(2)) as compared to films without SWNTs (0.6 mA/cm(2)). Likewise, the sensitivity at 5 mM glucose was significantly increased in the presence of SWNTs (74 μA/cm(2)·mM) as compared to control films (26 μA/cm(2)·mM). We demonstrate that the increase in the electrochemical and enzymatic response of these films depends on the amount of SWNTs incorporated and the method of SWNT incorporation. Furthermore, we report that the presence of SWNTs in thick films allows for more of the ferrocene redox centers to become accessible. The high current densities of the hydrogels should allow for the construction of miniature biosensors and enzymatic biofuel cells.

Structuring evolution: biochemical networks and metabolic diversification in birds.

PubMed

Morrison, Erin S; Badyaev, Alexander V

2016-08-25

Recurrence and predictability of evolution are thought to reflect the correspondence between genomic and phenotypic dimensions of organisms, and the connectivity in deterministic networks within these dimensions. Direct examination of the correspondence between opportunities for diversification imbedded in such networks and realized diversity is illuminating, but is empirically challenging because both the deterministic networks and phenotypic diversity are modified in the course of evolution. Here we overcome this problem by directly comparing the structure of a "global" carotenoid network - comprising of all known enzymatic reactions among naturally occurring carotenoids - with the patterns of evolutionary diversification in carotenoid-producing metabolic networks utilized by birds. We found that phenotypic diversification in carotenoid networks across 250 species was closely associated with enzymatic connectivity of the underlying biochemical network - compounds with greater connectivity occurred the most frequently across species and were the hotspots of metabolic pathway diversification. In contrast, we found no evidence for diversification along the metabolic pathways, corroborating findings that the utilization of the global carotenoid network was not strongly influenced by history in avian evolution. The finding that the diversification in species-specific carotenoid networks is qualitatively predictable from the connectivity of the underlying enzymatic network points to significant structural determinism in phenotypic evolution.

Amperometric sensing of hydrogen peroxide using glassy carbon electrode modified with copper nanoparticles

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

Sophia, J.; Muralidharan, G., E-mail: muraligru@gmail.com

2015-10-15

In this paper, fabrication of glassy carbon electrode (GCE) modified with nano copper particles is discussed. The modified electrode has been tested for the non-enzymatic electrochemical detection of hydrogen peroxide (H{sub 2}O{sub 2}). The copper nanoparticles (Cu NPs) were prepared employing a simple chemical reduction method. The presence of Cu NPs was confirmed through UV–visible (UV–vis) absorption spectroscopy and X-ray diffraction (XRD) analysis. The size and morphology of the particles were investigated using transmission electron microscopy (TEM). The electrochemical properties of the fabricated sensor were studied via cyclic voltammetry (CV), chronoamperometry and electrochemical impedance spectroscopy (EIS). The electrochemical sensor displayedmore » excellent performance features towards H{sub 2}O{sub 2} detection exhibiting wide linear range, low detection limit, swift response time, good reproducibility and stability.« less

The Development of Non-Enzymatic Glucose Biosensors Based on Electrochemically Prepared Polypyrrole-Chitosan-Titanium Dioxide Nanocomposite Films.

PubMed

Al-Mokaram, Ali M A Abdul Amir; Yahya, Rosiyah; Abdi, Mahnaz M; Mahmud, Habibun Nabi Muhammad Ekramul

2017-05-31

The performance of a modified electrode of nanocomposite films consisting of polypyrrole-chitosan-titanium dioxide (Ppy-CS-TiO₂) has been explored for the developing a non-enzymatic glucose biosensors. The synergy effect of TiO₂ nanoparticles (NPs) and conducting polymer on the current responses of the electrode resulted in greater sensitivity. The incorporation of TiO₂ NPs in the nanocomposite films was confirmed by X-ray photoelectron spectroscopy (XPS) spectra. FE-SEM and HR-TEM provided more evidence for the presence of TiO₂ in the Ppy-CS structure. Glucose biosensing properties were determined by amperommetry and cyclic voltammetry (CV). The interfacial properties of nanocomposite electrodes were studied by electrochemical impedance spectroscopy (EIS). The developed biosensors showed good sensitivity over a linear range of 1-14 mM with a detection limit of 614 μM for glucose. The modified electrode with Ppy-CS nanocomposite also exhibited good selectivity and long-term stability with no interference effect. The Ppy-CS-TiO₂ nanocomposites films presented high electron transfer kinetics. This work shows the role of nanomaterials in electrochemical biosensors and describes the process of their homogeneous distribution in composite films by a one-step electrochemical process, where all components are taken in a single solution in the electrochemical cell.

Changes in physicochemical properties and in vitro starch digestion of native and extruded maize flours subjected to branching enzyme and maltogenic α-amylase treatment.

PubMed

Román, Laura; Martínez, Mario M; Rosell, Cristina M; Gómez, Manuel

2017-08-01

Extrusion is an increasingly used type of processing which combined with enzymatic action could open extended possibilities for obtaining clean label modified flours. In this study, native and extruded maize flours were modified using branching enzyme (B) and a combination of branching enzyme and maltogenic α-amylase (BMA) in order to modulate their hydrolysis properties. The microstructure, pasting properties, in vitro starch hydrolysis and resistant starch content of the flours were investigated. Whereas BMA treatment led to greater number of holes on the granule surface in native samples, B and BMA extruded samples showed rougher surfaces with cavities. A reduction in the retrogradation trend was observed for B and BMA native flours, in opposition to the flat pasting profile of their extruded counterparts. The glucose release increased gradually for native flours as the time of reaction did, whereas for extruded flours a fast increase of glucose release was observed during the first minutes of reaction, and kept till the end, indicating a greater accessibility to their porous structure. These results suggested that, in enzymatically treated extruded samples, changes produced at larger hierarchical levels in their starch structure could have masked a slowdown in the starch digestion properties. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Metal resistance sequences and transgenic plants

DOEpatents

Meagher, Richard Brian; Summers, Anne O.; Rugh, Clayton L.

1999-10-12

The present invention provides nucleic acid sequences encoding a metal ion resistance protein, which are expressible in plant cells. The metal resistance protein provides for the enzymatic reduction of metal ions including but not limited to divalent Cu, divalent mercury, trivalent gold, divalent cadmium, lead ions and monovalent silver ions. Transgenic plants which express these coding sequences exhibit increased resistance to metal ions in the environment as compared with plants which have not been so genetically modified. Transgenic plants with improved resistance to organometals including alkylmercury compounds, among others, are provided by the further inclusion of plant-expressible organometal lyase coding sequences, as specifically exemplified by the plant-expressible merB coding sequence. Furthermore, these transgenic plants which have been genetically modified to express the metal resistance coding sequences of the present invention can participate in the bioremediation of metal contamination via the enzymatic reduction of metal ions. Transgenic plants resistant to organometals can further mediate remediation of organic metal compounds, for example, alkylmetal compounds including but not limited to methyl mercury, methyl lead compounds, methyl cadmium and methyl arsenic compounds, in the environment by causing the freeing of mercuric or other metal ions and the reduction of the ionic mercury or other metal ions to the less toxic elemental mercury or other metals.

Biochemical investigations and mapping of the calcium-binding sites of heparinase I from Flavobacterium heparinum.

PubMed

Shriver, Z; Liu, D; Hu, Y; Sasisekharan, R

1999-02-12

The heparinases from Flavobacterium heparinum are lyases that specifically cleave heparin-like glycosaminoglycans. Previously, amino acids located in the active site of heparinase I have been identified and mapped. In an effort to further understand the mechanism by which heparinase I cleaves its polymer substrate, we sought to understand the role of calcium, as a necessary cofactor, in the enzymatic activity of heparinase I. Specifically, we undertook a series of biochemical and biophysical experiments to answer the question of whether heparinase I binds to calcium and, if so, which regions of the protein are involved in calcium binding. Using the fluorescent calcium analog terbium, we found that heparinase I tightly bound divalent and trivalent cations. Furthermore, we established that this interaction was specific for ions that closely approximate the ionic radius of calcium. Through the use of the modification reagents N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, we showed that the interaction between heparinase I and calcium was essential for proper functioning of the enzyme. Preincubation with either calcium alone or calcium in the presence of heparin was able to protect the enzyme from inactivation by these modifying reagents. In addition, through mapping studies of Woodward's reagent K-modified heparinase I, we identified two putative calcium-binding sites, CB-1 (Glu207-Ala219) and CB-2 (Thr373-Arg384), in heparinase I that not only are specifically modified by Woodward's reagent K, leading to loss of enzymatic activity, but also conform to the calcium-coordinating consensus motif.

Enzymatic measurement of free and esterified cholesterol levels in plasma and other biological preparations using the oxygen electrode in a modified glucose analyzer.

PubMed

Dietschy, J M; Weeks, L E; Delente, J J

1976-12-01

A method is described for assaying free and esterified cholesterol using the oxygen electrode in a modified glucose analyzer to measure the relative amount of oxygen utilization taking place during oxydation of free cholesterol by the enzyme, cholesterol oxidase. A second enzyme, cholesterol ester hydrolase, is utilized to generate free cholesterol from cholesterol esters. This assay procedure is rapid, specific, reproducible and applicable to the measurement of free and esterified cholesterol carried in the major plasma lipoprotein fractions of man and the rat and, in addition, it can be utilized for the assay of sterols in subcellular fractions of cells.

Inhibitors of enzymes catalyzing modifications to histone lysine residues: structure, function and activity.

PubMed

Lillico, Ryan; Stesco, Nicholas; Khorshid Amhad, Tina; Cortes, Claudia; Namaka, Mike P; Lakowski, Ted M

2016-05-01

Gene expression is partly controlled by epigenetic mechanisms including histone-modifying enzymes. Some diseases are caused by changes in gene expression that can be mitigated by inhibiting histone-modifying enzymes. This review covers the enzyme inhibitors targeting histone lysine modifications. We summarize the enzymatic mechanisms of histone lysine acetylation, deacetylation, methylation and demethylation and discuss the biochemical roles of these modifications in gene expression and in disease. We discuss inhibitors of lysine acetylation, deacetylation, methylation and demethylation defining their structure-activity relationships and their potential mechanisms. We show that there are potentially indiscriminant off-target effects on gene expression even with the use of selective epigenetic enzyme inhibitors.

Determination of total dietary fiber in selected foods containing resistant maltodextrin by a simplified enzymatic-gravimetric method and liquid chromatography: interlaboratory study in China.

PubMed

Fu, Boqiang; Wang, Jing; Roturier, Jean Michel; Tang, Zhiyu; Li, Huan; Wei, Guangyan

2008-01-01

An interlaboratory study was conducted in China to validate the modified AOAC Official Method 2001.03 for the determination of total dietary fiber (TDF) in foods containing resistant maltodextrin (RMD), which will be adopted as the National Standard Method of China. The kind of buffer solution, the volume of filtrate evaporation, the volume of eluent for desalting and residual solution after evaporation, etc. were modified, which had been proved to have acceptable accuracy and precision in the routine assay. TDF contents in 3 representative foods and 2 kinds of RMD ingredient (i.e., NUTRIOSE 06 and NUTRIOSE 10) were measured using the modified method in 6 eligible laboratories representing commercial, industrial, and governmental laboratories in China. The results of the interlaboratory study indicated that the intralaboratory repeatability, interlaboratory reproducibility, and precision of the modified method are adequate for reliable analysis of TDF in food containing RMD, as well as resistant dextrin. Compared to AOAC Official Method 2001.03, the modified method is time- and cost-saving.

Glutathionylation regulates cytosolic NADP+-dependent isocitrate dehydrogenase activity.

PubMed

Shin, Seoung Woo; Oh, Chang Joo; Kil, In Sup; Park, Jeen-Woo

2009-04-01

Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) is susceptible to inactivation by numerous thiol-modifying reagents. This study now reports that Cys269 of IDPc is a target for S-glutathionylation and that this modification is reversed by dithiothreitol as well as enzymatically by cytosolic glutaredoxin in the presence of GSH. Glutathionylated IDPc was significantly less susceptible than native protein to peptide fragmentation by reactive oxygen species and proteolytic digestion. Glutathionylation may play a protective role in the degradation of protein through the structural alterations of IDPc. HEK293 cells treated with diamide displayed decreased IDPc activity and accumulated glutathionylated enzyme. Using immunoprecipitation with an anti-IDPc IgG and immunoblotting with an anti-GSH IgG, we purified and positively identified glutathionylated IDPc from the kidneys of mice subjected to ischemia/reperfusion injury and from the livers of ethanol-administered rats. These results suggest that IDPc activity is modulated through enzymatic glutathionylation and deglutathionylation during oxidative stress.

A non-equilibrium thermodynamic model for tumor extracellular matrix with enzymatic degradation

NASA Astrophysics Data System (ADS)

Xue, Shi-Lei; Li, Bo; Feng, Xi-Qiao; Gao, Huajian

2017-07-01

The extracellular matrix (ECM) of a solid tumor not only affords scaffolding to support tumor architecture and integrity but also plays an essential role in tumor growth, invasion, metastasis, and therapeutics. In this paper, a non-equilibrium thermodynamic theory is established to study the chemo-mechanical behaviors of tumor ECM, which is modeled as a poroelastic polyelectrolyte consisting of a collagen network and proteoglycans. By using the principle of maximum energy dissipation rate, we deduce a set of governing equations for drug transport and mechanosensitive enzymatic degradation in ECM. The results reveal that osmosis is primarily responsible for the compression resistance of ECM. It is suggested that a well-designed ECM degradation can effectively modify the tumor microenvironment for improved efficiency of cancer therapy. The theoretical predictions show a good agreement with relevant experimental observations. This study aimed to deepen our understanding of tumor ECM may be conducive to novel anticancer strategies.

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.

Activation of dioxygen by copper metalloproteins and insights from model complexes

PubMed Central

Quist, David A.; Diaz, Daniel E.; Liu, Jeffrey J.; Karlin, Kenneth D.

2017-01-01

Nature uses dioxygen as a key oxidant in the transformation of biomolecules. Among the enzymes that are utilized for these reactions are copper-containing met-alloenzymes, which are responsible for important biological functions such as the regulation of neurotransmitters, dioxygen transport, and cellular respiration. Enzymatic and model system studies work in tandem in order to gain an understanding of the fundamental reductive activation of dioxygen by copper complexes. This review covers the most recent advancements in the structures, spectroscopy, and reaction mechanisms for dioxygen-activating copper proteins and relevant synthetic models thereof. An emphasis has also been placed on cofactor biogenesis, a fundamentally important process whereby biomolecules are post-translationally modified by the pro-enzyme active site to generate cofactors which are essential for the catalytic enzymatic reaction. Significant questions remaining in copper-ion-mediated O2-activation in copper proteins are addressed. PMID:27921179

Biological Fuel Cells and Membranes.

PubMed

Ghassemi, Zahra; Slaughter, Gymama

2017-01-17

Biofuel cells have been widely used to generate bioelectricity. Early biofuel cells employ a semi-permeable membrane to separate the anodic and cathodic compartments. The impact of different membrane materials and compositions has also been explored. Some membrane materials are employed strictly as membrane separators, while some have gained significant attention in the immobilization of enzymes or microorganisms within or behind the membrane at the electrode surface. The membrane material affects the transfer rate of the chemical species (e.g., fuel, oxygen molecules, and products) involved in the chemical reaction, which in turn has an impact on the performance of the biofuel cell. For enzymatic biofuel cells, Nafion, modified Nafion, and chitosan membranes have been used widely and continue to hold great promise in the long-term stability of enzymes and microorganisms encapsulated within them. This article provides a review of the most widely used membrane materials in the development of enzymatic and microbial biofuel cells.

Facile hydrothermal synthesis of mn doped ZnO nanopencils for development of amperometric glucose biosensors

NASA Astrophysics Data System (ADS)

Shukla, Mayoorika; Pramila; Agrawal, Jitesh; Dixit, Tejendra; Palani, I. A.; Singh, Vipul

2018-05-01

Mn doped ZnO nanopencils were synthesized via low temperature hydrothermal process for fabrication of enzymatic electrochemical glucose biosensor. The KMnO4 was found to play a dual role in modifying morphology and inducing Mn doping. Interestingly, two different types of morphologies viz nanorods and nanopencils along with Mn doping in the later were obtained. Incorporation of Mn has shown a tremendous effect on the morphological variations, repression of defects and electrochemical charge transfer at electrode electrolyte interface. The possible reason behind obtained morphological changes has been proposed which in turn were responsible for the improvement in the different figure of merits of as fabricated enzymatic electrochemical biosensor. There has been a 17 fold enhancement in the sensitivity of the as fabricated glucose biosensor from ZnO nanorods to Mn doped ZnO nanopencils which can be attributed to morphological variation and Mn doping.

Biological Fuel Cells and Membranes

PubMed Central

Ghassemi, Zahra; Slaughter, Gymama

2017-01-01

Biofuel cells have been widely used to generate bioelectricity. Early biofuel cells employ a semi-permeable membrane to separate the anodic and cathodic compartments. The impact of different membrane materials and compositions has also been explored. Some membrane materials are employed strictly as membrane separators, while some have gained significant attention in the immobilization of enzymes or microorganisms within or behind the membrane at the electrode surface. The membrane material affects the transfer rate of the chemical species (e.g., fuel, oxygen molecules, and products) involved in the chemical reaction, which in turn has an impact on the performance of the biofuel cell. For enzymatic biofuel cells, Nafion, modified Nafion, and chitosan membranes have been used widely and continue to hold great promise in the long-term stability of enzymes and microorganisms encapsulated within them. This article provides a review of the most widely used membrane materials in the development of enzymatic and microbial biofuel cells. PMID:28106711

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.

Activation of dioxygen by copper metalloproteins and insights from model complexes.

PubMed

Quist, David A; Diaz, Daniel E; Liu, Jeffrey J; Karlin, Kenneth D

2017-04-01

Nature uses dioxygen as a key oxidant in the transformation of biomolecules. Among the enzymes that are utilized for these reactions are copper-containing metalloenzymes, which are responsible for important biological functions such as the regulation of neurotransmitters, dioxygen transport, and cellular respiration. Enzymatic and model system studies work in tandem in order to gain an understanding of the fundamental reductive activation of dioxygen by copper complexes. This review covers the most recent advancements in the structures, spectroscopy, and reaction mechanisms for dioxygen-activating copper proteins and relevant synthetic models thereof. An emphasis has also been placed on cofactor biogenesis, a fundamentally important process whereby biomolecules are post-translationally modified by the pro-enzyme active site to generate cofactors which are essential for the catalytic enzymatic reaction. Significant questions remaining in copper-ion-mediated O 2 -activation in copper proteins are addressed.

A DNA-scaffold platform enhances a multi-enzymatic cycling reaction.

PubMed

Mashimo, Yasumasa; Mie, Masayasu; Kobatake, Eiry

2018-04-01

We explored the co-localization of multiple enzymes on a DNA backbone via a DNA-binding protein, Gene-A* (A*-tag) to increase the efficiency of cascade enzymatic reactions. Firefly luciferase (FLuc) and pyruvate orthophosphate dikinase (PPDK) were genetically fused with A*-tag and modified with single-stranded (ss) DNA via A*-tag. The components were assembled on ssDNA by hybridization, thereby enhancing the efficiency of the cascading bioluminescent reaction producing light emission from pyrophosphate. The activity of A*-tag in each enzyme was investigated with dye-labeled DNA. Co-localization of the enzymes via hybridization was examined using a gel shift assay. The multi-enzyme complex showed significant improvement in the overall efficiency of the cascading reaction in comparison to a mixture of free enzymes. A*-tag is highly convenient for ssDNA modification of versatile enzymes, and it can be used for construction of functional DNA-enzyme complexes.

Enzymatic degradation of cell wall and related plant polysaccharides.

PubMed

Ward, O P; Moo-Young, M

1989-01-01

Polysaccharides such as starch, cellulose and other glucans, pectins, xylans, mannans, and fructans are present as major structural and storage materials in plants. These constituents may be degraded and modified by endogenous enzymes during plant growth and development. In plant pathogenesis by microorganisms, extracellular enzymes secreted by infected strains play a major role in plant tissue degradation and invasion of the host. Many of these polysaccharide-degrading enzymes are also produced by microorganisms widely used in industrial enzyme production. Most commerical enzyme preparations contain an array of secondary activities in addition to the one or two principal components which have standardized activities. In the processing of unpurified carbohydrate materials such as cereals, fruits, and tubers, these secondary enzyme activities offer major potential for improving process efficiency. Use of more defined combinations of industrial polysaccharases should allow final control of existing enzyme processes and should also lead to the development of novel enzymatic applications.

Artificial specific binders directly recovered from chemically modified nucleic acid libraries.

PubMed

Kasahara, Yuuya; Kuwahara, Masayasu

2012-01-01

Specific binders comprised of nucleic acids, that is, RNA/DNA aptamers, are attractive functional biopolymers owing to their potential broad application in medicine, food hygiene, environmental analysis, and biological research. Despite the large number of reports on selection of natural DNA/RNA aptamers, there are not many examples of direct screening of chemically modified nucleic acid aptamers. This is because of (i) the inferior efficiency and accuracy of polymerase reactions involving transcription/reverse-transcription of modified nucleotides compared with those of natural nucleotides, (ii) technical difficulties and additional time and effort required when using modified nucleic acid libraries, and (iii) ambiguous efficacies of chemical modifications in binding properties until recently; in contrast, the effects of chemical modifications on biostability are well studied using various nucleotide analogs. Although reports on the direct screening of a modified nucleic acid library remain in the minority, chemical modifications would be essential when further functional expansion of nucleic acid aptamers, in particular for medical and biological uses, is considered. This paper focuses on enzymatic production of chemically modified nucleic acids and their application to random screenings. In addition, recent advances and possible future research are also described.

Effect of modified pectin molecules on the growth of bone cells.

PubMed

Kokkonen, Hanna E; Ilvesaro, Joanna M; Morra, Marco; Schols, Henk A; Tuukkanen, Juha

2007-02-01

The aim of this study was to investigate molecular candidates for bone implant nanocoatings, which could improve biocompatibility of implant materials. Primary rat bone cells and murine preosteoblastic MC3T3-E1 cells were cultured on enzymatically modified hairy regions (MHR-A and MHR-B) of apple pectins. MHRs were covalently attached to tissue culture polystyrene (TCPS) or glass. Uncoated substrata or bone slices were used as controls. Cell attachment, proliferation, and differentiation were investigated with fluorescence and confocal microscopy. Bone cells seem to prefer MHR-B coating to MHR-A coating. On MHR-A samples, the overall numbers as well as proportions of active osteoclasts were diminished compared to those on MHR-B, TCPS, or bone. Focal adhesions indicating attachment of the osteoblastic cells were detected on MHR-B and uncoated controls but not on MHR-A. These results demonstrate the possibility to modify surfaces with pectin nanocoatings.

Direct determination of creatinine based on poly(ethyleneimine)/phosphotungstic acid multilayer modified electrode.

PubMed

Han, Ping; Xu, Shimei; Feng, Shun; Hao, Yanjun; Wang, Jide

2016-05-01

In this work, the direct determination of creatinine was achieved using a poly(ethyleneimine)/phosphotungstic acid multilayer modified electrode with the assistance of Copper(II) ions by cyclic voltammetry. The quantity of creatinine were determined by measuring the redox peak current of Cu(II)-creatinine complex/Cu(I)-creatinine complex. Factors affecting the response current of creatinine at the modified electrode were optimized. A linear relationship between the response current and the concentration of creatinine ranging from 0.125 to 62.5μM was obtained with a detection limit of 0.06μM. The proposed method was applied to determine creatinine in human urine, and satisfied results were gotten which was validated in accordance with high performance liquid chromatography. The proposed electrode provided a promising alternative in routine sensing for creatinine without enzymatic assistance. Copyright © 2016 Elsevier B.V. All rights reserved.

AGE-RELATED EFFECT ON THE CONCENTRATION OF COLLAGEN CROSSLINKS IN HUMAN OSTEONAL AND INTERSTITIAL BONE TISSUE

PubMed Central

Nyman, Jeffry S.; Roy, Anuradha; Acuna, Rae L.; Gayle, Heather J.; Reyes, Michael J.; Tyler, Jerrod H.; Dean, David D.; Wang, Xiaodu

2007-01-01

Collagen crosslinks are important to the quality of bone and may be contributors to the age-related increase in bone fracture. This study was performed to investigate whether age and gender effects on collagen crosslinks are similar in osteonal and interstitial bone tissues. Forty human cadaveric femurs were collected and divided into two age groups: Middle aged (42–63 years of age) and Elderly (69–90 years of age) with ten males and ten females in each group (n = 10). Micro-cores of bone tissue from both secondary osteons (newly formed) and interstitial regions (biologically old) in the medial quadrant of the diaphysis were extracted using a custom-modified, computer numerical controlled machine. The bone specimens were then analyzed using high performance liquid chromatography to determine the effects of age and gender on the concentration of mature, enzymatic crosslinks (hydroxylysyl-pyridinoline – HP and lysylpyridinoline – LP) and a non-enzymatic crosslink (pentosidine – PE) at these two bony sites. The results indicate that age has a significant effect on the concentration of LP and PE, while gender has a significant effect on HP and LP. In addition, the concentration of the crosslinks in the secondary osteons is significantly different from that in the interstitial bone regions. These results suggest that the rate of non-enzymatic crosslinking may increase while the formation of maturate enzymatic crosslinks may decrease with age. Such changes could potentially reduce the inherent quality of the bone tissue in the elderly skeleton. PMID:16962838

Over production of fermentable sugar for bioethanol production from carbohydrate-rich Malaysian food waste via sequential acid-enzymatic hydrolysis pretreatment.

PubMed

Hafid, Halimatun Saadiah; Nor 'Aini, Abdul Rahman; Mokhtar, Mohd Noriznan; Talib, Ahmad Tarmezee; Baharuddin, Azhari Samsu; Umi Kalsom, Md Shah

2017-09-01

In Malaysia, the amount of food waste produced is estimated at approximately 70% of total municipal solid waste generated and characterised by high amount of carbohydrate polymers such as starch, cellulose, and sugars. Considering the beneficial organic fraction contained, its utilization as an alternative substrate specifically for bioethanol production has receiving more attention. However, the sustainable production of bioethanol from food waste is linked to the efficient pretreatment needed for higher production of fermentable sugar prior to fermentation. In this work, a modified sequential acid-enzymatic hydrolysis process has been developed to produce high concentration of fermentable sugars; glucose, sucrose, fructose and maltose. The process started with hydrothermal and dilute acid pretreatment by hydrochloric acid (HCl) and sulphuric acid (H 2 SO 4 ) which aim to degrade larger molecules of polysaccharide before accessible for further steps of enzymatic hydrolysis by glucoamylase. A kinetic model is proposed to perform an optimal hydrolysis for obtaining high fermentable sugars. The results suggested that a significant increase in fermentable sugar production (2.04-folds) with conversion efficiency of 86.8% was observed via sequential acid-enzymatic pretreatment as compared to dilute acid pretreatment (∼42.4% conversion efficiency). The bioethanol production by Saccharomyces cerevisiae utilizing fermentable sugar obtained shows ethanol yield of 0.42g/g with conversion efficiency of 85.38% based on the theoretical yield was achieved. The finding indicates that food waste can be considered as a promising substrate for bioethanol production. Copyright © 2017. Published by Elsevier Ltd.

Chemical functionalization of surfaces for building three-dimensional engineered biosensors

NASA Astrophysics Data System (ADS)

Marques, Marco E.; Mansur, Alexandra A. P.; Mansur, Herman S.

2013-06-01

This study presents a new approach for developing biosensors based on enzymatic systems with designed three-dimensional structures. Silica glass slides were chemically functionalized at surfaces by reacting with organosilanes, 3-mercaptopropyltriethoxysilane (MPTES), and 3-aminopropyltriethoxysilane (APTES), using sol-gel process at room temperature. The functionalization of the supports was characterized by contact angle measurements and FTIR spectroscopy. The first enzyme layer was covalently immobilized to the support by a bi-functional linker (glutaraldehyde). The second enzyme layer was deposited using the protein conjugation method based on the high affinity "avidin-biotin" interactions. Each enzyme was biotinylated before being added to the nanostructured system and avidin was used as the binder between consecutive enzyme layers. The biochemical response was assayed at all stages to certify that the enzymatic bioactivity was retained throughout the entire layer-by-layer (LBL) process. The model of building 3D-enzymatic systems was evaluated using the enzymatic structure with glucose oxidase (GOx) and horseradish peroxidase (HRP). It was verified that the amino-modified support presented the highest bioactivity response compared to the other chemical functionalities. Moreover, the bienzyme nanostructure demonstrated relevant biochemical activity upon injecting the glucose substrate into the system. Finally, as a proof of concept, the bienzyme systems were assayed using real samples of regular and sugar-free soft drinks where they effectively behaved as structured biosensor for glucose with the built-in 3D hybrid architecture. Based on the results, it can be foreseen the development of promising new nanomaterials for several analytical applications such as monitoring the quality of food and beverages for nutrition purposes.

Bioinspired Design of Alcohol Dehydrogenase@nano TiO₂ Microreactors for Sustainable Cycling of NAD⁺/NADH Coenzyme.

PubMed

Lin, Sen; Sun, Shiyong; Wang, Ke; Shen, Kexuan; Ma, Biaobiao; Ren, Yuquan; Fan, Xiaoyu

2018-02-24

The bioinspired design and construction of enzyme@capsule microreactors with specific cell-like functionality has generated tremendous interest in recent years. Inspired by their fascinating complexity, scientists have endeavored to understand the essential aspects of a natural cell and create biomimicking microreactors so as to immobilize enzymes within the hierarchical structure of a microcapsule. In this study, simultaneous encapsulation of alcohol dehydrogenase (ADH) was achieved during the preparation of microcapsules by the Pickering emulsion method using amphiphilic modified TiO₂ nanoparticles (NPs) as building blocks for assembling the photocatalytic microcapsule membrane. The ADH@TiO₂ NP microreactors exhibited dual catalytic functions, i.e., spatially confined enzymatic catalysis and the membrane-associated photocatalytic oxidation under visible light. The sustainable cycling of nicotinamide adenine dinucleotide (NAD) coenzyme between NADH and NAD⁺ was realized by enzymatic regeneration of NADH from NAD⁺ reduction, and was provided in a form that enabled further photocatalytic oxidation to NAD⁺ under visible light. This bioinspired ADH@TiO₂ NP microreactor allowed the linking of a semiconductor mineral-based inorganic photosystem to enzymatic reactions. This is a first step toward the realization of sustainable biological cycling of NAD⁺/NADH coenzyme in synthetic functional microsystems operating under visible light irradiation.

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.

Designing cross-linked xylanase aggregates for bioconversion of agroindustrial waste biomass towards potential production of nutraceuticals.

PubMed

Hero, J S; Romero, C M; Pisa, J H; Perotti, N I; Olivaro, C; Martinez, M A

2018-05-01

Immobilized biocatalysts design has the potential to efficiently produce valuable bioproducts from lignocellulosic biomass. Among them, the carrier-free immobilization through the cross-linked enzyme aggregates technology is a simple and low-cost alternative. A two steps statistical approach was utilized to evaluate the synthesis of a cross-linked enzyme aggregate from a xylanolytic preparation, which was produced by Cohnella sp. AR92 grown in a peptone-based culture medium. The resulting immobilized biocatalyst, Xyl-CLEA, was significate more stable (25 to 45%) towards temperatures up to 50°C with respect to the free enzyme, and retained over 50% of its initial activity after 5 consecutive cycles of reuse. By means of infrared spectroscopy and electron microscopy, the Xyl-CLEA showed architectural features described as signature of type I and type II of protein aggregates. These, were the result of the simultaneous aggregation of a multiplicity of proteins from the crude enzymatic extract. The enzymatic activity was assessed using alkali pretreated sugar cane bagasse as substrate. Whereas the free enzymatic preparation released xylose as the main product, the immobilized xylanase produced xylooligosaccharides, thus showing that the immobilization procedure modified the potential application of the extracellular xylanase from Conhella sp. AR92. Copyright © 2018 Elsevier B.V. All rights reserved.

Electrochemical Reduction of Carbon Dioxide to Methanol by Direct Injection of Electrons into Immobilized Enzymes on a Modified Electrode.

PubMed

Schlager, Stefanie; Dumitru, Liviu Mihai; Haberbauer, Marianne; Fuchsbauer, Anita; Neugebauer, Helmut; Hiemetsberger, Daniela; Wagner, Annika; Portenkirchner, Engelbert; Sariciftci, Niyazi Serdar

2016-03-21

We present results for direct bio-electrocatalytic reduction of CO2 to C1 products using electrodes with immobilized enzymes. Enzymatic reduction reactions are well known from biological systems where CO2 is selectively reduced to formate, formaldehyde, or methanol at room temperature and ambient pressure. In the past, the use of such enzymatic reductions for CO2 was limited due to the necessity of a sacrificial co-enzyme, such as nicotinamide adenine dinucleotide (NADH), to supply electrons and the hydrogen equivalent. The method reported here in this paper operates without the co-enzyme NADH by directly injecting electrons from electrodes into immobilized enzymes. We demonstrate the immobilization of formate, formaldehyde, and alcohol dehydrogenases on one-and-the-same electrode for direct CO2 reduction. Carbon felt is used as working electrode material. An alginate-silicate hybrid gel matrix is used for the immobilization of the enzymes on the electrode. Generation of methanol is observed for the six-electron reduction with Faradaic efficiencies of around 10%. This method of immobilization of enzymes on electrodes offers the opportunity for electrochemical application of enzymatic electrodes to many reactions in which a substitution of the expensive sacrificial co-enzyme NADH is desired. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

PubMed Central

Valencia, Laura Carolina; García, Adriana; Ramírez-Pinilla, Martha Patricia; Fuentes, Jorge Luis

2011-01-01

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies. PMID:22215974

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae).

PubMed

Valencia, Laura Carolina; García, Adriana; Ramírez-Pinilla, Martha Patricia; Fuentes, Jorge Luis

2011-10-01

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.

Enzymatic synthesis of structured lipids.

PubMed

Iwasaki, Yugo; Yamane, Tsuneo

2004-01-01

Structured lipids (SLs) are defined as lipids that are modified chemically or enzymatically in order to change their structure. This review deals with structured triacylglycerols (STGs) and structured phospholipids (SPLs). The most typical STGs are MLM-type STGs, having medium chain fatty acids (FAs) at the 1- and 3-positions and a long chain fatty acid at the 2- position. MLM-type STGs are synthesized by: 1) 1,3-position-specific lipase-catalyzed acyl exchange of TG with FA or with FA ethylester (FAEt); 2) 1,3-position-specific lipase-catalyzed acylation of glycerol with FA, giving symmetric 1,3-diacyl-sn-glycerol, followed by chemical acylation at the sn-2 position, and; 3) 1,3-position-specific lipase-catalyzed deacylation of TG, giving 2-monoacylglycerol, followed by reacylation at the 1- and 3-positions with FA or with (FAEt). Enzymatic preparation of SPLs requires: 1) acyl group modification, and 2) head group modification of phospholipids. Acyl group modification is performed using lipases or phospholipase A2-mediated transesterification or ester synthesis to introduce arbitrary fatty acid to phospholipids. Head group modification is carried out by phospholipase D-catalyzed transphosphatidylation. A wide range of compounds can be introduced into the polar head of phospholipids, making it possible to prepare various SPLs.

In vitro study of antioxidant activity and phenolic content of Chrysanthemum balsamita varieties.

PubMed

Benedec, Daniela; Filip, Lorena; Vlase, Laurian; Bele, Constantin; Sevastre, Bogdan; Raita, Oana; Olah, Neli-Kinga; Hanganu, Daniela

2016-07-01

The purpose of our study was to identify the phenolic substances of two varieties of Chrysanthemum balsamita (balsamita and tanacetoides) and to measure the overall antioxidant activity. The phenolic compounds were determined by HPLC. The evaluation of the polyphenolic content was performed by colorimetric analysis. The antioxidant activity was measured by three in vitro assay models: the DPPH, the silver nanoparticles antioxidant capacity (SNPAC) and EPR radical detection. Using HPLC-MS analysis, phenolic acids, flavonoids and flavonoid aglycone were detected. The highest antioxidant activity was showed by Chrysanthemum balsamita var. balsamita, while the lowest for the Chrysanthemum balsamita var. tanacetoides extract, in accord with the polyphenolic content. The results show that Chrysanthemum balsamita var. balsamita might be a source of antioxidant flavonoids, especially rutin and isoquercitrin.

One new diphenylmethane glycoside from the leaves of Psidium guajava L.

PubMed

Shu, Ji-Cheng; Chou, Gui-Xin; Wang, Zheng-Tao

2012-11-01

To investigate the chemical constituents of Psidium guajava L, the EtOH/H(2)O extract of the fresh leaves was subjected to various chromatography. One diphenylmethane, one benzophenone, and eight flavonoids were isolated and elucidated as 2,6-dihydroxy-3-formaldehyde-5-methyl-4-O-(6″-O-galloyl-β-D-glucopyranosyl)-diphenylmethane (1), 2,6-dihydroxy-3,5-dimethyl-4-O-(6″-O-galloyl-β-D-glucopyranosyl)-benzophenone (2), kaempferol (3), quercetin (4), quercitrin (5), isoquercitrin (6), guaijaverin (7), avicularin (8), hyperoside (9), reynoutrin (10) by spectroscopic methods, including 1D and 2D NMR and HR-ESI-MS spectrometry as well as by comparison with published data. Compounds 5 and 10 are obtained from P. guajava for the first time, and compound 1 is a new diphenylmethane compound.

Liposome-based glioma targeted drug delivery enabled by stable peptide ligands.

PubMed

Wei, Xiaoli; Gao, Jie; Zhan, Changyou; Xie, Cao; Chai, Zhilan; Ran, Danni; Ying, Man; Zheng, Ping; Lu, Weiyue

2015-11-28

The treatment of glioma is one of the most challenging tasks in clinic. As an intracranial tumor, glioma exhibits many distinctive characteristics from other tumors. In particular, various barriers including enzymatic barriers in the blood and brain capillary endothelial cells, blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB) rigorously prevent drug and drug delivery systems from reaching the tumor site. To tackle this dilemma, we developed a liposomal formulation to circumvent multiple-barriers by modifying the liposome surface with proteolytically stable peptides, (D)CDX and c(RGDyK). (D)CDX is a D-peptide ligand of nicotine acetylcholine receptors (nAChRs) on the BBB, and c(RGDyK) is a ligand of integrin highly expressed on the BBTB and glioma cells. Lysosomal compartments of brain capillary endothelial cells are implicated in the transcytosis of those liposomes. However, both peptide ligands displayed exceptional stability in lysosomal homogenate, ensuring that intact ligands could exert subsequent exocytosis from brain capillary endothelial cells and glioma targeting. In the cellular uptake studies, dually labeled liposomes could target both brain capillary endothelial cells and tumor cells, effectively traversing the BBB and BBTB monolayers, overcoming enzymatic barrier and targeting three-dimensional tumor spheroids. Its targeting ability to intracranial glioma was further verified in vivo by ex vivo imaging and histological studies. As a result, doxorubicin liposomes modified with both (D)CDX and c(RGDyK) presented better anti-glioma effect with prolonged median survival of nude mice bearing glioma than did unmodified liposomes and liposomes modified with individual peptide ligand. In conclusion, the liposome suggested in the present study could effectively overcome multi-barriers and accomplish glioma targeted drug delivery, validating its potential value in improving the therapeutic efficacy of doxorubicin for glioma. Copyright © 2015 Elsevier B.V. All rights reserved.

Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System*

PubMed Central

Singh, Rajesh K.; Zerath, Sylvia; Kleifeld, Oded; Scheffner, Martin; Glickman, Michael H.; Fushman, David

2012-01-01

Of all ubiquitin-like proteins, Rub1 (Nedd8 in mammals) is the closest kin of ubiquitin. We show via NMR that structurally, Rub1 and ubiquitin are fundamentally similar as well. Despite these profound similarities, the prevalence of Rub1/Nedd8 and of ubiquitin as modifiers of the proteome is starkly different, and their attachments to specific substrates perform different functions. Recently, some proteins, including p53, p73, EGFR, caspase-7, and Parkin, have been shown to be modified by both Rub1/Nedd8 and ubiquitin within cells. To understand whether and how it might be possible to distinguish among the same target protein modified by Rub1 or ubiquitin or both, we examined whether ubiquitin receptors can differentiate between Rub1 and ubiquitin. Surprisingly, Rub1 interacts with proteasome ubiquitin-shuttle proteins comparably to ubiquitin but binds more weakly to a proteasomal ubiquitin receptor Rpn10. We identified Rub1-ubiquitin heteromers in yeast and Nedd8-Ub heteromers in human cells. We validate that in human cells and in vitro, human Rub1 (Nedd8) forms chains with ubiquitin where it acts as a chain terminator. Interestingly, enzymatically assembled K48-linked Rub1-ubiquitin heterodimers are recognized by various proteasomal ubiquitin shuttles and receptors comparably to K48-linked ubiquitin homodimers. Furthermore, these heterologous chains are cleaved by COP9 signalosome or 26S proteasome. A derubylation function of the proteasome expands the repertoire of its enzymatic activities. In contrast, Rub1 conjugates may be somewhat resilient to the actions of other canonical deubiquitinating enzymes. Taken together, these findings suggest that once Rub1/Nedd8 is channeled into ubiquitin pathways, it is recognized essentially like ubiquitin. PMID:23105008

Flavonoids: hemisynthesis, reactivity, characterization and free radical scavenging activity.

PubMed

Es-Safi, Nour-Eddine; Ghidouche, Souhila; Ducrot, Paul Henri

2007-09-26

Phenolic compounds form one of the main classes of secondary metabolites. They display a large range of structures and they are responsible for the major organoleptic characteristics of plant-derived-foods and beverages, particularly color and taste properties and they also contribute to the nutritional qualities of fruits and vegetables. Phenolic compounds are also highly unstable compounds which undergo numerous enzymatic and chemical reactions during postharvest food storage and processing thus adding to the complexity of plant polyphenol composition. Among these compounds flavonoids constitute one of the most ubiquitous groups of all plant phenolics. Owing to their importance in food organoleptic properties and in human health, a better understanding of their structures, their reactivity and chemical properties in addition to the mechanisms generating them appears essential to predict and control food quality. The purpose of this work is an overview of our findings concerning the hemisynthesis, the reactivity and the enzymatic oxidation of some flavonoids and shed light on the mechanisms involved in some of these processes and the structures of the resulting products. The free radical scavenging activity of some of the synthesized compounds is also presented and a structure-activity relationship is discussed. The first part of this review concerns the synthesis and structural characterization of modified monomeric flavanols. The use of these compounds as precursor for the preparation of natural and modified dimeric procyanidin derivatives was then explored through different coupling reactions. The full characterization of the synthesized compounds was achieved by concerted use of NMR and ESI-MS techniques. The free radical scavenging activity of some of the synthesized compounds was investigated. The second part of this review concerns the enzymatic oxidation of several flavonols by Trametes versicolor laccase. Most of the major oxidation products have been isolated as pure compounds and their structures unambiguously established through spectroscopic methods. Correlation between the structure of the oxidation product and the substitution pattern of the starting materials allows mechanistic features of this transformation to be elucidated.

Changes in hemolymph characteristics of ark shell Scapharaca broughtonii dealt with Vibrio anguillarum challenge in vivo and various of anticoagulants in vitro.

PubMed

Zhou, Liqing; Yang, Aiguo; Liu, Zhihong; Wu, Biao; Sun, Xiujun; Lv, Zhenming; Tian, Ji-Teng; Du, Meirong

2017-02-01

The ark shell Scapharca broughtonii is a commercially important shellfish in China. Alserver's solution (AS), modified Alserver's solution (MAS) and Heparin sodium solution (HSS) are common anticoagulants used for shellfish blood. To observe the immune response mediated by its hemocytes, we challenged in vivo S. broughtonii hemolymph with Vibrio anguillarum and dealt with the following three anticoagulants in vitro: Alserver's solution (AS), modified Alserver's solution (MAS) and Heparin sodium solution (HSS). The methodologies we used were immunostimulation with V. anguillarum, Wright-Giemsa staining, micro-examination, and flow cytometric and hydrolyzing enzyme activity analysis. The results showed that all three types of anticoagulants effectively prevented blood clotting in ark shellfish. The morphology of hemocytes did not significantly change 30 h after anticoagulant treatment, except for the shrinking of hemocytes after administering HSS. The size and permeability of hemocytes changed when treated with the anticoagulants and when stimulated with V. anguillarum. Both alkaline phosphatase (AKP) and acid phosphatase (ACP) in hemocytes and Plasma were measured at different times after they were stimulated with V. anguillarum in HSS and MAS. The AKP enzymatic activity in HSS was somewhat higher than in the MAS anticoagulant, but changes in response to V. anguillarum challenge of enzymatic activity were almost the same in HSS and MAS groups. In conclusion, all three types of anticoagulants may be used for ark shell blood preservation. They all changed the cell-surface characteristics of hemocytes to inhibit clot formation. The AS anticoagulant was appropriate for maintaining white and red cell shapes, while MAS was ideal for retaining throbus cell function. Lastly, HSS was appropriate for maintaining enzymatic activity in hemolymph and function of hemocytes. Following this investigation, we gained insight into the changes in hemolymph characteristic during immune response. Copyright © 2016. Published by Elsevier Ltd.

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.

Rhodium(II) metallopeptide catalyst design enables fine control in selective functionalization of natural SH3 domains.

PubMed

Vohidov, Farrukh; Coughlin, Jane M; Ball, Zachary T

2015-04-07

Chemically modified proteins are increasingly important for use in fundamental biophysical studies, chemical biology, therapeutic protein development, and biomaterials. However, chemical methods typically produce heterogeneous labeling and cannot approach the exquisite selectivity of enzymatic reactions. While bioengineered methods are sometimes an option, selective reactions of natural proteins remain an unsolved problem. Here we show that rhodium(II) metallopeptides combine molecular recognition with promiscuous catalytic activity to allow covalent decoration of natural SH3 domains, depending on choice of catalyst but independent of the specific residue present. A metallopeptide catalyst succeeds in modifying a single SH3-containing kinase at endogenous concentrations in prostate cancer (PC-3) cell lysate. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A solid-state control system for dynein-based ciliary/flagellar motility

PubMed Central

2013-01-01

Ciliary and flagellar beating requires the coordinated action of multiple dyneins with different enzymatic and motor properties. In this issue, Yamamoto et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201211048) identify the MIA (modifier of inner arms) complex within the Chlamydomonas reinhardtii axoneme that physically links to a known regulatory structure and provides a signaling conduit from the radial spokes to an inner arm dynein essential for waveform determination. PMID:23569213

Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid

NASA Astrophysics Data System (ADS)

Aljabali, Alaa A. A.; Barclay, J. Elaine; Steinmetz, Nicole F.; Lomonossoff, George P.; Evans, David J.

2012-08-01

Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors.Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors. Electronic supplementary information (ESI) available: Alternative conjugation strategies, agarose gel electrophoresis of CPMV and CPMV-HRP conjugates, UV-vis spectrum of HRP-ADHCPMV, agarose gel electrophoresis of GOX-ADHCPMV particles and corresponding TEM image, calibration curves for HRP-ADHCPMV and GOX-ADHCPMV, DLS data for GOX-ADHCPMV are made available. See DOI: 10.1039/c2nr31485a

Preparation and evaluation of dual-enzyme microreactor with co-immobilized trypsin and chymotrypsin.

PubMed

Meller, Kinga; Pomastowski, Paweł; Grzywiński, Damian; Szumski, Michał; Buszewski, Bogusław

2016-04-01

The preparation of capillary microfluidic reactor with co-immobilized trypsin and chymotrypsin with the use of a low-cost commercially available enzymatic reagent (containing these proteases) as well as the evaluation of its usefulness in proteomic research were presented. The monolithic copolymer synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) was used as a support. Firstly, the polymerization conditions were optimized and the monolithic bed was synthesized in the fused silica capillary modified with 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS). The polymer containing epoxy groups was then modified with 1,6-diaminohexane, followed by the attachment of glutaraldehyde and immobilization of enzymes. The efficiency of the prepared monolithic Immobilized Enzyme Microreactor (μ-IMER) with regard to trypsin activity was evaluated using the low-molecular mass compound (Nα-benzoyl-l-arginine ethyl ester, BAEE). The activities of both enzymes were investigated using a macromolecular protein (human transferrin, Tf) as a substrate. In the case of BAEE, the reaction product was separated from the substrate using the capillary liquid chromatography and the efficiency of the reaction was determined by the peak area of the substrate. The hydrolysis products of transferrin were analyzed with MALDI-TOF which allows for the verification of the prepared enzymatic system applicability in the field of proteomic research. Copyright © 2016 Elsevier B.V. All rights reserved.

The Development of Non-Enzymatic Glucose Biosensors Based on Electrochemically Prepared Polypyrrole–Chitosan–Titanium Dioxide Nanocomposite Films

PubMed Central

AL-Mokaram, Ali M. A. Abdul Amir; Yahya, Rosiyah; Abdi, Mahnaz M.; Mahmud, Habibun Nabi Muhammad Ekramul

2017-01-01

The performance of a modified electrode of nanocomposite films consisting of polypyrrole–chitosan–titanium dioxide (Ppy-CS-TiO2) has been explored for the developing a non-enzymatic glucose biosensors. The synergy effect of TiO2 nanoparticles (NPs) and conducting polymer on the current responses of the electrode resulted in greater sensitivity. The incorporation of TiO2 NPs in the nanocomposite films was confirmed by X-ray photoelectron spectroscopy (XPS) spectra. FE-SEM and HR-TEM provided more evidence for the presence of TiO2 in the Ppy-CS structure. Glucose biosensing properties were determined by amperommetry and cyclic voltammetry (CV). The interfacial properties of nanocomposite electrodes were studied by electrochemical impedance spectroscopy (EIS). The developed biosensors showed good sensitivity over a linear range of 1–14 mM with a detection limit of 614 μM for glucose. The modified electrode with Ppy-CS nanocomposite also exhibited good selectivity and long-term stability with no interference effect. The Ppy-CS-TiO2 nanocomposites films presented high electron transfer kinetics. This work shows the role of nanomaterials in electrochemical biosensors and describes the process of their homogeneous distribution in composite films by a one-step electrochemical process, where all components are taken in a single solution in the electrochemical cell. PMID:28561760

Electrochemical enzymatic fenitrothion sensor based on a tyrosinase/poly(2-hydroxybenzamide)-modified graphite electrode.

PubMed

Alves, Maria de Fátima; Corrêa, Ricardo Augusto Moreira de Souza; da Cruz, Filipe Soares; Franco, Diego Leoni; Ferreira, Lucas Franco

2018-07-15

This paper reports the electrosynthesis and characterisation of a polymeric film derived from 2-hydroxybenzamide over a graphite electrode and its application as an enzymatic biosensor for the determination and quantification of the pesticide fenitrothion. The material was analysed by scanning electron microscopy and its electrochemical properties characterised by cyclic voltammetry and electrochemical impedance spectroscopy. The enzyme tyrosinase was immobilised over the modified electrode by the drop and dry technique. Catechol was determined by direct reduction of biocatalytically formed o-quinone by employing the flow injection analysis technique. The analytical characteristics of the proposed sensor were optimised as follows: phosphate buffer 0.050 M at pH 6.5, flow rate 5.0 mL min -1 , sample injection volume 150 μL, catechol concentration 1.0 mM and maximum inhibition time by fenitrothion of 6 min. The biosensors showed a linear response to pesticide concentration from 0.018 to 3.60 μM. The limit of detection and limit of quantification were calculated as 4.70 nM and 15.9 nM (RSD < 2.7%), respectively. The intra- and inter-electrode RSDs were 3.35% (n = 15) and 8.70% (n = 7), respectively. In addition, water samples spiked with the pesticide showed an average recovery of 97.6% (±1.53). Copyright © 2018 Elsevier Inc. All rights reserved.

Strategies for an enzyme immobilization on electrodes: Structural and electrochemical characterizations

NASA Astrophysics Data System (ADS)

Ganesh, V.; Muthurasu, A.

2012-04-01

In this paper, we propose various strategies for an enzyme immobilization on electrodes (both metal and semiconductor electrodes). In general, the proposed methodology involves two critical steps viz., (1) chemical modification of substrates using functional monolayers [Langmuir - Blodgett (LB) films and/or self-assembled monolayers (SAMs)] and (2) anchoring of a target enzyme using specific chemical and physical interactions by attacking the terminal functionality of the modified films. Basically there are three ways to immobilize an enzyme on chemically modified electrodes. First method consists of an electrostatic interaction between the enzyme and terminal functional groups present within the chemically modified films. Second and third methods involve the introduction of nanomaterials followed by an enzyme immobilization using both the physical and chemical adsorption processes. As a proof of principle, in this work we demonstrate the sensing and catalytic activity of horseradish peroxidase (HRP) anchored onto SAM modified indium tin oxide (ITO) electrodes towards hydrogen peroxide (H2O2). Structural characterization of such modified electrodes is performed using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and contact angle measurements. The binding events and the enzymatic reactions are monitored using electrochemical techniques mainly cyclic voltammetry (CV).

Ultrasensitive determination of DNA oxidation products by gas chromatography-tandem mass spectrometry and the role of antioxidants in the prevention of oxidative damage.

PubMed

Dawbaa, Sam; Aybastıer, Önder; Demir, Cevdet

2017-04-15

Oxidative stress is considered as one of the significant causes of DNA damage which in turn contributes to cell death through a series of intermediate processes such as cancer formation, mutation, and aging. Natural sources such as plant and fruit products have provided us with interesting substances of antioxidant activity that could be recruited in protecting the genetic materials of the cells. This study is an effort to discover some of those antioxidants effects in their standard and natural forms by performing an ultrasensitive determination of the products of DNA oxidation using GC-MS/MS. Experiments were used to determine the direct antioxidant activity of the substances contained in the tendrils of Vitis vinifera (var. alphonse) by extracting them and achieving Folin-Ciocalteau and CHROMAC analyses to determine the total phenolic content (TPC) and the antioxidant capacity of the extract, respectively; results revealed a phenolic content of 11.39±0.30mg Gallic Acid Equivalent (GAE)/g of the plant's fresh weight (FW) by Folin-Ciocalteau and 8.17±0.49mg Trolox Equivalent (TE)/g FW by CHROMAC assays. The qualitative analysis of the plant extract by HPLC-DAD technique revealed that two flavonoid glycosides namely rutin and isoquercitrin in addition to chlorogenic acid were contained in the extract. The determination of the DNA oxidation products was performed after putting DNA, rutin and isoquercitrin standard samples with different concentration, and the extract's sample under oxidative stress. Eighteen DNA oxidation products were traced using GC-MS/MS with ultra-sensitivity and the experiments proved a significant decrease in the concentration of the DNA oxidation products when the extract was used as a protectant against the oxidative stress. It is believed by conclusion that the extract of V. vinifera's (var. alphonse) tendrils has a good antioxidant activity; hence it is recommended to be used as a part of the daily healthy food list if possible. Copyright © 2017. Published by Elsevier B.V.

[HPLC investigation of antioxidant components in Solidago herba].

PubMed

Apáti, Pál; Houghton, Peter J; Kéry, Agnes

2004-01-01

Representatives of Solidago species have been used in European phytotheraphy for centuries as a component of urological and antiphlogistical remedies. Solidago canadensis L. (Asteraceae) contains a wide range of active ingredients, such as flavonoids, saponins, hydroxycinnamates and mineral elements, which are responsible for its characteristic anti-inflammatory, spasmolytic and diuretic properties. Quality control of collected Solidaginis herba were performed according to the instructions of the X. German Pharmacopoea, while different LC-MS technologies were applied to evaluate the exact phenoloid composition. Three flavonol aglycons (quercetin, kaempferol and isorhamnetin) connected to several sugar components (glucose, rhamnose, galactose and rutinose), caffeoylquinic acid and a caffeoyl-shikimic acid glycoside were identified in the samples. Quercetin-3-O-beta-glucoside (isoquercitrin), quercetin-3-O-beta-galactoside (hyperoside), quercetin-3-O-beta-rhamnoside (quercitrin), quercetin-3-O-beta-rutinoside (rutin), kaempferol-3-O-beta-rhamnoside (afzelin), kaempferol-3-O-beta-rutinoside (nicotiflorin), caffeoil-quinic acid (chlorogenic acid) were identified in sample "A", while the presence of quercetin, quercetin-3-O-beta-glucoside (isoquercitrin), quercetin-3-/6"-O-acetyl-/-beta-glucopiranoside, quercetin-3-O-beta-rutinoside (rutin), kaempferol, kaempferol-3-O-beta-glucoside (astragalin), kaempferol-3-/6"-O-acetyl-/-beta-glucopiranoside, isorhamnetin, isorhamnetin-3-/6"-O-acetyl-/-beta-glucopiranoside, isorhamnetin-3-O-beta-rutinoside (narcissin), caffeoil-quinic acid (chlorogenic acid), caffeoil-shikimic acid-glucoside (dattelic acid-glucoside) were confirmed in sample "B". According to the occurrence of acetyl-glycosides and the diversity of sugar component of flavonoid glycosides Solidaginis herba samples chemotaxonomically were classified into different varieties. Incidence of acetyl-glycosidic flavonoids and absence of flavonoid galactosides and rhamnosides in the sample "B" together give support for the taxonomic recognition of varietases Solidago canadensis L. var. canadensis and var. scabra. Sample "A" was identified as Solidago canadensis L. var. canadensis, while sample "B" has proved to be belong to variety Solidago canadensis L. var. scabra. Due to the same flavonoid aglycons and the large amounts of flavonol glycosides occurring in each drug, phytochemical characteristics of investigated samples proved to be very similar.

Protonic transport through solitons in hydrogen-bonded systems

NASA Astrophysics Data System (ADS)

Kavitha, L.; Jayanthi, S.; Muniyappan, A.; Gopi, D.

2011-09-01

We offer an alternative route for investigating soliton solutions in hydrogen-bonded (HB) chains. We invoke the modified extended tangent hyperbolic function method coupled with symbolic computation to solve the governing equation of motion for proton dynamics. We investigate the dynamics of proton transfer in HB chains through bell-shaped soliton excitations, which trigger the bio-energy transport in most biological systems. This solitonic mechanism of proton transfer could play functional roles in muscular contraction, enzymatic activity and oxidative phosphorylation.

Effect of high hydrostatic pressure on the enzymatic hydrolysis of bovine serum albumin.

PubMed

De Maria, Serena; Ferrari, Giovanna; Maresca, Paola

2017-08-01

The extent of enzymatic proteolysis mainly depends on accessibility of the peptide bonds, which stabilize the protein structure. The high hydrostatic pressure (HHP) process is able to induce, at certain operating conditions, protein displacement, thus suggesting that this technology can be used to modify protein resistance to the enzymatic attack. This work aims at investigating the mechanism of enzymatic hydrolysis assisted by HHP performed under different processing conditions (pressure level, treatment time). Bovine serum albumin was selected for the experiments, solubilized in sodium phosphate buffer (25 mg mL -1 , pH 7.5) with α-chymotrypsin or trypsin (E/S ratio = 1/10) and HPP treatment (100-500 MPa, 15-25 min). HHP treatment enhanced the extent of the hydrolysis reaction of globular proteins, being more effective than conventional hydrolysis. At HHP treatment conditions maximizing the protein unfolding, the hydrolysis degree of proteins was increased as a consequence of the increased exposure of peptide bonds to the attack of proteolytic enzymes. The maximum hydrolysis degree (10% and 7% respectively for the samples hydrolyzed with α-chymotrypsin and trypsin) was observed for the samples processed at 400 MPa for 25 min. At pressure levels higher than 400 MPa the formation of aggregates was likely to occur; thus the degree of hydrolysis decreased. Protein unfolding represents the key factor controlling the efficiency of HHP-assisted hydrolysis treatments. The peptide produced under high pressure showed lower dimensions and a different structure with respect to those of the hydrolysates obtained when the hydrolysis was carried out at atmospheric pressure, thus opening new frontiers of application in food science and nutrition. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

An investigation of the decontamination of Siqveland matrix bands.

PubMed

Whitworth, C L; Davies, K; Palmer, N O A; Martin, M V

2007-02-24

This study investigated blood contamination of artificially and clinically contaminated Siqveland matrix bands and retainers. A modified version of the recognised Kastle-Meyer test for blood was used to compare the efficacy of enzymatic agents, a washer-disinfector and an instrument washer for pre-sterilisation cleaning of Siqveland matrix bands and retainers. Assembled Siqveland matrix bands were contaminated either artificially with horse blood or clinically during dental treatment. Contaminated assembled matrix bands and retainers were subjected to immersion in an enzymatic agent, automated processing in a washer-disinfector or instrument washer, or a combination of pre-soaking and automatic cleaning. Residual blood contamination from each band and retainer was measured and compared to the volume of blood recovered from an unprocessed control group of contaminated assembled matrix bands or retainers. Residual blood was recovered from every clinically contaminated assembled Siqveland matrix band and retainer. The volume of blood recovered from assembled Siqveland matrix bands ranged from 0.13-7.1 microl and from retainers, following removal of the matrix band, from 0.001-1.523 microl. The most effective method of pre-sterilisation cleaning for artificially contaminated assembled matrix bands was processing in the washer-disinfector. Conversely, the most effective method for cleaning clinically contaminated assembled matrix bands and retainers was pre-soaking in an enzymatic agent followed by a heavy-duty cycle in an instrument washer. It is not possible to clean assembled Siqveland matrix bands using any method currently available to dental practitioners. Matrix bands should be discarded after use on one patient. Once the band is removed, all detectable blood can be removed from the retainer by pre-soaking in an enzymatic detergent followed by processing in an instrument washer.

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.

Chloroquine Analog Interaction with C2- and Iota-Toxin in Vitro and in Living Cells.

PubMed

Kronhardt, Angelika; Beitzinger, Christoph; Barth, Holger; Benz, Roland

2016-08-10

C2-toxin from Clostridium botulinum and Iota-toxin from Clostridium perfringens belong both to the binary A-B-type of toxins consisting of two separately secreted components, an enzymatic subunit A and a binding component B that facilitates the entry of the corresponding enzymatic subunit into the target cells. The enzymatic subunits are in both cases actin ADP-ribosyltransferases that modify R177 of globular actin finally leading to cell death. Following their binding to host cells' receptors and internalization, the two binding components form heptameric channels in endosomal membranes which mediate the translocation of the enzymatic components Iota a and C2I from endosomes into the cytosol of the target cells. The binding components form ion-permeable channels in artificial and biological membranes. Chloroquine and related 4-aminoquinolines were able to block channel formation in vitro and intoxication of living cells. In this study, we extended our previous work to the use of different chloroquine analogs and demonstrate that positively charged aminoquinolinium salts are able to block channels formed in lipid bilayer membranes by the binding components of C2- and Iota-toxin. Similarly, these molecules protect cultured mammalian cells from intoxication with C2- and Iota-toxin. The aminoquinolinium salts did presumably not interfere with actin ADP-ribosylation or receptor binding but blocked the pores formed by C2IIa and Iota b in living cells and in vitro. The blocking efficiency of pores formed by Iota b and C2IIa by the chloroquine analogs showed interesting differences indicating structural variations between the types of protein-conducting nanochannels formed by Iota b and C2IIa.

Chloroquine Analog Interaction with C2- and Iota-Toxin in Vitro and in Living Cells

PubMed Central

Kronhardt, Angelika; Beitzinger, Christoph; Barth, Holger; Benz, Roland

2016-01-01

C2-toxin from Clostridium botulinum and Iota-toxin from Clostridium perfringens belong both to the binary A-B-type of toxins consisting of two separately secreted components, an enzymatic subunit A and a binding component B that facilitates the entry of the corresponding enzymatic subunit into the target cells. The enzymatic subunits are in both cases actin ADP-ribosyltransferases that modify R177 of globular actin finally leading to cell death. Following their binding to host cells’ receptors and internalization, the two binding components form heptameric channels in endosomal membranes which mediate the translocation of the enzymatic components Iota a and C2I from endosomes into the cytosol of the target cells. The binding components form ion-permeable channels in artificial and biological membranes. Chloroquine and related 4-aminoquinolines were able to block channel formation in vitro and intoxication of living cells. In this study, we extended our previous work to the use of different chloroquine analogs and demonstrate that positively charged aminoquinolinium salts are able to block channels formed in lipid bilayer membranes by the binding components of C2- and Iota-toxin. Similarly, these molecules protect cultured mammalian cells from intoxication with C2- and Iota-toxin. The aminoquinolinium salts did presumably not interfere with actin ADP-ribosylation or receptor binding but blocked the pores formed by C2IIa and Iota b in living cells and in vitro. The blocking efficiency of pores formed by Iota b and C2IIa by the chloroquine analogs showed interesting differences indicating structural variations between the types of protein-conducting nanochannels formed by Iota b and C2IIa. PMID:27517960

Enzyme-polyelectrolyte complexes in water-ethanol mixtures: negatively charged groups artificially introduced into alpha-chymotrypsin provide additional activation and stabilization effects.

PubMed

Kudryashova, E V; Gladilin, A K; Vakurov, A V; Heitz, F; Levashov, A V; Mozhaev, V V

1997-07-20

Formation of noncovalent complexes between alpha-chymotrypsin (CT) and a polyelectrolyte, polybrene (PB), has been shown to produce two major effects on enzymatic reactions in binary mixtures of polar organic cosolvents with water. (i) At moderate concentrations of organic cosolvents (10% to 30% v/v), enzymatic activity of CT is higher than in aqueous solutions, and this activation effect is more significant for CT in complex with PB (5- to 7-fold) than for free enzyme (1.5- to 2.5-fold). (ii) The range of cosolvent concentrations that the enzyme tolerates without complete loss of catalytic activity is much broader. For enhancement of enzyme stability in the complex with the polycation, the number of negatively charged groups in the protein has been artificially increased by using chemical modification with pyromellitic and succinic anhydrides. Additional activation effect at moderate concentrations of ethanol and enhanced resistance of the enzyme toward inactivation at high concentrations of the organic solvent have been observed for the modified preparations of CT in the complex with PB as compared with an analogous complex of the native enzyme. Structural changes behind alterations in enzyme activity in water-ethanol mixtures have been studied by the method of circular dichroism (CD). Protein conformation of all CT preparations has not changed significantly up to 30% v/v of ethanol where activation effects in enzymatic catalysis were most pronounced. At higher concentrations of ethanol, structural changes in the protein have been observed for different forms of CT that were well correlated with a decrease in enzymatic activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 267-277, 1997.

Applications and Emerging Trends of Hyaluronic Acid in Tissue Engineering, as a Dermal Filler, and in Osteoarthritis Treatment

PubMed Central

Fakhari, Amir; Berkland, Cory

2013-01-01

Hyaluronic acid (HA) is a naturally occurring biodegradable polymer with a variety of applications in medicine including scaffolding for tissue engineering, dermatological fillers, and viscosupplementation for osteoarthritis treatment. HA is available in most connective tissues in body fluids such as synovial fluid and the vitreous humor of the eye. HA is responsible for several structural properties of tissues as a component of extracellular matrix (ECM) and is involved in cellular signaling. Degradation of HA is a step-wise process that can occur via enzymatic or non-enzymatic reactions. A reduction in HA mass or molecular weight via degradation or slowing of synthesis affects physical and chemical properties such as tissue volume, viscosity, and elasticity. This review addresses the distribution, turnover, and tissue-specific properties of HA. This information is used as context for considering recent products and strategies for modifying the viscoelastic properties of HA in tissue engineering, as a dermal filler, and in osteoarthritis treatment. PMID:23507088

Identification of the cellular receptor for anthrax toxin

NASA Astrophysics Data System (ADS)

Bradley, Kenneth A.; Mogridge, Jeremy; Mourez, Michael; Collier, R. John; Young, John A. T.

2001-11-01

The tripartite toxin secreted by Bacillus anthracis, the causative agent of anthrax, helps the bacterium evade the immune system and can kill the host during a systemic infection. Two components of the toxin enzymatically modify substrates within the cytosol of mammalian cells: oedema factor (OF) is an adenylate cyclase that impairs host defences through a variety of mechanisms including inhibiting phagocytosis; lethal factor (LF) is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinase and causes lysis of macrophages. Protective antigen (PA), the third component, binds to a cellular receptor and mediates delivery of the enzymatic components to the cytosol. Here we describe the cloning of the human PA receptor using a genetic complementation approach. The receptor, termed ATR (anthrax toxin receptor), is a type I membrane protein with an extracellular von Willebrand factor A domain that binds directly to PA. In addition, a soluble version of this domain can protect cells from the action of the toxin.

Analysis of Resistant Starches in Rat Cecal Contents Using Fourier Transform Infrared Photoacoustic Spectroscopy

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

Anderson, Timothy J.; Ai, Yongfeng; Jones, Roger W.

Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) qualitatively and quantitatively measured resistant starch (RS) in rat cecal contents. Fisher 344 rats were fed diets of 55% (w/w, dry basis) starch for 8 weeks. Cecal contents were collected from sacrificed rats. A corn starch control was compared against three RS diets. The RS diets were high-amylose corn starch (HA7), HA7 chemically modified with octenyl succinic anhydride, and stearic-acid-complexed HA7 starch. To calibrate the FTIR-PAS analysis, samples from each diet were analyzed using an enzymatic assay. A partial least-squares cross-validation plot generated from the enzymatic assay and FTIR-PAS spectral results for starch fitmore » the ideal curve with a R2 of 0.997. A principal component analysis plot of components 1 and 2 showed that spectra from diets clustered significantly from each other. This study clearly showed that FTIR-PAS can accurately quantify starch content and identify the form of starch in complex matrices.« less

Enzymatic Synthesis of Lignin-Based Concrete Dispersing Agents.

PubMed

Jankowska, Dagmara; Heck, Tobias; Schubert, Mark; Yerlikaya, Alpaslan; Weymuth, Christophe; Rentsch, Daniel; Schober, Irene; Richter, Michael

2018-03-15

Lignin is the most abundant aromatic biopolymer, functioning as an integral component of woody materials. In its unmodified form it shows limited water solubility and is relatively unreactive, so biotechnological lignin valorisation for high-performance applications is greatly underexploited. Lignin can be obtained from the pulp and paper industry as a by-product. To expand its application, a new synthesis route to new dispersing agents for use as concrete additives was developed. The route is based on lignin functionalisation by enzymatic transformation. Screening of lignin-modifying systems resulted in functionalised lignin polymers with improved solubility in aqueous systems. Through grafting of sulfanilic acid or p-aminobenzoic acid by fungal laccases, lignin became soluble in water at pH≤4 or pH≤7, respectively. Products were analysed and evaluated in miniaturised application tests in cement paste and mortar. Their dispersing properties match the performance criteria of commercially available lignosulfonates. The study provides examples of new perspectives for the use of lignin. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Use of a biosynthetic intermediate to explore the chemical diversity of pseudo-natural fungal polyketides.

PubMed

Asai, Teigo; Tsukada, Kento; Ise, Satomi; Shirata, Naoki; Hashimoto, Makoto; Fujii, Isao; Gomi, Katsuya; Nakagawara, Kosuke; Kodama, Eiichi N; Oshima, Yoshiteru

2015-09-01

The structural complexity and diversity of natural products make them attractive sources for potential drug discovery, with their characteristics being derived from the multi-step combination of enzymatic and non-enzymatic conversions of intermediates in each biosynthetic pathway. Intermediates that exhibit multipotent behaviour have great potential for use as starting points in diversity-oriented synthesis. Inspired by the biosynthetic pathways that form complex metabolites from simple intermediates, we developed a semi-synthetic process that combines heterologous biosynthesis and artificial diversification. The heterologous biosynthesis of fungal polyketide intermediates led to the isolation of novel oligomers and provided evidence for ortho-quinonemethide equivalency in their isochromene form. The intrinsic reactivity of the isochromene polyketide enabled us to access various new chemical entities by modifying and remodelling the polyketide core and through coupling with indole molecules. We thus succeeded in generating exceptionally diverse pseudo-natural polyketides through this process and demonstrated an advanced method of using biosynthetic intermediates.

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray.

PubMed

Mao, Hailei; Wang, Huimin; Zhang, Donglei; Mao, Hongju; Zhao, Jianlong; Shi, Jian; Cui, Zhichu

2006-01-01

To establish a modified microarray method for detecting HBV gene mutations in the clinic. Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P < 0.01). Compared with a traditional fluorescence method, the colorimetry method exhibited the same level of sensitivity and reproducibility. An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.

Effect of enzymatic deamidation of soy protein by protein-glutaminase on the flavor-binding properties of the protein under aqueous conditions.

PubMed

Suppavorasatit, Inthawoot; Cadwallader, Keith R

2012-08-15

The effect of the enzymatic deamidation by protein-glutaminase (PG) on flavor-binding properties of soy protein isolate (SPI) under aqueous conditions was evaluated by a modified equilibrium dialysis (ultrafiltration) technique. Binding parameters, such as number of binding sites (n) and binding constants (K), were derived from Klotz plots. The partial deamidation of SPI by PG (43.7% degree of deamidation) decreased overall flavor-binding affinity (nK) at 25 °C for both vanillin and maltol by approximately 9- and 4-fold, respectively. The thermodynamic parameters of binding indicated that the flavor-protein interactions were spontaneous (negative ΔG°) and that the driving force of the interactions shifted from entropy to enthalpy driven as a result of deamidation. Deamidation of soy protein caused a change in the mechanism of binding from hydrophobic interactions or covalent bonding (Schiff base formation) to weaker van der Waals forces or hydrogen bonding.

Use of a biosynthetic intermediate to explore the chemical diversity of pseudo-natural fungal polyketides

NASA Astrophysics Data System (ADS)

Asai, Teigo; Tsukada, Kento; Ise, Satomi; Shirata, Naoki; Hashimoto, Makoto; Fujii, Isao; Gomi, Katsuya; Nakagawara, Kosuke; Kodama, Eiichi N.; Oshima, Yoshiteru

2015-09-01

The structural complexity and diversity of natural products make them attractive sources for potential drug discovery, with their characteristics being derived from the multi-step combination of enzymatic and non-enzymatic conversions of intermediates in each biosynthetic pathway. Intermediates that exhibit multipotent behaviour have great potential for use as starting points in diversity-oriented synthesis. Inspired by the biosynthetic pathways that form complex metabolites from simple intermediates, we developed a semi-synthetic process that combines heterologous biosynthesis and artificial diversification. The heterologous biosynthesis of fungal polyketide intermediates led to the isolation of novel oligomers and provided evidence for ortho-quinonemethide equivalency in their isochromene form. The intrinsic reactivity of the isochromene polyketide enabled us to access various new chemical entities by modifying and remodelling the polyketide core and through coupling with indole molecules. We thus succeeded in generating exceptionally diverse pseudo-natural polyketides through this process and demonstrated an advanced method of using biosynthetic intermediates.

Controlling enzymatic activity by immobilization on graphene oxide

NASA Astrophysics Data System (ADS)

Bolibok, Paulina; Wiśniewski, Marek; Roszek, Katarzyna; Terzyk, Artur P.

2017-04-01

In this study, graphene oxide (GO) has been applied as a matrix for enzyme immobilization. The protein adsorption capacity of GO is much higher than of other large surface area carbonaceous materials. Its structure and physicochemical properties are reported beneficial also for enzymatic activity modifications. The experimental proof was done here that GO-based biocatalytic systems with immobilized catalase are modifiable in terms of catalyzed reaction kinetic constants. It was found that activity and stability of catalase, considered here as model enzyme, closely depend on enzyme/GO ratio. The changes in kinetic parameters can be related to secondary structure alterations. The correlation between enzyme/GO ratio and kinetic and structure parameters is reported for the first time and enables the conscious control of biocatalytic processes and their extended applications. The biological activity of obtained biocatalytic systems was confirmed in vitro by the use of functional test. The addition of immobilized catalase improved the cells' viability after they were exposed to hydrogen peroxide and tert-butyl-hydroperoxide used as source of reactive oxygen species.

Identification of character impact odorants of different soybean lecithins.

PubMed

Stephan, A; Steinhart, H

1999-07-01

The potent odorants of standardized, enzymatically hydrolyzed, and deoiled soybean lecithins were characterized systematically by combined gas chromatography/mass spectrometry and olfactometry. Sixty-one odorants were identified; 53 of these odor-active compounds have not previously been reported as odorants of soybean lecithin flavor. By aroma extract dilution analysis and modified combined hedonic and response measurement the following odorants showed the highest flavor dilution factors and CHARM values: (E,E)-2, 4-decadienal (deep-fried), (E)-beta-damascenone (apple-like), 2, 3-diethyl-5-methylpyrazine (roasty, earthy), (E)-2-nonenal (cardboard-like), trans-4,5-epoxy-(E)-2-decenal (metallic), 1-nonen-3-one (mushroom-like), 2-ethyl-3,5-dimethylpyrazine (roasty, earthy), and 1-octen-3-one (mushroom-like). Enzymatic hydrolysis intensified especially the roasty sensation of 2, 3-diethyl-5-methylpyrazine, whereas deoiling effected a general significant decrease in olfactory perception on the nitrogen-containing compounds. In addition, sensory profiles of nasal and retronasal lecithin odor were performed.

Glycated cholecystokinin-8 has an enhanced satiating activity and is protected against enzymatic degradation.

PubMed

O'Harte, F P; Mooney, M H; Kelly, C M; Flatt, P R

1998-10-01

Monoglycated cholecystokinin octapeptide (CCK-8) (glucitol-Asp1 adduct) modified at the NH2-terminus was prepared under hyperglycemic conditions, purified by high-performance liquid chromatography, and characterized by mass spectrometry (Mr 1228.4 Da) and peptide sequencing. CCK-8 (100 nmol/kg, i.p.) significantly (P < 0.001) reduced voluntary food intake of fasted mice for up to 30 min after its administration, compared with saline-administered controls. Glycated CCK-8 reduced food intake at 30-120 min (P < 0.01 to P < 0.001) and significantly reduced feeding compared with CCK-8 from 60 to 120 min (P < 0.01). In vitro plasma degradation studies indicated that glycated CCK-8 was resistant to the normal rapid enzymatic conversion to CCK fragments. This study demonstrated that CCK-8 is a potent short-term inhibitor of food intake, and that structural modification of this peptide by amino-terminal glycation leads to enhanced satiating activity, partially due to increased resistance to serum aminopeptidase degradation.

Development of a nucleotide sugar purification method using a mixed mode column & mass spectrometry detection.

PubMed

Eastwood, Heather; Xia, Fang; Lo, Mei-Chu; Zhou, Jing; Jordan, John B; McCarter, John; Barnhart, Wesley W; Gahm, Kyung-Hyun

2015-11-10

Analysis of nucleotide sugars, nucleoside di- and triphosphates and sugar-phosphates is an essential step in the process of understanding enzymatic pathways. A facile and rapid separation method was developed to analyze these compounds present in an enzymatic reaction mixture utilized to produce nucleotide sugars. The Primesep SB column explored in this study utilizes hydrophobic interactions as well as electrostatic interactions with the phosphoric portion of the nucleotide sugars. Ammonium formate buffer was selected due to its compatibility with mass spectrometry. Negative ion mode mass spectrometry was adopted for detection of the sugar phosphate (fucose-1-phophate), as the compound is not amenable to UV detection. Various mobile phase conditions such as pH, buffer concentration and organic modifier were explored. The semi-preparative separation method was developed to prepare 30mg of the nucleotide sugar. (19)F NMR was utilized to determine purity of the purified fluorinated nucleotide sugar. The collected nucleotide sugar was found to be 99% pure. Published by Elsevier B.V.

A dual amplification strategy for DNA detection combining bio-barcode assay and metal-enhanced fluorescence modality.

PubMed

Zhou, Zhenpeng; Li, Tian; Huang, Hongduan; Chen, Yang; Liu, Feng; Huang, Chengzhi; Li, Na

2014-11-11

Silver-enhanced fluorescence was coupled with a bio-barcode assay to facilitate a dual amplification assay to demonstrate a non-enzymatic approach for simple and sensitive detection of DNA. In the assay design, magnetic nanoparticles seeded with silver nanoparticles were modified with the capture DNA, and silver nanoparticles were modified with the binding of ssDNA and the fluorescently labeled barcode dsDNA. Upon introduction of the target DNA, a sandwich structure was formed because of the hybridization reaction. By simple magnetic separation, silver-enhanced fluorescence of barcode DNAs could be readily measured without the need of a further step to liberate barcode DNAs from silver nanoparticles, endowing the method with simplicity and high sensitivity with a detection limit of 1 pM.

Peptide nanotube-modified electrodes for enzyme-biosensor applications.

PubMed

Yemini, Miri; Reches, Meital; Gazit, Ehud; Rishpon, Judith

2005-08-15

The fabrication and notably improved performance of composite electrodes based on modified self-assembled diphenylalanine peptide nanotubes is described. Peptide nanotubes were attached to gold electrodes, and we studied the resulting electrochemical behavior using cyclic voltammetry and chronoamperometry. The peptide nanotube-based electrodes demonstrated a direct and unmediated response to hydrogen peroxide and NADH at a potential of +0.4 V (vs SCE). This biosensor enables a sensitive determination of glucose by monitoring the hydrogen peroxide produced by an enzymatic reaction between the glucose oxidase attached to the peptide nanotubes and glucose. In addition, the marked electrocatalytic activity toward NADH enabled a sensitive detection of ethanol using ethanol dehydrogenase and NAD+. The peptide nanotube-based amperometric biosensor provides a potential new tool for sensitive biosensors and biomolecular diagnostics.

Compounds inhibiting the bioconversion of hydrothermally pretreated lignocellulose.

PubMed

Ko, Ja Kyong; Um, Youngsoon; Park, Yong-Cheol; Seo, Jin-Ho; Kim, Kyoung Heon

2015-05-01

Hydrothermal pretreatment using liquid hot water, steam explosion, or dilute acids enhances the enzymatic digestibility of cellulose by altering the chemical and/or physical structures of lignocellulosic biomass. However, compounds that inhibit both enzymes and microbial activity, including lignin-derived phenolics, soluble sugars, furan aldehydes, and weak acids, are also generated during pretreatment. Insoluble lignin, which predominantly remains within the pretreated solids, also acts as a significant inhibitor of cellulases during hydrolysis of cellulose. Exposed lignin, which is modified to be more recalcitrant to enzymes during pretreatment, adsorbs cellulase nonproductively and reduces the availability of active cellulase for hydrolysis of cellulose. Similarly, lignin-derived phenolics inhibit or deactivate cellulase and β-glucosidase via irreversible binding or precipitation. Meanwhile, the performance of fermenting microorganisms is negatively affected by phenolics, sugar degradation products, and weak acids. This review describes the current knowledge regarding the contributions of inhibitors present in whole pretreatment slurries to the enzymatic hydrolysis of cellulose and fermentation. Furthermore, we discuss various biological strategies to mitigate the effects of these inhibitors on enzymatic and microbial activity to improve the lignocellulose-to-biofuel process robustness. While the inhibitory effect of lignin on enzymes can be relieved through the use of lignin blockers and by genetically engineering the structure of lignin or of cellulase itself, soluble inhibitors, including phenolics, furan aldehydes, and weak acids, can be detoxified by microorganisms or laccase.

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

Glucose biosensor based on immobilization of glucose oxidase on a carbon paste electrode modified with microsphere-attached l-glycine.

PubMed

Donmez, Soner; Arslan, Fatma; Sarı, Nurşen; Hasanoğlu Özkan, Elvan; Arslan, Halit

2017-09-01

In the present study, a novel biosensor that is sensitive to glucose was prepared using the microspheres modified with (4-formyl-3-methoxyphenoxymethyl)polystyrene (FMPS) with l-glycine. Polymeric microspheres having Schiff bases were prepared from FMPS using the glycine condensation method. Glucose oxidase enzyme was immobilized onto modified carbon paste electrode by cross-linking with glutaraldehyde. Oxidation of enzymatically produced H 2 O 2 (+0.5 V vs. Ag/AgCl) was used for determination of glucose. Optimal temperature and pH were found as 50 °C and 8.0, respectively. The glucose biosensor showed a linear working range from 5.0 × 10 -4 to 1.0 × 10 -2 M, R 2 = 0.999. Storage and operational stability of the biosensor were also investigated. The biosensor gave perfect reproducible results after 20 measurements with 3.3% relative standard deviation. It also had good storage stability. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Cartilaginous extracellular matrix-modified chitosan hydrogels for cartilage tissue engineering.

PubMed

Choi, Bogyu; Kim, Soyon; Lin, Brian; Wu, Benjamin M; Lee, Min

2014-11-26

Cartilaginous extracellular matrix (ECM) components such as type-II collagen (Col II) and chondroitin sulfate (CS) play a crucial role in chondrogenesis. However, direct clinical use of natural Col II or CS as scaffolds for cartilage tissue engineering is limited by their instability and rapid enzymatic degradation. Here, we investigate the incorporation of Col II and CS into injectable chitosan hydrogels designed to gel upon initiation by exposure to visible blue light (VBL) in the presence of riboflavin. Unmodified chitosan hydrogel supported proliferation and deposition of cartilaginous ECM by encapsulated chondrocytes and mesenchymal stem cells. The incorporation of native Col II or CS into chitosan hydrogels further increased chondrogenesis. The incorporation of Col II, in particular, was found to be responsible for the enhanced cellular condensation and chondrogenesis observed in modified hydrogels. This was mediated by integrin α10 binding to Col II, increasing cell-matrix adhesion. These findings demonstrate the potential of cartilage ECM-modified chitosan hydrogels as biomaterials to promote cartilage regeneration.

A non-enzymatic sensor for hydrogen peroxide based on polyaniline, multiwalled carbon nanotubes and gold nanoparticles modified Au electrode.

PubMed

Narang, Jagriti; Chauhan, Nidhi; Pundir, C S

2011-11-07

We describe the construction of a polyaniline (PANI), multiwalled carbon nanotubes (MWCNTs) and gold nanoparticles (AuNPs) modified Au electrode for determination of hydrogen peroxide without using peroxidase (HRP). The AuNPs/MWCNT/PANI composite film deposited on Au electrode was characterized by Scanning Electron Microscopy (SEM) and electrochemical methods. Cyclic voltammetric (CV) studies of the electrode at different stages of construction demonstrated that the modified electrode had enhanced electrochemical oxidation of H(2)O(2), which offers a number of attractive features to develop amperometric sensors based on split of H(2)O(2). The amperometric response to H(2)O(2) showed a linear relationship in the range from 3.0 μM to 600.0 μM with a detection limit of 0.3 μM (S/N = 3) and with high sensitivity of 3.3 mA μM(-1). The sensor gave accurate and satisfactory results, when employed for determination of H(2)O(2) in milk and urine.

Development of modified starch technology (maltodextrin) from commercial tapioca on semi production scale using oil heater dextrinator

NASA Astrophysics Data System (ADS)

Triyono, Agus; Cecep Erwan Andriansyah, Raden; Luthfiyanti, Rohmah; Rahman, Taufik

2017-12-01

One way to improve functional starch is by modification of starch into dextrin or maltodextrin. Maltodextrin is used in the food industry as a food substitution. Development of enzymatically modified starch technology has been performed with the use of α-amylase at optimum pH of 5.5, temperature 75-85 °C, with enzyme activity of 135 KNU/g. The maltodextrin produced from commercial tapioca has the quality requirements for food according to SNI 1992. The yield of maltodextrin obtained is about 80%. The use of the optimum amount of the α-amylase enzyme is 0.07 % v/w and the substrate amount of tapioca starch is 35%. Analysis of the feasibility of modified starch with the assumption of production scale of 300 kg per day, the economic value of 10 years business, the price of starch is IDR 8,350/kg, from tapioca starch (tapioca) IDR 4,000 - IDR 4,500/kg.

Determination of dietary starch in animal feeds and pet food by an enzymatic-colorimetric method: collaborative study.

PubMed

Hall, Mary Beth

2015-01-01

Starch, glycogen, maltooligosaccharides, and other α-1,4- and α-1,6-linked glucose carbohydrates, exclusive of resistant starch, are collectively termed "dietary starch". This nutritionally important fraction is increasingly measured for use in diet formulation for animals as it can have positive or negative effects on animal performance and health by affecting energy supply, glycemic index, and formation of fermentation products by gut microbes. AOAC Method 920.40 that was used for measuring dietary starch in animal feeds was invalidated due to discontinued production of a required enzyme. As a replacement, an enzymatic-colorimetric starch assay developed in 1997 that had advantages in ease of sample handling and accuracy compared to other methods was considered. The assay was further modified to improve utilization of laboratory resources and reduce time required for the assay. The assay is quasi-empirical: glucose is the analyte detected, but its release is determined by run conditions and specification of enzymes. The modified assay was tested in an AOAC collaborative study to evaluate its accuracy and reliability for determination of dietary starch in animal feedstuffs and pet foods. In the assay, samples are incubated in screw cap tubes with thermostable α-amylase in pH 5.0 sodium acetate buffer for 1 h at 100°C with periodic mixing to gelatinize and partially hydrolyze α-glucan. Amyloglucosidase is added, and the reaction mixture is incubated at 50°C for 2 h and mixed once. After subsequent addition of water, mixing, clarification, and dilution as needed, free + enzymatically released glucose are measured. Values from a separate determination of free glucose are subtracted to give values for enzymatically released glucose. Dietary starch equals enzymatically released glucose multiplied by 162/180 (or 0.9) divided by the weight of the as received sample. Fifteen laboratories that represented feed company, regulatory, research, and commercial feed testing laboratories analyzed 10 homogenous test materials representing animal feedstuffs and pet foods in duplicate using the dietary starch assay. The test samples ranged from 1 to 70% in dietary starch content and included moist canned dog food, alfalfa pellets, distillers grains, ground corn grain, poultry feed, low starch horse feed, dry dog kibbles, complete dairy cattle feed, soybean meal, and corn silage. The average within-laboratory repeatability SD (sr) for percentage dietary starch in the test samples was 0.49 with a range of 0.03 to 1.56, and among-laboratory repeatability SDs (sR) averaged 0.96 with a range of 0.09 to 2.69. The HorRat averaged 2.0 for all test samples and 1.9 for test samples containing greater than 2% dietary starch. The HorRat results are comparable to those found for AOAC Method 996.11, which measures starch in cereal products. It is recommended that the dietary starch method be accepted for Official First Action status.

Vertically grown zinc oxide nanorods functionalized with ferric oxide for in vivo and non-enzymatic glucose detection

NASA Astrophysics Data System (ADS)

Marie, Mohammed; Manoharan, Anishkumar; Kuchuk, Andrian; Ang, Simon; Manasreh, M. O.

2018-03-01

An enzyme-free glucose sensor based on vertically grown zinc oxide nanorods (NRs) functionalized with ferric oxide (Fe2O3) is investigated. The well-aligned and high density ZnO NRs were synthesized on an FTO/glass substrate by a sol-gel and hydrothermal growth method. A dip-coating technique was utilized to modify the surface of the as-grown ZnO NRs with Fe2O3. The immobilized surface was coated with a layer of nafion membrane. The fabricated glucose sensor was characterized amperometrically at room temperature using three electrodes stationed in the phosphate buffer solution, where ZnO NRs/Fe2O3/nafion membrane was the sensing or working electrode, and platinum plate and silver/silver chloride were used as the counter and reference electrodes, respectively. The proposed non-enzymatic and modified glucose sensor exhibited a high sensitivity in the order of 0.052 μA cm-2 (mg/dL)-1, a lower detection limit of around 0.95 mmol L-1, a sharp and fast response time of ˜1 s, and a linear response to changes in glucose concentrations from 100-400 mg dL-1. The linear amperometric response of the sensor covers the physiological and clinical interest of glucose levels for diabetic patients. The device continues to function accurately after multiple measurements with a good reproducibility. The proposed glucose sensor is expected to be used clinically for in vivo monitoring of glucose.

Modified properties of a glycated and cross-linked soy protein isolate by transglutaminase and an oligochitosan of 5 kDa.

PubMed

Fu, Miao; Zhao, Xin-Huai

2017-01-01

Soy protein is an important protein ingredient for the food industry; however, its properties can be improved by enzymatic and chemical modifications. This study applied a new enzymatic glycation and cross-linking to modify soy protein isolate (SPI), using an oligochitosan of 5 kDa and transglutaminase. Properties of the obtained glycated and cross-linked SPI (GC-SPI) were unknown and thus assessed. GC-SPI contained glucosamine of 13.6 g kg -1 protein, but less reactable &bond;NH 2 than SPI (0.42 vs. 0.50 mol kg -1 protein). Infrared spectra and circular dichroism results showed that GC-SPI other than SPI and cross-linked SPI had more &bond;OH in molecules, and was more disordered in secondary structure. In comparison with SPI, GC-SPI showed enhanced water-binding capacity, could form aggregates with enlarged hydrodynamic radius (180.2 vs. 82.9 nm) and negative zeta-potential (-31.2 vs. -27.7 mV) in dispersion, but exhibited lower thermal stability (e.g. greater mass loss) upon heating at a temperature above 288 °C. GC-SPI also had lower in vitro proteolytic digestibility than SPI due to the protein cross-linking. Oligochitosan of 5 kDa and transglutaminase can be used to glycate and cross-link SPI. This approach is applicable to generate potential protein ingredient with good hydration and dispersive stabilisation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Synthesis and physico-chemical characterization of modified starches from banana (Musa AAB) and its biological activities in diabetic rats.

PubMed

Reddy, Chagam Koteswara; Suriya, M; Vidya, P V; Haripriya, Sundaramoorthy

2017-01-01

This study describes a simple method of preparation and physico-chemical properties of modified starches (type-3 resistant starches) from banana (Musa AAB), and the modified starches investigated as functional food with a beneficial effect on type-2 diabetes. RS3 was prepared using a method combined with debranching modification and physical modification; native and modifies starches were characterized by scanning electron microscope (SEM), powder X-ray diffraction (XRD), differential scanning calorimetry (DSC) and rapid visco analyzer (RVA). Use of the enzymatic and physical modification methodology, improved the yield of RS (26.62%) from Musa AAB. A reduced viscosity and swelling power; increased transition temperatures, water absorption capacity and solubility index with B-type crystalline pattern and loss of granular appearance were observed during the debranching modification and physical modification. The modified starches exhibited beneficial health effects in diabetic and HFD rats who consumed it. These results recommend that dietary feeding of RS3 was effective in the regulation of glucose and lipid profile in serum and suppressing the oxidative stress in rats under diabetic and HFD condition. This current study provides new bioactive starches, with potential applications in the food and non-food industries. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of alkali and oxidative treatments on the physicochemical, pasting, thermal and morphological properties of corn starch.

PubMed

Spier, Franciela; Zavareze, Elessandra da Rosa; Marques e Silva, Ricardo; Elias, Moacir Cardoso; Dias, Alvaro Renato Guerra

2013-07-01

Few studies on starch modifications using different chemical agents are available in the literature, and no reports were found on the combined effect of oxidation and alkaline treatment of corn starch. Thus this work evaluated the physicochemical, pasting, morphological, cystallinity and thermal properties of chemically modified corn starch, after either the isolated or combined action of alkaline (sodium hydroxide) and oxidative (sodium hypochlorite) treatments. The highest values for the sum of carbonyl and carboxyl and enzymatic hydrolysis occurred in starches submitted to oxidative treatment at high active chlorine concentrations. The alkali treatment in isolation modified the pasting properties, reduced the paste temperature and increased the peak viscosity, breakdown, final viscosity and setback of starches. Starch modified by the action of sodium hypochlorite and hydroxide in combination presented more severe damage on granule surfaces. The results show that corn starch modified by the combined action of oxidative and alkaline treatments should be studied more, especially at the concentration limit of sodium hydroxide where gelatinization occurs. Under these conditions the effect of oxidation can be more intense and thus allow the production of starches with different properties and an increase in their industrial applications. © 2013 Society of Chemical Industry.

Enzymatically Modified Starch Ameliorates Postprandial Serum Triglycerides and Lipid Metabolome in Growing Pigs.

PubMed

Metzler-Zebeli, Barbara U; Eberspächer, Eva; Grüll, Dietmar; Kowalczyk, Lidia; Molnar, Timea; Zebeli, Qendrim

2015-01-01

Developing host digestion-resistant starches to promote human health is of great research interest. Chemically modified starches (CMS) are widely used in processed foods and although the modification of the starch molecule allows specific reduction in digestibility, the metabolic effects of CMS have been less well described. This short-term study evaluated the impact of enzymatically modified starch (EMS) on fasting and postprandial profiles of blood glucose, insulin and lipids, and serum metabolome in growing pigs. Eight jugular-vein catheterized pigs (initial body weight, 37.4 kg; 4 months of age) were fed 2 diets containing 72% purified starch (EMS or waxy corn starch (control)) in a cross-over design for 7 days. On day 8, an 8-hour meal tolerance test (MTT) was performed with serial blood samplings. Besides biochemical analysis, serum was analysed for 201 metabolites through targeted mass spectrometry-based metabolomic approaches. Pigs fed the EMS diet showed increased (P<0.05) immediate serum insulin and plasma glucose response compared to pigs fed the control diet; however, area-under-the-curves for insulin and glucose were not different among diets. Results from MTT indicated reduced postprandial serum triglycerides with EMS versus control diet (P<0.05). Likewise, serum metabolome profiling identified characteristic changes in glycerophospholipid, lysophospholipids, sphingomyelins and amino acid metabolome profiles with EMS diet compared to control diet. Results showed rapid adaptations of blood metabolites to dietary starch shifts within 7 days. In conclusion, EMS ingestion showed potential to attenuate postprandial raise in serum lipids and suggested constant alteration in the synthesis or breakdown of sphingolipids and phospholipids which might be a health benefit of EMS consumption. Because serum insulin was not lowered, more research is warranted to reveal possible underlying mechanisms behind the observed changes in the profile of serum lipid metabolome in response to EMS consumption.

Enzymatically Modified Starch Ameliorates Postprandial Serum Triglycerides and Lipid Metabolome in Growing Pigs

PubMed Central

Metzler-Zebeli, Barbara U.; Eberspächer, Eva; Grüll, Dietmar; Kowalczyk, Lidia; Molnar, Timea; Zebeli, Qendrim

2015-01-01

Developing host digestion-resistant starches to promote human health is of great research interest. Chemically modified starches (CMS) are widely used in processed foods and although the modification of the starch molecule allows specific reduction in digestibility, the metabolic effects of CMS have been less well described. This short-term study evaluated the impact of enzymatically modified starch (EMS) on fasting and postprandial profiles of blood glucose, insulin and lipids, and serum metabolome in growing pigs. Eight jugular-vein catheterized pigs (initial body weight, 37.4 kg; 4 months of age) were fed 2 diets containing 72% purified starch (EMS or waxy corn starch (control)) in a cross-over design for 7 days. On day 8, an 8-hour meal tolerance test (MTT) was performed with serial blood samplings. Besides biochemical analysis, serum was analysed for 201 metabolites through targeted mass spectrometry-based metabolomic approaches. Pigs fed the EMS diet showed increased (P<0.05) immediate serum insulin and plasma glucose response compared to pigs fed the control diet; however, area-under-the-curves for insulin and glucose were not different among diets. Results from MTT indicated reduced postprandial serum triglycerides with EMS versus control diet (P<0.05). Likewise, serum metabolome profiling identified characteristic changes in glycerophospholipid, lysophospholipids, sphingomyelins and amino acid metabolome profiles with EMS diet compared to control diet. Results showed rapid adaptations of blood metabolites to dietary starch shifts within 7 days. In conclusion, EMS ingestion showed potential to attenuate postprandial raise in serum lipids and suggested constant alteration in the synthesis or breakdown of sphingolipids and phospholipids which might be a health benefit of EMS consumption. Because serum insulin was not lowered, more research is warranted to reveal possible underlying mechanisms behind the observed changes in the profile of serum lipid metabolome in response to EMS consumption. PMID:26076487

Flavonoids from Potentilla parvifolia Fisch. and Their Neuroprotective Effects in Human Neuroblastoma SH-SY5Y Cells in vitro.

PubMed

Yuan, Zhenzhen; Luan, Guangxiang; Wang, Zhenhua; Hao, Xueyan; Li, Ji; Suo, Yourui; Li, Gang; Wang, Honglun

2017-06-01

Potentilla parvifolia Fisch. (Rosaceae) is a traditional medicinal plant in P. R. China. In this study, seven flavonoids, ayanin (1), tricin (2), quercetin (3), tiliroside (4), miquelianin (5), isoquercitrin (6), and astragalin (7), were separated and purified from ethyl acetate extractive fractions from ethanol extracts of P. parvifolia using a combination of sevaral chromatographic methods. The human neuroblastoma SH-SY5Y cells were differentiated with all trans-retinoic acid and treated with okadaic acid to induce tau protein phosphorylation and synaptic atrophy, which could establish an Alzheimer's disease cell model. The neuroprotective effects of these flavonoids in cellular were evaluated in vitro by this cell model. Results from the Western blot and morphology analysis suggested that compounds 3 and 4 had the better neuroprotective effects. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

Phenolic Composition and Antioxidant Activity of Malus domestica Leaves

PubMed Central

Viškelis, Pranas; Uselis, Norbertas

2014-01-01

The aim of this study was to determine the composition and content of phenolic compounds in the ethanol extracts of apple leaves and to evaluate the antioxidant activity of these extracts. The total phenolic content was determined spectrophotometrically, as well as the total flavonoid content in the ethanol extracts of apple leaves and the antioxidant activity of these extracts, by the ABTS, DPPH, and FRAP assays. The highest amount of phenolic compounds and flavonoids as well as the highest antioxidant activity was determined in the ethanol extracts obtained from the apple leaves of the cv. Aldas. The analysis by the HPLC method revealed that phloridzin was a predominant component in the ethanol extracts of the apple leaves of all cultivars investigated. The following quercetin glycosides were identified and quantified in the ethanol extracts of apple leaves: hyperoside, isoquercitrin, avicularin, rutin, and quercitrin. Quercitrin was the major compound among quercetin glycosides. PMID:25302319

Flavonoids from Theobroma cacao down-regulate inflammatory mediators.

PubMed

Ramiro, Emma; Franch, Angels; Castellote, Cristina; Pérez-Cano, Francisco; Permanyer, Joan; Izquierdo-Pulido, Maria; Castell, Margarida

2005-11-02

In the present study, we report the effects of a cocoa extract on the secretion and RNA expression of various proinflammatory mediators by macrophages. Monocyte chemoattractant protein 1 and tumor necrosis factor alpha (TNFalpha) were significantly and dose-dependently diminished by cocoa extract, and this effect was higher than that produced by equivalent concentrations of epicatechin but was lower than that produced by isoquercitrin. Interestingly, cocoa extract added prior to cell activation resulted in a significantly greater inhibition of TNFalpha secretion. Both cocoa extract and epicatechin decreased TNFalpha, interleukin (IL) 1alpha, and IL-6 mRNA expression, suggesting that their inhibitory effect on cytokine secretion is produced, in part, at the transcriptional level. Cocoa extract also significantly decreased NO secretion in a dose-dependent manner and with a greater effect than that produced by epicatechin. In conclusion, our study shows that cocoa flavonoids not only inhibit NO release from macrophages but also down-regulate inflammatory cytokines and chemokines.

Anti-leishmanial, anti-inflammatory and antimicrobial activities of phenolic derivatives from Tibouchina paratropica.

PubMed

Tracanna, María I; Fortuna, Antonio M; Cárdenas, Angel V Contreras; Marr, Alexandra K; McMaster, W Robert; Gómez-Velasco, Anaximandro; Sánchez-Arreola, Eugenio; Hernández, Luis Ricardo; Bach, Horacio

2015-03-01

A new phenolic derivative, 2,8-dihydroxy-7H-furo[2,3-f]chromen-7-one (1), together with isoquercitrin (2), was isolated from the aerial parts of Tibouchina paratropica. Compound structures were elucidated by spectroscopic methods. Both compounds show antimicrobial activity towards a panel of bacterial and fungal pathogens, and compound 1 displayed potent anti-parasitic activity against Leishmania donovani (IC50  = 0.809 µg/mL). In addition, an 85% reduction in the secretion of the pro-inflammatory cytokine IL-6 was recorded when macrophages challenged with lipopolysaccharide were exposed to compound 1, but no effect on the anti-inflammatory IL-10 was observed. Compound 2 showed neither anti-parasitic nor anti-inflammatory properties. In addition, no cytotoxic activities were observed against the human-derived macrophage THP-1 cells. Copyright © 2014 John Wiley & Sons, Ltd.

Comparative polyphenolic content and antioxidant activities of Genista tinctoria L. and Genistella sagittalis (L.) Gams (Fabaceae).

PubMed

Hanganu, Daniela; Olah, Neli Kinga; Benedec, Daniela; Mocan, Andrei; Crisan, Gianina; Vlase, Laurian; Popica, Iulia; Oniga, Ilioara

2016-01-01

The aim of this study was focused on the polyphenolic composition and antioxidant capacity of Genista tinctoria L. and Genistella sagittalis (L.) Gams. A qualitative and quantitative characterization of the main phenolic compounds from the extracts were carried out using a HPLC-MS method. The total polyphenolic and flavonoid content was spectrophotometrically determined. The antioxidant activity towards various radicals generated in different systems was evaluated usingDPPH bleaching method, Trolox equivalent antioxidant capacity assay (TEAC) and Oxygen radical absorbance capacity (ORAC), and all indicated that G. tinctoria extract was more antioxidant than G. sagittalis extract.That was in good agreement with the total polyphenolic and flavonoidic content.Chlorogenic acid, p-coumaric acid, isoquercitrin and apigenin were identified in bothspecies. Caffeic acid, ferulic acid, hyperoside, rutin, quercitrin and luteolin were found only in G. tinctoria, while quercetin was determined in G. sagittalis.

Polyphenolic profile and biological activity of Chinese hawthorn (Crataegus pinnatifida BUNGE) fruits.

PubMed

Jurikova, Tunde; Sochor, Jiri; Rop, Otakar; Mlcek, Jiri; Balla, Stefan; Szekeres, Ladislav; Adam, Vojtech; Kizek, Rene

2012-12-06

Chinese hawthorn (Crataegus pinnatifida Bge.) fruits are rich in polyphenols (e.g., epicatechin, procyanidin B2, procyanidin B5, procyanidin C1, hyperoside, isoquercitrin and chlorogenic acid)--active compounds that exert beneficial effects. This review summarizes all information available on polyphenolic content and methods for their quantification in Chinese hawthorn berries and the relationships between individual polyphenolic compounds as well. The influence of species or cultivars, the locality of cultivation, the stage of maturity, and extract preparation conditions on the polyphenolic content were discussed as well. Currently, only fruits of C. pinnatifida and C. pinnatifida var. major are included in the Chinese Pharmacopoeia. Recent trials have demonstrated the efficacy of Chinese hawthorn fruit in lowering blood cholesterol and the risk of cardiovascular diseases. The fruit has also demonstrated anti-inflammatory and anti-tumour activities. This review deals mainly with the biological activity of the fruit related to its antioxidant properties.

Phenolic composition and antioxidant activity of Malus domestica leaves.

PubMed

Liaudanskas, Mindaugas; Viškelis, Pranas; Raudonis, Raimondas; Kviklys, Darius; Uselis, Norbertas; Janulis, Valdimaras

2014-01-01

The aim of this study was to determine the composition and content of phenolic compounds in the ethanol extracts of apple leaves and to evaluate the antioxidant activity of these extracts. The total phenolic content was determined spectrophotometrically, as well as the total flavonoid content in the ethanol extracts of apple leaves and the antioxidant activity of these extracts, by the ABTS, DPPH, and FRAP assays. The highest amount of phenolic compounds and flavonoids as well as the highest antioxidant activity was determined in the ethanol extracts obtained from the apple leaves of the cv. Aldas. The analysis by the HPLC method revealed that phloridzin was a predominant component in the ethanol extracts of the apple leaves of all cultivars investigated. The following quercetin glycosides were identified and quantified in the ethanol extracts of apple leaves: hyperoside, isoquercitrin, avicularin, rutin, and quercitrin. Quercitrin was the major compound among quercetin glycosides.

Membraneless enzymatic ethanol/O2 fuel cell: Transitioning from an air-breathing Pt-based cathode to a bilirubin oxidase-based biocathode

NASA Astrophysics Data System (ADS)

Aquino Neto, Sidney; Milton, Ross D.; Hickey, David P.; De Andrade, Adalgisa R.; Minteer, Shelley D.

2016-08-01

The bioelectrooxidation of ethanol was investigated in a fully enzymatic membraneless ethanol/O2 biofuel cell assembly using hybrid bioanodes containing multi-walled carbon nanotube (MWCNT)-decorated gold metallic nanoparticles with either a pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) enzyme or a nicotinamide adenine dinucleotide (NAD+)-dependent ADH enzyme. The biofuel cell anode was prepared with the PQQ-dependent enzyme and designed using either a direct electron transfer (DET) architecture or via a mediated electron transfer (MET) configuration through a redox polymer, 1,1‧-dimethylferrocene-modified linear polyethyleneimine (FcMe2-C3-LPEI). In the case of the bioanode containing the NAD+-dependent enzyme, only the mediated electron transfer mechanism was employed using an electropolymerized methylene green film to regenerate the NAD+ cofactor. Regardless of the enzyme being employed at the anode, a bilirubin oxidase-based biocathode prepared within a DET architecture afforded efficient electrocatalytic oxygen reduction in an ethanol/O2 biofuel cell. The power curves showed that DET-based bioanodes via the PQQ-dependent ADH still lack high current densities, whereas the MET architecture furnished maximum power density values as high as 226 ± 21 μW cm-2. Considering the complete membraneless enzymatic biofuel cell with the NAD+-dependent ADH-based bioanode, power densities as high as 111 ± 14 μW cm-2 were obtained. This shows the advantage of PQQ-dependent ADH for membraneless ethanol/O2 biofuel cell applications.

Identification of odor volatile compounds and deodorization of Paphia undulata enzymatic hydrolysate

NASA Astrophysics Data System (ADS)

Chen, Deke; Chen, Xin; Chen, Hua; Cai, Bingna; Wan, Peng; Zhu, Xiaolian; Sun, Han; Sun, Huili; Pan, Jianyu

2016-12-01

Unfavorable fishy odour is an inevitable problem in aquatic products. In the present study, headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) analysis of volatiles from untreated samples and three deodorized samples (under the optimal conditions) of Paphia undulata enzymatic hydrolysate revealed that the compounds contributing to the distinctive odor were 1-octen-3-ol, n-hexanal, n-heptanal, 2,4-heptadienal, and 2,4-decadienal, whereas n-pentanal, n-octanal, n-octanol, benzaldehyde, 2-ethylfuran and 2-pentylfuran were the main contributors to the aromatic flavor. The deodorizing effects of activated carbon (AC) adsorption, yeast extract (YE) masking and tea polyphenol (TP) treatment on a P. undulata enzymatic hydrolysate were investigated using orthogonal experiments with sensory evaluation as the index. The following optimized deodorization conditions were obtained: AC adsorption (35 mg mL-1, 80°C, 40 min), YE masking (7 mg mL-1, 45°C, 30 min) and TP treatment (0.4 mg mL-1, 40°C, 50 min). AC adsorption effectively removed off-flavor volatile aldehydes and ketones. YE masking modified the odor profile by increasing the relative contents of aromatic compounds and decreasing the relative contents of aldehydes and ketones. The TP treatment was not effective in reducing the odor score, but it significantly reduced the relative content of aldehydes while increasing that of alkanes. It is also notable that TP effectively suppressed trimethylamine (TMA) formation in a P. undulate hydrolysate solution for a period of 72 h.

Simple methods for the 3' biotinylation of RNA.

PubMed

Moritz, Bodo; Wahle, Elmar

2014-03-01

Biotinylation of RNA allows its tight coupling to streptavidin and is thus useful for many types of experiments, e.g., pull-downs. Here we describe three simple techniques for biotinylating the 3' ends of RNA molecules generated by chemical or enzymatic synthesis. First, extension with either the Schizosaccharomyces pombe noncanonical poly(A) polymerase Cid1 or Escherichia coli poly(A) polymerase and N6-biotin-ATP is simple, efficient, and generally applicable independently of the 3'-end sequences of the RNA molecule to be labeled. However, depending on the enzyme and the reaction conditions, several or many biotinylated nucleotides are incorporated. Second, conditions are reported under which splint-dependent ligation by T4 DNA ligase can be used to join biotinylated and, presumably, other chemically modified DNA oligonucleotides to RNA 3' ends even if these are heterogeneous as is typical for products of enzymatic synthesis. Third, we describe the use of 29 DNA polymerase for a template-directed fill-in reaction that uses biotin-dUTP and, thanks to the enzyme's proofreading activity, can cope with more extended 3' heterogeneities.

Influence of kaolinite on chiral hydrolysis of methyl dichlorprop enantiomers*

PubMed Central

Fang, Zhao-hua; Wen, Yue-zhong; Liu, Wei-ping

2005-01-01

The effect of kaolinite on the enzymatic chiral hydrolysis of methyl dichlorprop enantiomers ((R,S)-methyl-2-(2,4-dichlorophenoxy) propanoic acid, 2,4-DPM) was investigated using chiral gas chromatography. Compared with the control without kaolinite, the enantiomeric ratio (ER) increased from 1.35 to 8.33 and the residual ratio of 2,4-DPM decreased from 60.89% to 41.55% in the presence of kaolinite. Kaolinite likely had emotion influence on lipase activity and its enantioselectivity. Moreover, the amount of kaolinite added was also found to be a sensitive factor affecting the enantioselective hydrolysis of 2,4-DPM. Fourier transform infrared (FTIR) spectroscopy studies of the interaction of lipase with kaolinite provided insight into the molecular structure of the complex and offered explanation of the effects of kaolinite on enzymatic hydrolysis of 2,4-DPM. Spectra showed that the effect of kaolinite on the hydrolysis of 2,4-DPM was affected by adsorption of lipase on kaolinite and changes of adsorbed lipase conformation, which led to the modified enantioselectivity. PMID:16187418

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.

One-pot enzymatic glycan remodeling of a therapeutic monoclonal antibody by endoglycosidase S (Endo-S) from Streptococcus pyogenes.

PubMed

Tong, Xin; Li, Tiezheng; Orwenyo, Jared; Toonstra, Christian; Wang, Lai-Xi

2018-04-01

A facile, one-pot enzymatic glycan remodeling of antibody rituximab to produce homogeneous high-mannose and hybrid type antibody glycoforms is described. This method was based on the unique substrate specificity of the endoglycosidase S (Endo-S) from Streptococcus pyogenes. While Endo-S efficiently hydrolyzes the bi-antennary complex type IgG Fc N-glycans, we found that Endo-S did not hydrolyze the "ground state" high-mannose or hybrid glycoforms, and only slowly hydrolyzed the highly activated high-mannose or hybrid N-glycan oxazolines. Moreover, we found that wild-type Endo-S could efficiently use high-mannose or hybrid glycan oxazolines for transglycosylation without product hydrolysis. The combination of the remarkable difference in substrate specificity of Endo-S allows the deglycosylation of heterogeneous rituximab and the transglycosylation with glycan oxazoline to take place in one-pot without the need of isolating the deglycosylated intermediate or changing the enzyme to afford the high-mannose type, hybrid type, and some selectively modified truncated form of antibody glycoforms. Copyright © 2017 Elsevier Ltd. All rights reserved.

Response surface methodology for evaluation and optimization of process parameter and antioxidant capacity of rice flour modified by enzymatic extrusion.

PubMed

Xu, Enbo; Pan, Xiaowei; Wu, Zhengzong; Long, Jie; Li, Jingpeng; Xu, Xueming; Jin, Zhengyu; Jiao, Aiquan

2016-12-01

For the purpose of investigating the effect of enzyme concentration (EC), barrel temperature (BT), moisture content (MC), and screw speed (SS) on processing parameters (product temperature, die pressure and special mechanical energy (SME)) and product responses (extent of gelatinization (GE), retention rate of total phenolic content (TPC-RR)), rice flour extruded with thermostable α-amylase was analyzed by response surface methodology. Stepwise regression models were computed to generate response surface and contour plots, revealing that both TPC-RR and GE increased as increasing MC while expressed different sensitivities to BT during enzymatic extrusion. Phenolics preservation was benefited from low SME. According to multiple-factor optimization, the conditions required to obtain the target SME (10kJ/kg), GE (100%) and TPC-RR (85%) were: EC=1.37‰, BT=93.01°C, MC=44.30%, and SS=171.66rpm, with the actual values (9.49kJ/kg, 99.96% and 87.10%, respectively) showing a good fit to the predicted values. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Fluorescence Ratiometric Assay Strategy for Chemical Transmitter of Living Cells Using H2O2-Sensitive Conjugated Polymers.

PubMed

Wang, Yunxia; Li, Shengliang; Feng, Liheng; Nie, Chenyao; Liu, Libing; Lv, Fengting; Wang, Shu

2015-11-04

A new water-soluble conjugated poly(fluorene-co-phenylene) derivative (PFP-FB) modified with boronate-protected fluorescein (peroxyfluor-1) via PEG linker has been designed and synthesized. In the presence of H2O2, the peroxyfluor-1 group can transform into green fluorescent fluorescein by deprotecting the boronate protecting groups. In this case, upon selective excitation of PFP-FB backbone at 380 nm, efficient fluorescence resonance energy transfer (FRET) from PFP-FB backbone to fluorescein occurs, and accordingly, the fluorescence color of PFP-FB changes from blue to green. Furthermore, the emission color of PFP-FB and the FRET ratio change in a concentration-dependent manner. By taking advantage of PFP-FB, ratiometric detection of choline and acetylcholine (ACh) through cascade enzymatic reactions and further dynamic monitoring of the choline consumption process of cancer cells have been successfully realized. Thus, this new polymer probe promotes the development of enzymatic biosensors and provides a simpler and more effective way for detecting the chemical transmitter of living cells.

Simple and rapid enzymatic method for the synthesis of single-strand oligonucleotides containing trifluorothymidine.

PubMed

Suzuki, Norihiko; Fukushima, Masakazu

2010-11-01

To investigate the mechanism of trifluorothymidine (TFT)-induced DNA damage, we developed an enzymatic method for the synthesis of single-strand oligonucleotides containing TFT-monophosphate residues. Sixteen-mer oligonucleotides and 14-mer 5'-phosphorylated oligonucleotides were annealed to the template of 25-mer, so as to empty one nucleotide site. TFT-triphosphate was incorporated into the site by DNA polymerase and then ligated to 5'-phosphorylated oligonucleotides by DNA ligase. The synthesized 31-mer oligonucleotides containing TFT residues were isolated from the 25-mer complementary template by denaturing polyacrylamide electrophoresis. Using these single-strand oligonucleotides containing TFT residues, the cleavage of TFT residues from DNA, using mismatch uracil-DNA glycosylase (MUG) of E.coli origin, was compared with that of 5-fluorouracil (5FU) and 5-bromodeoxyuridine (BrdU). The TFT/A pair was not cleaved by MUG, while the other pairs, namely, 5FU/A, 5FU/G, BrdU/A, BrdU/G, and TFT/G, were easily cleaved from each synthesized DNA. Thus, this method is useful for obtaining some site-specifically modified oligonucleotides.

Total amino acid stabilization during cell-free protein synthesis reactions.

PubMed

Calhoun, Kara A; Swartz, James R

2006-05-17

Limitations in amino acid supply have been recognized as a substantial problem in cell-free protein synthesis reactions. Although enzymatic inhibitors and fed-batch techniques have been beneficial, the most robust way to stabilize amino acids is to remove the responsible enzymatic activities by genetically modifying the source strain used for cell extract preparation. Previous work showed this was possible for arginine, serine, and tryptophan, but cysteine degradation remained a major limitation in obtaining high protein synthesis yields. Through radiolabel techniques, we confirmed that cysteine degradation was caused by the activity of glutamate-cysteine ligase (gene gshA) in the cell extract. Next, we created Escherichia coli strain KC6 that combines a gshA deletion with previously described deletions for arginine, serine, and tryptophan stabilization. Strain KC6 grows well, and active cell extract can be produced from it for cell-free protein synthesis reactions. The extract from strain KC6 maintains stable amino acid concentrations of all 20 amino acids in a 3-h batch reaction. Yields for three different proteins improved 75-250% relative to cell-free expression using the control extract.

Cascade signal amplification for electrochemical immunosensing by integrating biobarcode probes, surface-initiated enzymatic polymerization and silver nanoparticle deposition.

PubMed

Lin, Dajie; Mei, Chengyang; Liu, Aili; Jin, Huile; Wang, Shun; Wang, Jichang

2015-04-15

A cascade signal amplification strategy through combining surface-initiated enzymatic polymerization (SIEP) and the subsequent deposition of strepavidin functionalized silver nanoparticles (AgNPs) was proposed. The first step of constructing the electrochemical immunosensor involves covalently immobilizing capture antibody on a chitosan modified glass carbon electrode, which then catalyzes DNA addition of deoxynucleotides (dNTP) at the 3'-OH group by terminal deoxynucleotidyl transferase (TdT), leading to the formation of long single-stranded DNAs labeled with numerous biotins. Following the deposition of numerous strepavidin functionalized AgNPs on those long DNA chains, electrochemical stripping signal of silver was used to monitor the immunoreaction in KCl solution. Using α-fetoprotein as a model analyte, this amplification strategy could detect fetoprotein down to 0.046pg/mL with a wide linear range from 0.1pg/mL to 1.0ng/mL. The achieved high sensitivity and good reproducibility suggest that this cascade signal amplification strategy has great potential for detecting biological samples and possibly clinical application. Copyright © 2014 Elsevier B.V. All rights reserved.

Modification of Sodium Release Using Porous Corn Starch and Lipoproteic Matrix.

PubMed

Christina, Josephine; Lee, Youngsoo

2016-04-01

Excessive sodium consumption can result in hypertension, diabetes, heart diseases, stroke, and kidney diseases. Various chips and extruded snacks, where salt is mainly applied on the product surface, accounted for almost 56% of snacks retail sales in 2010. Hence, it is important to target sodium reduction for those snack products. Past studies had shown that modifying the rate-release mechanism of sodium is a promising strategy for sodium reduction in the food industry. Encapsulation of salt can be a possible technique to control sodium release rate. Porous corn starch (PCS), created by enzymatic treatment and spray drying and lipoproteic matrix, created by gelation and freeze drying, were evaluated as carriers for controlled sodium release targeting topically applied salts. Both carriers encapsulated salt and their in vitro sodium release profiles were measured using a conductivity meter. The sodium release profiles of PCS treated with different enzymatic reaction times were not significantly different. Protein content and fat content altered sodium release profile from the lipoproteic matrix. The SEM images of PCS showed that most of the salt crystals coated the starch instead of being encapsulated in the pores while the SEM images and computed tomography scan of lipoproteic matrix showed salt dispersed throughout the matrix. Hence, PCS was found to have limitations as a sodium carrier as it could not effectively encapsulate salt inside its pores. The lipoproteic matrix was found to have a potential as a sodium carrier as it could effectively encapsulate salt and modify the sodium release profile. © 2016 Institute of Food Technologists®

Phytoremediation of Polychlorinated Biphenyls: New Trends and Promises

PubMed Central

Van Aken, Benoit; Correa, Paola A.; Schnoor, Jerald L.

2011-01-01

Transgenic plants and associated bacteria constitute a new generation of genetically modified organisms for efficient and environmental-friendly treatment of soil and water contaminated with polychlorinated biphenyls (PCBs). This review focuses on recent advances in phytoremediation for the treatment of PCBs, including the development of transgenic plants and associated bacteria. Phytoremediation, or the use of higher plants for rehabilitation of soil and groundwater, is a promising strategy for cost-effective treatment of sites contaminated by toxic compounds, including toxic PCBs. Plants can help mitigate environmental pollution by PCBs through a range of mechanisms: besides uptake from soil (phytoextraction), plants are capable of enzymatic transformation of PCBs (phytotransformation); by releasing a variety of secondary metabolites, plants also enhance the microbial activity in the root zone, improving biodegradation of PCBs (rhizoremediation). However, because of their hydrophobicity and chemical stability, PCBs are only slowly taken up and degraded by plants and associated bacteria, resulting in incomplete treatment and potential release of toxic metabolites into the environment. Moreover, naturally occurring plant-associated bacteria may not possess the enzymatic machinery necessary for PCB degradation. In order to overcome these limitations, bacterial genes involved in the metabolism of PCBs, such as biphenyl dioxygenases, have been introduced into higher plants, following a strategy similar to the development of transgenic crops. Similarly, bacteria have then been genetically modified that exhibit improved biodegradation capabilities and are able to maintain stable relationships with plants. Transgenic plants and associated bacteria bring hope for a broader and more efficient application of phytoremediation for the treatment of PCBs. PMID:20384372

A Nanostructured Sensor Based on Gold Nanoparticles and Nafion for Determination of Uric Acid

PubMed Central

Stozhko, Natalia; Bukharinova, Maria; Galperin, Leonid; Brainina, Khiena

2018-01-01

The paper discusses the mechanism of uric acid (UA) electrooxidation occurring on the surface of gold nanoparticles. It has been shown that the electrode process is purely electrochemical, uncomplicated with catalytic stages. The nanoeffects observed as the reduction of overvoltage and increased current of UA oxidation have been described. These nanoeffects are determined by the size of particles and do not depend on the method of particle preparation (citrate and “green” synthesis). The findings of these studies have been used to select a modifier for carbon screen-printed electrode (CSPE). It has been stated that CSPE modified with gold nanoparticles (5 nm) and 2.5% Nafion (Nf) may serve as non-enzymatic sensor for UA determination. The combination of the properties of nanoparticles and Nafion as a molecular sieve at the selected pH 5 phosphate buffer solution has significantly improved the resolution of the sensor compared to unmodified CSPE. A nanostructured sensor has demonstrated good selectivity in determining UA in the presence of ascorbic acid. The detection limit of UA is 0.25 μM. A linear calibration curve has been obtained over a range of 0.5–600 μM. The 2.5%Nf/Au(5nm)/CSPE has been successfully applied to determining UA in blood serum and milk samples. The accuracy and reliability of the obtained results have been confirmed by a good correlation with the enzymatic spectrophotometric analysis (R2 = 0.9938) and the “added−found” technique (recovery close to 100%). PMID:29509718

A large response range reflectometric urea biosensor made from silica-gel nanoparticles.

PubMed

Alqasaimeh, Muawia; Heng, Lee Yook; Ahmad, Musa; Raj, A S Santhana; Ling, Tan Ling

2014-07-22

A new silica-gel nanospheres (SiO2NPs) composition was formulated, followed by biochemical surface functionalization to examine its potential in urea biosensor development. The SiO2NPs were basically synthesized based on sol-gel chemistry using a modified Stober method. The SiO2NPs surfaces were modified with amine (-NH2) functional groups for urease immobilization in the presence of glutaric acid (GA) cross-linker. The chromoionophore pH-sensitive dye ETH 5294 was physically adsorbed on the functionalized SiO2NPs as pH transducer. The immobilized urease determined urea concentration reflectometrically based on the colour change of the immobilized chromoionophore as a result of the enzymatic hydrolysis of urea. The pH changes on the biosensor due to the catalytic enzyme reaction of immobilized urease were found to correlate with the urea concentrations over a linear response range of 50-500 mM (R2 = 0.96) with a detection limit of 10 mM urea. The biosensor response time was 9 min with reproducibility of less than 10% relative standard deviation (RSD). This optical urea biosensor did not show interferences by Na+, K+, Mg2+ and NH4+ ions. The biosensor performance has been validated using urine samples in comparison with a non-enzymatic method based on the use of p-dimethylaminobenzaldehyde (DMAB) reagent and demonstrated a good correlation between the two different methods (R2 = 0.996 and regression slope of 1.0307). The SiO2NPs-based reflectometric urea biosensor showed improved dynamic linear response range when compared to other nanoparticle-based optical urea biosensors.

Presenilin-Based Genetic Screens in Drosophila melanogaster Identify Novel Notch Pathway Modifiers

PubMed Central

Mahoney, Matt B.; Parks, Annette L.; Ruddy, David A.; Tiong, Stanley Y. K.; Esengil, Hanife; Phan, Alexander C.; Philandrinos, Panos; Winter, Christopher G.; Chatterjee, Runa; Huppert, Kari; Fisher, William W.; L'Archeveque, Lynn; Mapa, Felipa A.; Woo, Wendy; Ellis, Michael C.; Curtis, Daniel

2006-01-01

Presenilin is the enzymatic component of γ-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimer's disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for γ-tubulin in the pathway. PMID:16415372

Presenilin-based genetic screens in Drosophila melanogaster identify novel notch pathway modifiers.

PubMed

Mahoney, Matt B; Parks, Annette L; Ruddy, David A; Tiong, Stanley Y K; Esengil, Hanife; Phan, Alexander C; Philandrinos, Panos; Winter, Christopher G; Chatterjee, Runa; Huppert, Kari; Fisher, William W; L'Archeveque, Lynn; Mapa, Felipa A; Woo, Wendy; Ellis, Michael C; Curtis, Daniel

2006-04-01

Presenilin is the enzymatic component of gamma-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimer's disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for gamma-tubulin in the pathway.

Synthesis, hybridization characteristics, and fluorescence properties of oligonucleotides modified with nucleobase-functionalized locked nucleic acid adenosine and cytidine monomers.

PubMed

Kaura, Mamta; Kumar, Pawan; Hrdlicka, Patrick J

2014-07-03

Conformationally restricted nucleotides such as locked nucleic acid (LNA) are very popular as affinity-, specificity-, and stability-enhancing modifications in oligonucleotide chemistry to produce probes for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. Considerable efforts have been devoted in recent years to optimize the biophysical properties of LNA through additional modification of the sugar skeleton. We recently introduced C5-functionalization of LNA uridines as an alternative and synthetically more straightforward approach to improve the biophysical properties of LNA. In the present work, we set out to test the generality of this concept by studying the characteristics of oligonucleotides modified with four different C5-functionalized LNA cytidine and C8-functionalized LNA adenosine monomers. The results strongly suggest that C5-functionalization of LNA pyrimidines is indeed a viable approach for improving the binding affinity, target specificity, and/or enzymatic stability of LNA-modified ONs, whereas C8-functionalization of LNA adenosines is detrimental to binding affinity and specificity. These insights will impact the future design of conformationally restricted nucleotides for nucleic acid targeting applications.

Modified rare earth semiconductor oxide as a new nucleotide probe.

PubMed

Shrestha, S; Mills, C E; Lewington, J; Tsang, S C

2006-12-28

Recent rapid developments in biological analysis, medical diagnosis, pharmaceutical industry, and environmental control fuel the urgent need for recognition of particular DNA sequences from samples. Currently, DNA detection techniques use radiochemical, enzymatic, fluorescent, or electrochemiluminescent methods; however, these techniques require costly labeled DNA and highly skilled and cumbersome procedure, which prohibit any in-situ monitoring. Here, we report that hybridization of surface-immobilized single-stranded oligonucleotide on praseodymium oxide (evaluated as a biosensor surface for the first time) with complimentary strands in solution provokes a significant shift of electrical impedance curve. This shift is attributed to a change in electrical characteristics through modification of surface charge of the underlying modified praseodymium oxide upon hybridization with the complementary oligonucelotide strand. On the other hand, using a noncomplementary single strand in solution does not create an equivalent change in the impedance value. This result clearly suggests that a new and simple electrochemical technique based on the change in electrical properties of the modified praseodymium oxide semiconductor surface upon recognition and transduction of a biological event without using labeled species is revealed.

Attractor Metabolic Networks

PubMed Central

De la Fuente, Ildefonso M.; Cortes, Jesus M.; Pelta, David A.; Veguillas, Juan

2013-01-01

Background The experimental observations and numerical studies with dissipative metabolic networks have shown that cellular enzymatic activity self-organizes spontaneously leading to the emergence of a Systemic Metabolic Structure in the cell, characterized by a set of different enzymatic reactions always locked into active states (metabolic core) while the rest of the catalytic processes are only intermittently active. This global metabolic structure was verified for Escherichia coli, Helicobacter pylori and Saccharomyces cerevisiae, and it seems to be a common key feature to all cellular organisms. In concordance with these observations, the cell can be considered a complex metabolic network which mainly integrates a large ensemble of self-organized multienzymatic complexes interconnected by substrate fluxes and regulatory signals, where multiple autonomous oscillatory and quasi-stationary catalytic patterns simultaneously emerge. The network adjusts the internal metabolic activities to the external change by means of flux plasticity and structural plasticity. Methodology/Principal Findings In order to research the systemic mechanisms involved in the regulation of the cellular enzymatic activity we have studied different catalytic activities of a dissipative metabolic network under different external stimuli. The emergent biochemical data have been analysed using statistical mechanic tools, studying some macroscopic properties such as the global information and the energy of the system. We have also obtained an equivalent Hopfield network using a Boltzmann machine. Our main result shows that the dissipative metabolic network can behave as an attractor metabolic network. Conclusions/Significance We have found that the systemic enzymatic activities are governed by attractors with capacity to store functional metabolic patterns which can be correctly recovered from specific input stimuli. The network attractors regulate the catalytic patterns, modify the efficiency in the connection between the multienzymatic complexes, and stably retain these modifications. Here for the first time, we have introduced the general concept of attractor metabolic network, in which this dynamic behavior is observed. PMID:23554883

Exploiting enzyme catalysis in ultra-low ion strength media for impedance biosensing of avian influenza virus using a bare interdigitated electrode.

PubMed

Fu, Yingchun; Callaway, Zachary; Lum, Jacob; Wang, Ronghui; Lin, Jianhan; Li, Yanbin

2014-02-18

Enzyme catalysis is broadly used in various fields but generally applied in media with high ion strength. Here, we propose the exploitation of enzymatic catalysis in ultra-low ion strength media to induce ion strength increase for developing a novel impedance biosensing method. Avian influenza virus H5N1, a serious worldwide threat to poultry and human health, was adopted as the analyte. Magnetic beads were modified with H5N1-specific aptamer to capture the H5N1 virus. This was followed by binding concanavalin A (ConA), glucose oxidase (GOx), and Au nanoparticles (AuNPs) to create bionanocomposites through a ConA-glycan interaction. The yielded sandwich complex was transferred to a glucose solution to trigger an enzymatic reaction to produce gluconic acid, which ionized to increase the ion strength of the solution, thus decreasing the impedance on a screen-printed interdigitated array electrode. This method took advantages of the high efficiency of enzymatic catalysis and the high susceptibility of electrochemical impedance on the ion strength and endowed the biosensor with high sensitivity and a detection limit of 8 × 10(-4) HAU in 200 μL sample, which was magnitudes lower than that of some analogues based on biosensing methods. Furthermore, the proposed method required only a bare electrode for measurements of ion strength change and had negligible change on the surficial properties of the electrode, though some modification of magnetic beads/Au nanoparticles and the construction of a sandwich complex were still needed. This helped to avoid the drawbacks of commonly used electrode immobilization methods. The merit for this method makes it highly useful and promising for applications. The proposed method may create new possibilities in the broad and well-developed enzymatic catalysis fields and find applications in developing sensitive, rapid, low-cost, and easy-to-operate biosensing and biocatalysis devices.

Enhanced enzymatic stability and antitumor activity of daunorubicin-GnRH-III bioconjugates modified in position 4.

PubMed

Manea, Marilena; Leurs, Ulrike; Orbán, Erika; Baranyai, Zsuzsa; Öhlschläger, Peter; Marquardt, Andreas; Schulcz, Ákos; Tejeda, Miguel; Kapuvári, Bence; Tóvári, József; Mezo, Gábor

2011-07-20

Here, we report on the synthesis, enzymatic stability, and antitumor activity of novel bioconjugates containing the chemotherapeutic agent daunorubicin attached through an oxime bond to various gonadotropin-releasing hormone-III (GnRH-III) derivatives. In order to increase the enzymatic stability of the bioconjugates (in particular against chymotrypsin), (4)Ser was replaced by N-Me-Ser or Lys(Ac). A compound in which (4)Lys was not acetylated was also prepared, with the aim of investigating the influence of the free ε-amino group on the biochemical properties. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon, and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their stability/degradation (1) in human serum, (2) in the presence of rat liver lysosomal homogenate, and (3) in the presence of digestive enzymes (trypsin, chymotrypsin, and pepsin) was analyzed by liquid chromatography in combination with mass spectrometry. The results showed that (1) all synthesized bioconjugates had in vitro cytostatic effect, (2) they were stable in human serum at least for 24 h, and (3) they were hydrolyzed in the presence of lysosomal homogenate. All compounds were stable in the presence of (1) pepsin and (2) trypsin (except for the (4)Lys containing bioconjugate). In the presence of chymotrypsin, all bioconjugates were digested; the degradation rate strongly depending on their structure. The bioconjugates in which (4)Ser was replaced by N-Me-Ser or Lys(Ac) had the highest enzymatic stability, making them potential candidates for oral administration. In vivo tumor growth inhibitory effect of two selected bioconjugates was evaluated on orthotopically developed C26 murine colon carcinoma bearing mice. The results indicated that the compound containing Lys(Ac) in position 4 had significantly higher antitumor activity than the parent bioconjugate.

Surprising Alteration of Antibacterial Activity of 5″-Modified Neomycin against Resistant Bacteria

PubMed Central

Zhang, Jianjun; Chiang, Fang-I; Wu, Long; Czyryca, Przemyslaw Greg; Li, Ding; Chang, Cheng-Wei Tom

2009-01-01

A facile synthetic protocol for the production of neomycin B derivatives with various modifications at the 5″ position has been developed. Structural activity relationship (SAR) against aminoglycoside resistant bacteria equipped with various aminoglycoside-modifying enzymes (AME's) was investigated. Enzymatic and molecular modeling studies reveal that the superb substrate promiscuity of AME's allows the resistant bacteria to cope with diverse structural modifications despite the observation that several derivatives show enhanced antibacterial activity than the parent neomycin. Surprisingly, when testing synthetic neomycin derivatives against other human pathogens, two leads exhibit prominent activity against both Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) that are known to exert high level of resistance against clinically used aminoglycosides. These findings can be extremely useful in developing new aminoglycoside antibiotics against resistant bacteria. Our result also suggests that new biological and antimicrobial activities can be obtained by chemical modifications of old drugs. PMID:19012394

Macrophage Migration Inhibitory Factor is subjected to glucose modification and oxidation in Alzheimer’s Disease

PubMed Central

Kassaar, Omar; Pereira Morais, Marta; Xu, Suying; Adam, Emily L.; Chamberlain, Rosemary C.; Jenkins, Bryony; James, Tony; Francis, Paul T.; Ward, Stephen; Williams, Robert J.; van den Elsen, Jean

2017-01-01

Glucose and glucose metabolites are able to adversely modify proteins through a non-enzymatic reaction called glycation, which is associated with the pathology of Alzheimer’s Disease (AD) and is a characteristic of the hyperglycaemia induced by diabetes. However, the precise protein glycation profile that characterises AD is poorly defined and the molecular link between hyperglycaemia and AD is unknown. In this study, we define an early glycation profile of human brain using fluorescent phenylboronate gel electrophoresis and identify early glycation and oxidation of macrophage migration inhibitory factor (MIF) in AD brain. This modification inhibits MIF enzyme activity and ability to stimulate glial cells. MIF is involved in immune response and insulin regulation, hyperglycaemia, oxidative stress and glycation are all implicated in AD. Our study indicates that glucose modified and oxidised MIF could be a molecular link between hyperglycaemia and the dysregulation of the innate immune system in AD. PMID:28230058

Enzymatic biofuel cell based on electrodes modified with lipid liquid-crystalline cubic phases

NASA Astrophysics Data System (ADS)

Nazaruk, Ewa; Smoliński, Sławomir; Swatko-Ossor, Marta; Ginalska, Grażyna; Fiedurek, Jan; Rogalski, Jerzy; Bilewicz, Renata

Two glassy carbon electrodes modified with enzymes embedded in lyotropic liquid-crystalline cubic phase were used for the biofuel cell construction. The monoolein liquid-crystalline film allowed to avoid separators in the biofuel cell. Glucose and oxygen as fuels, and glucose oxidase and laccase as anode and cathode biocatalysts, respectively were used. The biofuel cell parameters were examined in McIlvaine buffer, pH 7 solution containing 15 mM of glucose and saturated with dioxygen. A series of mediators were tested taking into account their formal potentials, stability in the cubic phase and efficiency of mediation. Most stable was the biofuel cell based on tetrathiafulvalene (TTF) and 2,2‧-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as anode and cathode mediators, respectively. The open-circuit voltage was equal to 450 ± 40 mV. The power densities and current densities were measured for all the systems studied.

Enhancing saccharification of wheat straw by mixing enzymes from genetically-modified Trichoderma reesei and Aspergillus niger.

PubMed

Jiang, Yanping; Duarte, Alexandra Vivas; van den Brink, Joost; Wiebenga, Ad; Zou, Gen; Wang, Chengshu; de Vries, Ronald P; Zhou, Zhihua; Benoit, Isabelle

2016-01-01

To increase the efficiency of enzymatic hydrolysis for plant biomass conversion into renewable biofuel and chemicals. By overexpressing the point mutation A824 V transcriptional activator Xyr1 in Trichoderma reesei, carboxymethyl cellulase, cellobiosidase and β-D-glucosidase activities of the best mutant were increased from 1.8 IU/ml, 0.1 IU/ml and 0.05 IU/ml to 4.8 IU/ml, 0.4 IU/ml and 0.3 IU/ml, respectively. The sugar yield of wheat straw saccharification by combining enzymes from this mutant and the Aspergillus niger genetically modified strain ΔcreA/xlnR c/araR c was improved up to 7.5 mg/ml, a 229 % increase compared to the combination of wild type strains. Mixing enzymes from T. reesei and A. niger combined with the genetic modification of transcription factors is a promising strategy to increase saccharification efficiency.

Viral delivery of genome-modifying proteins for cellular reprogramming.

PubMed

Mikkelsen, Jacob Giehm

2018-06-18

Following the successful development of virus-based gene vehicles for genetic therapies, exploitation of viruses as carriers of genetic tools for cellular reprogramming and genome editing should be right up the street. However, whereas persistent, potentially life-long gene expression is the main goal of conventional genetic therapies, tools and bits for genome engineering should ideally be short-lived and active only for a limited time. Although viral vector systems have already been adapted for potent genome editing both in vitro and in vivo, regulatable gene expression systems or self-limiting expression circuits need to be implemented limiting exposure of chromatin to genome-modifying enzymes. As an alternative approach, emerging virus-based protein delivery technologies support direct protein delivery, providing a short, robust boost of enzymatic activity in transduced cells. Is this potentially the perfect way of shipping loads of cargo to many recipients and still maintain short-term activity? Copyright © 2018 Elsevier Ltd. All rights reserved.

Residual transglutaminase in collagen - effects, detection, quantification, and removal.

PubMed

Schloegl, W; Klein, A; Fürst, R; Leicht, U; Volkmer, E; Schieker, M; Jus, S; Guebitz, G M; Stachel, I; Meyer, M; Wiggenhorn, M; Friess, W

2012-02-01

In the present study, we developed an enzyme-linked immunosorbent assay (ELISA) for microbial transglutaminase (mTG) from Streptomyces mobaraensis to overcome the lack of a quantification method for mTG. We further performed a detailed follow-on-analysis of insoluble porcine collagen type I enzymatically modified with mTG primarily focusing on residuals of mTG. Repeated washing (4 ×) reduced mTG-levels in the washing fluids but did not quantitatively remove mTG from the material (p < 0.000001). Substantial amounts of up to 40% of the enzyme utilized in the crosslinking mixture remained associated with the modified collagen. Binding was non-covalent as could be demonstrated by Western blot analysis. Acidic and alkaline dialysis of mTG treated collagen material enabled complete removal the enzyme. Treatment with guanidinium chloride, urea, or sodium chloride was less effective in reducing the mTG content. Copyright © 2011 Elsevier B.V. All rights reserved.

New Method for Producing Significant Amounts of RNA Labeled at Specific Sites | Center for Cancer Research

Cancer.gov

Among biomacromolecules, RNA is the most versatile, and it plays indispensable roles in almost all aspects of biology. For example, in addition to serving as mRNAs coding for proteins, RNAs regulate gene expression, such as controlling where, when, and how efficiently a gene gets expressed, participate in RNA processing, encode the genetic information of some viruses, serve as scaffolds, and even possess enzymatic activity. To study these RNAs and their biological functions and to make use of those RNA activities for biomedical applications, researchers first need to make various types of RNA. For structural biologists incorporating modified or labeled nucleotides at specific sites in RNA molecules of interest is critical to gain structural insight into RNA functions. However, placing labeled or modified residue(s) in desired positions in a large RNA has not been possible until now.

Polysaccharide-based Noncovalent Assembly for Targeted Delivery of Taxol

NASA Astrophysics Data System (ADS)

Yang, Yang; Zhang, Ying-Ming; Chen, Yong; Chen, Jia-Tong; Liu, Yu

2016-01-01

The construction of synthetic straightforward, biocompatible and biodegradable targeted drug delivery system with fluorescent tracking abilities, high anticancer activities and low side effects is still a challenge in the field of biochemistry and material chemistry. In this work, we constructed targeted paclitaxel (Taxol) delivery nanoparticles composed of permethyl-β-cyclodextrin modified hyaluronic acid (HApCD) and porphyrin modified paclitaxel prodrug (PorTaxol), through host-guest and amphiphilic interactions. The obtained nanoparticles (HATXP) were biocompatible and enzymatic biodegradable due to their hydrophilic hyaluronic acid (HA) shell and hydrophobic Taxol core, and exhibited specific targeting internalization into cancer cells via HA receptor mediated endocytosis effects. The cytotoxicity experiments showed that the HATXP exhibited similar anticancer activities to, but much lower side effects than commercial anticancer drug Taxol. The present work would provide a platform for targeted paclitaxel drug delivery and a general protocol for the design of advanced multifunctional nanoscale biomaterials for targeted drug/gene delivery.

High-performance liquid chromatography/electrospray mass spectrometry for the analysis of modified bases in DNA: 7-(2-hydroxyethyl)guanine, the major ethylene oxide-DNA adduct.

PubMed

Leclercq, L; Laurent, C; De Pauw, E

1997-05-15

A method was developed for the analysis of 7-(2-hydroxyethyl)guanine (7HEG), the major DNA adduct formed after exposure to ethylene oxide (EO). The method is based on DNA neutral thermal hydrolysis, adduct micro-concentration, and final characterization and quantification by HPLC coupled to single-ion monitoring electrospray mass spectrometry (HPLC/SIR-ESMS). The method was found to be selective, sensitive, and easy to handle with no need for enzymatic digestion or previous sample derivatization. Detection limit was found to be close to 1 fmol of adduct injected (10(-10) M), thus allowing the detection of approximately three modified bases on 10(8) intact nucleotides in blood sample analysis. Quantification results are shown for 7HEG after calf thymus DNA and blood exposure to various doses of EO, in both cases obtaining clear dose-response relationships.

Highly sensitive electrochemical biosensor for bisphenol A detection based on a diazonium-functionalized boron-doped diamond electrode modified with a multi-walled carbon nanotube-tyrosinase hybrid film.

PubMed

Zehani, Nedjla; Fortgang, Philippe; Saddek Lachgar, Mohamed; Baraket, Abdoullatif; Arab, Madjid; Dzyadevych, Sergei V; Kherrat, Rochdi; Jaffrezic-Renault, Nicole

2015-12-15

A highly sensitive electrochemical biosensor for the detection of Bisphenol A (BPA) in water has been developed by immobilizing tyrosinase onto a diazonium-functionalized boron doped diamond electrode (BDD) modified with multi-walled carbon nanotubes (MWCNTs). The fabricated biosensor exhibits excellent electroactivity towards o-quinone, a product of this enzymatic reaction of BPA oxidation catalyzed by tyrosinase. The developed BPA biosensor displays a large linear range from 0.01 nM to 100 nM, with a detection limit (LOD) of 10 pM. The feasibility of the proposed biosensor has been demonstrated on BPA spiked water river samples. Therefore, it could be a promising and reliable analytical tool for on-site monitoring of BPA in waste water. Copyright © 2015 Elsevier B.V. All rights reserved.

Modification of Aspergillus niger by conducting polymer, Polypyrrole, and the evaluation of electrochemical properties of modified cells.

PubMed

Apetrei, Roxana-Mihaela; Carac, Geta; Bahrim, Gabriela; Ramanaviciene, Almira; Ramanavicius, Arunas

2018-06-01

The enhancement of bioelectrochemical properties of microorganism by in situ formation of conducting polymer within the cell structures (e.g. cell wall) was performed. The synthesis of polypyrrole (Ppy) within fungi (Aspergillus niger) cells was achieved. Two different Aspergillus niger strains were selected due to their ability to produce glucose oxidase, which initiated the Ppy formation through products of enzymatic reaction. The evolution of Ppy structural features was investigated by absorption spectroscopy, cyclic voltammetry and Fourier transform infrared spectroscopy. Copyright © 2018 Elsevier B.V. All rights reserved.

Enzymatically-stable oxetane-based dipeptide hydrogels.

PubMed

McDougall, Laura; Draper, Emily R; Beadle, Jonathan D; Shipman, Michael; Raubo, Piotr; Jamieson, Andrew G; Adams, Dave J

2018-02-13

Low molecular weight gelators that are not easily degraded by enzymes have a range of potential applications. Here, we report new Fmoc-protected dipeptides in which the amide carbonyl group has been replaced by an oxetane ring. Remarkably one of these peptidomimetics, but not the corresponding dipeptide, is an effective gelator, forming hydrogels at a concentration of 3 mg mL -1 . On assembly, there is a lack of beta-sheet structure, implying that there is no requirement for this motif in such a gel. Furthermore, the modified dipeptide is also stable to proteolysis compared to the parent dipeptide.

Automated glycan assembly of galactosylated xyloglucan oligosaccharides and their recognition by plant cell wall glycan-directed antibodies.

PubMed

Dallabernardina, Pietro; Ruprecht, Colin; Smith, Peter J; Hahn, Michael G; Urbanowicz, Breeanna R; Pfrengle, Fabian

2017-12-06

We report the automated glycan assembly of oligosaccharides related to the plant cell wall hemicellulosic polysaccharide xyloglucan. The synthesis of galactosylated xyloglucan oligosaccharides was enabled by introducing p-methoxybenzyl (PMB) as a temporary protecting group for automated glycan assembly. The generated oligosaccharides were printed as microarrays, and the binding of a collection of xyloglucan-directed monoclonal antibodies (mAbs) to the oligosaccharides was assessed. We also demonstrated that the printed glycans can be further enzymatically modified while appended to the microarray surface by Arabidopsis thaliana xyloglucan xylosyltransferase 2 (AtXXT2).

Separation of phospholipids in microfluidic chip device: application to high-throughput screening assays for lipid-modifying enzymes.

PubMed

Lin, Sansan; Fischl, Anthony S; Bi, Xiahui; Parce, Wally

2003-03-01

Phospholipid molecules such as ceramide and phosphoinositides play crucial roles in signal transduction pathways. Lipid-modifying enzymes including sphingomyelinase and phosphoinositide kinases regulate the generation and degradation of these lipid-signaling molecules and are important therapeutic targets in drug discovery. We now report a sensitive and convenient method to separate these lipids using microfluidic chip-based technology. The method takes advantage of the high-separation power of the microchips that separate lipids based on micellar electrokinetic capillary chromatography (MEKC) and the high sensitivity of fluorescence detection. We further exploited the method to develop a homogenous assay to monitor activities of lipid-modifying enzymes. The assay format consists of two steps: an on-plate enzymatic reaction using fluorescently labeled substrates followed by an on-chip MEKC separation of the reaction products from the substrates. The utility of the assay format for high-throughput screening (HTS) is demonstrated using phospholipase A(2) on the Caliper 250 HTS system: throughput of 80min per 384-well plate can be achieved with unattended running time of 5.4h. This enabling technology for assaying lipid-modifying enzymes is ideal for HTS because it avoids the use of radioactive substrates and complicated separation/washing steps and detects both substrate and product simultaneously.

Mechanisms for Covalent Immobilization of Horseradish Peroxidase on Ion-Beam-Treated Polyethylene

PubMed Central

Kondyurin, Alexey V.; Naseri, Pourandokht; Tilley, Jennifer M. R.; Nosworthy, Neil J.; Bilek, Marcela M. M.; McKenzie, David R.

2012-01-01

The surface of polyethylene was modified by plasma immersion ion implantation. Structure changes including carbonization and oxidation were observed. High surface energy of the modified polyethylene was attributed to the presence of free radicals on the surface. The surface energy decay with storage time after treatment was explained by a decay of the free radical concentration while the concentration of oxygen-containing groups increased with storage time. Horseradish peroxidase was covalently attached onto the modified surface by the reaction with free radicals. Appropriate blocking agents can block this reaction. All aminoacid residues can take part in the covalent attachment process, providing a universal mechanism of attachment for all proteins. The native conformation of attached protein is retained due to hydrophilic interactions in the interface region. The enzymatic activity of covalently attached protein remained high. The long-term activity of the modified layer to attach protein is explained by stabilisation of unpaired electrons in sp2 carbon structures. A high concentration of free radicals can give multiple covalent bonds to the protein molecule and destroy the native conformation and with it the catalytic activity. The universal mechanism of protein attachment to free radicals could be extended to various methods of radiation damage of polymers. PMID:24278665

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.

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.

Production of superparamagnetic nanobiocatalysts for green chemistry applications.

PubMed

Gasser, Christoph A; Ammann, Erik M; Schäffer, Andreas; Shahgaldian, Patrick; Corvini, Philippe F-X

2016-08-01

Immobilization of enzymes on solid supports is a convenient method for increasing enzymatic stability and enabling enzyme reuse. In the present work, a sorption-assisted surface conjugation method was developed and optimized to immobilize enzymes on the surface of superparamagnetic nanoparticles. An oxidative enzyme, i.e., laccase from Trametes versicolor was used as model enzyme. The immobilization method consists of the production of superparamagnetic nanoparticles by co-precipitation of FeCl2 and FeCl3. Subsequently, the particle surface is modified with an organosilane containing an amino group. Next, the enzymes are adsorbed on the particle surface before a cross-linking agent, i.e., glutaraldehyde is added which links the amino groups on the particle surface with the amino groups of the enzymes and leads to internal cross-linking of the enzymes as well. The method was optimized using response surface methodology regarding optimal enzyme and glutaraldehyde amounts, pH, and reaction times. Results allowed formulation of biocatalysts having high specific enzymatic activity and improved stability. The biocatalysts showed considerably higher stability compared with the dissolved enzymes over a pH range from 3 to 9 and in the presence of several chemical denaturants. To demonstrate the reusability of the immobilized enzymes, they were applied as catalysts for the production of a phenoxazinone dye. Virtually, 100 % of the precursor was transformed to the dye in each of the ten conducted reaction cycles while on average 84.5 % of the enzymatic activity present at the beginning of a reaction cycle was retained after each cycle highlighting the considerable potential of superparamagnetic biocatalysts for application in industrial processes.

Complex coacervate core micelles with a lysozyme-modified corona.

PubMed

Danial, Maarten; Klok, Harm-Anton; Norde, Willem; Stuart, Martien A Cohen

2007-07-17

This paper describes the preparation, characterization, and enzymatic activity of complex coacervate core micelles (C3Ms) composed of poly(acrylic acid) (PAA) and poly(N-methyl-2-vinyl pyridinium iodide)-b-poly(ethylene oxide) (PQ2VP-PEO) to which the antibacterial enzyme lysozyme is end-attached. C3Ms were prepared by polyelectrolyte complex formation between PAA and mixtures containing different ratios of aldehyde and hydroxyl end-functionalized PQ2VP-PEO. This resulted in the formation of C3Ms containing 0-40% (w/w) of the aldehyde end-functionalized PQ2VP-PEO block copolymer (PQ2VP-PEO-CHO). Chemical conjugation of lysozyme was achieved via reductive amination of the aldehyde groups, which are exposed at the surface of the C3M, with the amine groups present in the side chains of the lysine residues of the protein. Dynamic and static light scattering indicated that the conjugation of lysozyme to C3Ms prepared using 10 and 20% (w/w) PQ2VP-PEO-CHO resulted in the formation of unimicellar particles. Multimicellar aggregates, in contrast, were obtained when lysozyme was conjugated to C3Ms prepared using 30 or 40% (w/w) PQ2VP-PEO-CHO. The enzymatic activity of the unimicellar lysozyme-C3M conjugates toward the hydrolysis of the bacterial substrate Micrococcus lysodeikticus was comparable to that of free lysozyme. For the multimicellar particles, in contrast, significantly reduced enzymatic rates of hydrolysis, altered circular dichroism, and red-shifted tryptophan fluorescence spectra were measured. These results are attributed to the occlusion of lysozyme in the interior of the multimicellar conjugates.

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

Liu, Hongying, E-mail: liuhongying@hdu.edu.cn; Gu, Chunchuan; Li, Dujuan

Graphical abstract: A non-enzymatic H{sub 2}O{sub 2} sensor with high selectivity and sensitivity based on rose-shaped FeMoO{sub 4} synthesized by the convenient microwave-assisted hydrothermal method, was fabricated. - Highlights: • Rose-shaped FeMoO{sub 4} is synthesized within 10 min via microwave-assisted hydrothermal approach. • Non-enzymatic hydrogen peroxide biosensor based on FeMoO{sub 4} nanomaterials is fabricated. • The biosensor exhibits good performance. - Abstract: In this work, we demonstrated a simple, rapid and reliable microwave-assisted hydrothermal approach to synthesize the uniform rose-shaped FeMoO{sub 4} within 10 min. The morphologies of the synthesized materials were characterized by X-ray powder diffraction and scanning electronmore » microscopy. Moreover, a non-enzymatic amperometric sensor for the detection of hydrogen peroxide (H{sub 2}O{sub 2}) was fabricated on the basis of the FeMoO{sub 4} as electrocatalysis. The resulting FeMoO{sub 4} exhibited high sensitivity and good stability for the detection of H{sub 2}O{sub 2}, which may be attributed to the rose-shaped structure of the material and the catalytic property of FeMoO{sub 4}. Amperometric response showed that the modified electrode had a good response for H{sub 2}O{sub 2} with a linear range from 1 μM to 1.6 mM, a detection limit of 0.5 μM (S/N = 3), high selectivity and short response time. Additionally, good recoveries of analytes in real milk samples confirm the reliability of the prepared sensor in practical applications.« less

Integration of antimicrobial pectin-based edible coating and active modified atmosphere packaging to preserve the quality and microbial safety of fresh-cut persimmon (Diospyros kaki Thunb. cv. Rojo Brillante).

PubMed

Sanchís, Elena; Ghidelli, Christian; Sheth, Chirag C; Mateos, Milagros; Palou, Lluís; Pérez-Gago, María B

2017-01-01

The greatest hurdle to the commercial marketing of fresh-cut fruits is related to their higher susceptibility to enzymatic browning, tissue softening, and microbial growth. The aim of this study was to test the efficacy of a pectin-based edible coating and low oxygen modified atmosphere packaging (MAP) to control enzymatic browning and reduce microbial growth of fresh-cut 'Rojo Brillante' persimmon. The survival of Escherichia coli, Salmonella enteritidis and Listeria monocytogenes artificially inoculated on fresh-cut fruit was also assessed. The pectin coating was amended with 500 IU mL -1 nisin (NI) as antimicrobial agent and 10 g kg -1 citric acid and 10 g kg -1 calcium chloride as anti-browning and firming agents, respectively. Persimmon slices were dipped in the coating or in water (control) and packed under 5 kPa O 2 (MAP) or in ambient atmosphere for up to 9 days at 5 °C. Microbial growth, package gas composition, colour, firmness, polyphenol oxidase activity, visual quality and overall sensory flavour of persimmon slices were measured during storage. Coating application combined with active MAP significantly reduced the CO 2 emission and O 2 consumption in the package. The coating was effective in reducing browning and also inhibited the growth of mesophilic aerobic bacteria. Coating also reduced the populations of E. coli, S. enteritidis and L. monocytogenes. The combination of the pectin-based edible coating and active MAP proved to be the most effective treatment to maintain the sensory and microbiological quality of persimmon slices for more than 9 days of storage. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Carbamylation of vimentin is inducible by smoking and represents an independent autoantigen in rheumatoid arthritis

PubMed Central

Ospelt, Caroline; Bang, Holger; Feist, Eugen; Camici, Giovanni; Keller, Stephan; Detert, Jacqueline; Krämer, Anette; Gay, Steffen; Ghannam, Khetam; Burmester, Gerd R

2017-01-01

Objectives Smoking has been connected to citrullination of antigens and formation of anti-citrullinated peptide antibodies (ACPAs) in rheumatoid arthritis (RA). Since smoking can modify proteins by carbamylation (formation of homocitrulline), this study was conducted to investigate these effects on vimentin in animal models and RA. Methods The efficiency of enzymatic carbamylation of vimentin was characterised. B-cell response was investigated after immunisation of rabbits with different vimentin isoforms. Effects of tobacco smoke exposure on carbamylation of vimentin and formation of autoantibodies were analysed in mice. The antibody responses against isoforms of vimentin were characterised with respect to disease duration and smoking status of patients with RA. Results Enzymatic carbamylation of vimentin was efficiently achieved. Subsequent citrullination of vimentin was not disturbed by homocitrullination. Sera from rabbits immunised with carbamylated vimentin (carbVim), in addition to carbVim also recognised human IgG-Fc showing rheumatoid factor-like reactivity. Smoke-exposed mice contained detectable amounts of carbVim and developed a broad immune response against carbamylated antigens. Although the prevalence of anti-carbamylated antibodies in smokers and non-smokers was similar, the titres of carbamylated antibodies were significantly increased in sera of smoking compared with non-smoking RA. CarbVim antibodies were observed independently of ACPAs in early phases of disease and double-positive patients for anti-mutated citrullinated vimentin (MCV) and anti-carbVim antibodies showed an extended epitope recognition pattern towards MCV. Conclusions Carbamylation of vimentin is inducible by cigarette smoke exposure. The polyclonal immune response against modified antigens in patients with RA is not exclusively citrulline-specific and carbamylation of antigens could be involved in the pathogenesis of disease. Trial registration number ISRCTN36745608; EudraCT Number: 2006-003146-41. PMID:28183721

Increased Sensitivity to Testicular Toxicity of Transplacental Benzo[a]pyrene Exposure in Male Glutamate Cysteine Ligase Modifier Subunit Knockout (Gclm−/−) Mice

PubMed Central

Nakamura, Brooke N.; Mohar, Isaac; Lawson, Gregory W.; Cortés, Mabel M.; Hoang, Yvonne D.; Ortiz, Laura; Patel, Reshma; Rau, Bogdan A.; McConnachie, Lisa A.; Kavanagh, Terrance J.; Luderer, Ulrike

2012-01-01

Polycyclic aromatic hydrocarbons (PAHs), like benzo[a]pyrene (BaP), are ubiquitous environmental pollutants formed by the incomplete combustion of organic materials. The tripeptide glutathione (GSH) is a major antioxidant and is important in detoxification of PAH metabolites. Mice null for the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH concentrations. We investigated the effects of Gclm deletion alone on male fertility and spermatogenesis and its effect on the sensitivity of male embryos to the transplacental testicular toxicity of BaP. Gclm−/− males had dramatically decreased testicular and epididymal GCL enzymatic activity and total GSH concentrations compared with Gclm+/+ littermates. Ratios of reduced to oxidized GSH were significantly increased in Gclm−/− testes. GSH reductase enzymatic activity was increased in Gclm−/− epididymides. We observed no changes in fertility, testicular weights, testicular sperm head counts, or testicular histology and subtle changes in cauda epididymal sperm counts, motility, and morphology in Gclm−/− compared with Gclm+/+ males. Prenatal exposure to BaP from gestational day 7 to 16 was dose dependently associated with significantly decreased testicular and epididymal weights, testicular and epididymal sperm counts, and with vacuolated seminiferous tubules at 10 weeks of age. Gclm−/− males exposed prenatally to BaP had greater decreases in testicular weights, testicular sperm head counts, epididymal sperm counts, and epididymal sperm motility than Gclm+/+ littermates. These results show no effects of Gclm deletion alone on male fertility and testicular spermatogenesis and subtle epididymal effects but support increased sensitivity of Gclm−/− males to the transplacental testicular toxicity of BaP. PMID:22253057

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.

A Large Response Range Reflectometric Urea Biosensor Made from Silica-Gel Nanoparticles

PubMed Central

Alqasaimeh, Muawia; Heng, Lee Yook; Ahmad, Musa; Raj, A.S. Santhana; Ling, Tan Ling

2014-01-01

A new silica-gel nanospheres (SiO2NPs) composition was formulated, followed by biochemical surface functionalization to examine its potential in urea biosensor development. The SiO2NPs were basically synthesized based on sol–gel chemistry using a modified Stober method. The SiO2NPs surfaces were modified with amine (-NH2) functional groups for urease immobilization in the presence of glutaric acid (GA) cross-linker. The chromoionophore pH-sensitive dye ETH 5294 was physically adsorbed on the functionalized SiO2NPs as pH transducer. The immobilized urease determined urea concentration reflectometrically based on the colour change of the immobilized chromoionophore as a result of the enzymatic hydrolysis of urea. The pH changes on the biosensor due to the catalytic enzyme reaction of immobilized urease were found to correlate with the urea concentrations over a linear response range of 50–500 mM (R2 = 0.96) with a detection limit of 10 mM urea. The biosensor response time was 9 min with reproducibility of less than 10% relative standard deviation (RSD). This optical urea biosensor did not show interferences by Na+, K+, Mg2+ and NH4+ ions. The biosensor performance has been validated using urine samples in comparison with a non-enzymatic method based on the use of p-dimethylaminobenzaldehyde (DMAB) reagent and demonstrated a good correlation between the two different methods (R2 = 0.996 and regression slope of 1.0307). The SiO2NPs-based reflectometric urea biosensor showed improved dynamic linear response range when compared to other nanoparticle-based optical urea biosensors. PMID:25054632

Comparative study of DNA encapsulation into PLGA microparticles using modified double emulsion methods and spray drying techniques.

PubMed

Oster, C G; Kissel, T

2005-05-01

Recently, several research groups have shown the potential of microencapsulated DNA as adjuvant for DNA immunization and in tissue engineering approaches. Among techniques generally used for microencapsulation of hydrophilic drug substances into hydrophobic polymers, modified WOW double emulsion method and spray drying of water-in-oil dispersions take a prominent position. The key parameters for optimized microspheres are particle size, encapsulation efficiency, continuous DNA release and stabilization of DNA against enzymatic and mechanical degradation. This study investigates the possibility to encapsulate DNA avoiding shear forces which readily degrade DNA during this microencapsulation. DNA microparticles were prepared with polyethylenimine (PEI) as a complexation agent for DNA. Polycations are capable of stabilizing DNA against enzymatic, as well as mechanical degradation. Further, complexation was hypothesized to facilitate the encapsulation by reducing the size of the macromolecule. This study additionally evaluated the possibility of encapsulating lyophilized DNA and lyophilized DNA/PEI complexes. For this purpose, the spray drying and double emulsion techniques were compared. The size of the microparticles was characterized by laser diffractometry and the particles were visualized by scanning electron microscopy (SEM). DNA encapsulation efficiencies were investigated photometrically after complete hydrolysis of the particles. Finally, the DNA release characteristics from the particles were studied. Particles with a size of <10 microm which represent the threshold for phagocytic uptake could be prepared with these techniques. The encapsulation efficiency ranged from 100-35% for low theoretical DNA loadings. DNA complexation with PEI 25?kDa prior to the encapsulation process reduced the initial burst release of DNA for all techniques used. Spray-dried particles without PEI exhibited high burst releases, whereas double emulsion techniques showed continuous release rates.

Automated Solid-Phase Protein Modification with Integrated Enzymatic Digest for Reaction Validation: Application of a Compartmented Microfluidic Reactor for Rapid Optimization and Analysis of Protein Biotinylation

PubMed Central

Fraas, Regina; Diehm, Juliane; Franzreb, Matthias

2017-01-01

Protein modification by covalent coupling of small ligands or markers is an important prerequisite for the use of proteins in many applications. Well-known examples are the use of proteins with fluorescent markers in many in vivo experiments or the binding of biotinylated antibodies via biotin–streptavidin coupling in the frame of numerous bioassays. Multiple protocols were established for the coupling of the respective molecules, e.g., via the C and N-terminus, or via cysteines and lysines exposed at the protein surface. Still, in most cases the conditions of these standard protocols are only an initial guess. Optimization of the coupling parameters like reagent concentrations, pH, or temperature may strongly increase coupling yield and the biological activity of the modified protein. In order to facilitate the process of optimizing coupling conditions, a method was developed which uses a compartmented microfluidic reactor for the rapid screening of different coupling conditions. In addition, the system allows for the integration of an enzymatic digest of the modified protein directly after modification. In combination with a subsequent MALDI-TOF analysis of the resulting fragments, this gives a fast and detailed picture not only of the number and extent of the generated modifications but also of their position within the protein sequence. The described process was demonstrated for biotinylation of green fluorescent protein. Different biotin-excesses and different pH-values were tested in order to elucidate the influence on the modification extent and pattern. In addition, the results of solid-phase based modifications within the microfluidic reactor were compared to modification patterns resulting from coupling trials with unbound protein. As expected, modification patterns of immobilized proteins showed clear differences to the ones of dissolved proteins. PMID:29181376

Identification of RIP-II toxins by affinity enrichment, enzymatic digestion and LC-MS.

PubMed

Fredriksson, Sten-Åke; Artursson, Elisabet; Bergström, Tomas; Östin, Anders; Nilsson, Calle; Åstot, Crister

2015-01-20

Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.

RT-CaCCO process: an improved CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature.

PubMed

Shiroma, Riki; Park, Jeung-yil; Al-Haq, Muhammad Imran; Arakane, Mitsuhiro; Ike, Masakazu; Tokuyasu, Ken

2011-02-01

We improved the CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature (RT). We firstly optimized the RT-lime pretreatment for the lignocellulosic part. When the ratio of lime/dry-biomass was 0.2 (w/w), the RT lime-pretreatment for 7-d resulted in an effect on the enzymatic saccharification of cellulose and xylan equivalent to that of the pretreatment at 120°C for 1h. Sucrose, starch and β-1,3-1,4-glucan, which could be often detected in rice straw, were mostly stable under the RT-lime pretreatment condition. Then, the pretreatment condition in the conventional CaCCO process was modified by the adaptation of the optimized RT lime-pretreatment, resulting in significantly better carbohydrate recoveries via enzymatic saccharification than those of the CaCCO process (120°C for 1 h). Thus, the improved CaCCO process (the RT-CaCCO process) could preserve/pretreat the feedstock at RT in a wet form with minimum loss of carbohydrates. Copyright © 2010 Elsevier Ltd. All rights reserved.

A nanocomposite-based electrochemical sensor for non-enzymatic detection of hydrogen peroxide

PubMed Central

Du, Xin; Chen, Yuan; Dong, Wenhao; Han, Bingkai; Liu, Min; Chen, Qiang; Zhou, Jun

2017-01-01

Hydrogen peroxide (H2O2) plays important signaling roles in normal physiology and disease. However, analyzing the actions of H2O2 is often impeded by the difficulty in detecting this molecule. Herein, we report a novel nanocomposite-based electrochemical sensor for non-enzymatic detection of H2O2. Graphene oxide (GO) was selected as the dopant for the synthesis of polyaniline (PANI), leading to the successful fabrication of a water-soluble and stable GO-PANI composite. GO-PANI was subsequently subject to cyclic voltammetry to generate reduced GO-PANI (rGO-PANI), enhancing the conductivity of the material. Platinum nanoparticles (PtNPs) were then electrodeposited on the surface of the rGO-PANI-modified glassy carbon electrode (GCE) to form an electrochemical H2O2 sensor. Compared to previously reported sensors, the rGO-PANI-PtNP/GCE exhibited an expanded linear range, higher sensitivity, and lower detection limit in the quantification of H2O2. In addition, the sensor displayed outstanding reproducibility and selectivity in real-sample examination. Our study suggests that the rGO-PANI-PtNP/GCE may have broad utility in H2O2 detection under physiological and pathological conditions. PMID:28035076

Tumor Targeting and Drug Delivery by Anthrax Toxin.

PubMed

Bachran, Christopher; Leppla, Stephen H

2016-07-01

Anthrax toxin is a potent tripartite protein toxin from Bacillus anthracis. It is one of the two virulence factors and causes the disease anthrax. The receptor-binding component of the toxin, protective antigen, needs to be cleaved by furin-like proteases to be activated and to deliver the enzymatic moieties lethal factor and edema factor to the cytosol of cells. Alteration of the protease cleavage site allows the activation of the toxin selectively in response to the presence of tumor-associated proteases. This initial idea of re-targeting anthrax toxin to tumor cells was further elaborated in recent years and resulted in the design of many modifications of anthrax toxin, which resulted in successful tumor therapy in animal models. These modifications include the combination of different toxin variants that require activation by two different tumor-associated proteases for increased specificity of toxin activation. The anthrax toxin system has proved to be a versatile system for drug delivery of several enzymatic moieties into cells. This highly efficient delivery system has recently been further modified by introducing ubiquitin as a cytosolic cleavage site into lethal factor fusion proteins. This review article describes the latest developments in this field of tumor targeting and drug delivery.

Characterization and enzymatic hydrolysis of wood from transgenic Pinus taeda engineered with syringyl lignin or reduced lignin content

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

Edmunds, Charles W.; Peralta, Perry; Kelley, Stephen S.

Softwood is an abundant resource; however, currently its utilization for bioconversion to obtain platform sugars is limited. Pinus taeda trees which were genetically modified to either produce S lignin or to decrease lignin content were characterized with a suite of analytic techniques. Syringyl lignin was visualized in the secondary xylem of one genetic line with Maule staining. Solid-state nuclear magnetic resonance identified the S lignin units were coupled into the lignin through β-O-4 linkages, and thioacidolysis measured approximately 13% S lignin content in the same sample. Reductions of the lignin of as much as 33% were observed in the transgenics.more » To better understand how these modifications affect bioconversion, their amenability to hot water and dilute acid pretreatments and enzymatic hydrolysis was evaluated. Lignin reductions resulted in 1.9-3.2-fold increases in glucose release compared to the control. However, no apparent benefit was observed by S lignin incorporation at the concentrations reported in this study. Finally, these results highlight the potential for softwood cell wall properties to be improved for bioenergy/biochemical applications.« less

Colorimetric stack pad immunoassay for bacterial identification.

PubMed

Eltzov, Evgeni; Marks, Robert S

2017-01-15

A new colorimetric immunoassay concept, utilizing conventional lateral flow membranes (e.g., conjugation, sample, absorption and nitrocellulose), were placed in a different configuration in a stacking manner, where the liquid sample that may contain the analyte diffuses from the bottom to the upper-most layer. The key element of this proprietary technology is a capture layer, where a nitrocellulose membrane is modified with the target analyte of interest, namely in this study target Escherichia coli. During the immunoassay operation, samples contaminated with the target bacteria will conjugate to their corresponding HRP-antibodies laying in wait and the immune-target measurand complex flows by capillarity towards the upper-most layer to generate a colorimetric signal (positive answer) through an enzymatic reaction. In target-free samples, previously immobilized target bacteria on the capture layer will prevent the HRP-labeled anti-target antibodies from migrating to the upper-most layer, where the enzymatic substrate lays in wait. After optimization, the sensitivity of this approach was found to be 1,000 folds higher than ELISAs (10 2 cellsmL -1 ). The advantages of the stacked pad assay include: miniaturization, operational simplicity, fast response time (less than 5min), useful sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

Oxidant-antioxidant imbalance in horses infected with equine infectious anaemia virus.

PubMed

Bolfă, Pompei Florin; Leroux, Caroline; Pintea, Adela; Andrei, Sanda; Cătoi, Cornel; Taulescu, Marian; Tăbăran, Flaviu; Spînu, Marina

2012-06-01

This study assesses the impact of equine infectious anaemia virus (EIAV) infection on the oxidant/antioxidant equilibrium of horses. Blood samples from 96 Romanian horses aged 1-25 years, were divided into different groups according to their EIAV-infection status, age, and time post-seroconversion. The effect of infection on oxidative stress was estimated by measuring enzymatic antioxidants (superoxide dismutase [SOD], glutathione peroxidase [GPx] and catalase), non-enzymatic antioxidants (uric acid and carotenoids), and lipid peroxidation (malondialdehyde [MDA]). Infection modified the oxidant/antioxidant equilibrium in the horses, influencing GPx and uric acid levels (P<0.05). Time post-seroconversion also contributed to oxidative stress imbalance, exhibiting a significant influence on both SOD and MDA concentrations in the blood (P<0.05). Animal age did not have a significant influence on oxidative stress. Recently infected horses (<1 year following seroconversion), and horses >5 years old, represented the most vulnerable category in terms of oxidative stress, followed by recently infected animals <5 years old. The results of this study are novel in implicating EIAV infection in the development of oxidative stress in horses. Copyright © 2011 Elsevier Ltd. All rights reserved.

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?

Graphene oxide-labeled sandwich-type impedimetric immunoassay with sensitive enhancement based on enzymatic 4-chloro-1-naphthol oxidation.

PubMed

Hou, Li; Cui, Yuling; Xu, Mingdi; Gao, Zhuangqiang; Huang, Jianxin; Tang, Dianping

2013-09-15

A new sandwich-type impedimetric immunosensor based on functionalized graphene oxide nanosheets with a high ratio of horseradish peroxidase (HRP) and detection antibody was developed for the detection of carcinoembryonic antigen (CEA) by coupling with enzymatic biocatalytic precipitation of 4-chloro-1-naphthol (4-CN) on the captured antibody-modified glassy carbon electrode. Two molecular tags (with and without the graphene oxide nanosheets) were investigated for the detection of CEA and improved analytical features were acquired with the graphene-based labeling. With the labeling method, the performance and factors influencing the properties of the impedimetric immunosensors were also studied and evaluated. Under the optimal conditions, the dynamic concentration range of the impedimetric immunosensors spanned from 1.0pgmL(-1) to 80ngmL(-1) CEA with a detection limit (LOD) of 0.64pgmL(-1). Intra- and inter-assay coefficients of variation were less than 7.5% and 11%, respectively. Additionally, the methodology was evaluated for CEA analysis of 10 clinical serum samples and 5 diluted serum samples, receiving in a good accordance with the results obtained by the impedimetric immunoassay and the commercialized electrochemiluminescent method. Copyright © 2013 Elsevier B.V. All rights reserved.

Separating esterase targets of organophosphorus compounds in the brain by preparative chromatography.

PubMed

Mangas, I; Vilanova, E; Benabent, M; Estévez, J

2014-02-10

Low level exposure to organophosphorus esters (OPs) may cause long-term neurological effects and affect specific cognition domains in experimental animals and humans. Action on known targets cannot explain most of these effects by. Soluble carboxylesterases (EC 3.1.1.1) of chicken brain have been kinetically discriminated using paraoxon, mipafox and phenylmethyl sulfonylfluoride as inhibitors and phenyl valerate as a substrate. Three different enzymatic components were discriminated and called Eα, Eβ and Eγ. In this work, a fractionation procedure with various steps was developed using protein native separation methods by preparative HPLC. Gel permeation chromatography followed by ion exchange chromatography allowed enriched fractions with different kinetic behaviors. The soluble chicken brain fraction was fractionated, while total esterase activity, proteins and enzymatic components Eα, Eβ and Eγ were monitored in each subfraction. After the analysis, 13 fractions were pooled and conserved. Preincubation of the soluble chicken brain fraction of with the organophosphorus mipafox gave rise to a major change in the ion exchange chromatography profile, but not in the molecular exchanged chromatography profile, which suggest that mipafox permanently modifies the ionic properties of numerous proteins. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Wild Roman chamomile extracts and phenolic compounds: enzymatic assays and molecular modelling studies with VEGFR-2 tyrosine kinase.

PubMed

Guimarães, Rafaela; Calhelha, Ricardo C; Froufe, Hugo J C; Abreu, Rui M V; Carvalho, Ana Maria; Queiroz, Maria João R P; Ferreira, Isabel C F R

2016-01-01

Angiogenesis is a process by which new blood vessels are formed from the pre-existing vasculature, and it is a key process that leads to tumour development. Some studies have recognized phenolic compounds as chemopreventive agents; flavonoids, in particular, seem to suppress the growth of tumor cells modifying the cell cycle. Herein, the antiangiogenic activity of Roman chamomile (Chamaemelum nobile L.) extracts (methanolic extract and infusion) and the main phenolic compounds present (apigenin, apigenin-7-O-glucoside, caffeic acid, chlorogenic acid, luteolin, and luteolin-7-O-glucoside) was evaluated through enzymatic assays using the tyrosine kinase intracellular domain of the Vascular Endothelium Growth Factor Receptor-2 (VEGFR-2), which is a transmembrane receptor expressed fundamentally in endothelial cells involved in angiogenesis, and molecular modelling studies. The methanolic extract showed a lower IC50 value (concentration that provided 50% of VEGFR-2 inhibition) than the infusion, 269 and 301 μg mL(-1), respectively. Regarding phenolic compounds, luteolin and apigenin showed the highest capacity to inhibit the phosphorylation of VEGFR-2, leading us to believe that these compounds are involved in the activity revealed by the methanolic extract.

Characterization and enzymatic hydrolysis of wood from transgenic Pinus taeda engineered with syringyl lignin or reduced lignin content

DOE PAGES

Edmunds, Charles W.; Peralta, Perry; Kelley, Stephen S.; ...

2017-02-22

Softwood is an abundant resource; however, currently its utilization for bioconversion to obtain platform sugars is limited. Pinus taeda trees which were genetically modified to either produce S lignin or to decrease lignin content were characterized with a suite of analytic techniques. Syringyl lignin was visualized in the secondary xylem of one genetic line with Maule staining. Solid-state nuclear magnetic resonance identified the S lignin units were coupled into the lignin through β-O-4 linkages, and thioacidolysis measured approximately 13% S lignin content in the same sample. Reductions of the lignin of as much as 33% were observed in the transgenics.more » To better understand how these modifications affect bioconversion, their amenability to hot water and dilute acid pretreatments and enzymatic hydrolysis was evaluated. Lignin reductions resulted in 1.9-3.2-fold increases in glucose release compared to the control. However, no apparent benefit was observed by S lignin incorporation at the concentrations reported in this study. Finally, these results highlight the potential for softwood cell wall properties to be improved for bioenergy/biochemical applications.« less

Dentin biomodification potential depends on polyphenol source.

PubMed

Aguiar, T R; Vidal, C M P; Phansalkar, R S; Todorova, I; Napolitano, J G; McAlpine, J B; Chen, S N; Pauli, G F; Bedran-Russo, A K

2014-04-01

Although proanthocyanidins (PACs) modify dentin, the effectiveness of different PAC sources and the correlation with their specific chemical composition are still unknown. This study describes the chemical profiling of natural PAC-rich extracts from 7 plants using ultra high pressure/performance liquid chromatography (UHPLC) to determine the overall composition of these extracts and, in parallel, comprehensively evaluate their effect on dentin properties. The total polyphenol content of the extracts was determined (as gallic acid equivalents) using Folin-Ciocalteau assays. Dentin biomodification was assessed by the modulus of elasticity, mass change, and resistance to enzymatic biodegradation. Extracts with a high polyphenol and PAC content from Vitis vinifera, Theobroma cacao, Camellia sinensis, and Pinus massoniana induced a significant increase in modulus of elasticity and mass. The UHPLC analysis showed the presence of multiple types of polyphenols, ranging from simple phenolic acids to oligomeric PACs and highly condensed tannins. Protective effect against enzymatic degradation was observed for all experimental groups; however, statistically significant differences were observed between plant extracts. The findings provide clear evidence that the dentin bioactivities of PACs are source dependent, resulting from a combination of concentration and specific chemical constitution of the complex PAC mixtures.

Biotransformation of perfumery terpenoids, (−)-ambrox® by a fungal culture Macrophomina phaseolina and a plant cell suspension culture of Peganum harmala

PubMed Central

2012-01-01

Background Biotransformation offers chemo enzymatic system to modify the compounds into their novel analogues which are difficult to synthesize by chemical methods. This paper describes the biotransformational studies of ambrox, one of the most important components of natural Ambergris (wale sperm) with fungal and plant cell culture. Results Biotransformation of (−)-ambrox (1) with a fungal cell culture of Macrophomina phaseolina and a plant cell suspension cultures of Peganum harmala yielded oxygenated products, 3β-hydroxyambrox (2), 6β-hydroxyambrox (3), 1α-hydroxy-3oxoambrox (4), 1α,3β-dihydroxyambrox (5), 13,14,15,16-tetranorlabdane-3-oxo-8,12-diol (6), 3-oxoambrox (7), 2α-hydroxyambrox (8), 3β-hydroxysclareolide (9), and 2α,3β-dihydroxyambrox (10). Metabolite 4 was found to be new compound. These metabolites were structurally characterized on the basis of spectroscopic studies. Conclusion Nine oxygenated metabolites of (−)-ambrox (1) were obtained from Macrophomina phaseolina and Peganum harmala. Enzymatic system of screened organisms introduced hydroxyl and keto functionalities at various positions of compound 1 in a stereo- and regio-controlled manner. PMID:22863186

Immobilization of a mediator onto carbon cloth electrode and employment of the modified electrode to an electroenzymatic bioreactor.

PubMed

Jeong, Eun-Seon; Sathishkumar, Muthuswamy; Jayabalan, Rasu; Jeong, Su-Hyeon; Park, Song-Yie; Mun, Sung-Phil; Yun, Sei-Eok

2012-10-01

5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) was selected as an electron transfer mediator and was covalently immobilized onto high porosity carbon cloth to employ as a working electrode in an electrochemical NAD(+)-regeneration process, which was coupled to an enzymatic reaction. The voltammetric behavior of DTNB attached to carbon cloth resembled that of DTNB in buffered aqueous solution, and the electrocatalytic anodic current grew continuously upon addition of NADH at different concentrations, indicating that DTNB is immobilized to carbon cloth effectively and the immobilized DTNB is active as a soluble one. The bioelectrocatalytic NAD+ regeneration was coupled to the conversion of L-glutamate into alpha-ketoglutarate by L-glutamate dehydrogenase within the same microreactor. The conversion at 3 mM monosodium glutamate was very rapid, up to 12 h, to result in 90%, and then slow up to 24 h, showing 94%, followed by slight decrease. Low conversion was shown when substrate concentration exceeding 4 mM was tested, suggesting that L-glutamate dehydrogenase is inhibited by alpha-ketoglutarate. However, our electrochemical NAD+ regeneration procedure looks advantageous over the enzymatic procedure using NADH oxidase, from the viewpoint of reaction time to completion.

Analysis of non-enzymatically glycated peptides: neutral-loss-triggered MS3 versus multi-stage activation tandem mass spectrometry

PubMed Central

Zhang, Qibin; Petyuk, Vladislav A.; Schepmoes, Athena A.; Orton, Daniel J.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Metz, Thomas O.

2009-01-01

Non-enzymatic glycation of tissue proteins has important implications in the development of complications of diabetes mellitus. While electron transfer dissociation (ETD) has been shown to outperform collision-induced dissociation (CID) in sequencing glycated peptides by tandem mass spectrometry, ETD instrumentation is not yet widely available and often suffers from significantly lower sensitivity than CID. In this study, we evaluated different advanced CID techniques (i.e., neutral-loss-triggered MS3 and multi-stage activation) during liquid chromatography/multi-stage mass spectrometric (LC/MSn) analyses of Amadori-modified peptides enriched from human serum glycated in vitro. During neutral-loss-triggered MS3 experiments, MS3 scans triggered by neutral losses of 3 H2O or 3 H2O + HCHO produced similar results in terms of glycated peptide identifications. However, neutral losses of 3 H2O resulted in significantly more glycated peptide identifications during multi-stage activation experiments. Overall, the multi-stage activation approach produced more glycated peptide identifications, while the neutral-loss-triggered MS3 approach resulted in much higher specificity. Both techniques are viable alternatives to ETD for identifying glycated peptides. PMID:18763275

Modifying the red cell surface: towards an ABO-universal blood supply.

PubMed

Olsson, Martin L; Clausen, Henrik

2008-01-01

Eliminating the risk for ABO-incompatible transfusion errors and simplifying logistics by creating a universal blood inventory is a challenging idea. Goldstein and co-workers pioneered the field of enzymatic conversion of blood group A and B red blood cells (RBCs) to O (ECO). Using alpha-galactosidase from coffee beans to produce B-ECO RBCs, proof of principle for this revolutionary concept was achieved in clinical trials. However, because this enzyme has poor kinetic properties and low pH optimum the process was not economically viable. Conversion of group A RBCs was only achieved with the weak A2 subgroup with related enzymes having acidic pH optima. More recently, the identification of entirely new families of bacterial exoglycosidases with remarkably improved kinetic properties for cleaving A and B antigens has reinvigorated the field. Enzymatic conversion of groups A, B and AB RBCs with these novel enzymes resulting in ECO RBCs typing as O can now be achieved with low enzyme protein consumption, short incubation times and at neutral pH. Presently, clinical trials evaluating safety and efficacy of ECO RBCs are ongoing. Here, we review the status of the ECO technology, its impact and potential for introduction into clinical component preparation laboratories.

Enzymatic modification of corn starch with 4-α-glucanotransferase results in increasing slow digestible and resistant starch.

PubMed

Jiang, Huan; Miao, Ming; Ye, Fan; Jiang, Bo; Zhang, Tao

2014-04-01

In this study, partial 4-α-glucanotransferase (4αGT) treatment was used to modulate the fine structure responsible for the slow digestion and resistant property of starch. Normal corn starch modified using 4αGT for 4h showed an increase of slowly digestible starch from 9.40% to 20.92%, and resistant starch from 10.52 to 17.63%, respectively. The 4αGT treatment decreased the content of amylose from 32.6% to 26.8%. The molecular weight distribution and chain length distribution of 4αGT-treated starch showed a reduction of molecular weight and a great number of short (DP<13) and long (DP>30) chains through cleaving and reorganization of starch molecules. Both the short and long chain fractions of modified amylopectin were attributed to the low in vitro digestibility. The viscosity was inversely related to the digestibility of the 4αGT-treated starch. These results suggested that the 4αGT modified starch synthesized the novel amylopectin clusters with slow digestible and resistant character. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

Synthesis and characterization of graphene quantum dots/CoNiAl-layered double-hydroxide nanocomposite: Application as a glucose sensor.

PubMed

Samuei, Sara; Fakkar, Jila; Rezvani, Zolfaghar; Shomali, Ashkan; Habibi, Biuck

2017-03-15

In the present work, a novel nanocomposite based on the graphene quantum dots and CoNiAl-layered double-hydroxide was successfully synthesized by co-precipitation method. To achieve the morphological, structural and compositional information, the resulted nanocomposite was characterized by scanning electron microscopy X-ray diffraction, thermal gravimetric analysis, Fourier transform infrared spectroscopy, and photoluminescence. Then, the nanocomposite was used as a modifier to fabricate a modified carbon paste electrode as a non-enzymatic sensor for glucose determination. Electrochemical behavior and determination of glucose at the nanocomposite modified carbon paste electrode were investigated by cyclic voltammetry and chronoamperometry methods, respectively. The prepared sensor offered good electrocatalytic properties, fast response time, high reproducibility and stability. At the optimum conditions, the constructed sensor exhibits wide linear range; 0.01-14.0 mM with a detection limit of 6 μM (S/N = 3) and high sensitivity of 48.717 μAmM -1 . Finally, the sensor was successfully applied to determine the glucose in real samples which demonstrated its applicability. Copyright © 2017 Elsevier Inc. All rights reserved.

Methylglyoxal produces more changes in biochemical and biophysical properties of human IgG under high glucose compared to normal glucose level

PubMed Central

Khan, Mohd Adnan; Arif, Zarina; Khan, Mohd Asad; Moinuddin

2018-01-01

Hyperglycaemia triggers increased production of methylglyoxal which can cause gross modification in proteins’ structure vis-a-vis function though advanced glycation end products (AGEs). The AGEs may initiate vascular and nonvascular pathologies. In this study, we have examined the biochemical and biophysical changes in human IgG under normal and high glucose after introducing methylglyoxal into the assay mixture. This non-enzymatic reaction mainly engaged lysine residues as indicated by TNBS results. The UV results showed hyperchromicity in modified-IgG samples while fluorescence data supported AGEs formation during the course of reaction. Shift in amide I and amide II band position indicated perturbations in secondary structure. Increase carbonyl content and decrease in sulfhydryl suggests that the modification is accompanied by oxidative stress. All modified-IgG samples showed more thermostability than native IgG; the highest Tm was shown by IgG-high glucose-MGO variant. Results of ANS, Congo red and Thioflavin T dyes clearly suggest increase in hydrophobic patches and aggregation, respectively. SEM and TEM images support aggregates generation in modified-IgG samples. PMID:29351321

Increase of rutin antioxidant activity by generating Maillard reaction products with lysine.

PubMed

Zhang, Ru; Zhang, Bian-Ling; He, Ting; Yi, Ting; Yang, Ji-Ping; He, Bin

2016-06-01

Rutin exists in medicinal herbs, fruits, vegetables, and a number of plant-derived sources. Dietary sources containing rutin are considered beneficial because of their potential protective roles in multiple diseases related to oxidative stresses. In the present study, the change and antioxidation activity of rutin in Maillard reaction with lysine through a heating process were investigated. There is release of glucose and rhamnose that interact with lysine to give Maillard reaction products (MRPs), while rutin is converted to less-polar quercetin and a small quantity of isoquercitrin. Because of their high cell-membrane permeability, the rutin-lysine MRPs increase the free radical-scavenging activity in HepG2 cells, showing cellular antioxidant activity against Cu(2+)-induced oxidative stress higher than that of rutin. Furthermore, the MRPs significantly increased the Cu/Zn SOD (superoxide dismutase) activity and Cu/Zn SOD gene expression of HepG2 cells, consequently enhancing antioxidation activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Accumulation of Flavonoid Glycosides and UFGT Gene Expression in Mulberry Leaves (Morus alba L.) before and after Frost.

PubMed

Yu, Xiaofeng; Zhu, Yiling; Fan, Jingyi; Wang, Dujun; Gong, Xiaohui; Ouyang, Zhen

2017-08-01

In order to determine the molecular mechanism underlying the influence of frost on chemical changes in mulberry leaves, the UFGT activity, expression level, and accumulation of flavonoid glycosides in mulberry leaves (Morus alba L.) were studied. The expression of UFGT gene was investigated by quantitative real-time PCR (qRT-PCR) and the UFGT activity, accumulation of flavonoid glycosides was studied by high performance liquid chromatography. Then, the correlation between the expression level of UFGT, the UFGT activity, and the flavonoid glycosides accumulation with temperature was explored. The accumulation of isoquercitrin and astragalin is significantly positively correlated with UFGT gene expression and UFGT activity. On the contrary, the average temperature was significantly negatively correlated with the level of UFGT gene expression and UFGT activity. The results show that after frost, low temperature can induce the expression of UFGT gene in mulberry leaves, resulting in the accumulation of flavonoid glycosides. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

Phenolic contents and cellular antioxidant activity of Chinese hawthorn "Crataegus pinnatifida".

PubMed

Wen, Lingrong; Guo, Xingbo; Liu, Rui Hai; You, Lijun; Abbasi, Arshad Mehmood; Fu, Xiong

2015-11-01

It is evident from various epidemiological studies that consumption of fruits and vegetables is essential to maintain health and in the disease prevention. Present study was designed to examine phenolic contents and antioxidant properties of three varieties of Crataegus pinnatifida (Chinese hawthorn). Shanlihong variety exhibited elevated levels of total phenolics and flavonoid contents, including free and bond phenolics. Procyanidin B2 was most abundant phenolic compound in all samples, followed by epicatechin, chlorogenic acid, hyperoside, and isoquercitrin. The free ORAC values, and free hydro-PSC values were 398.3-555.8 μmol TE/g DW, and 299.1-370.9 μmol VCE/g DW, respectively. Moreover, the free cellular antioxidant activity (CAA) values were 678-1200 μmol of QE/100 g DW in the no PBS wash protocol, and 345.9-532.9 μmol of QE/100 g DW in the PBS wash protocol. C. pinnatifida fruit could be valuable to promote consumer health. Copyright © 2015 Elsevier Ltd. All rights reserved.

Antioxidant, xanthine oxidase and lipoxygenase inhibitory activities and phenolics of Bauhinia rufescens Lam. (Caesalpiniaceae).

PubMed

Compaoré, M; Lamien, C E; Lamien-Meda, A; Vlase, L; Kiendrebeogo, M; Ionescu, C; Nacoulma, O G

2012-01-01

An aqueous acetone extract of the stem with the leaves of Bauhinia rufescens and its fractions were analysed for their antioxidant and enzyme-inhibitory activities, as well as their phytochemical composition. For measurement of the antioxidant activities, the 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azinobis(3-ethylbenzoline-6-sulphonate) and the ferric-reducing methods were used. The results indicated that the aqueous acetone, its ethyl acetate and n-butanol fractions possessed considerable antioxidant activity. Further, the xanthine oxidase and lipoxygenase inhibitory assays showed that the n-butanol fraction possessed compounds that can inhibit both these enzymes. In the phytochemical analysis, the ethyl acetate and the n-butanol fractions of the aqueous acetone extract were screened by HPLC-MS for their phenolic content. The results indicated the presence of hyperoside, isoquercitrin, rutin quercetin, quercitrin, p-coumaric and ferulic acids in the non-hydrolysed fractions. In the hydrolysed fractions, kaempferol, p-coumaric and ferulic acids were identified.

Reversing DNA Methylation: Mechanisms, Genomics, and Biological Functions

PubMed Central

Wu, Hao; Zhang, Yi

2014-01-01

Methylation of cytosines in the mammalian genome represents a key epigenetic modification and is dynamically regulated during development. Compelling evidence now suggests that dynamic regulation of DNA methylation is mainly achieved through a cyclic enzymatic cascade comprised of cytosine methylation, iterative oxidation of methyl group by TET dioxygenases, and restoration of unmodified cytosines by either replication-dependent dilution or DNA glycosylase-initiated base excision repair. In this review, we discuss the mechanism and function of DNA demethylation in mammalian genomes, focusing particularly on how developmental modulation of the cytosine-modifying pathway is coupled to active reversal of DNA methylation in diverse biological processes. PMID:24439369

H2‐Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration

PubMed Central

Gutiérrez‐Sanz, Óscar; Natale, Paolo; Márquez, Ileana; Marques, Marta C.; Zacarias, Sonia; Pita, Marcos; Pereira, Inês A. C.

2016-01-01

Abstract ATP, the molecule used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1F0‐ATPase in a lipid membrane can couple the electrochemical oxidation of H2 to the synthesis of ATP. This electrode‐assisted conversion of H2 gas into ATP could serve to generate this biochemical fuel locally when required in biomedical devices or enzymatic synthesis of valuable products. PMID:26991333

An Electrochemical DNA Sensing System Using Modified Nanoparticle Probes for Detecting Methicillin-Resistant Staphylococcus aureus.

PubMed

Sakamoto, Hiroaki; Amano, Yoshihisa; Satomura, Takenori; Suye, Shin-Ichiro

2017-01-01

We have developed a novel, highly sensitive, biosensing system for detecting methicillin-resistant Staphylococcus aureus (MRSA). The system employs gold nanoparticles (AuNPs), magnetic nanoparticles (mNPs), and an electrochemical detection method. We have designed and synthesized ferrocene- and single-stranded DNA-conjugated nanoparticles that hybridize to MRSA DNA. Hybridized complexes are easily separated by taking advantage of mNPs. A current response could be obtained through the oxidation of ferrocene on the AuNP surface when a constant potential of +250 mV vs. Ag/AgCl is applied. The enzymatic reaction of L-proline dehydrogenase provides high signal amplification. This sensing system, using a nanoparticle-modified probe, has the ability to detect 10 pM of genomic DNA from MRSA without amplification by the polymerase chain reaction. Current responses are linearly related to the amount of genomic DNA in the range of 10-166 pM. Selectivity is confirmed by demonstrating that this sensing system could distinguish MRSA from Staphylococcus aureus (SA) DNA.

Pathogen trafficking pathways and host phosphoinositide metabolism.

PubMed

Weber, Stefan S; Ragaz, Curdin; Hilbi, Hubert

2009-03-01

Phosphoinositide (PI) glycerolipids are key regulators of eukaryotic signal transduction, cytoskeleton architecture and membrane dynamics. The host cell PI metabolism is targeted by intracellular bacterial pathogens, which evolved intricate strategies to modulate uptake processes and vesicle trafficking pathways. Upon entering eukaryotic host cells, pathogenic bacteria replicate in distinct vacuoles or in the host cytoplasm. Vacuolar pathogens manipulate PI levels to mimic or modify membranes of subcellular compartments and thereby establish their replicative niche. Legionella pneumophila, Brucella abortus, Mycobacterium tuberculosis and Salmonella enterica translocate effector proteins into the host cell, some of which anchor to the vacuolar membrane via PIs or enzymatically turnover PIs. Cytoplasmic pathogens target PI metabolism at the plasma membrane, thus modulating their uptake and antiapoptotic signalling pathways. Employing this strategy, Shigella flexneri directly injects a PI-modifying effector protein, while Listeria monocytogenes exploits PI metabolism indirectly by binding to transmembrane receptors. Thus, regardless of the intracellular lifestyle of the pathogen, PI metabolism is critically involved in the interactions with host cells.

Pretreatment of flaxseed protein isolate by high hydrostatic pressure: Impacts on protein structure, enzymatic hydrolysis and final hydrolysate antioxidant capacities.

PubMed

Perreault, Véronique; Hénaux, Loïc; Bazinet, Laurent; Doyen, Alain

2017-04-15

The effect of high hydrostatic pressure (HHP) on flaxseed protein structure and peptide profiles, obtained after protein hydrolysis, was investigated. Isolated flaxseed protein (1%, m/v) was subjected to HHP (600MPa, 5min or 20min at 20°C) prior to hydrolysis with trypsin only and trypsin-pronase. The results demonstrated that HHP treatment induced dissociation of flaxseed proteins and generated higher molecular weight aggregates as a function of processing duration. Fluorescence spectroscopy showed that HHP treatment, as well as processing duration, had an impact on flaxseed protein structure since exposition of hydrophobic amino acid tyrosine was modified. Except for some specific peptides, the concentrations of which were modified, similar peptide profiles were obtained after hydrolysis of pressure-treated proteins using trypsin. Finally, hydrolysates obtained using trypsin-pronase had a greater antioxidant capacity (ORAC) than control samples; these results confirmed that HHP enhanced the generation of antioxidant peptides. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ethanol production from horticultural waste treated by a modified organosolv method.

PubMed

Geng, Anli; Xin, Fengxue; Ip, Jun-yu

2012-01-01

In this study, we investigated the use of horticultural waste (HW) collected in Singapore as a renewable raw material for bioethanol production. A modified organosolv method using ethanol cooking under mild conditions followed by H(2)O(2) post-treatment was investigated for HW pretreatment. It was found that the addition of acid catalysts in the pretreatment process was not critical and post-treatment using H(2)O(2) was essential for the enhancement of HW digestibility. Enzymatic hydrolysis of the organosolv pretreated HW with 17.5% solid content, enzyme loading of 20 FPU/g HW of filter paper cellulase, and 80 CBU/g HW of β-glucosidase resulted in a HW hydrolysate containing 26.9 g/L reducing sugar after 72 h. Fermentation of the above hydrolysate medium produced 11.69 g/L ethanol at 8h using Saccharomyces cerevisiae. It proved that horticultural waste was a potential feedstock for fuel ethanol production and organosolv pretreatment method developed in this study was effective. Copyright © 2011 Elsevier Ltd. All rights reserved.

Secretory expression of the non-secretory-type Lentinula edodes laccase by Aspergillus oryzae.

PubMed

Yano, Akira; Kikuchi, Sayaka; Nakagawa, Yuko; Sakamoto, Yuichi; Sato, Toshitsugu

2009-01-01

The shiitake mushroom, Lentinula edodes, has an extracelluar secretory-type laccase, Lcc1, and a fruiting-body-accumulation-type laccase, Lcc4. We previously reported the production of Lcc1 by plant cells, but had difficulty producing Lcc4. Here, we report the production of Lcc1 and Lcc4 by Aspergillus oryzae and the extracellular secretory production of Lcc4 using a modified secretion signal peptide (SP) from Lcc1. Sp-Lcc4 produced by A. oryzae had biochemical activities similar to Lcc4 produced by L. edodes. Lcc1 did not react with beta-(3,4-dihydroxyphenol) alanine (DOPA), but Lcc4 from L. edodes and A. oryzae could oxidize DOPA. K(M) values for the substrates 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate), 2,6-dimethoxyphenol, guaiacol, pyrogallol, and catechol were similar for Lcc4 and Sp-Lcc4. In conclusion, a non-secretory-type fungal laccase is secreted into the culture media with its original enzymatic properties by exploiting modified secretory signal peptide. 2008 Elsevier GmbH.

Improvement of the stability and activity of immobilized glucose oxidase on modified iron oxide magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Abbasi, Mahboube; Amiri, Razieh; Bordbar, Abdol-Kalegh; Ranjbakhsh, Elnaz; Khosropour, Ahmad-Reza

2016-02-01

Immobilized proteins and enzymes are widely investigated in the medical field as well as the food and environmental fields. In this study, glucose oxidase (GOX) was covalently immobilized on the surface of modified iron oxide magnetic nanoparticles (MIMNs) to produce a bioconjugate complex. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to the size, shape and structure characterization of the MIMNs. Binding of GOX to these MIMNs was confirmed by using FT-IR spectroscopy. The stability of the immobilized and free enzyme at different temperature and pH values was investigated by measuring the enzymatic activity. These studies reveal that the enzyme's stability is enhanced by immobilization. Further experiments showed that the storage stability of the enzyme is improved upon binding to the MIMNs. The results of kinetic measurements suggest that the effect of the immobilization process on substrate and product diffusion is small. Such bioconjugates can be considered as a catalytic nanodevice for accelerating the glucose oxidation reaction for biotechnological purposes.

Effect of mineral elements on physicochemical properties of oxidised starches and generation of free radicals.

PubMed

Pietrzyk, Sławomir; Fortuna, Teresa; Królikowska, Karolina; Rogozińska, Ewelina; Labanowska, Maria; Kurdziel, Magdalena

2013-09-12

The objective of this study was to determine the effect of enrichment of oxidised starches with mineral compounds on their physicochemical properties and capability for free radical generation. Potato and spelt wheat starches were oxidised with sodium hypochlorite and, afterwards, modified with ions of potassium, magnesium and iron. The modified starches were analysed for: content of mineral elements, colour parameters (L*a*b*), water binding capacity solubility in water at temperature of 50 and 80 °C, and susceptibility to enzymatic hydrolysis with α-amylase. In addition, thermodynamic characteristics of gelatinisation was determined by differential scanning calorimetry (DSC), and the number and character of thermally generated free radicals was assayed using electron paramagnetic resonance (EPR). Based on the results achieved, it was concluded that the quantity of incorporated minerals and changes in the assayed physicochemical parameters depended not only on the botanical type of starch but also on the type of the incorporated mineral element. Copyright © 2013 Elsevier Ltd. All rights reserved.

Rheological Enhancement of Pork Myofibrillar Protein-Lipid Emulsion Composite Gels via Glucose Oxidase Oxidation/Transglutaminase Cross-Linking Pathway.

PubMed

Wang, Xu; Xiong, Youling L; Sato, Hiroaki

2017-09-27

Porcine myofibrillar protein (MP) was modified with glucose oxidase (GluOx)-iron that produces hydroxyl radicals then subjected to microbial transglutaminase (TGase) cross-linking in 0.6 M NaCl at 4 °C. The resulting aggregation and gel formation of MP were examined. The GluOx-mediated oxidation promoted the formation of both soluble and insoluble protein aggregates via disulfide bonds and occlusions of hydrophobic groups. The subsequent TGase treatment converted protein aggregates into highly cross-linked polymers. MP-lipid emulsion composite gels formed with such polymers exhibited markedly enhanced gelling capacity: up to 4.4-fold increases in gel firmness and 3.5-fold increases in gel elasticity over nontreated protein. Microstructural examination showed small oil droplets dispersed in a densely packed gel matrix when MP was oxidatively modified, and the TGase treatment further contributed to such packing. The enzymatic GluOx oxidation/TGase treatment shows promise to improve the textural properties of emulsified meat products.

Glassy carbon electrodes sequentially modified by cysteamine-capped gold nanoparticles and poly(amidoamine) dendrimers generation 4.5 for detecting uric acid in human serum without ascorbic acid interference.

PubMed

Ramírez-Segovia, A S; Banda-Alemán, J A; Gutiérrez-Granados, S; Rodríguez, A; Rodríguez, F J; Godínez, Luis A; Bustos, E; Manríquez, J

2014-02-17

Glassy carbon electrodes (GCE) were sequentially modified by cysteamine-capped gold nanoparticles (AuNp@cysteamine) and PAMAM dendrimers generation 4.5 bearing 128-COOH peripheral groups (GCE/AuNp@cysteamine/PAMAM), in order to explore their capabilities as electrochemical detectors of uric acid (UA) in human serum samples at pH 2. The results showed that concentrations of UA detected by cyclic voltammetry with GCE/AuNp@cysteamine/PAMAM were comparable (deviation <±10%; limits of detection (LOD) and quantification (LOQ) were 1.7×10(-4) and 5.8×10(-4) mg dL(-1), respectively) to those concentrations obtained using the uricase-based enzymatic-colorimetric method. It was also observed that the presence of dendrimers in the GCE/AuNp@cysteamine/PAMAM system minimizes ascorbic acid (AA) interference during UA oxidation, thus improving the electrocatalytic activity of the gold nanoparticles. Copyright © 2013 Elsevier B.V. All rights reserved.

Diatomite Modified Immobilized Delftia sp. for the Bio-Abiotic Removal of Antibiotics Amoxicillin in the Aqueous System

NASA Astrophysics Data System (ADS)

Gao, Lijuan; Sun, Jing; Guan, Kai; Shen, Tingting; Wang, Xikui

2017-05-01

Diatomite modified sodium alginate (Si/SA) immobilized Delftia sp. A2(2011) (STT01) was applied to degrade amoxicillin. The immobilized pellets provided a direct and visual probe for the degradation process due to their intrinsic bright colour. The results demonstrated that 100% of amoxicillin and 68.5% of CODcr removal were achieved after 72 h, comparing with the cases of sodium alginate (SA) system (81.2%, 46.9%) and the free cells system (60.5%, 35.5%). The degradation kinetics was in good agreement with Michaelis-Menten equation. The maximum rate (Vm ) and Michaelis constant (Km ) were calculated as 9.09 mg L-1 h-1 and 228 mg L-1, respectively. The results further revealed that diatomite not only acted as immobilization support to improve the mechanical strength and lifetime of the pellets but also as absorbent to promote the treatment efficiency. Therefore, both enzymatic catalysis and chemisorption were responsible for the removal of amoxicillin.

Encapsulation and delivery of food ingredients using starch based systems.

PubMed

Zhu, Fan

2017-08-15

Functional ingredients can be encapsulated by various wall materials for controlled release in food and digestion systems. Starch, as one of the most abundant natural carbohydrate polymers, is non-allergenic, GRAS, and cheap. There has been increasing interest of using starch in native and modified forms to encapsulate food ingredients such as flavours, lipids, polyphenols, carotenoids, vitamins, enzymes, and probiotics. Starches from various botanical sources in granular or amorphous forms are modified by chemical, physical, and/or enzymatic means to obtain the desired properties for targeted encapsulation. Other wall materials are also employed in combination with starch to facilitate some types of encapsulation. Various methods of crafting the starch-based encapsulation such as electrospinning, spray drying, antisolvent, amylose inclusion complexation, and nano-emulsification are introduced in this mini-review. The physicochemical and structural properties of the particles are described. The encapsulation systems can positively influence the controlled release of food ingredients in food and nutritional applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Immobilization of Glucose Oxidase on a Carbon Nanotubes/Dendrimer-Ferrocene Modified Electrode for Reagentless Glucose Biosensing.

PubMed

Zhou, Juan; Li, Huan; Yang, Huasong; Cheng, Hui; Lai, Guosong

2017-01-01

Ferrocene-grafted dendrimer was covalently linked to the surface of a carbon nanotubes (CNTs)-chitosan (CS) nanocomposite modified electrode for immobilizing high-content glucose oxidase (GOx), which resulted in the successful development a novel reagentless glucose biosensor. Electrochemical impedance spectroscopy, cyclic voltammetry, and amperometry were used to characterize the preparation process and the enzymatically catalytic response of this biosensor. Due to the excellent electron transfer acceleration of the CNTs and the high-content loading of the GOx biomolecule and ferrocene mediator on the electrode matrix, this biosensor showed excellent analytical performance such as fast response time less than 10 s, wide linear range from 0.02 to 2.91 mM and low detection limit down to 7.5 μM as well as satisfactory stability and reproducibility toward the amperometric glucose determination. In addition, satisfactory result was obtained when it was used for the glucose measurements in human blood samples. Thus this biosensor provides great potentials for practical applications.

Chemical methods for encoding and decoding of posttranslational modifications

PubMed Central

Chuh, Kelly N.; Batt, Anna R.; Pratt, Matthew R.

2016-01-01

A large array of posttranslational modifications can dramatically change the properties of proteins and influence different aspects of their biological function such as enzymatic activity, binding interactions, and proteostasis. Despite the significant knowledge that has been gained about the function of posttranslational modifications using traditional biological techniques, the analysis of the site-specific effects of a particular modification, the identification of the full compliment of modified proteins in the proteome, and the detection of new types of modifications remains challenging. Over the years, chemical methods have contributed significantly in both of these areas of research. This review highlights several posttranslational modifications where chemistry-based approaches have made significant contributions to our ability to both prepare homogeneously modified proteins and identify and characterize particular modifications in complex biological settings. As the number and chemical diversity of documented posttranslational modifications continues to rise, we believe that chemical strategies will be essential to advance the field in years to come. PMID:26933738

Lipases as biocatalysts for the synthesis of structured lipids.

PubMed

Jala, Ram Chandra Reddy; Hu, Peng; Yang, Tiankui; Jiang, Yuanrong; Zheng, Yan; Xu, Xuebing

2012-01-01

Structured lipids (SL) are broadly referred to as modified or synthetic oils and fats or lipids with functional or pharmaceutical applications. Some structured lipids, such as triglycerides that contain both long-chain (mainly essential) fatty acids and medium- or short-chain fatty acids and also artificial products that mimic the structure of natural materials, namely human milk fat substitutes and cocoa butter equivalents, have been discussed. Further, other modified or synthetic lipids, such as structured phospholipids and synthetic phenolic lipids are also included in this chapter. For all the products described in this chapter, enzymatic production in industry has been already conducted in one way or another. Cocoa butter equivalents, healthy oil containing medium-chain fatty acids, phosphatidyl serine, and phenol lipids from enzyme technology have been reported for commercial operation. As the demand for better quality functional lipids is increasing, the production of structured lipids becomes an interesting area. Thus, in this chapter we have discussed latest developments as well as present industrial situation of all commercially important structured lipids.

Ectoparasite Caligus rogercresseyi modifies the lactate response in Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynchus kisutch).

PubMed

Vargas-Chacoff, L; Muñoz, J L P; Hawes, C; Oyarzún, R; Pontigo, J P; Saravia, J; González, M P; Mardones, O; Labbé, B S; Morera, F J; Bertrán, C; Pino, J; Wadsworth, S; Yáñez, A

2017-08-30

Although Caligus rogercresseyi negatively impacts Chilean salmon farming, the metabolic effects of infection by this sea louse have never been completely characterized. Therefore, this study analyzed lactate responses in the plasma, as well as the liver/muscle lactate dehydrogenase (LDH) activity and gene expression, in Salmo salar and Oncorhynchus kisutch infested by C. rogercresseyi. The lactate responses of Atlantic and Coho salmon were modified by the ectoparasite. Both salmon species showed increasing in plasma levels, whereas enzymatic activity increased in the muscle but decreased in the liver. Gene expression was overexpressed in both Coho salmon tissues but only in the liver for Atlantic salmon. These results suggest that salmonids need more energy to adapt to infection, resulting in increased gene expression, plasma levels, and enzyme activity in the muscles. The responses differed between both salmon species and over the course of infection, suggesting potential species-specific responses to sea-lice infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of glutamine and glutamic acid in mammalian cell cultures using tetrathiafulvalene modified enzyme electrodes.

PubMed

Mulchandani, A; Bassi, A S

1996-01-01

Tetrathiafulvalene (TTF) mediated amperometric enzyme electrodes have been developed for the monitoring of L-glutamine and L-glutamic acid in growing mammalian cell cultures. The detection of glutamine was accomplished by a coupled enzyme system comprised of glutaminase plus glutamate oxidase, while the detection of glutamic acid was carried out by a single enzyme, glutamate oxidase. The appropriate enzyme(s) were immoblized on the Triton-X treated surface of tetrathiafulvalene modified carbon paste electrodes by adsorption, in conjunction with entrapment by an electrochemically deposited copolymer film of 1,3-phenylenediamine and resorcinol. Operating conditions for the glutamine enzyme electrode were optimized with respect to the amount of enzymes immoblized, pH, temperature and mobile phase flow rate for operation in a flow injection (FIA) system. When applied to glutamine and glutamic acid measurements in mammalian cell culture in FIA, the results obtained with enzyme electrodes were in excellent agreement with those determined by enzymatic analysis.

Catalytic and immunochemical properties of arylsulphatase A from urine, modified by potassium ferrate.

PubMed

Laidler, P M; Steczko, J

1986-01-01

Arylsulphatase A (EC 3.1.6.1.) from urine was inactivated with potassium ferrate, a strong oxidizing agent. The inhibition could be prevented by competitive inhibitors, tetraborate and orthophosphate. Tetraborate which was shown to be a powerful competitive inhibitor (determined Ki = 4 X 10(-5) M) gave more efficient protection. The partially inactivated enzyme exhibited a Km value similar to that of the unmodified arylsulphatase A, and its Vmax decreased in proportion to the loss of enzymatic activity. The partially modified enzyme did not lose its ability to catalyse hydrolysis of p-nitrocatechol sulphate according to the "anomalous kinetics" exhibited towards this substrate and characteristic for arylsulphatase A. The immunochemical properties of arylsulphatase A either fully or partially inactivated were similar to those of the native enzyme. The results allow to conclude that ferrate reacts with arylsulphatase A in its active site. Thus ferrate seems to be a very sensitive probe for amino acid residues essential for catalytic activity of arylsulphatase A.

Attainable region analysis for continuous production of second generation bioethanol

PubMed Central

2013-01-01

Background Despite its semi-commercial status, ethanol production from lignocellulosics presents many complexities not yet fully solved. Since the pretreatment stage has been recognized as a complex and yield-determining step, it has been extensively studied. However, economic success of the production process also requires optimization of the biochemical conversion stage. This work addresses the search of bioreactor configurations with improved residence times for continuous enzymatic saccharification and fermentation operations. Instead of analyzing each possible configuration through simulation, we apply graphical methods to optimize the residence time of reactor networks composed of steady-state reactors. Although this can be easily made for processes described by a single kinetic expression, reactions under analysis do not exhibit this feature. Hence, the attainable region method, able to handle multiple species and its reactions, was applied for continuous reactors. Additionally, the effects of the sugars contained in the pretreatment liquor over the enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) were assessed. Results We obtained candidate attainable regions for separate enzymatic hydrolysis and fermentation (SHF) and SSF operations, both fed with pretreated corn stover. Results show that, despite the complexity of the reaction networks and underlying kinetics, the reactor networks that minimize the residence time can be constructed by using plug flow reactors and continuous stirred tank reactors. Regarding the effect of soluble solids in the feed stream to the reactor network, for SHF higher glucose concentration and yield are achieved for enzymatic hydrolysis with washed solids. Similarly, for SSF, higher yields and bioethanol titers are obtained using this substrate. Conclusions In this work, we demonstrated the capabilities of the attainable region analysis as a tool to assess the optimal reactor network with minimum residence time applied to the SHF and SSF operations for lignocellulosic ethanol production. The methodology can be readily modified to evaluate other kinetic models of different substrates, enzymes and microorganisms when available. From the obtained results, the most suitable reactor configuration considering residence time and rheological aspects is a continuous stirred tank reactor followed by a plug flow reactor (both in SSF mode) using washed solids as substrate. PMID:24286451

Attainable region analysis for continuous production of second generation bioethanol.

PubMed

Scott, Felipe; Conejeros, Raúl; Aroca, Germán

2013-11-29

Despite its semi-commercial status, ethanol production from lignocellulosics presents many complexities not yet fully solved. Since the pretreatment stage has been recognized as a complex and yield-determining step, it has been extensively studied. However, economic success of the production process also requires optimization of the biochemical conversion stage. This work addresses the search of bioreactor configurations with improved residence times for continuous enzymatic saccharification and fermentation operations. Instead of analyzing each possible configuration through simulation, we apply graphical methods to optimize the residence time of reactor networks composed of steady-state reactors. Although this can be easily made for processes described by a single kinetic expression, reactions under analysis do not exhibit this feature. Hence, the attainable region method, able to handle multiple species and its reactions, was applied for continuous reactors. Additionally, the effects of the sugars contained in the pretreatment liquor over the enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) were assessed. We obtained candidate attainable regions for separate enzymatic hydrolysis and fermentation (SHF) and SSF operations, both fed with pretreated corn stover. Results show that, despite the complexity of the reaction networks and underlying kinetics, the reactor networks that minimize the residence time can be constructed by using plug flow reactors and continuous stirred tank reactors. Regarding the effect of soluble solids in the feed stream to the reactor network, for SHF higher glucose concentration and yield are achieved for enzymatic hydrolysis with washed solids. Similarly, for SSF, higher yields and bioethanol titers are obtained using this substrate. In this work, we demonstrated the capabilities of the attainable region analysis as a tool to assess the optimal reactor network with minimum residence time applied to the SHF and SSF operations for lignocellulosic ethanol production. The methodology can be readily modified to evaluate other kinetic models of different substrates, enzymes and microorganisms when available. From the obtained results, the most suitable reactor configuration considering residence time and rheological aspects is a continuous stirred tank reactor followed by a plug flow reactor (both in SSF mode) using washed solids as substrate.

Modification of the nanostructure of lignocellulose cell walls via a non-enzymatic lignocellulose deconstruction system in brown rot wood-decay fungi.

PubMed

Goodell, Barry; Zhu, Yuan; Kim, Seong; Kafle, Kabindra; Eastwood, Daniel; Daniel, Geoffrey; Jellison, Jody; Yoshida, Makoto; Groom, Leslie; Pingali, Sai Venkatesh; O'Neill, Hugh

2017-01-01

Wood decayed by brown rot fungi and wood treated with the chelator-mediated Fenton (CMF) reaction, either alone or together with a cellulose enzyme cocktail, was analyzed by small angle neutron scattering (SANS), sum frequency generation (SFG) spectroscopy, Fourier transform infrared (FTIR) analysis, X-ray diffraction (XRD), atomic force microscopy (AFM), and transmission electron microscopy (TEM). Results showed that the CMF mechanism mimicked brown rot fungal attack for both holocellulose and lignin components of the wood. Crystalline cellulose and lignin were both depolymerized by the CMF reaction. Porosity of the softwood cell wall did not increase during CMF treatment, enzymes secreted by the fungi did not penetrate the decayed wood. The enzymes in the cellulose cocktail also did not appear to alter the effects of the CMF-treated wood relative to enhancing cell wall deconstruction. This suggests a rethinking of current brown rot decay models and supports a model where monomeric sugars and oligosaccharides diffuse from the softwood cell walls during non-enzymatic action. In this regard, the CMF mechanism should not be thought of as a "pretreatment" used to permit enzymatic penetration into softwood cell walls, but instead it enhances polysaccharide components diffusing to fungal enzymes located in wood cell lumen environments during decay. SANS and other data are consistent with a model for repolymerization and aggregation of at least some portion of the lignin within the cell wall, and this is supported by AFM and TEM data. The data suggest that new approaches for conversion of wood substrates to platform chemicals in biorefineries could be achieved using the CMF mechanism with >75% solubilization of lignocellulose, but that a more selective suite of enzymes and other downstream treatments may be required to work when using CMF deconstruction technology. Strategies to enhance polysaccharide release from lignocellulose substrates for enhanced enzymatic action and fermentation of the released fraction would also aid in the efficient recovery of the more uniform modified lignin fraction that the CMF reaction generates to enhance biorefinery profitability.

Enzymatic corn wet milling: engineering process and cost model

PubMed Central

Ramírez, Edna C; Johnston, David B; McAloon, Andrew J; Singh, Vijay

2009-01-01

Background Enzymatic corn wet milling (E-milling) is a process derived from conventional wet milling for the recovery and purification of starch and co-products using proteases to eliminate the need for sulfites and decrease the steeping time. In 2006, the total starch production in USA by conventional wet milling equaled 23 billion kilograms, including modified starches and starches used for sweeteners and ethanol production [1]. Process engineering and cost models for an E-milling process have been developed for a processing plant with a capacity of 2.54 million kg of corn per day (100,000 bu/day). These models are based on the previously published models for a traditional wet milling plant with the same capacity. The E-milling process includes grain cleaning, pretreatment, enzymatic treatment, germ separation and recovery, fiber separation and recovery, gluten separation and recovery and starch separation. Information for the development of the conventional models was obtained from a variety of technical sources including commercial wet milling companies, industry experts and equipment suppliers. Additional information for the present models was obtained from our own experience with the development of the E-milling process and trials in the laboratory and at the pilot plant scale. The models were developed using process and cost simulation software (SuperPro Designer®) and include processing information such as composition and flow rates of the various process streams, descriptions of the various unit operations and detailed breakdowns of the operating and capital cost of the facility. Results Based on the information from the model, we can estimate the cost of production per kilogram of starch using the input prices for corn, enzyme and other wet milling co-products. The work presented here describes the E-milling process and compares the process, the operation and costs with the conventional process. Conclusion The E-milling process was found to be cost competitive with the conventional process during periods of high corn feedstock costs since the enzymatic process enhances the yields of the products in a corn wet milling process. This model is available upon request from the authors for educational, research and non-commercial uses. PMID:19154623

Stereospecific enzymatic transformation of alpha-ketoglutarate to (2S,3R)-3-methyl glutamate during acidic lipopeptide biosynthesis.

PubMed

Mahlert, Christoph; Kopp, Florian; Thirlway, Jenny; Micklefield, Jason; Marahiel, Mohamed A

2007-10-03

The acidic lipopeptides, including the calcium-dependent antibiotics (CDA), daptomycin, and A54145, are important macrocyclic peptide natural products produced by Streptomyces species. All three compounds contain a 3-methyl glutamate (3-MeGlu) as the penultimate C-terminal residue, which is important for bioactivity. Here, biochemical in vitro reconstitution of the 3-MeGlu biosynthetic pathway is presented, using exclusively enzymes from the CDA producer Streptomyces coelicolor. It is shown that the predicted 3-MeGlu methyltransferase GlmT and its homologues DptI from the daptomycin producer Streptomyces roseosporus and LptI from the A54145 producer Streptomyces fradiae do not methylate free glutamic acid, PCP-bound glutamate, or Glu-containing CDA in vitro. Instead, GlmT, DptI, and LptI are S-adenosyl methionine (SAM)-dependent alpha-ketoglutarate methyltransferases that catalyze the stereospecific methylation of alpha-ketoglutarate (alphaKG) leading to (3R)-3-methyl-2-oxoglutarate. Subsequent enzyme screening identified the branched chain amino acid transaminase IlvE (SCO5523) as an efficient catalyst for the transformation of (3R)-3-methyl-2-oxoglutarate into (2S,3R)-3-MeGlu. Comparison of reversed-phase HPLC retention time of dabsylated 3-MeGlu generated by the coupled enzymatic reaction with dabsylated synthetic standards confirmed complete stereocontrol during enzymatic catalysis. This stereospecific two-step conversion of alphaKG to (2S,3R)-3-MeGlu completes our understanding of the biosynthesis and incorporation of beta-methylated amino acids into the nonribosomal lipopeptides. Finally, understanding this pathway may provide new possibilities for the production of modified peptides in engineered microbes.

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

Daily rhythms of catalase and glutathione peroxidase expression and activity are endogenously driven in the hippocampus and are modified by a vitamin A-free diet.

PubMed

Navigatore-Fonzo, Lorena S; Delgado, Silvia M; Gimenez, Maria Sofia; Anzulovich, Ana C

2014-01-01

Alterations in enzymatic antioxidant defense systems lead to a deficit of cognitive functions and altered hippocampal synaptic plasticity. The objectives of this study were to investigate endogenous rhythms of catalase (CAT) and glutathione peroxidase (GPx) expression and activity, as well as CREB1 mRNA, in the rat hippocampus, and to evaluate to which extent the vitamin A deficiency could affect those temporal patterns. Rats from control and vitamin A-deficient (VAD) groups received a diet containing 4000 IU of vitamin A/kg diet, or the same diet devoid of vitamin A, respectively, during 3 months. Rats were maintained under 12-hour-dark conditions, during 10 days before the sacrifice. Circadian rhythms of CAT, GPx, RXRγ, and CREB1 mRNA levels were determined by reverse transcriptrase polymerase chain reaction in hippocampus samples isolated every 4 hours during a 24-hour period. CAT and GPx enzymatic activities were also determined by kinetic assays. Regulatory regions of clock and antioxidant enzymes genes were scanned for E-box, RXRE, and CRE sites. E-box, RXRE, and CRE sites were found on regulatory regions of GPx and CAT genes, which display a circadian expression in the rat hippocampus. VAD phase shifted CAT, GPx, and RXRγ endogenous rhythms without affecting circadian expression of CREB1. CAT and GPx expression and enzymatic activity are circadian in the rat hippocampus. The VAD affected the temporal patterns antioxidant genes expression, probably by altering circadian rhythms of its RXR receptors and clock factors; thus, it would impair the temporal orchestration of hippocampal daily cognitive performance.

Evaluation of Enzymatically Modified Soy Protein Isolate Film Forming Solution and Film at Different Manufacturing Conditions.

PubMed

Mohammad Zadeh, Elham; O'Keefe, Sean F; Kim, Young-Teck; Cho, Jin-Hun

2018-04-01

The effects of transglutaminase on soy protein isolate (SPI) film forming solution and films were investigated by rheological behavior and physicochemical properties based on different manufacturing conditions (enzyme treatments, enzyme incubation times, and protein denaturation temperatures). Enzymatic crosslinking reaction and changes in molecular weight distribution were confirmed by viscosity measurement and SDS-PAGE, respectively, compared to 2 controls: the nonenzyme treated and the deactivated enzyme treated. Films treated with both the enzyme and the deactivated enzyme showed significant increase in tensile strength (TS), percent elongation (%E), and initial contact angle of films compared to the nonenzyme control film due to the bulk stabilizers in the commercial enzyme. Water absorption property, protein solubility, Fourier transform infrared (FTIR) and X-ray diffraction (XRD) spectroscopy revealed that enzyme treated SPI film matrix in the molecular structure level, resulted in the changes in physicochemical properties. Based on our observation, the enzymatic treatment at appropriate conditions is a practical and feasible way to control the physical properties of protein based biopolymeric film for many different scientific and industrial areas. Enzymes can make bridges selectively among different amino acids in the structure of protein matrix. Therefore, protein network is changed after enzyme treatment. The behavior of biopolymeric materials is dependent on the network structure to be suitable in different applications such as bioplastics applied in food and pharmaceutical products. In the current research, transglutaminase, as an enzyme, applied in soy protein matrix in different types of forms, activated and deactivated, and different preparation conditions to investigate its effects on different properties of the new bioplastic film. © 2018 Institute of Food Technologists®.

Characterization of chemically and enzymatically treated hemp fibres using atomic force microscopy and spectroscopy

NASA Astrophysics Data System (ADS)

George, Michael; Mussone, Paolo G.; Abboud, Zeinab; Bressler, David C.

2014-09-01

The mechanical and moisture resistance properties of natural fibre reinforced composites are dependent on the adhesion between the matrix of choice and the fibre. The main goal of this study was to investigate the effect of NaOH swelling of hemp fibres prior to enzymatic treatment and a novel chemical sulfonic acid method on the physical properties of hemp fibres. The colloidal properties of treated hemp fibres were studied exclusively using an atomic force microscope. AFM imaging in tapping mode revealed that each treatment rendered the surface topography of the hemp fibres clean and exposed the individual fibre bundles. Hemp fibres treated with laccase had no effect on the surface adhesion forces measured. Interestingly, mercerization prior to xylanase + cellulase and laccase treatments resulted in greater enzyme access evident in the increased adhesion force measurements. Hemp fibres treated with sulfonic acid showed an increase in surface de-fibrillation and smoothness. A decrease in adhesion forces for 4-aminotoulene-3-sulfonic acid (AT3S) treated fibres suggested a reduction in surface polarity. This work demonstrated that AFM can be used as a tool to estimate the surface forces and roughness for modified fibres and that enzymatic coupled with chemical methods can be used to improve the surface properties of natural fibres for composite applications. Further, this work is one of the first that offers some insight into the effect of mercerization prior to enzymes and the effect on the surface topography. AFM will be used to selectively screen treated fibres for composite applications based on the adhesion forces associated with the colloidal interface between the AFM tip and the fibre surfaces.

The use of electrochemistry for the synthesis of 17 alpha-hydroxyprogesterone by a fusion protein containing P450c17.

PubMed

Estabrook, R W; Shet, M S; Faulkner, K; Fisher, C W

1996-11-01

A method has been developed for the commercial application of the unique oxygen chemistry catalyzed by various cytochrome P450s. This is illustrated here for the synthesis of hydroxylated steroids. This method requires the preparation of large amounts of enzymatically functional P450 proteins that can serve as catalysts and a technique for providing electrons at an economically acceptable cost. To generate large amounts of enzymatically active recombinant P450s we have engineered the cDNAs for various P450s, including bovine adrenal P450c17, by linking them to a modified cDNA for rat NADPH-P450 reductase and placing them in the plasmid pCWori+. Transformation of E. coli results in the high level expression of an enzymatically active protein that can be easily purified by affinity chromatography. Incubation of the purified enzyme with steroid in a reaction vessel containing a platinum electrode and a Ag/AgCl electrode couple poised at -650 mV, together with the electromotively active redox mediator, cobalt sepulchrate, results in the 17 alpha-hydroxylation of progesterone at rates as high as 25 nmoles of progesterone hydroxylated/min/nmole of P450. Thus, high concentrations of hydroxylated steroids can be produced with incubation conditions of hours duration without the use of costly NADPH. Similar experiments have been carried out for the generation of the 6 beta-hydroxylation product of testosterone (using a fusion protein containing human P450 3A4). It is apparent that this method is applicable to many other P450 catalyzed reactions for the synthesis of large amounts of hydroxylated steroid metabolites. The electrochemical system is also applicable to drug discovery studies for the characterization of drug metabolites.

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.

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.

Dietary Antioxidants as Modifiers of Physiologic Adaptations to Exercise

PubMed Central

Mankowski, Robert T.; Anton, Stephen D.; Buford, Thomas W.; Leeuwenburgh, Christiaan

2015-01-01

Adaptive responses to exercise training (ET) are crucial in maintaining physiological homeostasis and health span. Exercise-induced aerobic bioenergetic reactions in mitochondria and cytosol increase production of reactive oxygen species (ROSs), where excess of ROS can be scavenged by enzymatic as well as non-enzymatic antioxidants to protect against deleterious oxidative stress. Free radicals, however, have recently been recognized as crucial signaling agents that promote adaptive mechanisms to ET, such as mitochondrial biogenesis, antioxidant (AO) enzyme activity defense system upregulation, insulin sensitivity, and glucose uptake in skeletal muscle. Commonly used non-enzymatic AO supplements, such as vitamins C and E, a-lipoic acid, and polyphenols, in combination with ET, have been proposed as ways to prevent exercise-induced oxidative stress and hence improve adaptation responses to endurance training. Preclinical and clinical studies to date have shown inconsistent results indicating either positive or negative effects of endurance training combined with different blends of AO supplements (mostly vitamins C and E and a-lipoic acid) on redox status, mitochondrial biogenesis pathways, and insulin sensitivity. Preclinical reports on ET combined with resveratrol, however, have shown consistent positive effects on exercise performance, mitochondrial biogenesis, and insulin sensitivity, with clinical trials reporting mixed effects. Relevant clinical studies have been few and have used inconsistent results and methodology (types of compounds, combinations, and supplementation time). The future studies would investigate the effects of specific antioxidants and other popular supplements, such as a-lipoic acid and resveratrol, on training effects in humans. Of particular importance are older adults who may be at higher risk of age-related increased oxidative stress, an impaired AO enzyme defense system, and comorbidities such as hypertension, insulin resistance, and diabetes. PMID:25606815

Anti-arthritic Effects of Total Flavonoids from Juniperus sabina on Complete Freund's Adjuvant Induced Arthritis in Rats

PubMed Central

Zhao, Jun; Liu, Tao; Xu, Fang; You, Shuping; Xu, Fang; Li, Chenyang; Gu, Zhengyi

2016-01-01

Context: Twigs and leaves of Juniperus sabina L. have been traditionally used as the medicinal herb in China for the treatment of many ailments including rheumatoid arthritis (RA). Aims: To confirm the therapeutic effect of total flavonoids from J. sabina (JSTF) on RA-induced by Complete Freund's Adjuvant (CFA) in rats. Settings and Design: Wistar rats (200 ± 20 g) were immunized by intradermal injection of 0.1 mL of CFA into the right hind metatarsal footpad. JSTF was administered orally at the dose of 125,250 and 500 mg/kg on 14 days after the induction of adjuvant arthritis. Tripterygium glycoside (20 mg/kg) was used as a positive control. Paw swelling, arthritic score, body weight loss, serum cytokines, inflammatory mediators, and histological change were measured. Results: We found that JSTF could ameliorate paw swelling of CFA rats, and significantly inhibit arthritic score (P < 0.05). The overproduction of tumor necrosis factor alpha and interleukin 1beta were remarkably suppressed in the serum of JSTF (125,500 mg/kg) treated rats (P < 0.05). Histopathological studies also showed a marked decrease of synovial inflammatory infiltration and synovial lining hyperplasia in the joints of JSTF-treated animals. Six flavonoids were isolated and from JSTF by various chromatographic methods and identified as follows: Catechin, quercitrin, isoquercitrin, isoscutellarein 7-O-β-D-xylopyranoside, isoscutellarein 7-O-β-D-xylopyranose-(1 → 3)-α-L-rhamnoside, and rutin. Conclusions: These results suggest the potential therapeutically effect of JSTF as an anti-arthritis agent toward CFA-induced arthritis in rats, and verified therapeutic applications of J. sabina on RA in folk medicine. SUMMARY Twigs and leaves of Juniperus sabina L. have been traditionally used as the medicinal herb in China for the treatment of rheumatoid arthritisJSTF could ameliorate paw swelling of CFA rats, and significantly inhibit arthritic scoreHistopathological studies showed a marked decrease of synovial inflammatory infiltration and synovial lining hyperplasia in the joints of JSTF-treated animalsSix flavonoids were isolated and from JSTF including: Catechin, quercitrin, isoquercitrin, isoscutellarein 7-O-β-D-xylopyranoside, isoscutellarein 7-O-β-D-xylopyranose-(1 → 3)-α-L- rhamnoside, and rutin. Abbreviations used: JSTF: Total flavonoids from Juniperus sabina; CFA: Complete Freund's Adjuvant; TG: Tripterygium glycoside; TNF-α: Tumor necrosis factor alpha; IL-1β: Interleukin 1beta; IL-6: Interleukin 6; H and E: Hematoxylin and eosin. PMID:27601846

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.

Effect of Grinding at Modified Atmosphere or Vacuum on Browning, Antioxidant Capacities, and Oxidative Enzyme Activities of Apple.

PubMed

Kim, Ah-Na; Lee, Kyo-Yeon; Kim, Hyun-Jin; Chun, Jiyeon; Kerr, William L; Choi, Sung-Gil

2018-01-01

This study evaluated the effects of grinding at atmospheric pressure (control), under vacuum (∼2.67 kPa), or with modified atmosphere (N 2 and CO 2 ) on the browning, antioxidant activity, phenolics, and oxidative enzyme activity of apples as a function of time. The control group was affected most, showing distinct browning and losing most of the antioxidant activity and concentrations of the main phenolic compounds. The modified atmosphere groups retained color, antioxidant activity, and phenolic compounds better than the control group. Least changes were obtained with vacuum grinding, particularly in terms of preventing enzymatic browning and oxidation of antioxidants apples. At 12 h after grinding, vacuum-ground apples retained total phenolic contents 5.32, 1.54, and 1.49 times higher than control, nitrogen gas, and carbon dioxide gas-ground samples, respectively. The oxidative enzyme activity, including that of polyphenol oxidase and peroxidase, decreased in the control and modified atmosphere group, but they were maintained in the samples ground under the vacuum. In this study, we found that grinding with modified atmosphere or vacuum conditions could effectively prevent browning as well as loss of phenolic compounds and antioxidant activity of ground apples. These results can help scientists and engineers build better grinding systems for retaining nutrient and quality factors of ground apples. In addition, these results may be useful to other fruit and vegetable industries that wish to retain fresh-like quality and nutritional value during grinding and storage. © 2017 Institute of Food Technologists®.

Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2',4'-bridged nucleosides.

PubMed

Kuwahara, Masayasu; Obika, Satoshi; Nagashima, Jun-ichi; Ohta, Yuki; Suto, Yoshiyuki; Ozaki, Hiroaki; Sawai, Hiroaki; Imanishi, Takeshi

2008-08-01

In order to systematically analyze the effects of nucleoside modification of sugar moieties in DNA polymerase reactions, we synthesized 16 modified templates containing 2',4'-bridged nucleotides and three types of 2',4'-bridged nucleoside-5'-triphospates with different bridging structures. Among the five types of thermostable DNA polymerases used, Taq, Phusion HF, Vent(exo-), KOD Dash and KOD(exo-), the KOD Dash and KOD(exo-) DNA polymerases could smoothly read through the modified templates containing 2'-O,4'-C-methylene-linked nucleotides at intervals of a few nucleotides, even at standard enzyme concentrations for 5 min. Although the Vent(exo-) DNA polymerase also read through these modified templates, kinetic study indicates that the KOD(exo-) DNA polymerase was found to be far superior to the Vent(exo-) DNA polymerase in accurate incorporation of nucleotides. When either of the DNA polymerase was used, the presence of 2',4'-bridged nucleotides on a template strand substantially decreased the reaction rates of nucleotide incorporations. The modified templates containing sequences of seven successive 2',4'-bridged nucleotides could not be completely transcribed by any of the DNA polymerases used; yields of longer elongated products decreased in the order of steric bulkiness of the modified sugars. Successive incorporation of 2',4'-bridged nucleotides into extending strands using 2',4'-bridged nucleoside-5'-triphospates was much more difficult. These data indicate that the sugar modification would have a greater effect on the polymerase reaction when it is adjacent to the elongation terminus than when it is on the template as well, as in base modification.

Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter; He, Yufan; Lu, Maolin; Cao, Jin; Guo, Qing

2014-03-01

Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced and magnetic tweezers-correlated 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 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. Our results 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.

Enhanced non-enzymatic glucose biosensor of Ga-doped ZnO nanorods

NASA Astrophysics Data System (ADS)

Peng, Wan-Chan; Wang, Zi-Hao; Yang, Chih-Chiang; Huang, Chien-Sheng; Su, Yan-Kuin; Ruan, Jian-Long

2017-04-01

In this work gallium (Ga)-Doped ZnO nanorods (GZO NRs) successfully applied for the development of enzyme free glucose. GZO NRs synthesized by using the hydrothermal on ZnO seed layer was subsequently deposited onto the glass substrate. The GZO NRs electrode has peak currents increasing from 620 to 941μA with glucose concentration (6, 8 and 10 mM) in cyclic voltammograms. GZO NRs electrode sensitivity of the sensor to glucose oxidation was 33.4 (μA/mM-cm2). The GZO NRs modified electrode showed a greatly enhanced electrocatalytic property toward glucose oxidation, as well as an excellent anti-interference and a good stability.

Recent advances of mesoporous materials in sample preparation.

PubMed

Zhao, Liang; Qin, Hongqiang; Wu, Ren'an; Zou, Hanfa

2012-03-09

Sample preparation has been playing an important role in the analysis of complex samples. Mesoporous materials as the promising adsorbents have gained increasing research interest in sample preparation due to their desirable characteristics of high surface area, large pore volume, tunable mesoporous channels with well defined pore-size distribution, controllable wall composition, as well as modifiable surface properties. The aim of this paper is to review the recent advances of mesoporous materials in sample preparation with emphases on extraction of metal ions, adsorption of organic compounds, size selective enrichment of peptides/proteins, specific capture of post-translational peptides/proteins and enzymatic reactor for protein digestion. Copyright © 2011 Elsevier B.V. All rights reserved.

Bienzymatic Biosensor for Rapid Detection of Aspartame by Flow Injection Analysis

PubMed Central

Radulescu, Maria-Cristina; Bucur, Bogdan; Bucur, Madalina-Petruta; Radu, Gabriel Lucian

2014-01-01

A rapid, simple and stable biosensor for aspartame detection was developed. Alcohol oxidase (AOX), carboxyl esterase (CaE) and bovine serum albumin (BSA) were immobilised with glutaraldehyde (GA) onto screen-printed electrodes modified with cobalt-phthalocyanine (CoPC). The biosensor response was fast. The sample throughput using a flow injection analysis (FIA) system was 40 h−1 with an RSD of 2.7%. The detection limits for both batch and FIA measurements were 0.1 μM for methanol and 0.2 μM for aspartame, respectively. The enzymatic biosensor was successfully applied for aspartame determination in different sample matrices/commercial products (liquid and solid samples) without any pre-treatment step prior to measurement. PMID:24412899

Bienzymatic biosensor for rapid detection of aspartame by flow injection analysis.

PubMed

Radulescu, Maria-Cristina; Bucur, Bogdan; Bucur, Madalina-Petruta; Radu, Gabriel Lucian

2014-01-09

A rapid, simple and stable biosensor for aspartame detection was developed. Alcohol oxidase (AOX), carboxyl esterase (CaE) and bovine serum albumin (BSA) were immobilised with glutaraldehyde (GA) onto screen-printed electrodes modified with cobalt-phthalocyanine (CoPC). The biosensor response was fast. The sample throughput using a flow injection analysis (FIA) system was 40 h⁻¹ with an RSD of 2.7%. The detection limits for both batch and FIA measurements were 0.1 µM for methanol and 0.2 µM for aspartame, respectively. The enzymatic biosensor was successfully applied for aspartame determination in different sample matrices/commercial products (liquid and solid samples) without any pre-treatment step prior to measurement.

Self-supported fibrin-polyvinyl alcohol interpenetrating polymer networks: an easily handled and rehydratable biomaterial.

PubMed

Bidault, Laurent; Deneufchatel, Marie; Vancaeyzeele, Cédric; Fichet, Odile; Larreta-Garde, Véronique

2013-11-11

A fibrin hydrogel at physiological concentration (5 mg/mL) was associated with polyvinyl alcohol (PVA) inside an interpenetrating polymer networks (IPN) architecture. Previously, PVA has been modified with methacrylate functions in order to cross-link it by free-radical polymerization. The fibrin network was synthesized by the enzymatic hydrolysis of fibrinogen by thrombin. The resulting self-supported materials simultaneously exhibit the properties of the fibrin hydrogel and those of the synthetic polymer network. Their storage modulus is 50-fold higher than that of the fibrin hydrogel and they are completely rehydratable. These materials are noncytotoxic toward human fibroblast and the fibrin present on the surface of PVAm-based IPNs favors cell development.

Spectroscopic probing of ortho-nitrophenol localization in phospholipid bilayers.

PubMed

Sánchez, Julieta M; Turina, Anahí del V; Perillo, María A

2007-11-12

In the present study we estimated the localization of ortho-nitrophenol (ONP) within model membranes through its efficiency to quench and to modify the anisotropy of DPH and TMA-DPH fluorescence. These fluorescent probes are known to sense the hydrocarbon core and the polar head group region of membranes, respectively. TMA-DPH fluorescence in MLVs was more efficiently quenched than DPH (K(q,TMA-DPH)=2.36 and K(q,DPH)=1.07 mM ns(-1)). Moreover, these results demonstrated the interfacial localization of ONP and may contribute to understand membrane-mediated mechanisms of ONP-induced toxicity and the behavior of ONP as a product of several enzymatic reactions occurring in the presence of lipid-water interfaces.

Amadori glycated proteins: role in production of autoantibodies in diabetes mellitus and effect of inhibitors on non-enzymatic glycation.

PubMed

Ansari, Nadeem A; Dash, Debabrata

2013-02-01

Nonenzymatic glycation of macromolecules, especially proteins leading to their oxidation is increased in diabetes mellitus due to hyperglycemia and play an important role in associated complications of the disease. The glycation primarily occurs at intrachain lysine residues of proteins and results in the formation of an early stage stable product as Amadori-lysine which undergo further irreversible chemical reactions to form advanced glycation endproducts. This review deals with the role of Amadori modified proteins in pathogenesis of diabetes. We aim to explain immunogenicity of Amadori-glycated proteins, which might be involve in production of serum autoantibodies in the diabetic patients, and effect of inhibitors on the glycation process.

Use of synthetic peptides and site-specific antibodies to localize a diphtheria toxin sequence associated with ADP-ribosyltransferase activity.

PubMed Central

Olson, J C

1993-01-01

Diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A have the same molecular mechanism of toxicity; both toxins ADP-ribosylate a modified histidine residue in elongation factor 2. To help identify amino acids involved in this reaction, sequences in DT that share homology with P. aeruginosa exotoxin A were synthesized and examined for a role in the ADP-ribosyltransferase reaction. By using this approach, residues 32 to 54 of DT were found to define an epitope associated with antibody-mediated inhibition of DT enzyme activity. This lends further support to the notion that residues in this region of DT are involved in the enzymatic reaction. PMID:8423159

More Genetic Engineering With Cloned Hemoglobin Genes

NASA Technical Reports Server (NTRS)

Bailey, James E.

1992-01-01

Cells modified to enhance growth and production of proteins. Method for enhancing both growth of micro-organisms in vitro and production of various proteins or metalbolites in these micro-organisms provides for incorporation of selected chromosomal or extrachormosomal deoxyribonucleic acid (DNA) sequences into micro-organisms from other cells or from artificial sources. Incorporated DNA includes parts encoding desired product(s) or characteristic(s) of cells and parts that control expression of productor characteristic-encoding parts in response to variations in environment. Extended method enables increased research into growth of organisms in oxygen-poor environments. Industrial applications found in enhancement of processing steps requiring oxygen in fermentation, enzymatic degradation, treatment of wastes containing toxic chemicals, brewing, and some oxidative chemical reactions.

How Do Enzymes 'Meet' Nanoparticles and Nanomaterials?

PubMed

Chen, Ming; Zeng, Guangming; Xu, Piao; Lai, Cui; Tang, Lin

2017-11-01

Enzymes are fundamental biological catalysts responsible for biological regulation and metabolism. The opportunity for enzymes to 'meet' nanoparticles and nanomaterials is rapidly increasing due to growing demands for applications in nanomaterial design, environmental monitoring, biochemical engineering, and biomedicine. Therefore, understanding the nature of nanomaterial-enzyme interactions is becoming important. Since 2014, enzymes have been used to modify, degrade, or make nanoparticles/nanomaterials, while numerous nanoparticles/nanomaterials have been used as materials for enzymatic immobilization and biosensors and as enzyme mimicry. Among the various nanoparticles and nanomaterials, metal nanoparticles and carbon nanomaterials have received extensive attention due to their fascinating properties. This review provides an overview about how enzymes meet nanoparticles and nanomaterials. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fabrication of nitric oxide-releasing polyurethane glucose sensor membranes

PubMed Central

Koh, Ahyeon; Riccio, Daniel A.; Sun, Bin; Carpenter, Alexis W.; Nichols, Scott P.; Schoenfisch, Mark H.

2011-01-01

Despite clear evidence that polymeric nitric oxide (NO) release coatings reduce the foreign body response (FBR) and may thus improve the analytical performance of in vivo continuous glucose monitoring devices when used as sensor membranes, the compatibility of the NO release chemistry with that required for enzymatic glucose sensing remains unclear. Herein, we describe the fabrication and characterization of NO-releasing polyurethane sensor membranes using NO donor-modified silica vehicles embedded within the polymer. In addition to demonstrating tunable NO release as a function of the NO donor silica scaffold and polymer compositions and concentrations, we describe the impact of the NO release vehicle and its release kinetics on glucose sensor performance. PMID:21795038

An Overview of the Chemistry and Biology of Reactive Aldehydes

PubMed Central

Fritz, Kristofer S.; Petersen, Dennis R.

2012-01-01

The non-enzymatic free radical generation of reactive aldehydes is known to contribute to diseases of sustained oxidative stress including rheumatoid arthritis, atherosclerosis, neurodegenerative and a number of liver diseases. At the same time, the accumulation of lipid electrophiles has been demonstrated to play a role in cell signaling events through modification of proteins critical for cellular homeostasis. Given the broad scope of reactivity profiles and the ability to modify numerous proteomic and genomic processes, new emphasis is being placed on a systems-based analysis of the consequences of electrophilic adduction. This review focuses on the generation and chemical reactivity of lipid-derived aldehydes with a special focus on the homeostatic responses to electrophilic stress. PMID:22750507

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.

Controlled release of an extract of Calendula officinalis flowers from a system based on the incorporation of gelatin-collagen microparticles into collagen I scaffolds: design and in vitro performance.

PubMed

Jiménez, Ronald A; Millán, Diana; Suesca, Edward; Sosnik, Alejandro; Fontanilla, Marta R

2015-06-01

Aiming to develop biological skin dresses with improved performance in the treatment of skin wounds, acellular collagen I scaffolds were modified with polymeric microparticles and the subsequent loading of a hydroglycolic extract of Calendula officinalis flowers. Microparticles made of gelatin-collagen were produced by a water-in-oil emulsion/cross-linking method. Thereafter, these microparticles were mixed with collagen suspensions at three increasing concentrations and the resulting mixtures lyophilized to make microparticle-loaded porous collagen scaffolds. Resistance to enzymatic degradation, ability to associate with the C. officinalis extract, and the extract release profile of the three gelatin-collagen microparticle-scaffold prototypes were assessed in vitro and compared to collagen scaffolds without microparticles used as control. Data indicated that the incorporation of gelatin-collagen microparticles increased the resistance of the scaffolds to in vitro enzymatic degradation, as well as their association with the C. officinalis flower extract. In addition, a sharp decrease in cytotoxicity, as well as more prolonged release of the extract, was attained. Overall results support the potential of these systems to develop innovative dermal substitutes with improved features. Furthermore, the gelatin-collagen mixture represents a low-cost and scalable alternative with high clinical transferability, especially appealing in developing countries.

Elasticity, biodegradability and cell adhesive properties of chitosan/hyaluronan multilayer films

NASA Astrophysics Data System (ADS)

Schneider, Aurore; Richert, Ludovic; Francius, Gregory; Voegel, Jean-Claude; Picart, Catherine

2007-03-01

In the bioengineering field, a recent and promising approach to modifying biomaterial surfaces is the layer-by-layer (LbL) technique used to build thin polyelectrolyte multilayer films. In this work, we focused on polyelectrolyte multilayer films made of two polysaccharides, chitosan (CHI) and hyaluronan (HA), and on the control of their physico-chemical and cell adhesive properties by chemical cross-linking. CHI/HA films were cross-linked using a water soluble carbodiimide and observed by confocal laser scanning microscopy (CLSM) with a fluorescently labeled CHI. Film thicknesses were similar for native and cross-linked films. The film nanometer roughness was measured by atomic force microscopy and was found to be higher for cross-linked films. Cross-linking the films also leads to a drastic change in film stiffness. The elastic modulus of the films (Young's modulus) as measured by AFM nano-indentation was about tenfold increased for cross-linked films as compared to native ones. From a biological point of view, cross-liked films are more resistant to enzymatic degradation by hyaluronidase. Furthermore, the increase in film stiffness has a favorable effect on the adhesion and spreading of chondrosarcoma cells. Thus, the CHI/HA cross-linked films could be used for various applications due to their adhesive properties and to their mechanical properties (including stability in enzymatic media).

Noncanonical substrate preference of lambda exonuclease for 5'-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction.

PubMed

Wu, Tongbo; Yang, Yufei; Chen, Wei; Wang, Jiayu; Yang, Ziyu; Wang, Shenlin; Xiao, Xianjin; Li, Mengyuan; Zhao, Meiping

2018-04-06

Lambda exonuclease (λ exo) plays an important role in the resection of DNA ends for DNA repair. Currently, it is also a widely used enzymatic tool in genetic engineering, DNA-binding protein mapping, nanopore sequencing and biosensing. Herein, we disclose two noncanonical properties of this enzyme and suggest a previously undescribed hydrophobic interaction model between λ exo and DNA substrates. We demonstrate that the length of the free portion of the substrate strand in the dsDNA plays an essential role in the initiation of digestion reactions by λ exo. A dsDNA with a 5' non-phosphorylated, two-nucleotide-protruding end can be digested by λ exo with very high efficiency. Moreover, we show that when a conjugated structure is covalently attached to an internal base of the dsDNA, the presence of a single mismatched base pair at the 5' side of the modified base may significantly accelerate the process of digestion by λ exo. A detailed comparison study revealed additional π-π stacking interactions between the attached label and the amino acid residues of the enzyme. These new findings not only broaden our knowledge of the enzyme but will also be very useful for research on DNA repair and in vitro processing of nucleic acids.

Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts.

PubMed Central

MacMillan-Crow, L A; Crow, J P; Kerby, J D; Beckman, J S; Thompson, J A

1996-01-01

Inflammatory processes in chronic rejection remain a serious clinical problem in organ transplantation. Activated cellular infiltrate produces high levels of both superoxide and nitric oxide. These reactive oxygen species interact to form peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. We identified enhanced immunostaining for nitrotyrosine localized to tubular epithelium of chronically rejected human renal allografts. Western blot analysis of rejected tissue demonstrated that tyrosine nitration was restricted to a few specific polypeptides. Immunoprecipitation and amino acid sequencing techniques identified manganese superoxide dismutase, the major antioxidant enzyme in mitochondria, as one of the targets of tyrosine nitration. Total manganese superoxide dismutase protein was increased in rejected kidney, particularly in the tubular epithelium; however, enzymatic activity was significantly decreased. Exposure of recombinant human manganese superoxide dismutase to peroxynitrite resulted in a dose-dependent (IC50 = 10 microM) decrease in enzymatic activity and concomitant increase in tyrosine nitration. Collectively, these observations suggest a role for peroxynitrite during development and progression of chronic rejection in human renal allografts. In addition, inactivation of manganese superoxide dismutase by peroxynitrite may represent a general mechanism that progressively increases the production of peroxynitrite, leading to irreversible oxidative injury to mitochondria. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8876227

Green coconut fiber: a novel carrier for the immobilization of commercial laccase by covalent attachment for textile dyes decolourization.

PubMed

Cristóvão, Raquel O; Silvério, Sara C; Tavares, Ana P M; Brígida, Ana Iraidy S; Loureiro, José M; Boaventura, Rui A R; Macedo, Eugénia A; Coelho, Maria Alice Z

2012-09-01

Commercial laccase formulation was immobilized on modified green coconut fiber silanized with 3-glycidoxypropyltrimethoxysilane, aiming to achieve a cheap and effective biocatalyst. Two different strategies were followed: one point (pH 7.0) and multipoint (pH 10.0) covalent attachment. The influence of immobilization time on enzymatic activity and the final reduction with sodium borohydride were evaluated. The highest activities were achieved after 2 h of contact time in all situations. Commercial laccase immobilized at pH 7.0 was found to have higher activity and higher affinity to the substrate. However, the immobilization by multipoint covalent attachment improved the biocatalyst thermal stability at 50 °C, when compared to soluble enzyme and to the immobilized enzyme at pH 7.0. The Schiff's bases reduction by sodium borohydride, in spite of causing a decrease in enzyme activity, showed to contribute to the increase of operational stability through bonds stabilization. Finally, these immobilized enzymes showed high efficiency in the continuous decolourization of reactive textile dyes. In the first cycle, the decolourization is mainly due to dyes adsorption on the support. However, when working in successive cycles, the adsorption capacity of the support decreases (saturation) and the enzymatic action increases, indicating the applicability of this biocatalyst for textile wastewater treatment.

[Method of component assay of alpha-glucosyltransferase-treated stevia (enzymatically modified stevia) products using enzymatic hydrolysis].

PubMed

Hirata, Keiko; Shimamura, Yasuhiro; Suzuki, Keiko; Sadamasu, Yuki; Ito, Koichi

2005-12-01

We have developed an analytical method for components of alpha-glucosyltransferase-treated stevia, a food additive product. Suitable conditions to separate additional sugar from alpha-glucosyltransferase-treated stevia by using glucoamylase were found (55 degrees C for 3 hr with 250 U of glucoamylase in 10 mL of reaction solution). By solid-phase extraction using a C18 cartridge column, polysaccharides were excluded from the sample, and the glycosides and sugar obtained after hydrolysis with glucoamylase were separated on another C18 cartridge column. The glycosides and sugar contents were determined by HPLC. By this method, additional sugar was detected in all of three product samples tested and the sugar was glucose. The contents of glucose and total glycosides (minus unreacted glycoside) were 25-42% and 35.7-52.5%, respectively. In alpha-glucosyltransferase-treated stevia, the sum of total glycosides and glucose amounted to 77.5-80.4% of the total and their recoveries from samples from which polysaccharide had been excluded by C18 cartridge column processing were over 85%. The contents of alpha-glucosyltransferase-treated stevia obtained by multiplying the sugar content by the coefficient (0.9) for hydrolysis and converting on dry weight basis were all over 80.0% and met the standard set by the Japan Food Additives Association.

Production and partial characterization of lipases from a newly isolated Penicillium sp. using experimental design.

PubMed

Wolski, E; Rigo, E; Di Luccio, M; Oliveira, J V; de Oliveira, D; Treichel, H

2009-07-01

The objective of this work was to investigate the lipase production by a newly isolated Penicillium sp., using experimental design technique, in submerged fermentation using a medium based on peptone, yeast extract, NaCl and olive oil, as well as to characterize the crude enzymatic extracts obtained. Lipase activity values of 9.5 U ml(-1) in 96 h of fermentation was obtained at the maximized operational conditions of peptone, yeast extract, NaCl and olive oil concentrations (g l(-1)) of 20.0, 5.0, 5.0 and of 10.0 respectively. The partial characterization of crude enzymatic extract obtained by submerged fermentation showed optimum activity at pH range from 4.9 to 5.5 and temperature from 37 degrees C to 42 degrees C. The crude extract maintained its initial activity at freezing temperatures up to 100 days. A newly isolated strain of Penicillium sp. used in this work yielded good lipase activities compared to the literature. The growing interest in lipase production is related to the potential biotechnological applications that these enzymes present. New lipase producers are relevant to finding enzymes with different catalytic properties of commercial interest could be obtained, without using genetically modified organisms (GMO).

Nucleoside adducts from the in vitro reaction of benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide or benzo[a]pyrene 4,5-oxide with nucleic acids.

PubMed

Jennette, K W; Jeffrey, A M; Blobstein, S H; Beland, F A; Harvey, R G; Weinstein, I B

1977-03-08

The covalent binding of benzo[a]pyrene 4,5-oxide and benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide isomer I and isomer II to nucleic acids in aqueous acetone solution has been investigated. Benzo[a]pyrene 4,5-oxide reacted preferentially with guanosine residues. On the other hand, benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide isomer I and II reacted extensively with guanosine, adenosine, and cytidine residues. Time course studies showed that the reactivity of isomer I or isomer II with homopolyribonucleotides followed the order poly(G) greater than poly(A) greater than poly(C). Alkaline or enzymatic hydrolysis of the modified nucleic acids and subsequent chromatography on Sephadex LH-20 columns yielded benzo[a]pyrene-nucleotide adducts. These were enzymatically converted to the corresponding nucleosides which were resolved into several distinct components by high-pressure liquid chromatography. Evidence was obtained for the presence of multiple nucleoside adducts of guanosine, adenosine, cytidine, deoxyguanosine, deoxyadenosine, and deoxycytidine. The HPLC profiles of adducts formed with isomer I were different from the corresponding profiles of adducts formed with isomer II. Structural aspects of these nucleoside adducts are discussed.

A novel three-dimensional carbonized PANI1600@CNTs network for enhanced enzymatic biofuel cell.

PubMed

Kang, Zepeng; Jiao, Kailong; Cheng, Jin; Peng, Ruiyun; Jiao, Shuqiang; Hu, Zongqian

2018-03-15

A novel three-dimensional (3D) carbon composite of PANI 1600 @CNTs with rhizobium-like structure is prepared by in-situ polymerization of aniline monomers around and along the functionalized carbon nanotubes (CNTs) and then carbonized at 1600°C for enzymatic biofuel cells (EBFCs). The SEM and TEM images clearly show that the carbonized PANI grew seamlessly on the surface of CNTs and presented the rhizobium-like structure. The carbonized PANI acts like conductive "glue" and connects the adjacent tubes together, which can assemble the CNTs into a 3D network. The PANI 1600 @CNTs composite modified glassy carbon electrodes based on glucose oxidase (GOx) and laccase (Lac) exhibit high electrochemical performance. A glucose//O 2 EBFC constitutes of the fabricated anode and cathode performs a maximum power density of 1.12mWcm -2 at 0.45V. Furthermore, three of the fabricated EBFCs in series are able to lightening up a yellow light-emitting diode (LED) whose turn-on voltage is about at 1.8V. This work may be helpful for exploiting novel substrates by carbonizing the composites of conducting polymer with nano materials at high-temperature for immobilization of enzymes in the EBFCs or biosensor fields. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of ethanol as a co-solvent on the aerosol performance and stability of spray-dried lysozyme.

PubMed

Ji, Shuying; Thulstrup, Peter Waaben; Mu, Huiling; Hansen, Steen Honoré; van de Weert, Marco; Rantanen, Jukka; Yang, Mingshi

2016-11-20

In the spray drying process, organic solvents can be added to facilitate drying, accommodate certain functional excipients, and modify the final particle characteristics. In this study, lysozyme was used as a model pharmaceutical protein to study the effect of ethanol as a co-solvent on the stability and aerosol performance of spray-dried protein. Lysozyme was dissolved in solutions with various ratios of ethanol and water, and subsequently spray-dried. A change from spherical particles into wrinkled and folded particles was observed upon increasing the ratio of ethanol in the feed. The aerosol performance of the spray-dried lysozyme from ethanol-water solution was improved compared to that from pure water. The conformation of lysozyme in the ethanol-water solution and spray dried powder was altered, but the native structure of lysozyme was restored upon reconstitution in water after the spray drying process. The enzymatic activities of the spray-dried lysozyme showed no significant impact of ethanol; however, the lysozyme enzymatic activity was ca. 25% lower compared to the starting material. In conclusion, the addition of ethanol as a co-solvent in the spray drying feed for lysozyme did not compromise the conformation of the protein after drying, while it improved the inhaled aerosol performance. Copyright © 2016 Elsevier B.V. All rights reserved.

Microfluidic Assembly of a Multifunctional Tailorable Composite System Designed for Site Specific Combined Oral Delivery of Peptide Drugs.

PubMed

Araújo, Francisca; Shrestha, Neha; Shahbazi, Mohammad-Ali; Liu, Dongfei; Herranz-Blanco, Bárbara; Mäkilä, Ermei M; Salonen, Jarno J; Hirvonen, Jouni T; Granja, Pedro L; Sarmento, Bruno; Santos, Hélder A

2015-08-25

Multifunctional tailorable composite systems, specifically designed for oral dual-delivery of a peptide (glucagon-like peptide-1) and an enzymatic inhibitor (dipeptidyl peptidase 4 (DPP4)), were assembled through the microfluidics technique. Both drugs were coloaded into these systems for a synergistic therapeutic effect. The systems were composed of chitosan and cell-penetrating peptide modified poly(lactide-co-glycolide) and porous silicon nanoparticles as nanomatrices, further encapsulated in an enteric hydroxypropylmethylcellulose acetylsuccinate polymer. The developed multifunctional systems were pH-sensitive, inherited by the enteric polymer, enabling the release of the nanoparticles only in the simulated intestinal conditions. Moreover, the encapsulation into this polymer prevented the degradation of the nanoparticles' modifications. These nanoparticles showed strong and higher interactions with the intestinal cells in comparison with the nonmodified ones. The presence of DPP4 inhibitor enhanced the peptide permeability across intestinal cell monolayers. Overall, this is a promising platform for simultaneously delivering two drugs from a single formulation. Through this approach peptides are expected to increase their bioavailability and efficiency in vivo both by their specific release at the intestinal level and also by the reduced enzymatic activity. The use of this platform, specifically in combination of the two antidiabetic drugs, has clinical potential for the therapy of type 2 diabetes mellitus.

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

Effect of glycation derived from α-dicarbonyl compounds on the in vitro digestibility of β-casein and β-lactoglobulin: A model study with glyoxal, methylglyoxal and butanedione.

PubMed

Zhao, Di; Le, Thao T; Larsen, Lotte Bach; Li, Lin; Qin, Dan; Su, Guoying; Li, Bing

2017-12-01

α-Dicarbonyl compounds, which are widely found in common consumed food, are one of the precursors of advanced glycation end products (AGEs). In this study, the effect of glycation derived from glyoxal (GO), methylglyoxal (MGO) or butanedione (BU) on the in vitro digestibility of β-casein (β-CN) and β-lactoglobulin (β-Lg) was investigated. Glycation from α-dicarbonyl compounds reduced the in vitro digestibility of studied proteins in both gastric and intestinal stage. In addition, glycation substantially altered the peptides released through gastric and gastrointestinal digestion, as detected by liquid chromatography electrospray-ionization tandem mass spectrometry (LC-ESI-MS/MS). Crosslinked glycation structures derived from BU considerably reduced the sensitivity of glycated β-Lg towards digestive proteases, albeit to a lesser degree in glycated β-CN due to its intrinsic unordered structure. By contrast, non-crosslinked AGEs that formed adjacent to enzymatic cleavage sites did not block the enzymatic reaction in several cases, as evidenced by the corresponding digested peptides modified with glycation structures. These findings expand our understanding of the nutritional influence of α-dicarbonyl compounds and health impact of relevant dietary AGEs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Engineering of vault nanocapsules with enzymatic and fluorescent properties

PubMed Central

Kickhoefer, Valerie A.; Garcia, Yvette; Mikyas, Yeshi; Johansson, Erik; Zhou, Jing C.; Raval-Fernandes, Sujna; Minoofar, Payam; Zink, Jeffrey I.; Dunn, Bruce; Stewart, Phoebe L.; Rome, Leonard H.

2005-01-01

One of the central issues facing the emerging field of nanotechnology is cellular compatibility. Nanoparticles have been proposed for diagnostic and therapeutic applications, including drug delivery, gene therapy, biological sensors, and controlled catalysis. Viruses, liposomes, peptides, and synthetic and natural polymers have been engineered for these applications, yet significant limitations continue to prevent their use. Avoidance of the body's natural immune system, lack of targeting specificity, and the inability to control packaging and release are remaining obstacles. We have explored the use of a naturally occurring cellular nanoparticle known as the vault, which is named for its morphology with multiple arches reminiscent of cathedral ceilings. Vaults are 13-MDa ribonucleoprotein particles with an internal cavity large enough to sequester hundreds of proteins. Here, we report a strategy to target and sequester biologically active materials within the vault cavity. Attachment of a vault-targeting peptide to two proteins, luciferase and a variant of GFP, resulted in their sequestration within the vault cavity. The targeted proteins confer enzymatic and fluorescent properties on the recombinant vaults, both of which can be detected by their emission of light. The modified vaults are compatible with living cells. The ability to engineer vault particles with designed properties and functionalities represents an important step toward development of a biocompatible nanocapsule. PMID:15753293

Engineering of vault nanocapsules with enzymatic and fluorescent properties.

PubMed

Kickhoefer, Valerie A; Garcia, Yvette; Mikyas, Yeshi; Johansson, Erik; Zhou, Jing C; Raval-Fernandes, Sujna; Minoofar, Payam; Zink, Jeffrey I; Dunn, Bruce; Stewart, Phoebe L; Rome, Leonard H

2005-03-22

One of the central issues facing the emerging field of nanotechnology is cellular compatibility. Nanoparticles have been proposed for diagnostic and therapeutic applications, including drug delivery, gene therapy, biological sensors, and controlled catalysis. Viruses, liposomes, peptides, and synthetic and natural polymers have been engineered for these applications, yet significant limitations continue to prevent their use. Avoidance of the body's natural immune system, lack of targeting specificity, and the inability to control packaging and release are remaining obstacles. We have explored the use of a naturally occurring cellular nanoparticle known as the vault, which is named for its morphology with multiple arches reminiscent of cathedral ceilings. Vaults are 13-MDa ribonucleoprotein particles with an internal cavity large enough to sequester hundreds of proteins. Here, we report a strategy to target and sequester biologically active materials within the vault cavity. Attachment of a vault-targeting peptide to two proteins, luciferase and a variant of GFP, resulted in their sequestration within the vault cavity. The targeted proteins confer enzymatic and fluorescent properties on the recombinant vaults, both of which can be detected by their emission of light. The modified vaults are compatible with living cells. The ability to engineer vault particles with designed properties and functionalities represents an important step toward development of a biocompatible nanocapsule.

Film Self-Assembly of Oppositely Charged Macromolecules Triggered by Electrochemistry through a Morphogenic Approach.

PubMed

Dochter, Alexandre; Garnier, Tony; Pardieu, Elodie; Chau, Nguyet Trang Thanh; Maerten, Clément; Senger, Bernard; Schaaf, Pierre; Jierry, Loïc; Boulmedais, Fouzia

2015-09-22

The development of new surface functionalization methods that are easy to use, versatile, and allow local deposition represents a real scientific challenge. Overcoming this challenge, we present here a one-pot process that consists in self-assembling, by electrochemistry on an electrode, films made of oppositely charged macromolecules. This method relies on a charge-shifting polyanion, dimethylmaleic-modified poly(allylamine) (PAHd), that undergoes hydrolysis at acidic pH, leading to an overall switching of its charge. When a mixture of the two polyanions, PAHd and poly(styrenesulfonate) (PSS), is placed in contact with an electrode, where the pH is decreased locally by electrochemistry, the transformation of PAHd into a polycation (PAH) leads to the continuous self-assembly of a nanometric PAH/PSS film by electrostatic interactions. The pH decrease is obtained by the electrochemical oxidation of hydroquinone, which produces protons locally over nanometric distances. Using a negatively charged enzyme, alkaline phosphatase (AP), instead of PSS, this one-pot process allows the creation of enzymatically active films. Under mild conditions, self-assembled PAH/AP films have an enzymatic activity which is adjustable simply by controlling the self-assembly time. The selective functionalization of microelectrode arrays by PAH/AP was achieved, opening the route toward miniaturized biosensors.

Investigation of a submerged membrane reactor for continuous biomass hydrolysis

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

Malmali, Mohammadmahdi; Stickel, Jonathan; Wickramasinghe, S. Ranil

Enzymatic hydrolysis of cellulose is one of the most costly steps in the bioconversion of lignocellulosic biomass. Use of a submerged membrane reactor has been investigated for continuous enzymatic hydrolysis of cellulose thus allowing for greater use of the enzyme compared to a batch process. Moreover, the submerged 0.65 μm polyethersulfone microfiltration membrane avoids the need to pump a cellulose slurry through an external loop. Permeate containing glucose is withdrawn at pressures slightly below atmospheric pressure. The membrane rejects cellulose particles and cellulase enzyme bound to cellulose. Our proof-of-concept experiments have been conducted using a modified, commercially available membrane filtrationmore » cell under low fluxes around 75 L/(m2 h). The operating flux is determined by the rate of glucose production. Maximizing the rate of glucose production involves optimizing mixing, reactor holding time, and the time the feed is held in the reactor prior to commencement of membrane filtration and continuous operation. When we maximize glucose production rates it will require that we operate it at low glucose concentration in order to minimize the adverse effects of product inhibition. Consequently practical submerged membrane systems will require a combined sugar concentration step in order to concentrate the product sugar stream prior to fermentation.« less

Hydrolase stabilization via entanglement in poly(propylene sulfide) nanoparticles: stability towards reactive oxygen species.

PubMed

Allen, Brett L; Johnson, Jermaine D; Walker, Jeremy P

2012-07-27

In the advancement of green syntheses and sustainable reactions, enzymatic biocatalysis offers extremely high reaction rates and selectivity that goes far beyond the reach of chemical catalysts; however, these enzymes suffer from typical environmental constraints, e.g. operational temperature, pH and tolerance to oxidative environments. A common hydrolase enzyme, diisopropylfluorophosphatase (DFPase, EC 3.1.8.2), has demonstrated a pronounced efficacy for the hydrolysis of a variety of substrates for potential toxin remediation, but suffers from the aforementioned limitations. As a means to enhance DFPase's stability in oxidative environments, enzymatic covalent immobilization within the polymeric matrix of poly(propylene sulfide) (PPS) nanoparticles was performed. By modifying the enzyme's exposed lysine residues via thiolation, DFPase is utilized as a comonomer/crosslinker in a mild emulsion polymerization. The resultant polymeric polysulfide shell acts as a 'sacrificial barrier' by first oxidizing to polysulfoxides and polysulfones, rendering DFPase in an active state. DFPase-PPS nanoparticles thus retain activity upon exposure to as high as 50 parts per million (ppm) of hypochlorous acid (HOCl), while native DFPase is observed as inactive at 500 parts per billion (ppb). This trend is also confirmed by enzyme-generated (chloroperoxidase (CPO), EC 1.11.1.10) reactive oxygen species (ROS) including both HOCl (3 ppm) and ClO(2) (100 ppm).

Continuous monitoring of L-glutamate released from cultured nerve cells by an online sensor coupled with micro-capillary sampling.

PubMed

Niwa, O; Horiuchi, T; Torimitsu, K

1997-01-01

A small volume L-glutamate online sensor was developed in order to monitor changes in the local concentration of L-glutamate released from cultured nerve cells. Syringe pump in the suction mode is used to sample extracellular fluid continuously from a glass micro-capillary and the concentration of L-glutamate can be determined by using a glassy carbon (GC) electrode modified with an Os-polyvinylpyridine mediator bottom film containing horseradish peroxidase and a bovine serum albumin top layer containing L-glutamate oxidase. The overall efficiency of L-glutamate detection with a sensor is 71% under optimum conditions due to an efficient enzymatic reaction at the modified electrode in the thin layer radial flow cell. As a result, we achieved a detection limit of 7-15 nM and a linear range of 50 nM to 10 microM. In an in vitro experiment, the extracellular fluid near a particular nerve cell can be sampled with this micro-pipet and continuously introduced into the modified GC electrode in the radial flow cell via suction provided by a syringe pump. The nerve cells are stimulated by the KCl in a glass capillary and the L-glutamate concentration change can be monitored by changing the distance between the sampling pipet and the nerve cells.

Flow injection amperometric sensor with a carbon nanotube modified screen printed electrode for determination of hydroquinone.

PubMed

Upan, Jantima; Reanpang, Preeyaporn; Chailapakul, Orawon; Jakmunee, Jaroon

2016-01-01

Flow injection amperometric (FI-Amp) sensor was developed for sensitive and selective determination of hydroquinone. A simple screen printed carbon electrode (SPCE) was modified with various nanomaterials for improvement of sensitivity on the determination of quinone. As a result, the appropriate sensitivity is obtained from the SPCE modified with carbon nanotube (CNT) which indicated that CNT contributed to the transfer of electron to quinone. The reproducibility (n=9) and repeatability (n=111) of SPCE-CNT were obtained at 4.4% and 3.6%RSD, respectively. The SPCE-CNT electrode and enzymatic column were incorporated to the FI-Amp system to determine hydroquinone. Laccase was immobilized on silica gel using a cross-linking method by glutaraldehyde modification and then packed in the column. The laccase column has high efficiency for catalytic oxidation of hydroquinone to quinone, which further detects by amperometric detection. Parameters affecting response of the proposed sensor, i.e., pH, ionic strength, and temperature have been optimized. The proposed system provided a wide linear range between 1 and 50 µM with detection limit of 0.1 µM. Satisfactory recoveries in the range of 91.2-103.8% were obtained for the analysis of water sample. Copyright © 2015 Elsevier B.V. All rights reserved.

Cost Analysis of Cot-Side Screening Methods for Neonatal Hypoglycaemia.

PubMed

Glasgow, Matthew J; Harding, Jane E; Edlin, Richard

2018-06-12

Babies at risk of neonatal hypoglycaemia are often screened using cot-side glucometers, but non-enzymatic glucometers are inaccurate, potentially resulting in over-treatment and under-treatment, and low values require laboratory confirmation. More accurate enzymatic glucometers are available but at apparently higher costs. Our objective was to compare the cost of screening for neonatal hypoglycaemia using point-of-care enzymatic and non-enzymatic glucometers. We used a decision tree to model costs, including consumables and staff time. Sensitivity analyses assessed the impact of staff time, staff costs, probability that low results are confirmed via laboratory testing, false-positive and false-negative rates of non-enzymatic glucometers, and the blood glucose concentration threshold. In the primary analysis, screening using an enzymatic glucometer cost NZD 86.94 (USD 63.47) while using a non-enzymatic glucometer cost NZD 97.08 (USD 70.87) per baby. Sensitivity analyses showed that using an enzymatic glucometer is cost saving with wide variations in staff time and costs, irrespective of the false-positive level of non-enzymatic glucometers, and where ≥78% of low values are laboratory confirmed. Where non-enzymatic glucometers may be less costly (e.g., false-negative rate exceeds 15%), instances of hypoglycaemia will be missed. Reducing the blood glucose concentration threshold to 1.94 mmol/L reduced the incidence of hypoglycaemia from 52 to 13%, and the cost of screening using a non-enzymatic glucometer to NZD 47.71 (USD 34.83). In view of their lower cost in most circumstances and greater accuracy, enzymatic glucometers should be routinely utilised for point-of-care screening for neonatal hypoglycaemia. © 2018 S. Karger AG, Basel.

Cftr gene targeting in mouse embryonic stem cells mediated by Small Fragment Homologous Replacement (SFHR).

PubMed

Sangiuolo, Federica; Scaldaferri, Maria Lucia; Filareto, Antonio; Spitalieri, Paola; Guerra, Lorenzo; Favia, Maria; Caroppo, Rosa; Mango, Ruggiero; Bruscia, Emanuela; Gruenert, Dieter C; Casavola, Valeria; De Felici, Massimo; Novelli, Giuseppe

2008-01-01

Different gene targeting approaches have been developed to modify endogenous genomic DNA in both human and mouse cells. Briefly, the process involves the targeting of a specific mutation in situ leading to the gene correction and the restoration of a normal gene function. Most of these protocols with therapeutic potential are oligonucleotide based, and rely on endogenous enzymatic pathways. One gene targeting approach, "Small Fragment Homologous Replacement (SFHR)", has been found to be effective in modifying genomic DNA. This approach uses small DNA fragments (SDF) to target specific genomic loci and induce sequence and subsequent phenotypic alterations. This study shows that SFHR can stably introduce a 3-bp deletion (deltaF508, the most frequent cystic fibrosis (CF) mutation) into the Cftr (CF Transmembrane Conductance Regulator) locus in the mouse embryonic stem (ES) cell genome. After transfection of deltaF508-SDF into murine ES cells, SFHR-mediated modification was evaluated at the molecular levels on DNA and mRNA obtained from transfected ES cells. About 12% of transcript corresponding to deleted allele was detected, while 60% of the electroporated cells completely lost any measurable CFTR-dependent chloride efflux. The data indicate that the SFHR technique can be used to effectively target and modify genomic sequences in ES cells. Once the SFHR-modified ES cells differentiate into different cell lineages they can be useful for elucidating tissue-specific gene function and for the development of transplantation-based cellular and therapeutic protocols.

Stimulation and inhibition of enzymatic hydrolysis by organosolv lignins as determined by zeta potential and hydrophobicity

Treesearch

Yang Huang; Shaolong Sun; Chen Huang; Qiang Yong; Thomas Elder; Maobing Tu

2017-01-01

Background: Lignin typically inhibits enzymatic hydrolysis of cellulosic biomass, but certain organosolv lignins or lignosulfonates enhance enzymatic hydrolysis. The hydrophobic and electrostatic interactions between lignin and cellulases play critical roles in the enzymatic hydrolysis process. However, how to incorporate these two...

Nucleophilic modification of human complement protein C3: correlation of conformational changes with acquisition of C3b-like functional properties.

PubMed

Isenman, D E; Kells, D I; Cooper, N R; Müller-Eberhard, H J; Pangburn, M K

1981-07-21

Inactivation of C3 by enzymatic cleavage, nucleophilic addition, or slow freezing and thawing resulted in the acquisition of similar end-state conformations as judged by near-UV circular dichroism. Although inactivation by the two nonenzymatic processes involves no peptide bond scission, the inactivated C3 resembled C3b in that it possessed a free sulfhydryl group not present in the native protein and an increased surface hydrophobicity as evidenced by enhanced binding of the fluorophore 8-anilino-1-naphthalensulfonate (ANS). The C3b-like functional properties of modified C3 [Pangburn, M. K., & Müller-Eberhard, H. J. (1980) J. Exp. Med. 152, 1102-1114] may thus be understood in terms of the similarity of its conformation to that of C3b. The rate of the conformational change following proteolytic cleavage was fast and appeared to be limited by the rate of the enzymatic reaction. In contrast, the rate of conformational change following addition of methylamine was slow and rate limited by the conformational rearrangement itself, not by the chemical modification. A kinetic analysis of the changes in circular dichroism and ANS fluorescence enhancement suggested that the nucleophilic addition was spectroscopically undetectable and was followed by a minimally biphasic, spectroscopically demonstrable conformational rearrangement. The appearance of C3b-like functional activity in nucleophile-modified C3 largely parallels the time course of the spectroscopically detectable conformational change but is distinctly slower than the rate at which hemolytic activity is lost. While fully transconformed methylamine-inactivated C3 can bind factor B and is susceptible to cleavage by C3b inactivator and its cofactor beta 1H, this cleavage occurs at a substantially slower rate than the equivalent process in C3b. The implications of these findings in terms of the mechanism through which the alterative pathway of complement is initiated are discussed.

Detection of Catalase as a major protein target of the lipid peroxidation product 4-HNE and the lack of its genetic association as a risk factor in SLE

PubMed Central

D'souza, Anil; Kurien, Biji T; Rodgers, Rosalie; Shenoi, Jaideep; Kurono, Sadamu; Matsumoto, Hiroyuki; Hensley, Kenneth; Nath, Swapan K; Scofield, R Hal

2008-01-01

Background Systemic lupus erythematosus (SLE) is a multifactorial disorder characterized by the presence of autoantibodies. We and others have implicated free radical mediated peroxidative damage in the pathogenesis of SLE. Since harmful free radical products are formed during this oxidative process, including 4-hydroxy 2-nonenol (4-HNE) and malondialdehyde (MDA), we hypothesized that specific HNE-protein adducts would be present in SLE red blood cell (RBC) membranes. Catalase is located on chromosome 11p13 where linkage analysis has revealed a marker in the same region of the genome among families with thrombocytopenia, a clinical manifestation associated with severe lupus in SLE affected pedigrees. Moreover, SLE afflicts African-Americans three times more frequently than their European-American counterparts. Hence we investigated the effects of a genetic polymorphism of catalase on risk and severity of SLE in 48 pedigrees with African American ancestry. Methods Tryptic digestion followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis was used to identify the protein modified by HNE, following Coomassie staining to visualize the bands on the acrylamide gels. Genotyping analysis for the C → T, -262 bp polymorphism in the promoter region of catalase was performed by PCR-RFLP and direct PCR-sequencing. We used a "pedigree disequilibrium test" for the family based association analysis, implemented in the PDT program to analyze the genotyping results. Results We found two proteins to be HNE-modified, migrating around 80 and 50 kD respectively. Tryptic digestion followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of the Coomassie stained 80 kD band revealed that the target of HNE modification was catalase, a protein shown to associate with RBC membrane proteins. All the test statistics carried out on the genotyping analysis for the C → T, -262 bp polymorphism in the promoter region of catalase were non-significant (p > 0.05) in our data, which suggested that this SNP is not associated with SLE. Conclusion Our results indicate that catalase is one of the proteins modified due to oxidative stress. However, catalase may not be a susceptibility gene for SLE. Nonetheless, catalase is oxidatively modified among SLE patients. This suggests a possible role between oxidative modification of catalase and its affects on enzymatic activity in SLE. An oxidatively modified catalase could be one of the reasons for lower enzymatic activity among SLE subjects, which in turn could favor the accumulation of deleterious hydrogen peroxide. Furthermore, HNE-products are potential neoantigens and could be involved in the pathogenesis of SLE. Decrease in catalase activity could affect the oxidant-antioxidant balance. Chronic disturbance of this balance in patients with SLE may work favorably for the premature onset of atherogenesis with severe vascular effect. PMID:18606005

Low concentration of sodium bicarbonate improves the bioactive compound levels and antioxidant and α-glucosidase inhibitory activities of tartary buckwheat sprouts.

PubMed

Qin, Peiyou; Wei, Aichun; Zhao, Degang; Yao, Yang; Yang, Xiushi; Dun, Baoqing; Ren, Guixing

2017-06-01

This study aimed to investigate the effects of different concentrations of sodium bicarbonate (NaHCO 3 ) on the accumulation of flavonoids, total phenolics and d-chiro-inositol (DCI), as well as the antioxidant and α-glucosidase inhibitory activities, in tartary buckwheat sprouts. Treatment with low concentrations of NaHCO 3 (0.05, 0.1, and 0.2%) resulted in an increase in flavonoids, total phenolic compounds and DCI concentrations, and improved DPPH radical-scavenging and α-glucosidase inhibition activities compared with the control (0%). The highest levels of total flavonoids (26.69mg/g DW), individual flavonoids (rutin, isoquercitrin, quercetin, and kaempferol), total phenolic compounds (29.31mg/g DW), DCI (12.56mg/g DW), as well as antioxidant and α-glucosidase inhibition activities, were observed in tartary buckwheat sprouts treated with 0.05% NaHCO 3 for 96h. These results indicated that appropriate treatment with NaHCO 3 could improve the healthy benefits of tartary buckwheat sprouts. Copyright © 2016 Elsevier Ltd. All rights reserved.

Application of mixed cloud point extraction for the analysis of six flavonoids in Apocynum venetum leaf samples by high performance liquid chromatography.

PubMed

Zhou, Jun; Sun, Jiang Bing; Xu, Xin Yu; Cheng, Zhao Hui; Zeng, Ping; Wang, Feng Qiao; Zhang, Qiong

2015-03-25

A simple, inexpensive and efficient method based on the mixed cloud point extraction (MCPE) combined with high performance liquid chromatography was developed for the simultaneous separation and determination of six flavonoids (rutin, hyperoside, quercetin-3-O-sophoroside, isoquercitrin, astragalin and quercetin) in Apocynum venetum leaf samples. The non-ionic surfactant Genapol X-080 and cetyl-trimethyl ammonium bromide (CTAB) was chosen as the mixed extracting solvent. Parameters that affect the MCPE processes, such as the content of Genapol X-080 and CTAB, pH, salt content, extraction temperature and time were investigated and optimized. Under the optimized conditions, the calibration curve for six flavonoids were all linear with the correlation coefficients greater than 0.9994. The intra-day and inter-day precision (RSD) were below 8.1% and the limits of detection (LOD) for the six flavonoids were 1.2-5.0 ng mL(-1) (S/N=3). The proposed method was successfully used to separate and determine the six flavonoids in A. venetum leaf samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Preparative isolation and purification of seven main antioxidants from Eucommia ulmoides Oliv. (Du-zhong) leaves using HSCCC guided by DPPH-HPLC experiment.

PubMed

Dai, Xingping; Huang, Qiong; Zhou, Boting; Gong, Zhicheng; Liu, Zhaoqian; Shi, Shuyun

2013-08-15

Seven antioxidants were purified from Eucommia ulmoides Oliv. leaves using HSCCC guided by DPPH-HPLC experiment. HSCCC was successfully used to separate target antioxidants by three runs with different solvent systems after D101 column chromatography fractionation. Ethyl acetate-n-butanol-water (1:2:3, v/v/v) was selected as the optimum solvent system to purify geniposidic acid. Ethyl acetate-ethanol-water (4:1:5, v/v/v) was used to isolate caffeic acid, chlorogenic acid and ferulic acid. While three flavonoids, quercetin-3-O-sambubioside, rutin and isoquercitrin were purified by petroleum ether-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v). The structures were identified by MS and NMR. Antioxidant activities were assessed, and compounds 2-7 showed strong antioxidant activities. This is the first report about separation of antioxidants from E. ulmoides leaves by HSCCC. The results indicated that the combinative methods using DPPH-HPLC and HSCCC could be widely applied for screening and isolation of antioxidants from complex extracts. Copyright © 2013 Elsevier Ltd. All rights reserved.

Annurca peel extract: from the chemical composition, through the functional activity, to the formulation and characterisation of a topical oil-in-water emulsion.

PubMed

Sansone, Francesca; Esposito, Tiziana; Mencherini, Teresa; Piccinelli, Anna Lisa; Gazzerro, Patrizia; Picerno, Patrizia; Russo, Paola; Del Gaudio, Pasquale; Essolito, Massimilano; Campiglia, Pietro; Aquino, Rita P

2016-06-01

The aim of this study was to produce a hydro-alcoholic safe antioxidant Malus pumila Miller cv Annurca peel extract (APE) useful as functional ingredient in an oil-in-water emulsion. Results showed that APE contains a hydroxycinnamic acid (chlorogenic acid), flavonol glycosides (quercetin derivatives) and a dihydrochalcone, phloridzin (phloretin-2-O-glucoside). The isoquercitrin (quercetin-3-O-glucoside) content was quantified in 0.3% w/w of extract. APE showed a significant and concentration-dependent free-radical scavenging activity correlated to its polyphenols content. No cytotoxic effect was observed in primary human epidermal keratinocyte adults and dermal fibroblast cell lines. The formulative approach led to produce a stable emulsion able to load a high amount of APE, up to 6.0% w/w. The homogenous distribution of APE in the emulsion was clearly demonstrated by fluorescence microscopy analysis. The emulsion resulted able to enhance the in vitro release rate of APE through synthetic membranes with respect to the raw material.

Foliar phenolics in sugar maple (Acer saccharum) as a potential indicator of tropospheric ozone pollution.

PubMed

Sager, E P S; Hutchinson, T C; Croley, T R

2005-06-01

Tropospheric O3 has been implicated in the declining health of forest ecosystems in Europe and North America and has been shown to have negative consequences on human health. We have measured tropospheric ozone (O3) in the lower canopy through the use of passive monitors located in five woodlots along a 150 km urban-rural transect, originating in the large urban complex of Toronto, Canada. We also sampled foliage from 10 mature sugar maple trees in each woodlot and measured the concentration of a number of phenolic compounds and macronutrients. O3 concentrations were highest in the two rural woodlots, located approximately 150 km downwind of Toronto, when compared to the woodlots found within the Greater Toronto Area. Foliar concentrations of three flavonoids, avicularin, isoquercitrin, and quercitrin, were significantly greater and nitrogen concentrations significantly lower at these same rural woodlots, suggesting some physiological disruption is occurring in those sites where exposure to tropospheric O3 is greater. We suggest that foliar phenolics of sugar maple may be a biochemical indicator of tropospheric ozone exposure.

Quantitative determination of plant phenolics in Urtica dioica extracts by high-performance liquid chromatography coupled with tandem mass spectrometric detection.

PubMed

Orčić, Dejan; Francišković, Marina; Bekvalac, Kristina; Svirčev, Emilija; Beara, Ivana; Lesjak, Marija; Mimica-Dukić, Neda

2014-01-15

A method for quantification of 45 plant phenolics (including benzoic acids, cinnamic acids, flavonoid aglycones, C- and O-glycosides, coumarins, and lignans) in plant extracts was developed, based on reversed phase HPLC separation of extract components, followed by tandem mass spectrometric detection. The phenolic profile of 80% MeOH extracts of the stinging nettle (Urtica dioica L.) herb, root, stem, leaf and inflorescence was obtained by using this method. Twenty-one of the investigated compounds were present at levels above the reliable quantification limit, with 5-O-caffeoylquinic acid, rutin and isoquercitrin as the most abundant. The inflorescence extracts were by far the richest in phenolics, with the investigated compounds amounting 2.5-5.1% by weight. As opposed to this, the root extracts were poor in phenolics, with only several acids and derivatives being present in significant amounts. The results obtained by the developed method represent the most detailed U. dioica chemical profile so far. Copyright © 2013 Elsevier Ltd. All rights reserved.

HDAC inhibition modulates hippocampus-dependent long-term memory for object location in a CBP-dependent manner

PubMed Central

Haettig, Jakob; Stefanko, Daniel P.; Multani, Monica L.; Figueroa, Dario X.; McQuown, Susan C.; Wood, Marcelo A.

2011-01-01

Transcription of genes required for long-term memory not only involves transcription factors, but also enzymatic protein complexes that modify chromatin structure. Chromatin-modifying enzymes, such as the histone acetyltransferase (HAT) CREB (cyclic-AMP response element binding) binding protein (CBP), are pivotal for the transcriptional regulation required for long-term memory. Several studies have shown that CBP and histone acetylation are necessary for hippocampus-dependent long-term memory and hippocampal long-term potentiation (LTP). Importantly, every genetically modified Cbp mutant mouse exhibits long-term memory impairments in object recognition. However, the role of the hippocampus in object recognition is controversial. To better understand how chromatin-modifying enzymes modulate long-term memory for object recognition, we first examined the role of the hippocampus in retrieval of long-term memory for object recognition or object location. Muscimol inactivation of the dorsal hippocampus prior to retrieval had no effect on long-term memory for object recognition, but completely blocked long-term memory for object location. This was consistent with experiments showing that muscimol inactivation of the hippocampus had no effect on long-term memory for the object itself, supporting the idea that the hippocampus encodes spatial information about an object (such as location or context), whereas cortical areas (such as the perirhinal or insular cortex) encode information about the object itself. Using location-dependent object recognition tasks that engage the hippocampus, we demonstrate that CBP is essential for the modulation of long-term memory via HDAC inhibition. Together, these results indicate that HDAC inhibition modulates memory in the hippocampus via CBP and that different brain regions utilize different chromatin-modifying enzymes to regulate learning and memory. PMID:21224411

Evaluation of physical structural features on influencing enzymatic hydrolysis efficiency of micronized wood

Treesearch

Jinxue Jiang; Jinwu Wang; Xiao Zhang; Michael Wolcott

2016-01-01

Enzymatic hydrolysis of lignocellulosic biomass is highly dependent on the changes in structural features after pretreatment. Mechanical milling pretreatment is an effective approach to alter the physical structure of biomass and thus improve enzymatic hydrolysis. This study examined the influence of structural characteristics on the enzymatic hydrolysis of micronized...

Biotin determination in food supplements by an electrochemical magneto biosensor.

PubMed

Kergaravat, Silvina V; Gómez, Gabriel A; Fabiano, Silvia N; Laube Chávez, Tamara I; Pividori, María I; Hernández, Silvia R

2012-08-15

An electrochemical magneto biosensor for the rapid determination of biotin in food samples is reported. The affinity reaction was performed on streptavidin-modified magnetic microbeads as a solid support in a direct competitive format. The biotinylated horseradish peroxidase enzyme (biotin-HRP) competes with free biotin in the sample for the binding sites of streptavidin on the magnetic microbeads. The modified magnetic beads were then easily captured by a magneto graphite-epoxy composite electrode and the electrochemical signal was based on the enzymatic activity of the HRP enzyme under the addition of H(2)O(2) as the substrate and o-phenilendiamine as cosubstrate. The response was electrochemically detected by square wave voltammetry. The limit of detection was 8.4×10(-8) mol L(--1) of biotin (20 μg L(--1)) with a dynamic range from 0.94 to 2.4×10(-7) mol L(--1). Biotin-fortified commercial dietary supplement and infant formula samples were evaluated obtaining good performances in the results. Total time of analysis was 40 min per 20 assays. Copyright © 2012 Elsevier B.V. All rights reserved.

Development of a simple, low cost chronoamperometric assay for fructose based on a commercial graphite-nanoparticle modified screen-printed carbon electrode.

PubMed

Nicholas, Phil; Pittson, Robin; Hart, John P

2018-02-15

This paper describes the development of a simple, low cost chronoamperometric assay, for the measurement of fructose, using a graphite-nanoparticle modified screen-printed electrode (SPCE-G-COOH). Cyclic voltammetry showed that the response of the SPCE-G-COOH enhanced the sensitivity and precision, towards the enzymatically generated ferrocyanide species, over a plain SPCE; therefore the former was employed in subsequent studies. Calibration studies were carried out using chronoamperometry with a 40µl mixture containing fructose, mediator and FDH, deposited onto the SPCE-G-COOH. The response was linear from 0.1mM to 1.0mM. A commercial fruit juice sample was analysed using the developed assay and the fructose concentration was calculated to be 477mM with a precision of 3.03% (n=5). Following fortification (477mM fructose) the mean recovery was found to be 97.12% with a coefficient of variation of 6.42% (n=5); consequently, the method holds promise for the analysis of commercial fruit juices. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Snail repressor recruits EZH2 to specific genomic sites through the enrollment of the lncRNA HOTAIR in epithelial-to-mesenchymal transition

PubMed Central

Battistelli, C; Cicchini, C; Santangelo, L; Tramontano, A; Grassi, L; Gonzalez, F J; de Nonno, V; Grassi, G; Amicone, L; Tripodi, M

2017-01-01

The transcription factor Snail is a master regulator of cellular identity and epithelial-to-mesenchymal transition (EMT) directly repressing a broad repertoire of epithelial genes. How chromatin modifiers instrumental to its activity are recruited to Snail-specific binding sites is unclear. Here we report that the long non-coding RNA (lncRNA) HOTAIR (for HOX Transcript Antisense Intergenic RNA) mediates a physical interaction between Snail and enhancer of zeste homolog 2 (EZH2), an enzymatic subunit of the polycomb-repressive complex 2 and the main writer of chromatin-repressive marks. The Snail-repressive activity, here monitored on genes with a pivotal function in epithelial and hepatic morphogenesis, differentiation and cell-type identity, depends on the formation of a tripartite Snail/HOTAIR/EZH2 complex. These results demonstrate an lncRNA-mediated mechanism by which a transcriptional factor conveys a general chromatin modifier to specific genes, thereby allowing the execution of hepatocyte transdifferentiation; moreover, they highlight HOTAIR as a crucial player in the Snail-mediated EMT. PMID:27452518

Photo-switchable microbial fuel-cells.

PubMed

Schlesinger, Orr; Dandela, Rambabu; Bhagat, Ashok; Adepu, Raju; Meijler, Michael M; Xia, Lin; Alfonta, Lital

2018-05-01

Regulation of Bio-systems in a clean, simple, and efficient way is important for the design of smart bio-interfaces and bioelectronic devices. Light as a non-invasive mean to control the activity of a protein enables spatial and temporal control far superior to other chemical and physical methods. The ability to regulate the activity of a catalytic enzyme in a biofuel-cell reduces the waste of resources and energy and turns the fuel-cell into a smart and more efficient device for power generation. Here we present a microbial-fuel-cell based on a surface displayed, photo-switchable alcohol dehydrogenase. The enzyme was modified near the active site using non-canonical amino acids and a small photo-reactive molecule, which enables reversible control of enzymatic activity. Depending on the modification site, the enzyme exhibits reversible behavior upon irradiation with UV and visible light, in both biochemical, and electrochemical assays. The change observed in power output of a microbial fuel cell utilizing the modified enzyme was almost five-fold, between inactive and active states. © 2018 Wiley Periodicals, Inc.

A cascading activity-based probe sequentially targets E1–E2–E3 ubiquitin enzymes

PubMed Central

Mulder, Monique P.C.; Witting, Katharina; Berlin, Ilana; Pruneda, Jonathan N.; Wu, Kuen-Phon; Chang, Jer-Gung; Merkx, Remco; Bialas, Johanna; Groettrup, Marcus; Vertegaal, Alfred C.O.; Schulman, Brenda A.; Komander, David; Neefjes, Jacques; Oualid, Farid El; Ovaa, Huib

2016-01-01

Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers, orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a staggering breadth of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Akin to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe ‘hops’ and ‘traps’ catalytically active ubiquitin-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activities in living cells, presents novel and versatile tools to interrogate the Ub/Ubl cascades. PMID:27182664

Biocompatible electrochemiluminescent biosensor for choline based on enzyme/titanate nanotubes/chitosan composite modified electrode.

PubMed

Dai, Hong; Chi, Yuwu; Wu, Xiaoping; Wang, Youmei; Wei, Mingdeng; Chen, Guonan

2010-02-15

A new biocompatible ECL biosensor based on enzyme/titanate nanotubes/chitosan composite film was developed for the determination of analytes in biological samples. In the fabrication of the new ECL biosensor, biocompatible titanate nanotubes (TNTs) and a model enzyme, i.e., choline oxidase (ChOX), were immobilized on a chitosan modified glassy carbon electrode (GCE) via electrostatic adsorption and covalent interaction, respectively. By this ECL biosensor, choline was enzymatically oxidized to hydrogen peroxide and detected by a sensitive luminol ECL system. The use of TNTs not only provided a biocompatible microenvironment for the immobilized enzyme, which resulted in an excellent stability and long lifetime of the ECL biosensor, but also exhibited great enhancement towards luminol ECL and thus led to a significant improvement in sensitivity of ECL biosensor. Satisfactory results were obtained when employing this biosensor in assaying the total choline in milk samples. The work would provide a common platform to develop various sensitive, selective and biocompatible ECL biosensors based on using enzyme/TNTs/CHIT composite films. Copyright 2009 Elsevier B.V. All rights reserved.

Assembly of a biocompatible triazole-linked gene by one-pot click-DNA ligation

NASA Astrophysics Data System (ADS)

Kukwikila, Mikiembo; Gale, Nittaya; El-Sagheer, Afaf H.; Brown, Tom; Tavassoli, Ali

2017-11-01

The chemical synthesis of oligonucleotides and their enzyme-mediated assembly into genes and genomes has significantly advanced multiple scientific disciplines. However, these approaches are not without their shortcomings; enzymatic amplification and ligation of oligonucleotides into genes and genomes makes automation challenging, and site-specific incorporation of epigenetic information and/or modified bases into large constructs is not feasible. Here we present a fully chemical one-pot method for the assembly of oligonucleotides into a gene by click-DNA ligation. We synthesize the 335 base-pair gene that encodes the green fluorescent protein iLOV from ten functionalized oligonucleotides that contain 5ʹ-azide and 3ʹ-alkyne units. The resulting click-linked iLOV gene contains eight triazoles at the sites of chemical ligation, and yet is fully biocompatible; it is replicated by DNA polymerases in vitro and encodes a functional iLOV protein in Escherichia coli. We demonstrate the power and potential of our one-pot gene-assembly method by preparing an epigenetically modified variant of the iLOV gene.

[Parametabolism as Non-Specific Modifier of Supramolecular Interactions in Living Systems].

PubMed

Kozlov, V A; Sapozhnikov, S P; Sheptuhina, A I; Golenkov, A V

2015-01-01

As it became known recently, in addition to the enzyme (enzymes and/or ribozymes) in living organisms occur a large number of ordinary chemical reactions without the participation of biological catalysts. These reactions are distinguished by low speed and, as a rule, the irreversibility. For example, along with diabetes mellitus, glycation and fructosilation of proteins are observed resulted in posttranslational modification with the low- or nonfunctioning protein formation which is poorly exposed to enzymatic proteolysis and therefore accumulates in the body. In addition, the known processes such as the nonenzymatic carbomoylation, pyridoxylation and thiamiation proteins. There is a reasonable basis to believe that alcoholic injury also realized through parametabolic secondary metabolites synthesis such as acetaldehyde. At the same time, the progress in supramolecular chemistry proves that in biological objects there is another large group ofparametabolic reactions caused by the formation of supramolecular complexes. Obviously, known parameterizes interactions can modify the formation of supramolecular complexes in living objects. These processes are of considerable interest for fundamental biology and fundamental and practical medicine, but they remain unexplored due to a lack of awareness of a wide range of researchers.

Employing the promiscuity of lantibiotic biosynthetic machineries to produce novel antimicrobials.

PubMed

Montalbán-López, Manuel; van Heel, Auke J; Kuipers, Oscar P

2017-01-01

As the number of new antibiotics that reach the market is decreasing and the demand for them is rising, alternative sources of novel antimicrobials are needed. Lantibiotics are potent peptide antimicrobials that are ribosomally synthesized and stabilized by post-translationally introduced lanthionine rings. Their ribosomal synthesis and enzymatic modifications provide excellent opportunities to design and engineer a large variety of novel antimicrobial compounds. The research conducted in this area demonstrates that the modularity present in both the peptidic rings as well as in the combination of promiscuous modification enzymes can be exploited to further increase the diversity of lantibiotics. Various approaches, where the modifying enzymes and corresponding leader peptides are decoupled from their natural core peptide and integrated in designed plug-and-play production systems, enable the production of modified peptides that are either derived from vast genomic data or designed using functional parts from a wide diversity of core peptides. These approaches constitute a powerful discovery platform to develop novel antimicrobials with high therapeutic potential. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Polyamide 6/chitosan nanofibers as support for the immobilization of Trametes versicolor laccase for the elimination of endocrine disrupting chemicals.

PubMed

Maryšková, Milena; Ardao, Inés; García-González, Carlos A; Martinová, Lenka; Rotková, Jana; Ševců, Alena

2016-07-01

In recent years, there has been an increase in efforts to improve wastewater treatment as the concentration of dangerous pollutants, such as endocrine disrupting chemicals, in wastewater increases. These compounds, which mimic the effect of hormones, have a negative impact on human health and are not easily removed from water. One way to effectively eliminate these pollutants is to use enzymatically activated materials. In this study, we report on the use of laccase from the white rot fungus Trametes versicolor immobilized onto polyamide 6/chitosan (PA6/CHIT) nanofibers modified using two different spacers (bovine serum albumin and hexamethylenediamine). We then tested the ability of the PA6/CHIT-laccase biocatalysts to eliminate a mixture containing 50μM of two endocrine disrupting chemicals: bisphenol A and 17α-ethinylestradiol. The PA6/CHIT nanofiber matrix used in this study not only proved to be a suitable carrier for immobilized and modified laccase but was also efficient in the removal of a mixture of endocrine disrupting chemicals in three treatment cycles. Copyright © 2016 Elsevier Inc. All rights reserved.

Enzymatic membrane reactors for biodegradation of recalcitrant compounds. Application to dye decolourisation.

PubMed

López, C; Mielgo, I; Moreira, M T; Feijoo, G; Lema, J M

2002-11-13

Membrane bioreactors are being increasingly used in enzymatic catalysed transformations. However, the application of enzymatic-based treatment systems in the environmental field is rather unusual. The aim of this paper is to overview the application of enzymatic membrane reactors to wastewater treatment, more specifically to dye decolourisation. Firstly, the basic aspects such as different configurations of enzymatic reactors, advantages and disadvantages associated to their utilisation are revised as well as the application of this technology to wastewater treatment. Secondly, dye decolourisation by white-rot fungi and their oxidative enzymes are discussed, presenting an overall view from for in vivo and in vitro systems. Finally, dye decolourisation by manganese peroxidase in an enzymatic membrane reactor in continuous operation is presented.

Effects of Al(III) and Nano-Al13 Species on Malate Dehydrogenase Activity

PubMed Central

Yang, Xiaodi; Cai, Ling; Peng, Yu; Li, Huihui; Chen, Rong Fu; Shen, Ren Fang

2011-01-01

The effects of different aluminum species on malate dehydrogenase (MDH) activity were investigated by monitoring amperometric i-t curves for the oxidation of NADH at low overpotential using a functionalized multi-wall nanotube (MWNT) modified glass carbon electrode (GCE). The results showed that Al(III) and Al13 can activate the enzymatic activity of MDH, and the activation reaches maximum levels as the Al(III) and Al13 concentration increase. Our study also found that the effects of Al(III) and Al13 on the activity of MDH depended on the pH value and aluminum speciation. Electrochemical and circular dichroism spectra methods were applied to study the effects of nano-sized aluminum compounds on biomolecules. PMID:22163924

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.

Transglutaminase reactivity with gelatine: perspective applications in tissue engineering.

PubMed

Bertoni, F; Barbani, N; Giusti, P; Ciardelli, G

2006-05-01

Gelatine was crosslinked by means of an enzymatic treatment using tissue transglutaminase (tTGase) (Sigma) and microbial transglutaminase (mTGase) (Ajinomoto) which catalyses the formation of isopeptide bonds between the gamma-carbonyl group of a glutamine residue and the epsilon-amino group of a lysine residue. The reaction is an interesting alternative to the traditional glutaraldehyde crosslinking, which has several drawbacks (e.g., in medical application) due to the toxicity of the chemical reagent. To further investigate the possibility to utilize the modified protein for tissue engineering application, TGase crosslinked gelatine was incorporated in a gellan matrix, a polysaccharide, to enhance the stability in aqueous media. Films obtained by casting were characterized by thermal analysis, chemical imaging, swelling behaviour and cell adhesion.

Effects of Al(III) and nano-Al13 species on malate dehydrogenase activity.

PubMed

Yang, Xiaodi; Cai, Ling; Peng, Yu; Li, Huihui; Chen, Rong Fu; Shen, Ren Fang

2011-01-01

The effects of different aluminum species on malate dehydrogenase (MDH) activity were investigated by monitoring amperometric i-t curves for the oxidation of NADH at low overpotential using a functionalized multi-wall nanotube (MWNT) modified glass carbon electrode (GCE). The results showed that Al(III) and Al(13) can activate the enzymatic activity of MDH, and the activation reaches maximum levels as the Al(III) and Al(13) concentration increase. Our study also found that the effects of Al(III) and Al(13) on the activity of MDH depended on the pH value and aluminum speciation. Electrochemical and circular dichroism spectra methods were applied to study the effects of nano-sized aluminum compounds on biomolecules.

Determination of Organophosphate Pesticides at a Carbon Nanotube/Organophosphorus Hydrolase Electrochemical Biosensor

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

Deo, R P.; Wang, Joseph; Block, I

2005-02-08

An amperometric biosensor for organophosphorus (OP) pesticides based on a carbon-nanotube (CNT) modified transducer and an organophosphorus hydrolase (OPH) biocatalyst is described. A bilayer approach with the OPH layer atop of the CNT film was used for preparing the CNT/OPH biosensor. The CNT layer leads to a greatly improved anodic detection of the enzymatically-generated p-nitrophenol product, including higher sensitivity and stability. The sensor performance was optimized with respect to the surface modification and operating conditions. Under the optimal conditions the biosensor was used to measure as low as 0.15 {micro}M paraoxon and 0.8 {micro}M methyl parathion with sensitivities of 25more » and 6 nA/{micro}M, respectively.« less

Electrodeposited gels prepared from protein alloys

PubMed Central

Lin, Yinan; Wang, Siran; Chen, Ying; Wang, Qianrui; Burke, Kelly A; Spedden, Elise M; Staii, Cristian; Weiss, Anthony S; Kaplan, David L

2015-01-01

Aim Silk-tropoelastin alloys, composed of recombinant human tropoelastin and regenerated Bombyx mori silk fibroin, are an emerging, versatile class of biomaterials endowed with tunable combinations of physical and biological properties. Electrodeposition of these alloys provides a programmable means to assemble functional gels with both spatial and temporal controllability. Materials & methods Tropoelastin-modified silk was prepared by enzymatic coupling between tyrosine residues. Hydrogel coatings were electrodeposited using two wire electrodes. Results & discussion Mechanical characterization and in vitro cell culture revealed enhanced adhesive capability and cellular response of these alloy gels as compared with electrogelled silk alone. Conclusion These electro-depositable silk-tropoelastin alloys constitute a suitable coating material for nanoparticle-based drug carriers and offer a novel opportunity for on-demand encapsulation/release of nanomedicine. PMID:25816881

Biocompatibility of nano-hydroxyapatite/polyetheretherketone composite materials with osteoblasts cultured in vitro

NASA Astrophysics Data System (ADS)

Wang, Lin; Huang, Feijuan; Wu, Zhengzhi; Ma, Rui

2017-04-01

The biocompatibility of the Sprague Dawley (SD) rat osteoblasts, which were cultured on the surfaces of nano-hydroxyapatite/polyetheretherketone (n-HA/PEEK) composites were investigated in this work. The osteoblasts of 24- hour old SD rats were cultured and identified by modified enzymatic digestion in vitro. The morphology and proliferation of cells were observed in CCK-8 regent staining, inverted microscopes, and by scanning electron microscopy (SEM) respectively. The results show that n-HA/PEEK composites have good biocompatibility with SD osteoblasts and that they can promote the growth of the cells that were cultured on the surfaces of the composites. The content of HA in n- HA/PEEK composites plays an important role in cell proliferation.

Amadori Glycated Proteins: Role in Production of Autoantibodies in Diabetes Mellitus and Effect of Inhibitors on Non-Enzymatic Glycation

PubMed Central

Ansari, Nadeem A.; Dash, Debabrata

2012-01-01

Nonenzymatic glycation of macromolecules, especially proteins leading to their oxidation is increased in diabetes mellitus due to hyperglycemia and play an important role in associated complications of the disease. The glycation primarily occurs at intrachain lysine residues of proteins and results in the formation of an early stage stable product as Amadori-lysine which undergo further irreversible chemical reactions to form advanced glycation endproducts. This review deals with the role of Amadori modified proteins in pathogenesis of diabetes. We aim to explain immunogenicity of Amadori-glycated proteins, which might be involve in production of serum autoantibodies in the diabetic patients, and effect of inhibitors on the glycation process. PMID:23423609

Tyrosinase-catalyzed site-specific immobilization of engineered C-phycocyanin to surface

PubMed Central

Faccio, Greta; Kämpf, Michael M.; Piatti, Chiara; Thöny-Meyer, Linda; Richter, Michael

2014-01-01

Enzymatic crosslinking of proteins is often limited by the steric availability of the target residues, as of tyrosyl side chains in the case of tyrosinase. Carrying an N-terminal peptide-tag containing two tyrosine residues, the fluorescent protein C-phycocyanin HisCPC from Synechocystis sp. PCC6803 was crosslinked to fluorescent high-molecular weight forms with tyrosinase. Crosslinking with tyrosinase in the presence of L-tyrosine produced non fluorescent high-molecular weight products. Incubated in the presence of tyrosinase, HisCPC could also be immobilized to amino-modified polystyrene beads thus conferring a blue fluorescence. Crosslinking and immobilization were site-specific as both processes required the presence of the N-terminal peptide in HisCPC. PMID:24947668

Improvement of soil characteristics and growth of Dorycnium pentaphyllum by amendment with agrowastes and inoculation with AM fungi and/or the yeast Yarowia lipolytica.

PubMed

Medina, A; Vassileva, M; Caravaca, F; Roldán, A; Azcón, R

2004-08-01

The effectiveness of two microbiologically treated agrowastes [dry olive cake (DOC) and/or sugar beet (SB)] on plant growth, soil enzymatic activities and other soil characteristics was determined in a natural soil from a desertified area. Dorycnium pentaphyllum, a legume plant adapted to stress situations, was the test plant to evaluate the effect of inoculation of native arbuscular mycorrhizal (AM) fungi and/or Yarowia lipolytica (a dry soil adapted yeast) on amended and non-amended soils. Plant growth and nutrition, symbiotic developments and soil enzymatic activities were limited in non-amended soil where microbial inoculations did not improve plant development. The lack of nodules formation and AM colonization can explain the limited plant growth in this natural soil. The effectiveness and performance of inocula applied was only evident in amended soils. AM colonization and spores number in natural soil were increased by amendments and the inoculation with Y. lipolytica promoted this value. The effect of the inoculations on plant N-acquisition was only important in AM-inoculated plants growing in SB medium. Enzymatic activities as urease and protease activities were particularly increased in DOC amended soil meanwhile dehydrogenase activity was greatest in treatments inoculated with Y. lipolytica in SB added soil. The biological activities in rhizosphere of agrowaste amended soil, used as indices of changes in soil properties and fertility, were affected not only by the nature of amendments but also by the inoculant applied. All these results show that the lignocellulosic agrowastes treated with a selected microorganism and its further interaction with beneficial microbial groups (native AM fungi and/or Y. lipolytica) is a useful tool to modify soil physico-chemical, biological and fertility properties that enhance the plant performance probably by making nutrients more available to plants.

Organophosphate-induced changes in the PKA regulatory function of Swiss Cheese/NTE lead to behavioral deficits and neurodegeneration.

PubMed

Wentzell, Jill S; Cassar, Marlène; Kretzschmar, Doris

2014-01-01

Organophosphate-induced delayed neuropathy (OPIDN) is a Wallerian-type axonopathy that occurs weeks after exposure to certain organophosphates (OPs). OPs have been shown to bind to Neuropathy Target Esterase (NTE), thereby inhibiting its enzymatic activity. However, only OPs that also induce the so-called aging reaction cause OPIDN. This reaction results in the release and possible transfer of a side group from the bound OP to NTE and it has been suggested that this induces an unknown toxic function of NTE. To further investigate the mechanisms of aging OPs, we used Drosophila, which expresses a functionally conserved orthologue of NTE named Swiss Cheese (SWS). Treating flies with the organophosporous compound tri-ortho-cresyl phosphate (TOCP) resulted in behavioral deficits and neurodegeneration two weeks after exposure, symptoms similar to the delayed effects observed in other models. In addition, we found that primary neurons showed signs of axonal degeneration within an hour after treatment. Surprisingly, increasing the levels of SWS, and thereby its enzymatic activity after exposure, did not ameliorate these phenotypes. In contrast, reducing SWS levels protected from TOCP-induced degeneration and behavioral deficits but did not affect the axonopathy observed in cell culture. Besides its enzymatic activity as a phospholipase, SWS also acts as regulatory PKA subunit, binding and inhibiting the C3 catalytic subunit. Measuring PKA activity in TOCP treated flies revealed a significant decrease that was also confirmed in treated rat hippocampal neurons. Flies expressing additional PKA-C3 were protected from the behavioral and degenerative phenotypes caused by TOCP exposure whereas primary neurons were not. In addition, knocking-down PKA-C3 caused similar behavioral and degenerative phenotypes as TOCP treatment. We therefore propose a model in which OP-modified SWS cannot release PKA-C3 and that the resulting loss of PKA-C3 activity plays a crucial role in developing the delayed symptoms of OPIDN but not in the acute toxicity.

Organophosphate-Induced Changes in the PKA Regulatory Function of Swiss Cheese/NTE Lead to Behavioral Deficits and Neurodegeneration

PubMed Central

Kretzschmar, Doris

2014-01-01

Organophosphate-induced delayed neuropathy (OPIDN) is a Wallerian-type axonopathy that occurs weeks after exposure to certain organophosphates (OPs). OPs have been shown to bind to Neuropathy Target Esterase (NTE), thereby inhibiting its enzymatic activity. However, only OPs that also induce the so-called aging reaction cause OPIDN. This reaction results in the release and possible transfer of a side group from the bound OP to NTE and it has been suggested that this induces an unknown toxic function of NTE. To further investigate the mechanisms of aging OPs, we used Drosophila, which expresses a functionally conserved orthologue of NTE named Swiss Cheese (SWS). Treating flies with the organophosporous compound tri-ortho-cresyl phosphate (TOCP) resulted in behavioral deficits and neurodegeneration two weeks after exposure, symptoms similar to the delayed effects observed in other models. In addition, we found that primary neurons showed signs of axonal degeneration within an hour after treatment. Surprisingly, increasing the levels of SWS, and thereby its enzymatic activity after exposure, did not ameliorate these phenotypes. In contrast, reducing SWS levels protected from TOCP-induced degeneration and behavioral deficits but did not affect the axonopathy observed in cell culture. Besides its enzymatic activity as a phospholipase, SWS also acts as regulatory PKA subunit, binding and inhibiting the C3 catalytic subunit. Measuring PKA activity in TOCP treated flies revealed a significant decrease that was also confirmed in treated rat hippocampal neurons. Flies expressing additional PKA-C3 were protected from the behavioral and degenerative phenotypes caused by TOCP exposure whereas primary neurons were not. In addition, knocking-down PKA-C3 caused similar behavioral and degenerative phenotypes as TOCP treatment. We therefore propose a model in which OP-modified SWS cannot release PKA-C3 and that the resulting loss of PKA-C3 activity plays a crucial role in developing the delayed symptoms of OPIDN but not in the acute toxicity. PMID:24558370

Randomized DNA libraries construction tool: a new 3-bp 'frequent cutter' TthHB27I/sinefungin endonuclease with chemically-induced specificity.

PubMed

Krefft, Daria; Papkov, Aliaksei; Prusinowski, Maciej; Zylicz-Stachula, Agnieszka; Skowron, Piotr M

2018-05-11

Acoustic or hydrodynamic shearing, sonication and enzymatic digestion are used to fragment DNA. However, these methods have several disadvantages, such as DNA damage, difficulties in fragmentation control, irreproducibility and under-representation of some DNA segments. The DNA fragmentation tool would be a gentle enzymatic method, offering cleavage frequency high enough to eliminate DNA fragments distribution bias and allow for easy control of partial digests. Only three such frequently cleaving natural restriction endonucleases (REases) were discovered: CviJI, SetI and FaiI. Therefore, we have previously developed two artificial enzymatic specificities, cleaving DNA approximately every ~ 3-bp: TspGWI/sinefungin (SIN) and TaqII/SIN. In this paper we present the third developed specificity: TthHB27I/SIN(SAM) - a new genomic tool, based on Type IIS/IIC/IIG Thermus-family REases-methyltransferases (MTases). In the presence of dimethyl sulfoxide (DMSO) and S-adenosyl-L-methionine (SAM) or its analogue SIN, the 6-bp cognate TthHB27I recognition sequence 5'-CAARCA-3' is converted into a combined 3.2-3.0-bp 'site' or its statistical equivalent, while a cleavage distance of 11/9 nt is retained. Protocols for various modes of limited DNA digestions were developed. In the presence of DMSO and SAM or SIN, TthHB27I is transformed from rare 6-bp cutter to a very frequent one, approximately 3-bp. Thus, TthHB27I/SIN(SAM) comprises a new tool in the very low-represented segment of such prototype REases specificities. Moreover, this modified TthHB27I enzyme is uniquely suited for controlled DNA fragmentation, due to partial DNA cleavage, which is an inherent feature of the Thermus-family enzymes. Such tool can be used for quasi-random libraries generation as well as for other DNA manipulations, requiring high frequency cleavage and uniform distribution of cuts along DNA.

Dithiothreitol-Regulated Coverage of Oligonucleotide-Modified Gold Nanoparticles To Achieve Optimized Biosensor Performance.

PubMed

Liang, Pingping; Canoura, Juan; Yu, Haixiang; Alkhamis, Obtin; Xiao, Yi

2018-01-31

DNA-modified gold nanoparticles (AuNPs) are useful signal-reporters for detecting diverse molecules through various hybridization- and enzyme-based assays. However, their performance is heavily dependent on the probe DNA surface coverage, which can influence both target binding and enzymatic processing of the bound probes. Current methods used to adjust the surface coverage of DNA-modified AuNPs require the production of multiple batches of AuNPs under different conditions, which is costly and laborious. We here develop a single-step assay utilizing dithiothreitol (DTT) to fine-tune the surface coverage of DNA-modified AuNPs. DTT is superior to the commonly used surface diluent, mercaptohexanol, as it is less volatile, allowing for the rapid and reproducible controlling of surface coverage on AuNPs with only micromolar concentrations of DTT. Upon adsorption, DTT forms a dense monolayer on gold surfaces, which provides antifouling capabilities. Furthermore, surface-bound DTT adopts a cyclic conformation, which reorients DNA probes into an upright position and provides ample space to promote DNA hybridization, aptamer assembly, and nuclease digestion. We demonstrate the effects of surface coverage on AuNP-based sensors using DTT-regulated DNA-modified AuNPs. We then use these AuNPs to visually detect DNA and cocaine in colorimetric assays based on enzyme-mediated AuNP aggregation. We determine that DTT-regulated AuNPs with lower surface coverage achieve shorter reaction times and lower detection limits relative to those for assays using untreated AuNPs or DTT-regulated AuNPs with high surface coverage. Additionally, we demonstrate that our DTT-regulated AuNPs can perform cocaine detection in 50% urine without any significant matrix effects. We believe that DTT regulation of surface coverage can be broadly employed for optimizing DNA-modified AuNP performance for use in biosensors as well as drug delivery and therapeutic applications.

7-Deazapurine containing DNA: efficiency of c7GdTP, c7AdTP and c7IdTP incorporation during PCR-amplification and protection from endodeoxyribonuclease hydrolysis.

PubMed Central

Seela, F; Röling, A

1992-01-01

The enzymatic synthesis of 7-deazapurine nucleoside containing DNA (501 bp) is performed by PCR-amplification (Taq polymerase) using a pUC18 plasmid DNA as template and the triphosphates of 7-deaza-2'-deoxyguanosine (c7Gd), -adenosine (c7Ad) and -inosine (c7Id). c7GdTP can fully replace dGTP resulting in a completely modified DNA-fragment of defined size and sequence. The other two 7-deazapurine triphosphates (c7AdTP) and (c7IdTP) require the presence of the parent purine 2'-deoxyribonucleotides. In purine/7-deazapurine nucleotide mixtures Taq polymerase prefers purine over 7-deazapurine nucleotides but accepts c7GdTP much better than c7AdTP or c7IdTP. As incorporation of 7-deazapurine nucleotides represents a modification of the major groove of DNA it can be used to probe DNA/protein interaction. Regioselective phosphodiester hydrolysis of the modified DNA-fragments was studied with 28 endodeoxyribonucleases. c7Gd is able to protect the DNA from the phosphodiester hydrolysis in more than 20 cases, only a few enzymes (Mae III, Rsa I, Hind III, Pvu II or Taq I) do still hydrolyze the modified DNA. c7Ad protects DNA less efficiently, as this DNA could only be modified in part. The absence of N-7 as potential binding position or a geometric distortion of the recognition duplex caused by the 7-deazapurine base can account for protection of hydrolysis. Images PMID:1738604

Gold nanoparticles on titanium and interaction with prototype protein.

PubMed

Padmos, J Daniel; Duchesne, Paul; Dunbar, Michael; Zhang, Peng

2010-10-01

Modifying titanium (Ti) implant surfaces with functional proteins can strengthen the interface between prosthesis and bone. A prototype system was developed using gold nanoparticles (AuNPs) to immobilize proteins onto Ti. An electroless (galvanic displacement) deposition method was first used to form AuNPs of controlled size and coverage on commercial Ti foil (giving Ti-AuNPs). Parameters were then modified to create two groups of discs (n = 26) with different average AuNP diameters. Scanning electron microscopy and X-ray photoelectron spectroscopy were used to characterize the morphology and surface structure of Ti-AuNPs. To study the interaction of Ti-AuNPs with proteins, Ti discs (n = 8) modified with plain AuNPs and discs (n = 8) modified with thiol (HS--R--COOH)-functionalized AuNPs were treated with lysozyme solution. The amount and activity of the lysozyme on the discs were examined with Micro-BCA and enzymatic assays. Lysozyme was immobilized onto the discs, and the assays showed that the discs with thiol-functionalized AuNPs, discs with bare AuNPs, and Ti controls had average lysozyme adsorptions of 23 x 10(4), 2.3 x 10(4), and 5.7 x 10(4) microg/m2, respectively. The activity assays showed that 21.5, 18.4, and 12.5% of the adsorbed lysozyme was active on the discs with thiol-functionalized AuNPs, discs with bare AuNPs, and Ti controls, respectively. This technique holds promise for binding functional biomolecules to surgical implants, hence possibly creating implant surfaces that react to their local environment. Copyright 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010.

Plant regeneration from leaf protoplasts of Solanum torvum.

PubMed

Guri, A; Volokita, M; Sink, K C

1987-07-01

A protocol to obtain regenerated plants from protoplasts of Solanum torvum Sw a wild species of eggplant resistant to Verticillium wilt is reported. Leaf protoplasts were enzymatically isolated from six-week old seedlings grown in a controlled environment chamber. Protoplasts were plated on modified KM medium (0.4 M glucose)+(mg/l): 1.0 p-chlorophenoxyacetic acid (CPA)+1.0 naphthaleneacetic acid (NAA)+0.5 6-benzylaminopurine (BAP) and 0.02 abscisic acid (ABA). The protoplast density was 5×10(4) per ml with 5 ml placed in each of two quadrants in X-dishes (100×15 mm). The reservoir medium was modified KM+(mg/l): 0.1 NAA+0.5 BAP+0.1 M sucrose+0.1 M mannitol+0.6% washed agar+1% activated charcoal. Dishes were initially placed in the dark at 27°C. Protoplast division was initiated in 1-2 weeks and 4 weeks later p-calli were 1-3 mm. Plating efficiency was 11% when measured at 3 weeks. Six-week old p-calli were transferred individually onto Whatman No. 1 filter paper layered on modified KM (0.15 M sucrose)+mg/l: 2.0 indoleacetic acid (IAA)+2.0 zeatin+0.5% washed agar for 2 weeks. Subsequently, shoots occurred within 4 weeks at 70% efficiency on MS+30 g/l sucrose+2 mg/l zeatin. Shoots were rooted on half strength MS+10 g/l sucrose.

Biofuel cells based on direct enzyme-electrode contacts using PQQ-dependent glucose dehydrogenase/bilirubin oxidase and modified carbon nanotube materials.

PubMed

Scherbahn, V; Putze, M T; Dietzel, B; Heinlein, T; Schneider, J J; Lisdat, F

2014-11-15

Two types of carbon nanotube electrodes (1) buckypaper (BP) and (2) vertically aligned carbon nanotubes (vaCNT) have been used for elaboration of glucose/O2 enzymatic fuel cells exploiting direct electron transfer. For the anode pyrroloquinoline quinone dependent glucose dehydrogenase ((PQQ)GDH) has been immobilized on [poly(3-aminobenzoic acid-co-2-methoxyaniline-5-sulfonic acid), PABMSA]-modified electrodes. For the cathode bilirubin oxidase (BOD) has been immobilized on PQQ-modified electrodes. PABMSA and PQQ act as promoter for enzyme bioelectrocatalysis. The voltammetric characterization of each electrode shows current densities in the range of 0.7-1.3 mA/cm(2). The BP-based fuel cell exhibits maximal power density of about 107 µW/cm(2) (at 490 mV). The vaCNT-based fuel cell achieves a maximal power density of 122 µW/cm(2) (at 540 mV). Even after three days and several runs of load a power density over 110 µW/cm(2) is retained with the second system (10mM glucose). Due to a better power exhibition and an enhanced stability of the vaCNT-based fuel cells they have been studied in human serum samples and a maximal power density of 41 µW/cm(2) (390 mV) can be achieved. Copyright © 2014 Elsevier B.V. All rights reserved.

Optical tracking of organically modified silica nanoparticles as DNA carriers: A nonviral, nanomedicine approach for gene delivery

NASA Astrophysics Data System (ADS)

Roy, Indrajit; Ohulchanskyy, Tymish Y.; Bharali, Dhruba J.; Pudavar, Haridas E.; Mistretta, Ruth A.; Kaur, Navjot; Prasad, Paras N.

2005-01-01

This article reports a multidisciplinary approach to produce fluorescently labeled organically modified silica nanoparticles as a nonviral vector for gene delivery and biophotonics methods to optically monitor intracellular trafficking and gene transfection. Highly monodispersed, stable aqueous suspensions of organically modified silica nanoparticles, encapsulating fluorescent dyes and surface functionalized by cationic-amino groups, are produced by micellar nanochemistry. Gel-electrophoresis studies reveal that the particles efficiently complex with DNA and protect it from enzymatic digestion of DNase 1. The electrostatic binding of DNA onto the surface of the nanoparticles, due to positively charged amino groups, is also shown by intercalating an appropriate dye into the DNA and observing the Förster (fluorescence) resonance energy transfer between the dye (energy donor) intercalated in DNA on the surface of nanoparticles and a second dye (energy acceptor) inside the nanoparticles. Imaging by fluorescence confocal microscopy shows that cells efficiently take up the nanoparticles in vitro in the cytoplasm, and the nanoparticles deliver DNA to the nucleus. The use of plasmid encoding enhanced GFP allowed us to demonstrate the process of gene transfection in cultured cells. Our work shows that the nanomedicine approach, with nanoparticles acting as a drug-delivery platform combining multiple optical and other types of probes, provides a promising direction for targeted therapy with enhanced efficacy as well as for real-time monitoring of drug action. nonviral vector | ORMOSIL nanoparticles | confocal microscopy

Metabolic De-Isotoping for Improved LC-MS Characterization of Modified RNAs

NASA Astrophysics Data System (ADS)

Wetzel, Collin; Li, Siwei; Limbach, Patrick A.

2014-07-01

Mapping, sequencing, and quantifying individual noncoding ribonucleic acids (ncRNAs), including post-transcriptionally modified nucleosides, by mass spectrometry is a challenge that often requires rigorous sample preparation prior to analysis. Previously, we have described a simplified method for the comparative analysis of RNA digests (CARD) that is applicable to relatively complex mixtures of ncRNAs. In the CARD approach for transfer RNA (tRNA) analysis, two complete sets of digestion products from total tRNA are compared using the enzymatic incorporation of 16O/18O isotopic labels. This approach allows one to rapidly screen total tRNAs from gene deletion mutants or comparatively sequence total tRNA from two related bacterial organisms. However, data analysis can be challenging because of convoluted mass spectra arising from the natural 13C and 15 N isotopes present in the ribonuclease-digested tRNA samples. Here, we demonstrate that culturing in 12C-enriched/13C-depleted media significantly reduces the isotope patterns that must be interpreted during the CARD experiment. Improvements in data quality yield a 35 % improvement in detection of tRNA digestion products that can be uniquely assigned to particular tRNAs. These mass spectral improvements lead to a significant reduction in data processing attributable to the ease of spectral identification of labeled digestion products and will enable improvements in the relative quantification of modified RNAs by the 16O/18O differential labeling approach.

GRP78 enabled micelle-based glioma targeted drug delivery.

PubMed

Ran, Danni; Mao, Jiani; Shen, Qing; Xie, Cao; Zhan, Changyou; Wang, Ruifeng; Lu, Weiyue

2017-06-10

GRP78, a specific cancer cell-surface marker, is implicated in cancer cells proliferation, apoptosis resistance, metastasis and drug resistance. l-VAP (SNTRVAP) is a tumor homing peptide exhibiting high binding affinity in vitro to GRP78 protein overexpressed on glioma, glioma stem cells, vasculogenic mimicry and neovasculature. Even though short peptides are often non-immunogenic and demonstrate high affinity to tumor cells, their targeting efficacy is always undermined by rapid blood clearance and enzymatic degradation. In the present study, two d peptides RI-VAP (retro inverso isomer of l-VAP) and d-VAP (retro isomer of l-VAP) were developed by structure-guided peptide design and retro-inverso isomerization technique for glioma targeting. RI-VAP and d-VAP were predicted to bind their receptor GRP78 protein with similar binding affinity, which was experimentally confirmed. The results of in vivo imaging demonstrated that RI-VAP and d-VAP had remarkably advantage over l-VAP for tumor accumulation. In addition, RI-VAP and d-VAP modified paclitaxel-loaded polymeric micelle had better anti-tumor efficacy in comparison to taxol, paclitaxel-loaded plain micelles and l-VAP modified micelles. Overall, the VAP modified micelles suggested in the present study could effectively achieve glioma-targeted drug delivery, validating the potential of the stable VAP peptides in improving the therapeutic efficacy of paclitaxel for glioma. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

PubMed Central

Ren, Xiaomei; El-Sagheer, Afaf H.; Brown, Tom

2016-01-01

A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX. PMID:26819406

The Apicomplexa-specific glucosamine-6-phosphate N-acetyltransferase gene family encodes a key enzyme for glycoconjugate synthesis with potential as therapeutic target.

PubMed

Cova, Marta; López-Gutiérrez, Borja; Artigas-Jerónimo, Sara; González-Díaz, Aida; Bandini, Giulia; Maere, Steven; Carretero-Paulet, Lorenzo; Izquierdo, Luis

2018-03-05

Apicomplexa form a phylum of obligate parasitic protozoa of great clinical and veterinary importance. These parasites synthesize glycoconjugates for their survival and infectivity, but the enzymatic steps required to generate the glycosylation precursors are not completely characterized. In particular, glucosamine-phosphate N-acetyltransferase (GNA1) activity, needed to produce the essential UDP-N-acetylglucosamine (UDP-GlcNAc) donor, has not been identified in any Apicomplexa. We scanned the genomes of Plasmodium falciparum and representatives from six additional main lineages of the phylum for proteins containing the Gcn5-related N-acetyltransferase (GNAT) domain. One family of GNAT-domain containing proteins, composed by a P. falciparum sequence and its six apicomplexan orthologs, rescued the growth of a yeast temperature-sensitive GNA1 mutant. Heterologous expression and in vitro assays confirmed the GNA1 enzymatic activity in all lineages. Sequence, phylogenetic and synteny analyses suggest an independent origin of the Apicomplexa-specific GNA1 family, parallel to the evolution of a different GNA1 family in other eukaryotes. The inability to disrupt an otherwise modifiable gene target suggests that the enzyme is essential for P. falciparum growth. The relevance of UDP-GlcNAc for parasite viability, together with the independent evolution and unique sequence features of Apicomplexa GNA1, highlights the potential of this enzyme as a selective therapeutic target against apicomplexans.

Noncanonical substrate preference of lambda exonuclease for 5′-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction

PubMed Central

Yang, Yufei; Chen, Wei; Wang, Jiayu; Yang, Ziyu; Wang, Shenlin; Xiao, Xianjin; Li, Mengyuan

2018-01-01

Abstract Lambda exonuclease (λ exo) plays an important role in the resection of DNA ends for DNA repair. Currently, it is also a widely used enzymatic tool in genetic engineering, DNA-binding protein mapping, nanopore sequencing and biosensing. Herein, we disclose two noncanonical properties of this enzyme and suggest a previously undescribed hydrophobic interaction model between λ exo and DNA substrates. We demonstrate that the length of the free portion of the substrate strand in the dsDNA plays an essential role in the initiation of digestion reactions by λ exo. A dsDNA with a 5′ non-phosphorylated, two-nucleotide-protruding end can be digested by λ exo with very high efficiency. Moreover, we show that when a conjugated structure is covalently attached to an internal base of the dsDNA, the presence of a single mismatched base pair at the 5′ side of the modified base may significantly accelerate the process of digestion by λ exo. A detailed comparison study revealed additional π–π stacking interactions between the attached label and the amino acid residues of the enzyme. These new findings not only broaden our knowledge of the enzyme but will also be very useful for research on DNA repair and in vitro processing of nucleic acids. PMID:29490081

Comparison of NiS2 and α-NiS hollow spheres for supercapacitors, non-enzymatic glucose sensors and water treatment.

PubMed

Wei, Chengzhen; Cheng, Cheng; Cheng, Yanyan; Wang, Yan; Xu, Yazhou; Du, Weimin; Pang, Huan

2015-10-21

NiS2 hollow spheres are successfully prepared by a one-step template free method. Meanwhile, α-NiS hollow spheres can also be synthesized via the calcination of the pre-obtained NiS2 hollow spheres at 400 °C for 1 h in air. The electrochemical performances of the as-prepared NiS2 and α-NiS hollow sphere products are evaluated. When used for supercapacitors, compared with NiS2 hollow spheres, the α-NiS hollow sphere electrode shows a large specific capacitance of 717.3 F g(-1) at 0.6 A g(-1) and a good cycle life. Furthermore, NiS2 and α-NiS hollow spheres are successfully applied to fabricate non-enzymatic glucose sensors. In particular, the α-NiS hollow spheres exhibit good catalytic activity for the oxidation of glucose, a fast amperometric response time of less than 5 s, and the detection limit is estimated to be 0.08 μM. More importantly, compared with other normally co-existing interfering species, such as ascorbic acid, uric acid and dopamine, the electrode modified with α-NiS hollow spheres shows good selectivity. Moreover, the α-NiS hollow spheres also present good capacity to remove Congo red organic pollutants from wastewater by their surface adsorption ability.

Functional Characterization of Lpt3 and Lpt6, the Inner-Core Lipooligosaccharide Phosphoethanolamine Transferases from Neisseria meningitidis▿

PubMed Central

Wenzel, Cory Q.; St. Michael, Frank; Stupak, Jacek; Li, Jianjun; Cox, Andrew D.; Richards, James C.

2010-01-01

The lipooligosaccharide (LOS) of Neisseria meningitidis contains heptose (Hep) residues that are modified with phosphoethanolamine (PEtn) at the 3 (3-PEtn) and/or 6 (6-PEtn) position. The lpt3 (NMB2010) and lpt6 (NMA0408) genes of N. meningitidis, which are proposed to encode the required HepII 3- and 6-PEtn transferases, respectively, were cloned and overexpressed as C-terminally polyhistidine-tagged fusion proteins in Escherichia coli and found to localize to the inner membrane, based on sucrose density gradient centrifugation. Lpt3-His6 and Lpt6-His6 were purified from Triton X-100-solubilized membranes by nickel chelation chromatography, and dot blot analysis of enzymatic reactions with 3-PEtn- and 6-PEtn-specific monoclonal antibodies demonstrated conclusively that Lpt3 and Lpt6 are phosphatidylethanolamine-dependent LOS HepII 3- and 6-PEtn transferases, respectively, and that both enzymes are capable of transferring PEtn to both fully acylated LOS and de-O-acylated (de-O-Ac) LOS. Further enzymatic studies using capillary electrophoresis-mass spectrometry (MS) demonstrated that both Lpt3 and Lpt6 are capable of transferring PEtn to de-O-Ac LOS molecules already containing PEtn at the 6 and 3 positions of HepII, respectively, demonstrating that there is no obligate order of PEtn addition in the generation of 3,6-di-PEtn LOS moieties in vitro. PMID:19854897

Monotopic modifications derived from in vitro glycation of albumin with ribose.

PubMed

Pataridis, Statis; Stastná, Zdeňka; Sedláková, Pavla; Mikšík, Ivan

2013-06-01

Post-translational modifications are significant reactions that occur to proteins. One of these modifications is a non-enzymatic reaction between the oxo-group(s) of sugars and amino-group(s) of protein - glycation. This reaction plays an important role in the chronic complications of diabetes mellitus, or in the aging process of organisms, that is, it has an important role in the pathophysiology and "normal" physiology of animals. In the work presented here, we studied the glycation of albumins (HSA and BSA). Methodologically, we used nano-LC coupled to a QTOF mass spectrometer. In vitro-modified proteins were cleaved by trypsin and the arising peptides were separated on a C(18) nano column with a trap-column. Peptides and their modifications were analysed with a high-resolution QTOF mass spectrometer with a mass determination precision of better than 5 ppm. Non-enzymatic in vitro reaction products between albumin and ribose were identified. Besides well-known carboxymethyl lysine, new modifications were determined - creating mass shifts of 78 and 218. The origin of the first modification is discussed and its possible structure is presented. In addition, a mass shift of 132 belonging to a Schiff base was also identified. The location of all the modifications within the structure of the proteins was determined and their reactivity to various oxo-compounds was also examined. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Surface plasmon resonance biosensor for enzymatic detection of small analytes

NASA Astrophysics Data System (ADS)

Massumi Miyazaki, Celina; Makoto Shimizu, Flávio; Mejía-Salazar, J. R.; Oliveira, Osvaldo N., Jr.; Ferreira, Marystela

2017-04-01

Surface plasmon resonance (SPR) biosensing is based on the detection of small changes in the refractive index on a gold surface modified with molecular recognition materials, thus being mostly limited to detecting large molecules. In this paper, we report on a SPR biosensing platform suitable to detect small molecules by making use of the mediator-type enzyme microperoxidase-11 (MP11) in layer-by-layer films. By depositing a top layer of glucose oxidase or uricase, we were able to detect glucose or uric acid with limits of detection of 3.4 and 0.27 μmol l-1, respectively. Measurable SPR signals could be achieved because of the changes in polarizability of MP11, as it is oxidized upon interaction with the analyte. Confirmation of this hypothesis was obtained with finite difference time domain simulations, which also allowed us to discard the possible effects from film roughness changes observed in atomic force microscopy images. The main advantage of this mediator-type enzyme approach is in the simplicity of the experimental method that does not require an external potential, unlike similar approaches for SPR biosensing of small molecules. The detection limits reported here were achieved without optimizing the film architecture, and therefore the performance can in principle be further enhanced, while the proposed SPR platform may be extended to any system where hydrogen peroxide is generated in enzymatic reactions.

The relation between xyr1 overexpression in Trichoderma harzianum and sugarcane bagasse saccharification performance.

PubMed

da Silva Delabona, Priscila; Rodrigues, Gisele Nunes; Zubieta, Mariane Paludetti; Ramoni, Jonas; Codima, Carla Aloia; Lima, Deise Juliana; Farinas, Cristiane Sanchez; da Cruz Pradella, José Geraldo; Seiboth, Bernhard

2017-03-20

This work investigates the influence of the positive regulator XYR1 of Trichoderma harzianum on the production of cellulolytic enzymes, using sugarcane bagasse as carbon source. Constitutive expression of xyr1 was achieved under the control of the strong Trichoderma reesei pki1 promoter. Five clones with xyr1 overexpression achieved higher xyr1 expression and greater enzymatic productivity when cultivated under submerged fermentation, hence validating the genetic construction for T. harzianum. Clone 5 presented a relative expression of xyr1 26-fold higher than the parent strain and exhibited 66, 37, and 36% higher values for filter paper activity, xylanase activity, and β-glucosidase activity, respectively, during cultivation in a stirred-tank bioreactor. The overexpression of xyr1 in T. harzianum resulted in an enzymatic complex with significantly improved performance in sugarcane bagasse saccharification, with an enhancement of 25% in the first 24h. Our results also show that constitutive overexpression of xyr1 leads to the induction of several important players in biomass degradation at early (24h) and also late (48h) timepoints of inoculation. However, we also observed that the carbon catabolite repressor CRE1 was upregulated in xyr1 overexpression mutants. These findings demonstrate the feasibility of improving cellulase production by modifying regulator expression and suggest an attractive approach for increasing total cellulase productivity in T. harzianum. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparison of various types of stationary phases in non-aqueous reversed-phase high-performance liquid chromatography-mass spectrometry of glycerolipids in blackcurrant oil and its enzymatic hydrolysis mixture.

PubMed

Lísa, Miroslav; Holcapek, Michal; Sovová, Helena

2009-11-20

The selection of column packing during the development of high-performance liquid chromatography method is a crucial step to achieve sufficient chromatographic resolution of analyzed species in complex mixtures. Various stationary phases are tested in this paper for the analysis of complex mixture of triacylglycerols (TGs) in blackcurrant oil using non-aqueous reversed-phase (NARP) system with acetonitrile-2-propanol mobile phase. Conventional C(18) column in the total length of 45 cm is used for the separation of TGs according to their equivalent carbon number, the number and positions of double bonds and acyl chain lengths. The separation of TGs and their more polar hydrolysis products after the partial enzymatic hydrolysis of blackcurrant oil in one chromatographic run is achieved using conventional C(18) column. Retention times of TGs are reduced almost 10 times without the loss of the chromatographic resolution using ultra high-performance liquid chromatography with 1.7 microm C(18) particles. The separation in NARP system on C(30) column shows an unusual phenomenon, because the retention order of TGs changes depending on the column temperature, which is reported for the first time. The commercial monolithic column modified with C(18) is used for the fast analysis of TGs to increase the sample throughput but at cost of low resolution.

Physicochemical properties of cross-linked and acetylated starches and products of their hydrolysis in continuous recycle membrane reactor.

PubMed

Prochaska, Krystyna; Konował, Emilia; Sulej-Chojnacka, Joanna; Lewandowicz, Grazyna

2009-11-01

The aim of the present work was to study the physicochemical properties of doubly modified, by cross-linking and acetylating, starches as well as the products of their enzymatic hydrolysis. A two step procedure of hydrolysis, including the batch and membrane reactors, were investigated. The second step of enzymatic processes were carried out in a continuous recycle membrane reactor (CRMR). Three kinds of commercial starches--two preparations of acetylated distarch adipate E1422 of different degrees of cross-linking, as well as one preparation of acetylated distarch phosphate E1414 were examined. It was found that the degree of substitution of acetyl groups in the macromolecules of starch did not influence the effectiveness of hydrolysis. However, the degree of cross-linking with adipate groups slightly decreased the efficiency of processing in the CRMR. Additionally, the relationship between the type of hydrocolloid and its adsorption activity in the air/water and oil/water systems was considered. All obtained derivatives revealed adsorption properties and reduced the surface/interface tension in the air/water and oil/water systems. The efficiency and effectiveness of adsorption of the investigated hydrocolloids were affected by the type of modification as well as the degree of substitution of acetyl groups in the macromolecules of starch. Particle size distributions formed in aqueous solutions for all investigated hydrolyses were determined and compared with results obtained for commercial products.

Immobilization of α-amylase onto a calix[4]arene derivative: Evaluation of its enzymatic activity.

PubMed

Veesar, Irshad Ali; Solangi, Imam Bakhsh; Memon, Shahabuddin

2015-06-01

In order to enhance the cost-effectiveness practicability of enzymes in many industries such as pharmaceutical, food, medical and some other technological processes, there is great need to immobilize them onto a solid supports. In this study, a new and efficient immobilization of α-amylase from Saccharomyces cerevisiae has been developed by using the surface functionalization of calix[4]arene as support. A glutaraldehyde-containing amino group functionalized calix[4]arene was used to immobilize α-amylase covalently. In this procedure, imide bonds are formed between amino groups on the protein and aldehyde groups on the calix[4]arene surface. The surface modified support was characterized using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM). The effect of various preparation conditions on the immobilized α-amylase process such as immobilization time, enzyme concentration, temperature and pH were investigated. The influence of pH and temperature on the activity of free and immobilized α-amylase was also studied using starch as substrate. The optimum reaction temperature and pH value for the enzymatic conversion catalyzed by the immobilized α-amylase were 25°C and 7, respectively. Compared to the free enzyme, the immobilized α-amylase retained 85% of its original activity and exhibited significant thermal stability than the free one and excellent durability. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization of the activity and stability of amylase from saliva and detergent: laboratory practicals for studying the activity and stability of amylase from saliva and various commercial detergents.

PubMed

Valls, Cristina; Rojas, Cristina; Pujadas, Gerard; Garcia-Vallve, Santi; Mulero, Miquel

2012-07-01

This article presents two integrated laboratory exercises intended to show students the role of α-amylases (AAMYs) in saliva and detergents. These laboratory practicals are based on the determination of the enzymatic activity of amylase from saliva and different detergents using the Phadebas test (quantitative) and the Lugol test (qualitative) under different conditions (e.g. variations in temperature and alkalinity). This work also proposes the study of enzyme stability in the presence of several surfactants and oxidizing agents using the same technical approach. The proposed laboratory exercises promote the understanding of the physiological function of this enzyme and the biotechnological applications of AAMYs in the detergent industry. The exercises also promote the understanding that the enzymatic stability and performance are dependent on the organism of origin, and if necessary, these properties could be modified by genetic engineering. In addition, this article reinforces the development of laboratory skills, problem-solving capabilities, and the ability to write a laboratory report. The exercises are proposed primarily as an undergraduate project for advanced students in the biochemical and biotechnological sciences. These laboratory practicals are complementary to the previously published BAMBED article (Biochemistry and Molecular Biology Education Vol. 39, No. 4, pp. 280-290, 2011) on detergent proteases. Copyright © 2012 Wiley Periodicals, Inc.

Ecological guild and enzyme activities of rhizosphere soil microbial communities associated with Bt-maize cultivation under field conditions in North West Province of South Africa.

PubMed

van Wyk, Deidré A B; Adeleke, Rasheed; Rhode, Owen H J; Bezuidenhout, Carlos C; Mienie, Charlotte

2017-09-01

Insecticidal proteins expressed by genetically modified Bt maize may alter the enzymatic and microbial communities associated with rhizosphere soil. This study investigated the structure and enzymatic activity of rhizosphere soil microbial communities associated with field grown Bt and non-Bt maize. Rhizosphere soil samples were collected from Bt and non-Bt fields under dryland and irrigated conditions. Samples were subjected to chemical tests, enzyme analyses, and next generation sequencing. Results showed that nitrate and phosphorus concentrations were significantly higher in non-Bt maize dryland soils, while organic carbon was significantly higher in non-Bt maize irrigated field soil. Acid phosphatase and β-glucosidase activities were significantly reduced in soils under Bt maize cultivation. The species diversity differed between fields and Bt and non-Bt maize soils. Results revealed that Actinobacteria, Proteobacteria, and Acidobacteria were the dominant phyla present in these soils. Redundancy analyses indicated that some chemical properties and enzyme activities could explain differences in bacterial community structures. Variances existed in microbial community structures between Bt and non-Bt maize fields. There were also differences between the chemical and biochemical properties of rhizosphere soils under Bt and non-Bt maize cultivation. These differences could be related to agricultural practices and cultivar type. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An innovative method for immobilizing sucrose isomerase on ε-poly-L-lysine modified mesoporous TiO2.

PubMed

Wu, Lingtian; Liu, Yi; Chi, Bo; Xu, Zheng; Feng, Xiaohai; Li, Sha; Xu, Hong

2015-11-15

Sucrose isomerase (SIase) is the key enzyme in the enzymatic synthesis of isomaltulose. Mesoporous titanium dioxide (M-TiO2) and ε-poly-L-lysine-functionalized M-TiO2 (EPL-M-TiO2) were prepared as carriers for immobilizing SIase. SIase was effectively immobilized on EPL-M-TiO2 (SI-EPL-M-TiO2) with an enzyme activity of 39.41 U/g, and the enzymatic activity recovery rate up to 93.26%. The optimal pH and temperature of immobilized SIase were 6.0 and 30° C, respectively. SI-EPL-M-TiO2 was more stable in pH and thermal tests than SIase immobilized on M-TiO2 and free SIase. K(m) of SI-EPL-M-TiO2 was 204.92 mmol/L, and vmax was 45.7 μmol/L/s. Batch catalysis reaction of sucrose by SI-EPL-M-TiO2 was performed under the optimal conditions. The half-life period of SI-EPL-M-TiO2 under continuous reaction was 114 h, and the conversion rate of sucrose after 16 batches consistently remained at around 95%, which indicates that SI-EPL-M-TiO2 has good operational stability. Thus, SI-EPL-M-TiO2 can be used as a biocatalyst in food industries. Copyright © 2015. Published by Elsevier Ltd.

Effect of enzymatic orientation through the use of syringaldazine molecules on multiple multi-copper oxidase enzymes.

PubMed

Ulyanova, Yevgenia; Babanova, Sofia; Pinchon, Erica; Matanovic, Ivana; Singhal, Sameer; Atanassov, Plamen

2014-07-14

The effect of proper enzyme orientation at the electrode surface was explored for two multi-copper oxygen reducing enzymes: Bilirubin Oxidase (BOx) and Laccase (Lac). Simultaneous utilization of "tethering" agent (1-pyrenebutanoic acid, succinimidyl ester; PBSE), for stable enzyme immobilization, and syringaldazine (Syr), for enzyme orientation, of both Lac and BOx led to a notable enhancement of the electrode performance. For Lac cathodes tested in solution it was established that PBSE-Lac and PBSE-Syr-Lac modified cathodes demonstrated approximately 6 and 9 times increase in current density, respectively, compared to physically adsorbed and randomly oriented Lac cathodes. Further testing in solution utilizing BOx showed an even higher increase in achievable current densities, thus BOx was chosen for additional testing in air-breathing mode. In subsequent air-breathing experiments the incorporation of PBSE and Syr with BOx resulted in current densities of 0.65 ± 0.1 mA cm(-2); 2.5 times higher when compared to an unmodified BOx cathode. A fully tethered/oriented BOx cathode was combined with a NAD-dependent Glucose Dehydrogenase anode for the fabrication of a complete enzymatic membraneless fuel cell. A maximum power of 1.03 ± 0.06 mW cm(-2) was recorded for the complete fuel cell. The observed significant enhancement in the performance of "oriented" cathodes was a result of proper enzyme orientation, leading to facilitated enzyme/electrode interface interactions.

Miniaturised enzymatic conductometric biosensor with Nafion membrane for the direct determination of formaldehyde in water samples.

PubMed

Nguyen-Boisse, Thanh-Thuy; Saulnier, Joëlle; Jaffrezic-Renault, Nicole; Lagarde, Florence

2014-02-01

A new conductometric enzyme-based biosensor was developed for the determination of formaldehyde (FA) in aqueous solutions. The biosensor was prepared by cross-linking formaldehyde dehydrogenase from Pseudomonas putida with bovine serum albumin in saturated glutaraldehyde vapours (GA) at the surface of interdigitated gold microelectrodes. Nicotinamide adenine dinucleotide cofactor (NAD(+)) was added in solution at each measurement to maintain enzyme activity. Addition of a Nafion layer over the enzyme modified electrode resulted in a significant increase of biosensor signal due to enhanced accumulation of protons generated by enzymatic reaction at the electrode surface. Different parameters affecting enzyme activity or playing a role in ionic transfer through the Nafion membrane were optimised. In optimal conditions (0.045 mg enzyme, 30 min exposure to GA, 0.3 μL of a 1% (v/v) Nafion solution deposit, measurement in 5 mM phosphate buffer pH 7 containing 20 μM NAD(+)), the biosensor signal was linear up to 10 mM FA, and the detection limit was 18 μM. Relative standard deviations calculated from five consecutive replicates of FA solutions were lower than 5% in the 1-10 mM range. The biosensor was successfully applied to the determination of FA in spiked water samples (tap water and Rhone river water), with recoveries in the 95-110% range.

Reticulated vitreous carbon as a scaffold for enzymatic fuel cell designing.

PubMed

Kizling, Michal; Dzwonek, Maciej; Olszewski, Bartłomiej; Bącal, Paweł; Tymecki, Łukasz; Więckowska, Agnieszka; Stolarczyk, Krzysztof; Bilewicz, Renata

2017-09-15

Three - dimensional (3D) electrodes are successfully used to overcome the limitations of the low space - time yield and low normalized space velocity obtained in electrochemical processes with two - dimensional electrodes. In this study, we developed a three - dimensional reticulated vitreous carbon - gold (RVC-Au) sponge as a scaffold for enzymatic fuel cells (EFC). The structure of gold and the real electrode surface area can be controlled by the parameters of metal electrodeposition. In particular, a 3D RVC-Au sponge provides a large accessible surface area for immobilization of enzyme and electron mediators, moreover, effective mass diffusion can also take place through the uniform macro - porous scaffold. To efficiently bind the enzyme to the electrode and enhance electron transfer parameters the gold surface was modified with ultrasmall gold nanoparticles stabilized with glutathione. These quantum sized nanoparticles exhibit specific electronic properties and also expand the working surface of the electrode. Significantly, at the steady state of power generation, the EFC device with RVC-Au electrodes provided high volumetric power density of 1.18±0.14mWcm -3 (41.3±3.8µWcm -2 ) calculated based on the volume of electrode material with OCV 0.741±0.021V. These new 3D RVC-Au electrodes showed great promise for improving the power generation of EFC devices. Copyright © 2017 Elsevier B.V. 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

In vitro and in vivo assessment of meadowsweet (Filipendula ulmaria) as anti-inflammatory agent.

PubMed

Katanić, Jelena; Boroja, Tatjana; Mihailović, Vladimir; Nikles, Stefanie; Pan, San-Po; Rosić, Gvozden; Selaković, Dragica; Joksimović, Jovana; Mitrović, Slobodanka; Bauer, Rudolf

2016-12-04

Meadowsweet (Filipendula ulmaria (L.) Maxim, Rosaceae) has been traditionally used in most European countries for the treatment of inflammatory diseases due to its antipyretic, analgesic, astringent, and anti-rheumatic properties. However, there is little scientific evidence on F. ulmaria anti-inflammatory effects regarding its impact on cyclooxygenases enzymatic activity and in vivo assessment of anti-inflammatory potential. This study aims to reveal the anti-inflammatory activity of methanolic extracts from the aerial parts (FUA) and roots (FUR) of F. ulmaria, both in in vitro and in vivo conditions. The characteristic phenolic compounds in F. ulmaria extracts were monitored via high performance thin layer chromatography (HPTLC). The in vitro anti-inflammatory activity of F. ulmaria extracts was evaluated using cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzyme assays, and an assay for determining COX-2 gene expression. The in vivo anti-inflammatory effect of F. ulmaria extracts was determined in two doses (100 and 200 mg/kg b.w.) with hot plate test and carrageenan-induced paw edema test in rats. Inflammation was also evaluated by histopathological and immunohistochemical analysis. FUA extract showed the presence of rutoside, spiraeoside, and isoquercitrin. Both F. ulmaria extracts at a concentration of 50μg/mL were able to inhibit COX-1 and -2 enzyme activities, whereby FUA extract (62.84% and 46.43% inhibition, respectively) was double as effective as the root extract (32.11% and 20.20%, respectively). Extracts hardly inhibited the level of COX-2 gene expression in THP-1 cells at a concentration of 25μg/mL (10.19% inhibition by FUA and 8.54% by FUR). In the hot plate test, both extracts in two doses (100 and 200mg/kg b.w.), exhibited an increase in latency time when compared with the control group (p<0.05). In the carrageenan-induced acute inflammation test, FUA at doses of 100 and 200mg/kg b.w., and FUR at 200mg/kg, were able to significantly reduce the mean maximal swelling of rat paw until 6h of treatment. Indomethacin, FUA, and FUR extracts significantly decreased inflammation score and this effect was more pronounced after 24h, compared to the control group (p<0.05). The observed results of in vitro and, for the first time, in vivo anti-inflammatory activity of meadowsweet extracts, provide support of the traditional use of this plant in the treatment of different inflammatory conditions. Further investigation of the anti-inflammatory compounds could reveal the mechanism of anti-inflammatory action of these extracts. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Enzymatic hydrolysis of sodium dodecyl sulphate (SDS)-pretreated newspaper for cellulosic ethanol production by Saccharomyces cerevisiae and Pichia stipitis.

PubMed

Xin, Fengxue; Geng, Anli; Chen, Ming Li; Gum, Ming Jun Marcus

2010-10-01

Fermentation of enzymatic hydrolysate of waste newspaper was investigated for cellulosic ethanol production in this study. Various nonionic and ionic surfactants were applied for waste newspaper pretreatment to increase the enzymatic digestibility. The surfactant-pretreated newspaper was enzymatically digested in 0.05 M sodium citrate buffer (pH 4.8) with varying solid content, filter paper unit loading (FPU/g newspaper), and ratio of filter paper unit/beta-glucosidase unit (FPU/CBU). Newspaper pretreated with the anionic surfactant sodium dodecyl sulphate (SDS) demonstrated the highest sugar yield. The addition of Tween-80 in the enzymatic hydrolysis process enhanced the enzymatic digestibility of newspaper pretreated with all of the surfactants. Enzymatic hydrolysis of SDS-pretreated newspaper with 15% solid content, 15 FPU/g newspaper, and FPU/CBU of 1:4 resulted in a newspaper hydrolysate conditioning 29.07 g/L glucose and 4.08 g/L xylose after 72 h of incubation at 50 degrees C. The fermentation of the enzymatic hydrolysate with Saccharomyces cerevisiae, Pichia stipitis, and their co-culture produced 14.29, 13.45, and 14.03 g/L of ethanol, respectively. Their corresponding ethanol yields were 0.43, 0.41, and 0.42 g/g.

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.

[Enzymatic analysis of the quality of foodstuffs].

PubMed

Kolesnov, A Iu

1997-01-01

Enzymatic analysis is an independent and separate branch of enzymology and analytical chemistry. It has become one of the most important methodologies used in food analysis. Enzymatic analysis allows the quick, reliable determination of many food ingredients. Often these contents cannot be determined by conventional methods, or if methods are available, they are determined only with limited accuracy. Today, methods of enzymatic analysis are being increasingly used in the investigation of foodstuffs. Enzymatic measurement techniques are used in industry, scientific and food inspection laboratories for quality analysis. This article describes the requirements of an optimal analytical method: specificity, sample preparation, assay performance, precision, sensitivity, time requirement, analysis cost, safety of reagents.

Bioelectrocatalytic NAD+/NADH inter-conversion: transformation of an enzymatic fuel cell into an enzymatic redox flow battery.

PubMed

Quah, Timothy; Milton, Ross D; Abdellaoui, Sofiene; Minteer, Shelley D

2017-07-25

Diaphorase and a benzylpropylviologen redox polymer were combined to create a bioelectrode that can both oxidize NADH and reduce NAD + . We demonstrate how bioelectrocatalytic NAD + /NADH inter-conversion can transform a glucose/O 2 enzymatic fuel cell (EFC) with an open circuit potential (OCP) of 1.1 V into an enzymatic redox flow battery (ERFB), which can be rapidly recharged by operation as an EFC.

[Chosen non-enzymatic substances that participate in a protection against overproduction of free radicals].

PubMed

Gałecka, Elzbieta; Mrowicka, Małgorzata; Malinowska, Katarzyna; Gałecki, Piotr

2008-09-01

Free radicals are substantial elements that take part in proper function of metabolic pathways of human cells and tissues in hydrophobic as well as in hydrophilic environment. Nevertheless overproduction of above molecules causes oxidative stress, a process which is very harmful for lipids, proteins, and others molecules what reduces their normal function. To protect against adverse effects of free radicals and theirs derivatives to human body there is a group of antioxidants divided into enzymatic and non-enzymatic substances. Enzymatic antioxidants are represented mainly by enzymes such as: copper-zinc superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR). Glutathione (GSH), thioredoxin (Trx), vitamins, melatonin, polyphenols, trace elements, albumin, and others function as non-enzymatic free radicals scavengers. This work in a brief way describes properties of chosen representants of non-enzymatic antioxidant system.

Bioethanol production: an integrated process of low substrate loading hydrolysis-high sugars liquid fermentation and solid state fermentation of enzymatic hydrolysis residue.

PubMed

Chu, Qiulu; Li, Xin; Ma, Bin; Xu, Yong; Ouyang, Jia; Zhu, Junjun; Yu, Shiyuan; Yong, Qiang

2012-11-01

An integrated process of enzymatic hydrolysis and fermentation was investigated for high ethanol production. The combination of enzymatic hydrolysis at low substrate loading, liquid fermentation of high sugars concentration and solid state fermentation of enzymatic hydrolysis residue was beneficial for conversion of steam explosion pretreated corn stover to ethanol. The results suggested that low substrate loading hydrolysis caused a high enzymatic hydrolysis yield; the liquid fermentation of about 200g/L glucose by Saccharomyces cerevisiae provided a high ethanol concentration which could significantly decrease cost of the subsequent ethanol distillation. A solid state fermentation of enzymatic hydrolysis residue was combined, which was available to enhance ethanol production and cellulose-to-ethanol conversion. The results of solid state fermentation demonstrated that the solid state fermentation process accompanied by simultaneous saccharification and fermentation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Enzymatic hydrolysis of biomimetic bacterial cellulose-hemicellulose composites.

PubMed

Penttilä, Paavo A; Imai, Tomoya; Hemming, Jarl; Willför, Stefan; Sugiyama, Junji

2018-06-15

The production of biofuels and other chemicals from lignocellulosic biomass is limited by the inefficiency of enzymatic hydrolysis. Here a biomimetic composite material consisting of bacterial cellulose and wood-based hemicelluloses was used to study the effects of hemicelluloses on the enzymatic hydrolysis with a commercial cellulase mixture. Bacterial cellulose synthesized in the presence of hemicelluloses, especially xylan, was found to be more susceptible to enzymatic hydrolysis than hemicellulose-free bacterial cellulose. The reason for the easier hydrolysis could be related to the nanoscale structure of the substrate, particularly the packing of cellulose microfibrils into ribbons or bundles. In addition, small-angle X-ray scattering was used to show that the average nanoscale morphology of bacterial cellulose remained unchanged during the enzymatic hydrolysis. The reported easier enzymatic hydrolysis of bacterial cellulose produced in the presence of wood-based xylan offers new insights to overcome biomass recalcitrance through genetic engineering. Copyright © 2018 Elsevier Ltd. All rights reserved.

Photoelectrochemical enzymatic biosensors.

PubMed

Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

2017-06-15

Enzymatic biosensors have been valuable bioanalytical devices for analysis of diverse targets in disease diagnosis, biological and biomedical research, etc. Photoelectrochemical (PEC) bioanalysis is a recently emerged method that promptly becoming a subject of new research interests due to its attractive potential for future bioanalysis with high sensitivity and specificity. PEC enzymatic biosensors integrate the inherent sensitivities of PEC bioanalysis and the selectivity of enzymes and thus share their both advantages. Currently, PEC enzymatic biosensors have become a hot topic of significant research and the recent impetus has grown rapidly as demonstrated by increased research papers. Given the pace of advances in this area, this review will make a thorough discussion and survey on the fundamentals, sensing strategies, applications and the state of the art in PEC enzymatic biosensors, followed by future prospects based on our own opinions. We hope this work could provide an accessible introduction to PEC enzymatic biosensors for any scientist. Copyright © 2016 Elsevier B.V. All rights reserved.

Organosolv pretreatment by crude glycerol from oleochemicals industry for enzymatic hydrolysis of wheat straw.

PubMed

Sun, Fubao; Chen, Hongzhang

2008-09-01

In order to defray the cost of biodiesel production, the ensuing work was to further investigate utilization of the crude glycerol (CG) from oleochemicals industry in the atmospheric autocatalytic organosolv pretreatment (AAOP) to enhance enzymatic hydrolysis. The AAOP-CG enabled wheat straw to achieve with reasonable enzymatic hydrolysis yields, reaching 75% for the wet substrate and 63% for the dried. Lipophilic compounds from the CG formed pitch deposition on the fiber, which was responsible for low delignification (30%) and also troublesome in practical operation. Pitch deposits itself had no significant role on enzymatic hydrolysis. A striking finding of the lignin recondensation and/or lignin-carbohydrate complex helped explain why dried pretreated wheat straw had a low enzymatic hydrolysis yield. The CG was suitable for the AAOP to enhance enzymatic hydrolysis of lignocellulosic biomass. But it was advisable to remove lipophilic compounds from crude glycerol before utilization.

Enzymatic regeneration of adenosine triphosphate cofactor

NASA Technical Reports Server (NTRS)

Marshall, D. L.

1974-01-01

Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

A comparison of the distribution of enzymatically and non-enzymatically produced lead phosphate in insect flight muscle.

PubMed

Tice, L W

1969-01-01

Lead phosphate precipitates were produced in indirect flight muscles of Phormia regina by sequential incubation in solutions containing lead and inorganic phosphate and their distribution was compared with those produced by ATP hydrolysis in the presence of lead. Enzymatically produced precipitates were associated almost exclusively with thick filaments. Non-enzymatically produced precipitates were associated with thick filaments but were also found associated with thin filaments in significant numbers.

Enzymatic debridement of deeply burned faces: Healing and early scarring based on tissue preservation compared to traditional surgical debridement.

PubMed

Schulz, Alexandra; Fuchs, Paul Christian; Rothermundt, Irene; Hoffmann, Alexandra; Rosenberg, Lior; Shoham, Yaron; Oberländer, Henrik; Schiefer, Jennifer

2017-09-01

Facial burns occur frequently and depending on the injured skin layers often heal with scars which may cause permanent functional and cosmetic sequelae. Preservation of the sensitive facial skin layers, especially of the dermis is essential for scarless epithelialisation. Enzymatic debridement of deep thermal burns has already been shown to assist with preserving viable dermis. However, up to date, there are no published reports on wound healing and in the long term aesthetic outcome after enzymatic debridement of facial burns. Therefore we performed a-single centre clinical trial that included 26 subjects aged 18-78 years with facial burns clinically evaluated as deep dermal or deeper. Burns were treated either with enzymatic debridement or excisional surgical debridement. Then we compared both groups regarding debridement selectivity, wound closure and scar quality after more than 12 months. Enzymatic debridement significantly reduced time to complete wound closure after admission (19.85 days versus 42.23 days, p=0.002), and after enzymatic eschar removal (18.92 days versus 35.62 days, p=0.042). The number of procedures to complete debridement were significantly lower in the enzymatic debridement group (1.00 versus 1.77, p=0.003). 77% of facial burns that had been debrided enzymatically were found to be more superficially burned than initially estimated. Wounds undergoing autografting of any size were significantly reduced by enzymatic debridement (15% versus 77%, p=0.002). Scar quality after enzymatic debridement was superior compared to surgical debridement after 12 months regarding pigmentation (p=0.016), thickness (p=0.16), relief (p=0.10), pliability (p=0.01), surface area (p=0.004), stiffness (p=0.023), thickness (0.011) and scar irregularity (p=0.011). Regarding erythema and melanin, viscoelasticity and pliability, trans-epidermal water loss or laser tissue oxygen saturation, haemoglobin level and microcirculation we found no significant differences for treated and untreated skin in the EDNX group. In our current study we found Bromelain based enzymatic debridement better in some aspects of tissue preservation in deep dermal facial burn. Copyright © 2017 Elsevier Ltd and ISBI. All rights reserved.

Selective biosensing of Staphylococcus aureus using chitosan quantum dots

NASA Astrophysics Data System (ADS)

Abdelhamid, Hani Nasser; Wu, Hui-Fen

2018-01-01

Selective biosensing of Staphylococcus aureus (S. aureus) using chitosan modified quantum dots (CTS@CdS QDs) in the presence of hydrogen peroxide is reported. The method is based on the intrinsic positive catalase activity of S. aureus. CTS@CdS quantum dots provide high dispersion in aqueous media with high fluorescence emission. Staphylococcus aureus causes a selective quenching of the fluorescence emission of CTS@CdS QDs in the presence of H2O2 compared to other pathogens such as Escherichia coli and Pseudomonas aeruginosa. The intrinsic enzymatic character of S. aureus (catalase positive) offers selective and fast biosensing. The present method is highly selective for positive catalase species and requires no expensive reagents such as antibodies, aptamers or microbeads. It could be extended for other species that are positive catalase.

Simple and cost-effective fluorescent labeling of 5-hydroxymethylcytosine

NASA Astrophysics Data System (ADS)

Shahal, Tamar; Green, Ori; Hananel, Uri; Michaeli, Yael; Shabat, Doron; Ebenstein, Yuval

2016-12-01

The nucleobase 5-hydroxymethylcytosine (5-hmC), a modified form of cytosine, is an important epigenetic mark related to regulation of gene expression. 5-hmC levels are highly dynamic during early development and are modulated during the progression of neurodegenerative disease and cancer. We describe a spectroscopic method for the global quantification of 5-hmC in genomic DNA. This method relies on the enzymatic glucosylation of 5-hmC, followed by a glucose oxidation step that results in the formation of aldehyde moieties that are covalently linked to a fluorescent reporter by oxime ligation. The fluorescence intensity of the labeled sample is directly proportional to its 5-hmC content. We show that this simple and cost-effective technique is suitable for quantification of 5-hmC content in different mouse tissues.

Positive And Negative Feedback Loops Coupled By Common Transcription Activator And Repressor

NASA Astrophysics Data System (ADS)

Sielewiesiuk, Jan; Łopaciuk, Agata

2015-03-01

Dynamical systems consisting of two interlocked loops with negative and positive feedback have been studied using the linear analysis of stability and numerical solutions. Conditions for saddle-node bifurcation were formulated in a general form. Conditions for Hopf bifurcations were found in a few symmetrical cases. Auto-oscillations, when they exist, are generated by the negative feedback repressive loop. This loop determines the frequency and amplitude of oscillations. The positive feedback loop of activation slightly modifies the oscillations. Oscillations are possible when the difference between Hilll's coefficients of the repression and activation is sufficiently high. The highly cooperative activation loop with a fast turnover slows down or even makes the oscillations impossible. The system under consideration can constitute a component of epigenetic or enzymatic regulation network.

Maternal T-cell engraftment impedes with diagnosis of a SCID-ADA patient.

PubMed

Lanfranchi, Arnalda; Lougaris, Vassilios; Notarangelo, Lucia Dora; Soncini, Elena; Comini, Marta; Beghin, Alessandra; Bolda, Federica; Montanelli, Alessandro; Imberti, Luisa; Porta, Fulvio

2018-02-02

We describe the case of a child affected by severe combined immunodeficiency (SCID) with adenosine deaminase (ADA) deficiency showing a maternal T-cell engraftment, a finding that has never been reported before. The presence of engrafted maternal T cells was misleading. Although ADA enzymatic levels were suggestive of ADA-SCID, the child did not present the classical signs of ADA deficiency; therefore, the initial diagnosis was of a conventional SCID. However, ADA toxic metabolites and molecular characterization confirmed this diagnosis. Polyethylene glycol-modified bovine (PEG) ADA therapy progressively decreased the number of maternal engrafted T cells. The child was grafted with full bone marrow from a matched unrelated donor, after a reduced conditioning regimen, and the result was the complete immunological reconstitution. Copyright © 2018 Elsevier Inc. All rights reserved.

The role of chitosan on oral delivery of peptide-loaded nanoparticle formulation.

PubMed

Wong, Chun Y; Al-Salami, Hani; Dass, Crispin R

2017-12-01

Therapeutic peptides are conventionally administered via subcutaneous injection. Chitosan-based nanoparticles are gaining increased attention for their ability to serve as a carrier for oral delivery of peptides and vaccination. They offered superior biocompatibiltiy, controlled drug release profile and facilitated gastrointestinal (GI) absorption. The encapsulated peptides can withstand enzymatic degradation and various pH. Chitosan-based nanoparticles can also be modified by ligand conjugation to the surface of nanoparticle for transcellular absorption and specific-targeted delivery of macromolecules to the tissue of interest. Current research suggests that chitosan-based nanoparticles can deliver therapeutic peptide for the treatment of several medical conditions such as diabetes, bacterial infection and cancer. This review summarises the role of chitosan in oral nanoparticle delivery and identifies the clinical application of peptide-loaded chitosan-based nanoparticles.

Host cell recruitment patterns by bone morphogenetic protein-2 releasing hyaluronic acid hydrogels in a mouse subcutaneous environment.

PubMed

Todeschi, Maria R; El Backly, Rania M; Varghese, Oommen P; Hilborn, Jöns; Cancedda, Ranieri; Mastrogiacomo, Maddalena

2017-07-01

This study aimed to identify host cell recruitment patterns in a mouse model in response to rhBMP-2 releasing hyaluronic acid hydrogels and influence of added nano-hydroxyapatite particles on rhBMP-2 release and pattern of bone formation. Implanted gels were retrieved after implantation and cells were enzymatically dissociated for flow cytometric analysis. Percentages of macrophages, progenitor endothelial cells and putative mesenchymal stem cells were measured. Implants were evaluated for BMP-2 release by ELISA and by histology to monitor tissue formation. Hyaluronic acid+BMP-2 gels influenced the inflammatory response in the bone healing microenvironment. Host-derived putative mesenchymal stem cells were major contributors. Addition of hydroxyapatite nanoparticles modified the release pattern of rhBMP-2, resulting in enhanced bone formation.

Capillary electrophoresis of chitooligosaccharides in acidic solution: simple determination using a quaternary-ammonium-modified column and indirect photometric detection with crystal violet.

PubMed

Hattori, Toshiaki; Anraku, Nobuhiro; Kato, Ryo

2010-02-01

Five chitosan oligosaccharides were separated in acidic aqueous solution by capillary electrophoresis (CE) with indirect photometric detection using a positively coated capillary. Electrophoretic mobility of the chitooligosaccharides (COSs) depended on the number of monomer units in acidic aqueous solution, similar to other polyelectrolyte oligomers. The separation was developed in nitric acid aqueous solution at pH 3.0 with 1 mM Crystal Violet, using a capillary positively coated with N-trimethoxypropyl-N,N,N-trimethylammonium chloride. The limit of the detection for chitooligosaccharides with two to six saccharide chains was less than 5 microM. CE determination of an enzymatically hydrolyzed COS agreed with results from HPLC. 2009 Elsevier B.V. All rights reserved.

Field-effect amperometric immuno-detection of protein biomarker.

PubMed

Wang, Jiapeng; Yau, Siu-Tung

2011-11-15

The field-effect enzymatic detection technique has been applied to the amperometric immunoassay of the cancer biomarker, carcinoma antigen 125 (CA 125). The detection adopted a reagentless approach, in which the analyte, CA 125, was immobilized on the detecting electrode, which was modified using carbon nanotubes, and the detection signal was obtained by measuring the reduction peak current of the enzyme that was used to label the antibody. A gating voltage was applied to the detecting electrode, inducing increase in the signal current and therefore providing amplification of the detection signal. The voltage-controlled signal amplification of the detection system has increased the sensitivity and lowered the detection limit of the system. A detection limit of 0.9U/ml was obtained in the work. Copyright © 2011 Elsevier B.V. All rights reserved.

Label-free electrical detection of pyrophosphate generated from DNA polymerase reactions on field-effect devices.

PubMed

Credo, Grace M; Su, Xing; Wu, Kai; Elibol, Oguz H; Liu, David J; Reddy, Bobby; Tsai, Ta-Wei; Dorvel, Brian R; Daniels, Jonathan S; Bashir, Rashid; Varma, Madoo

2012-03-21

We introduce a label-free approach for sensing polymerase reactions on deoxyribonucleic acid (DNA) using a chelator-modified silicon-on-insulator field-effect transistor (SOI-FET) that exhibits selective and reversible electrical response to pyrophosphate anions. The chemical modification of the sensor surface was designed to include rolling-circle amplification (RCA) DNA colonies for locally enhanced pyrophosphate (PPi) signal generation and sensors with immobilized chelators for capture and surface-sensitive detection of diffusible reaction by-products. While detecting arrays of enzymatic base incorporation reactions is typically accomplished using optical fluorescence or chemiluminescence techniques, our results suggest that it is possible to develop scalable and portable PPi-specific sensors and platforms for broad biomedical applications such as DNA sequencing and microbe detection using surface-sensitive electrical readout techniques.

Enzymatic Fuel Cells: Integrating Flow-Through Anode and Air-Breathing Cathode into a Membrane-Less Biofuel Cell Design (Postprint)

DTIC Science & Technology

2011-06-01

AFRL-RX-TY-TP-2011-0081 ENZYMATIC FUEL CELLS: INTEGRATING FLOW- THROUGH ANODE AND AIR-BREATHING CATHODE INTO A MEMBRANE-LESS BIOFUEL CELL...RESPONSIBLE PERSON 19b. TELEPHONE NUMBER (Include area code) 01-JUN-2011 Journal Article (POSTPRINT) 01-JAN-2010 -- 31-JAN-2011 Enzymatic Fuel Cells...unlimited. Ref Public Affairs Case # 88ABW-2011-2228, 14 Apr 11. Document contains color images. One of the key goals of enzymatic biofuel cells

Alcohol biosensing by polyamidoamine (PAMAM)/cysteamine/alcohol oxidase-modified gold electrode.

PubMed

Akin, Mehriban; Yuksel, Merve; Geyik, Caner; Odaci, Dilek; Bluma, Arne; Höpfner, Tim; Beutel, Sascha; Scheper, Thomas; Timur, Suna

2010-01-01

A highly stable and sensitive amperometric alcohol biosensor was developed by immobilizing alcohol oxidase (AOX) through Polyamidoamine (PAMAM) dendrimers on a cysteamine-modified gold electrode surface. Ethanol determination is based on the consumption of dissolved oxygen content due to the enzymatic reaction. The decrease in oxygen level was monitored at -0.7 V vs. Ag/AgCl and correlated with ethanol concentration. Optimization of variables affecting the system was performed. The optimized ethanol biosensor showed a wide linearity from 0.025 to 1.0 mM with 100 s response time and detection limit of (LOD) 0.016 mM. In the characterization studies, besides linearity some parameters such as operational and storage stability, reproducibility, repeatability, and substrate specificity were studied in detail. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 67% of its initial sensitivity was kept after 1 month storage at 4 degrees C. The analytical characteristics of the system were also evaluated for alcohol determination in flow injection analysis (FIA) mode. Finally, proposed biosensor was applied for ethanol analysis in various alcoholic beverage as well as offline monitoring of alcohol production through the yeast cultivation. Copyright 2010 American Institute of Chemical Engineers

Simultaneous enzymatic and SERS properties of bifunctional chitosan-modified popcorn-like Au-Ag nanoparticles for high sensitive detection of melamine in milk powder.

PubMed

Li, Junrong; Zhang, Guannan; Wang, Lihua; Shen, Aiguo; Hu, Jiming

2015-08-01

In this work, we suggest a chitosan-modified popcorn-like Au-Ag nanoparticles (CSPNPs) based assay for high sensitive detection of melamine, in which CSPNPs not only provide with an intrinsic peroxidase-like activity but also act as surface enhanced Raman scattering (SERS) substrates. CSPNPs can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 to the charge transfer complex (CTC), which contributes to a tremendous surface-enhanced resonant Raman scattering (SERRS) signals with 632.8 nm laser excitation. The target molecule melamine can generate an additional compound with H2O2, which means the available amount of H2O2 for the oxidation of TMB reduced. Correspondingly, the SERRS intensity of CTC is decreased. The decreased Raman intensity is proportional to the concentration of melamine over a wide range from 10 nM to 50 μM (R(2)=0.989), with a limit of detection (LOD) of 8.51 nM. Moreover, the proposed highly selective method is fully capable of rapid, separation-free detection of melamine in milk powder. Copyright © 2015 Elsevier B.V. All rights reserved.

Detection of RNA nucleoside modifications with the uridine-specific ribonuclease MC1 from Momordica charantia

PubMed Central

Addepalli, Balasubrahmanym; Lesner, Nicholas P.; Limbach, Patrick A.

2015-01-01

A codon-optimized recombinant ribonuclease, MC1 is characterized for its uridine-specific cleavage ability to map nucleoside modifications in RNA. The published MC1 amino acid sequence, as noted in a previous study, was used as a template to construct a synthetic gene with a natural codon bias favoring expression in Escherichia coli. Following optimization of various expression conditions, the active recombinant ribonuclease was successfully purified as a C-terminal His-tag fusion protein from E. coli [Rosetta 2(DE3)] cells. The isolated protein was tested for its ribonuclease activity against oligoribonucleotides and commercially available E. coli tRNATyr I. Analysis of MC1 digestion products by ion-pairing reverse phase liquid-chromatography coupled with mass spectrometry (IP-RP-LC-MS) revealed enzymatic cleavage of RNA at the 5′-termini of uridine and pseudouridine, but cleavage was absent if the uridine was chemically modified or preceded by a nucleoside with a bulky modification. Furthermore, the utility of this enzyme to generate complementary digestion products to other common endonucleases, such as RNase T1, which enables the unambiguous mapping of modified residues in RNA is demonstrated. PMID:26221047

Endocrinological effects of single daily ketoconazole administration in male beagle dogs.

PubMed

De Coster, R; Beerens, D; Dom, J; Willemsens, G

1984-10-01

Some endocrinological effects of single daily oral administration of 150 mg ketoconazole for 15 days were investigated in 4 male beagle dogs. Plasma testosterone fell markedly within 3-4 h and then progressively returned to control concentrations by 10 h after drug administration. On the other hand, plasma 17 alpha-hydroxyprogesterone, progesterone and 17 alpha, 20 alpha-dihydroxyprogesterone increased within 3-10 h before returning to basal values after 24 h. Plasma LH did not rise significantly though some high individual levels were noted. Plasma cortisol and oestradiol-17 alpha levels were not significantly modified by the treatment. These results confirm that a high therapeutic dose of ketoconazole, given orally once a day, transiently inhibits in vivo the 17-20 lyase enzyme of the testis, without modifying basal cortisol and oestradiol-17 beta plasma concentrations and that enzymatic inhibition still occurs after daily treatment for up to 2 weeks but remains transient and parallels the resorption profile of the drug so that normal plasma testosterone levels are observed from 10 to 24 h after drug intake. However, permanent inhibition of androgen biosynthesis might be obtained by the administration of high doses of ketoconazole given several times a day.

S-Geranyl-2-thiouridine wobble nucleosides of bacterial tRNAs; chemical and enzymatic synthesis of S-geranylated-RNAs and their physicochemical characterization

PubMed Central

Sierant, Malgorzata; Leszczynska, Grazyna; Sadowska, Klaudia; Dziergowska, Agnieszka; Rozanski, Michal; Sochacka, Elzbieta; Nawrot, Barbara

2016-01-01

Recently, highly lipophilic S-geranylated derivatives of 5-methylaminomethyl-2-thiouridine (mnm5geS2U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5geS2U) were found at the first (wobble) anticodon position in bacterial tRNAs specific for Lys, Glu and Gln. The function and cellular biogenesis of these unique tRNAs remain poorly understood. Here, we present one direct and two post-synthetic chemical routes for preparing model geS2U-RNAs. Our experimental data demonstrate that geS2U-RNAs are more lipophilic than their parent S2U-RNAs as well as non-modified U-RNAs. Thermodynamic studies revealed that the S-geranyl-2-thiouridine-containing RNA has higher affinity toward complementary RNA strand with G opposite the modified unit than with A. Recombinant tRNA selenouridine synthase (SelU) exhibits sulfur-specific geranylation activity toward model S2U-RNA, which is composed of the anticodon-stem-loop (ASL) from the human tRNALys3 sequence. In addition, the presence of magnesium ions is required to achieve appreciable geranylation efficiencies. PMID:27566149