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Sample records for active recombinant enzyme

  1. Expression, purification and characterization of recombinant human choline acetyltransferase: phosphorylation of the enzyme regulates catalytic activity.

    PubMed Central

    Dobransky, T; Davis, W L; Xiao, G H; Rylett, R J

    2000-01-01

    Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons and, in humans, may be produced in 82- and 69-kDa forms. In this study, recombinant choline acetyltransferase from baculovirus and bacterial expression systems was used to identify protein isoforms by two-dimensional SDS/PAGE and as substrate for protein kinases. Whereas hexa-histidine-tagged 82- and 69-kDa enzymes did not resolve as individual isoforms on two-dimensional gels, separation of wild-type choline acetyltransferase expressed in insect cells revealed at least nine isoforms for the 69-kDa enzyme and at least six isoforms for the 82-kDa enzyme. Non-phosphorylated wild-type choline acetyltransferase expressed in Escherichia coli yielded six (69 kDa) and four isoforms (82 kDa) respectively. Immunofluorescent labelling of insect cells expressing enzyme showed differential subcellular localization with the 69-kDa enzyme localized adjacent to plasma membrane and the 82-kDa enzyme being cytoplasmic at 24 h. By 64 h, the 69-kDa form was in cytoplasm and the 82-kDa form was only present in nucleus. Studies in vitro showed that recombinant 69-kDa enzyme was a substrate for protein kinase C (PKC), casein kinase II (CK2) and alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaM kinase), but not for cAMP-dependent protein kinase (PKA); phosphorylation by PKC and CK2 enhanced enzyme activity. The 82-kDa enzyme was a substrate for PKC and CK2 but not for PKA or alpha-CaM kinase, with only PKC yielding increased enzyme activity. Dephosphorylation of both forms of enzyme by alkaline phosphatase decreased enzymic activity. These studies are of functional significance as they report for the first time that phosphorylation enhances choline acetyltransferase catalytic activity. PMID:10861222

  2. Recombinant L-asparaginase 1 from Saccharomyces cerevisiae: an allosteric enzyme with antineoplastic activity

    PubMed Central

    Costa, Iris Munhoz; Schultz, Leonardo; de Araujo Bianchi Pedra, Beatriz; Leite, Mariana Silva Moreira; Farsky, Sandra H. P.; de Oliveira, Marcos Antonio; Pessoa, Adalberto; Monteiro, Gisele

    2016-01-01

    L-asparaginase (L-ASNase) (EC 3.5.1.1) is an important enzyme for the treatment of acute lymphoblastic leukaemia. Currently, the enzyme is obtained from bacteria, Escherichia coli and Erwinia chrysanthemi. The bacterial enzymes family is subdivided in type I and type II; nevertheless, only type II have been employed in therapeutic proceedings. However, bacterial enzymes are susceptible to induce immune responses, leading to a high incidence of adverse effects compromising the effectiveness of the treatment. Therefore, alternative sources of L-ASNase may be useful to reduce toxicity and enhance efficacy. The yeast Saccharomyces cerevisiae has the ASP1 gene responsible for encoding L-asparaginase 1 (ScASNase1), an enzyme predicted as type II, like bacterial therapeutic isoforms, but it has been poorly studied. Here we characterised ScASNase1 using a recombinant enzyme purified by affinity chromatography. ScASNase1 has specific activity of 196.2 U/mg and allosteric behaviour, like type I enzymes, but with a low K0.5 = 75 μM like therapeutic type II. We showed through site-directed mutagenesis that the T64-Y78-T141-K215 residues are involved in catalysis. Furthermore, ScASNase1 showed cytotoxicity for the MOLT-4 leukemic cell lineage. Our data show that ScASNase1 has characteristics described for the two subfamilies of l-asparaginase, types I and II, and may have promising antineoplastic properties. PMID:27824095

  3. Rat acid phosphatase: overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties, and crystallization.

    PubMed Central

    Vihko, P; Kurkela, R; Porvari, K; Herrala, A; Lindfors, A; Lindqvist, Y; Schneider, G

    1993-01-01

    Rat prostatic acid phosphatase (rPAP; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was expressed in the baculovirus expression vector system. Recombinant protein was secreted into the medium at a high yield by infected insect cells, which were cultured at high density in a 30-liter bioreactor allowing high oxygen content for rapidly growing cells. About 20% of the cell protein produced was rPAP. Partial sequence determination of the N terminus of the purified recombinant secreted protein revealed identity to the native secreted protein, showing that the signal peptide is recognized and properly cleaved in insect cells. The enzyme was purified by using L-(+)-tartrate affinity chromatography. The purified protein had a high specific activity of 2620 mumol.min-1.mg-1 with p-nitrophenyl phosphate at the substrate, and it also showed phosphotyrosine phosphatase activity. The molecular mass of the recombinant rPAP was 155 kDa. Two subunits of 46 kDa and 48 kDa could be detected in SDS/PAGE, but only one subunit of 41 kDa was present after digestion with N-glycosidase. The active enzyme is a trimer of subunits differing only in glycosylation. When recombinant rPAP was crystallized with polyethylene glycol 6000 as the precipitant, the crystals were trigonal (space group P3(1)21) with cell dimensions a = 89.4 A and c = 152.0 A. The observed diffraction pattern extends to a resolution of at least 3 A. Images PMID:8430088

  4. Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation

    PubMed Central

    Balamurugan, Appakalai N.; Green, Michael L.; Breite, Andrew G.; Loganathan, Gopalakrishnan; Wilhelm, Joshua J.; Tweed, Benjamin; Vargova, Lenka; Lockridge, Amber; Kuriti, Manikya; Hughes, Michael G.; Williams, Stuart K.; Hering, Bernhard J.; Dwulet, Francis E.; McCarthy, Robert C.

    2016-01-01

    Background Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation. Methods We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42). Results Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas. Conclusions A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival

  5. Human Recombinant Cytochrome P450 Enzymes Display Distinct Hydrogen Peroxide Generating Activities During Substrate Independent NADPH Oxidase Reactions

    PubMed Central

    Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-01-01

    Microsomal enzymes generate H2O2 in the presence of NADPH. In this reaction, referred to as “oxidase” activity, H2O2 is generated directly or indirectly via the formation of superoxide anion. In the presence of redox active transition metals, H2O2 can form highly toxic hydroxyl radicals and, depending on the “oxidase” activity of individual cytochrome P450 isoenzymes, this can compromise cellular functioning and contribute to tissue injury. In the present studies, we compared the initial rates of H2O2 generating activity of microsomal preparations containing various human recombinant cytochromes P450s. In the absence of cytochrome P450s the human recombinant NADPH cytochrome P450 reductase (CPR) generated low, but detectable amounts of H2O2 (∼0.04 nmol H2O2/min/100 units of reductase). Significantly greater activity was detected in preparations containing individual cytochrome P450s coexpressed with CPR (from 6.0 nmol H2O2/min/nmol P450 to 0.2 nmol/min/nmol P450); CYP1A1 was the most active, followed by CYP2D6, CYP3A4, CYP2E1, CYP4A11, CYP1A2, and CYP2C subfamily enzymes. H2O2 generating activity of the cytochrome P450s was independent of the ratio of CYP/CPR. Thus, similar H2O2 generating activity was noted with the same cytochrome P450s (CYP3A4, CYP2E1, and CYP2C9) expressed at or near the ratio of CYP/CPR in human liver microsomes (5–7), and when CPR was present in excess (CYP/CPR = 0.2–0.3). Because CYP3A4/5/7 represent up to 40% of total cytochrome P450 in the liver, these data indicate that these enzymes are the major source of H2O2 in human liver microsomes. PMID:25061110

  6. Dramatic Differences in Organophosphorus Hydrolase Activity between Human and Chimeric Recombinant Mammalian Paraoxonase-1 Enzymes

    DTIC Science & Technology

    2009-01-01

    unpredictable effects on the substrate specificities and possibly the hydrolytic mechanisms of HuPON1, G2E6, and G3C9. The replacement of residue H115 in the... mechanism †This work was supported byNIHCounterACTCenter of Excellence Grant U54 NS058183 [to D.E.L. (Center PI), D.M.C., and T.J.M.] and by the Defense...terminal 6-His tags. The enzymes were expressed in Origami B (DE3) cells (Novagen, Madison, WI). When bacterial cultures reached an A600 of 0.8, they

  7. Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme.

    PubMed

    Pellegrini, Vanessa O A; Serpa, Viviane Isabel; Godoy, Andre S; Camilo, Cesar M; Bernardes, Amanda; Rezende, Camila A; Junior, Nei Pereira; Franco Cairo, João Paulo L; Squina, Fabio M; Polikarpov, Igor

    2015-11-01

    Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

  8. Characterization of a recombinant glutaminase-free L-asparaginase (ansA3) enzyme with high catalytic activity from Bacillus licheniformis.

    PubMed

    Sudhir, Ankit P; Dave, Bhaumik R; Prajapati, Anil S; Panchal, Ketankumar; Patel, Darshan; Subramanian, R B

    2014-12-01

    L-Asparaginase (3.5.1.1) is an enzyme widely used to treat the acute lymphoblastic leukemia. Two genes coding for L-asparaginase (ansA1 and ansA3) from Bacillus licheniformis MTCC 429 were cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were purified to homogeneity by one-step purification process and further characterized for various biochemical parameters. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that both the enzymes are monomers of ∼37 kDa. Recombinant ansA1 was found to be highly unstable, and recombinant ansA3 was catalytically active and stable, which showed an optimum activity of 407.65 IU/mg at 37 °C and pH 8. Recombinant ansA3 showed higher substrate specificity for L-asparagine with negligible glutaminase activity. Kinetic parameters like K m , V max, k cat, and k cat/K m were calculated for recombinant ansA3.

  9. Enzyme replacement therapy for Morquio A: an active recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in Escherichia coli BL21.

    PubMed

    Rodríguez, Alexander; Espejo, Angela J; Hernández, Alejandra; Velásquez, Olga L; Lizaraso, Lina M; Cordoba, Henry A; Sánchez, Oscar F; Alméciga-Díaz, Carlos J; Barrera, Luis A

    2010-11-01

    Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. Currently no effective therapies exist for MPS IVA. In this work, production of a recombinant GALNS enzyme (rGALNS) in Escherichia coli BL21 strain was studied. At shake scale, the effect of glucose concentration on microorganism growth, and microorganism culture and induction times on rGALNS production were evaluated. At bench scale, the effect of aeration and agitation on microorganism growth, and culture and induction times were evaluated. The highest enzyme activity levels at shake scale were observed in 12 h culture after 2-4 h induction. At bench scale the highest enzyme activity levels were observed after 2 h induction. rGALNS amounts in inclusion bodies fraction were up to 17-fold higher than those observed in the soluble fraction. However, the highest levels of active enzyme were found in the soluble fraction. Western blot analysis showed the presence of a 50-kDa band, in both soluble and inclusion bodies fractions. These results show for the first time the feasibility and potential of production of active rGALNS in a prokaryotic system for development of enzyme replacement therapy for MPS IVA disease.

  10. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  11. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  12. Strong enhancement of recombinant cytosine deaminase activity in Bifidobacterium longum for tumor-targeting enzyme/prodrug therapy.

    PubMed

    Hamaji, Yoshinori; Fujimori, Minoru; Sasaki, Takayuki; Matsuhashi, Hitomi; Matsui-Seki, Keiichi; Shimatani-Shibata, Yuko; Kano, Yasunobu; Amano, Jun; Taniguchi, Shun'ichiro

    2007-04-01

    In our previous studies, a strain of the nonpathogenic, anaerobic, intestinal bacterium, Bifidobacterium longum (B. longum), was found to be localized selectively and to proliferate within solid tumors after systemic administration. In addition, B. longum transformed with the shuttle-plasmid encoding the cytosine deaminase (CD) gene expressed active CD, which deaminated the prodrug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU). We also reported antitumor efficacy with the same plasmid in several animal experiments. In this study, we constructed a novel shuttle-plasmid, pAV001-HU-eCD-M968, which included the mutant CD gene with a mutation at the active site to increase the enzymatic activity. In addition, the plasmid-transformed B. longum produces mutant CD and strongly increased (by 10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid increases the effectiveness of our enzyme/prodrug strategy.

  13. Identification of cellular genes induced in human cells after activation of the OAS/RNaseL pathway by vaccinia virus recombinants expressing these antiviral enzymes.

    PubMed

    Domingo-Gil, Elena; González, José Manuel; Esteban, Mariano

    2010-03-01

    Interferon (IFN) type I induces the expression of antiviral proteins such as 2',5'-oligoadenylate synthetases (OAS). The enzyme OAS is activated by dsRNA to produce 5'-phosphorylated, 2-5-linked oligoadenylates (2-5A) that activate RNaseL which, in turn, triggers RNA breakdown, leading to multiple biological functions. Although RNaseL is required for IFN antiviral function, there are many aspects of the molecular mechanisms that remain obscure. Here, we have used microarray analyses from human HeLa cells infected with vaccinia virus (VACV) recombinants expressing OAS-RNaseL enzymes (referred as 2-5A system) with the aim to identify host genes that are up- or down-regulated in the course of infection by the activation of this antiviral pathway. We found that activation of the 2-5A system from VACV recombinants produces a remarkable stimulation of transcription for genes that regulate many cellular processes, like those that promote cell growth arrest, GADD45B and KCTD11, apoptosis as CUL2, PDCD6, and TNFAIP8L2, IFN-stimulated genes as IFI6, and related to tumor suppression as PLA2G2A. The 2-5A system activation produces down-regulation of transcription of some genes that promote cell growth as RUNX2 and ESR2 and of genes in charge to maintain mitochondria homeostasis as MIPEP and COX5A. These results reveal new genes induced in response to the activation of the 2-5A system with roles in apoptosis, translational control, cell growth arrest, and tumor suppression.

  14. Enzymic properties of recombinant BACE2.

    PubMed

    Kim, Yong-Tae; Downs, Deborah; Wu, Shili; Dashti, Azar; Pan, Yujun; Zhai, Peng; Wang, Xinjuan; Zhang, Xuejun C; Lin, Xinli

    2002-11-01

    BACE2 (Memapsin 1) is a membrane-bound aspartic protease that is highly homologous with BACE1 (Memapsin 2). While BACE1 processes the amyloid precursor protein (APP) at a key step in generating the beta-amyloid peptide and presumably causes Alzheimer's disease (AD), BACE2 has not been demonstrated to be directly involved in APP processing, and its physiological functions remain to be determined. In vivo, BACE2 is expressed as a precursor protein containing pre-, pro-, protease, transmembrane, and cytosolic domains/peptides. To determine the enzymatic properties of BACE2, two variants of its pro-protease domain, pro-BACE2-T1 (PB2-T1) and pro-BACE2-T2 (PB2-T2), were constructed. They have been expressed in Escherichia coli as inclusion bodies, refolded and purified. These two recombinant proteins have the same N terminus but differ at their C-terminal ends: PB2-T1 ends at Pro466, on the boundary of the postulated transmembrane domain, and PB2-T2 ends at Ser431, close to the homologous ends of other aspartic proteases such as pepsin. While PB2-T1 shares similar substrate specificities with BACE1 and other 'general' aspartic proteases, the specificity of PB2-T2 is more constrained, apparently preferring to cleave at the NH2-terminal side of paired basic residues. Unlike other 'typical' aspartic proteases, which are active only under acidic conditions, the recombinant BACE2, PB2-T1, was active at a broad pH range. In addition, pro-BACE2 can be processed at its in vivo maturation site by BACE1.

  15. Food-processing enzymes from recombinant microorganisms--a review.

    PubMed

    Olempska-Beer, Zofia S; Merker, Robert I; Ditto, Mary D; DiNovi, Michael J

    2006-07-01

    Enzymes are commonly used in food processing and in the production of food ingredients. Enzymes traditionally isolated from culturable microorganisms, plants, and mammalian tissues are often not well-adapted to the conditions used in modern food production methods. The use of recombinant DNA technology has made it possible to manufacture novel enzymes suitable for specific food-processing conditions. Such enzymes may be discovered by screening microorganisms sampled from diverse environments or developed by modification of known enzymes using modern methods of protein engineering or molecular evolution. As a result, several important food-processing enzymes such as amylases and lipases with properties tailored to particular food applications have become available. Another important achievement is improvement of microbial production strains. For example, several microbial strains recently developed for enzyme production have been engineered to increase enzyme yield by deleting native genes encoding extracellular proteases. Moreover, certain fungal production strains have been modified to reduce or eliminate their potential for production of toxic secondary metabolites. In this article, we discuss the safety of microorganisms used as hosts for enzyme-encoding genes, the construction of recombinant production strains, and methods of improving enzyme properties. We also briefly describe the manufacture and safety assessment of enzyme preparations and summarize options for submitting information on enzyme preparations to the US Food and Drug Administration.

  16. 'Super-perfect' enzymes: Structural stabilities and activities of recombinant triose phosphate isomerases from Pyrococcus furiosus and Thermococcus onnurineus produced in Escherichia coli.

    PubMed

    Sharma, Prerna; Guptasarma, Purnananda

    2015-05-08

    Triose phosphate isomerases (TIMs) are considered to be 'kinetically perfect' enzymes, limited in their activity only by the rates of diffusion of substrate and product molecules. Most studies conducted thus far have been on mesophile-derived TIMs. Here, we report studies of two extremophile-derived TIMs produced in Escherichia coli: (i) TonTIM, sourced from the genome of the thermophile archaeon, Thermococcus onnurineus, and (ii) PfuTIM, sourced from the genome of the hyperthermophile archaeon, Pyrococcus furiosus (PfuTIM). Although these enzymes are presumed to have evolved to function optimally at temperatures close to the boiling point of water, we find that TonTIM and PfuTIM display second-order rate-constants of activity (k(cat)/K(m) values) comparable to mesophile-derived TIMs, at 25 °C. At 90 °C, TonTIM and PfuTIM reach maximum velocities of reaction of ∼ 10(6)-10(7) μmol/s/mg, and display k(cat)/K(m) values in the range of ∼ 10(10)-10(11) M(-1) s(-1), which are three orders of magnitude higher than those reported for mesophile TIMs. Further, the two enzymes display no signs of having undergone any structural unfolding at 90 °C. Such enzymes could thus probably be called 'super-perfect' enzymes.

  17. Enhanced enzyme activities of inclusion bodies of recombinant beta-galactosidase via the addition of inducer analog after L-arabinose induction in the araBAD promoter system of Escherichia coli.

    PubMed

    Jung, Kyung-Hwan

    2008-03-01

    We observed that an inclusion body (IB) of recombinant beta-galactosidase that was produced by the araBAD promoter system in Escherichia coli (E. coli) showed enzyme activity. In order to improve its activity, the lowering of the transcription rate of the beta-galactosidase structural gene was attempted through competition between an inducer (L-arabinose) and an inducer analog (D-fucose). In the deep-well microtiter plate culture and lab-scale fermentor culture, it was demonstrated that the addition of D-fucose caused an improvement in specific beta-galactosidase production, although beta-galactosidase was produced as an IB. In particular, the addition of D-fucose after induction led to an increase in the specific activity of beta-galactosidase IB. Finally, we confirmed that the addition of D-fucose after induction caused changes in the structure of beta-galactosidase IB, with higher enzyme activity. Based on these results, we expect that an improved enzyme IB will be used as a biocatalyst of the enzyme bioprocess, because an enzyme IB can be purified easily and has physical durability.

  18. Photoperiodism and Enzyme Activity

    PubMed Central

    Queiroz, Orlando; Morel, Claudine

    1974-01-01

    Metabolic readjustments after a change from long days to short days appear, in Kalanchoe blossfeldiana, to be achieved through the operation of two main mechanisms: variation in enzyme capacity, and circadian rhythmicity. After a lag time, capacity in phosphoenolpyruvate carboxylase and capacity in aspartate aminotransferase increase exponentially and appear to be allometrically linked during 50 to 60 short days; then a sudden fall takes place in the activity of the former. Malic enzyme and alanine aminotransferase behave differently. Thus, the operation of the two sections of the pathway (before and after the malate step) give rise to a continuously changing functional compartmentation in the pathway. Circadian rhythmicity, on the other hand, produces time compartmentation through phase shifts and variation in amplitude, independently for each enzyme. These characteristics suggest that the operation of a so-called biological clock would be involved. We propose the hypothesis that feedback regulation would be more accurate and efficient when applied to an already oscillating, clock-controlled enzyme system. PMID:16658749

  19. N-linked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verruculosum and its effect on the enzyme activity.

    PubMed

    Dotsenko, Anna S; Gusakov, Alexander V; Volkov, Pavel V; Rozhkova, Aleksandra M; Sinitsyn, Arkady P

    2016-02-01

    Cellobiohydrolase I from Penicillium verruculosum (PvCel7A) has four potential N-glycosylation sites at its catalytic module: Asn45, Asn194, Asn388, and Asn430. In order to investigate how the N-glycosylation influences the activity and other properties of the enzyme, the wild type (wt) PvCel7A and its mutant forms, carrying Asn to Ala substitutions, were cloned into Penicillium canescens PCA10 (niaD-) strain, a fungal host for production of heterologous proteins. The rPvCel7A-wt and N45A, N194A, N388A mutants were successfully expressed and purified for characterization, whereas the expression of N430A mutant was not achieved. The MALDI-TOF mass spectrometry fingerprinting of peptides, obtained as a result of digestion of rPvCel7A forms with specific proteases, showed that the N-linked glycans represent variable high-mannose oligosaccharides and the products of their sequential enzymatic trimming, according to the formula (Man)0-13 (GlcNAc)2 , or a single GlcNAc residue. Mutations had no notable effect on pH-optimum of PvCel7A activity and enzyme thermostability. However, the mutations influenced both the enzyme adsorption ability on Avicel and its activity against natural and synthetic substrates. In particular, the N45A mutation led to a significant increase in the rate of Avicel and milled aspen wood hydrolysis, while the substrate digestion rates in the case of N194A and N388A mutants were notably lower relative to rPvCel7A-wt. These data, together with data of 3D structural modeling of the PvCel7A catalytic module, indicate that the N-linked glycans are an important part of the processive catalytic machinery of PvCel7A.

  20. Expression of a Bacillus phytase C gene in Pichia pastoris and properties of the recombinant enzyme.

    PubMed

    Guerrero-Olazarán, Martha; Rodríguez-Blanco, Lilí; Carreon-Treviño, J Gerardo; Gallegos-López, Juan A; Viader-Salvadó, José M

    2010-08-01

    The cloning and expression of a native gene encoding a Bacillus subtilis phytase using Pichia pastoris as the host is described. In addition, the influence of N-glycosylation on the biochemical properties of the B. subtilis phytase, the influence of pH on the thermostability of the recombinant and native B. subtilis phytases, and the resistance of both phytases to shrimp digestive enzymes and porcine trypsin are also described. After 48 h of methanol induction in shake flasks, a selected recombinant strain produced and secreted 0.82 U/ml (71 mg/liter) recombinant phytase. This phytase was N-glycosylated, had a molecular mass of 39 kDa after N-deglycosylation, exhibited activity within a pH range of 2.5 to 9 and at temperatures of 25 to 70 degrees C, had high residual activity (85% +/- 2%) after 10 min of heat treatment at 80 degrees C and pH 5.5 in the presence of 5 mM CaCl(2), and was resistant to shrimp digestive enzymes and porcine trypsin. Although the recombinant Bacillus phytase had pH and temperature activity profiles that were similar to those of the corresponding nonglycosylated native phytase, the thermal stabilities of the recombinant and native phytases were different, although both were calcium concentration and pH dependent.

  1. Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes.

    PubMed Central

    Pitson, S M; D'andrea, R J; Vandeleur, L; Moretti, P A; Xia, P; Gamble, J R; Vadas, M A; Wattenberg, B W

    2000-01-01

    Sphingosine 1-phosphate (S1P) is a novel lipid messenger that has important roles in a wide variety of mammalian cellular processes including growth, differentiation and death. Basal levels of S1P in mammalian cells are generally low, but can increase rapidly and transiently when cells are exposed to mitogenic agents and other stimuli. This increase is largely due to increased activity of sphingosine kinase (SK), the enzyme that catalyses its formation. In the current study we have purified, cloned and characterized the first human SK to obtain a better understanding of its biochemical activity and possible activation mechanisms. The enzyme was purified to homogeneity from human placenta using ammonium sulphate precipitation, anion-exchange chromatography, calmodulin-affinity chromatography and gel-filtration chromatography. This resulted in a purification of over 10(6)-fold from the original placenta extract. The enzyme was cloned and expressed in active form in both HEK-293T cells and Escherichia coli, and the recombinant E. coli-derived SK purified to homogeneity. To establish whether post-translational modifications lead to activation of human SK activity we characterized both the purified placental enzyme and the purified recombinant SK produced in E. coli, where such modifications would not occur. The premise for this study was that post-translational modifications are likely to cause conformational changes in the structure of SK, which may result in detectable changes in the physico-chemical or catalytic properties of the enzyme. Thus the enzymes were characterized with respect to substrate specificity and kinetics, inhibition kinetics and various other physico-chemical properties. In all cases, both the native and recombinant SKs displayed remarkably similar properties, indicating that post-translational modifications are not required for basal activity of human SK. PMID:10947957

  2. Are there differences in the catalytic activity per unit enzyme of recombinantly expressed and human liver microsomal cytochrome P450 2C9? A systematic investigation into inter-system extrapolation factors.

    PubMed

    Crewe, H K; Barter, Z E; Yeo, K Rowland; Rostami-Hodjegan, A

    2011-09-01

    The 'relative activity factor' (RAF) compares the activity per unit of microsomal protein in recombinantly expressed cytochrome P450 enzymes (rhCYP) and human liver without separating the potential sources of variation (i.e. abundance of enzyme per mg of protein or variation of activity per unit enzyme). The dimensionless 'inter-system extrapolation factor' (ISEF) dissects differences in activity from those in CYP abundance. Detailed protocols for the determination of this scalar, which is used in population in vitro-in vivo extrapolation (IVIVE), are currently lacking. The present study determined an ISEF for CYP2C9 and, for the first time, systematically evaluated the effects of probe substrate, cytochrome b5 and methods for assessing the intrinsic clearance (CL(int) ). Values of ISEF for S-warfarin, tolbutamide and diclofenac were 0.75 ± 0.18, 0.57 ± 0.07 and 0.37 ± 0.07, respectively, using CL(int) values derived from the kinetic values V(max) and K(m) of metabolite formation in rhCYP2C9 + reductase + b5 BD Supersomes™. The ISEF values obtained using rhCYP2C9 + reductase BD Supersomes™ were more variable, with values of 7.16 ± 1.25, 0.89 ± 0.52 and 0.50 ± 0.05 for S-warfarin, tolbutamide and diclofenac, respectively. Although the ISEF values obtained from rhCYP2C9 + reductase + b5 for the three probe substrates were statistically different (p < 0.001), the use of the mean value of 0.54 resulted in predicted oral clearance values for all three substrates within 1.4 fold of the observed literature values. For consistency in the relative activity across substrates, use of a b5 expressing recombinant system, with the intrinsic clearance calculated from full kinetic data is recommended for generation of the CYP2C9 ISEF. Furthermore, as ISEFs have been found to be sensitive to differences in accessory proteins, rhCYP system specific ISEFs are recommended.

  3. Effects of cryopreservation and hypothermic storage on cell viability and enzyme activity in recombinant encapsulated cells overexpressing alpha-L-iduronidase.

    PubMed

    Mayer, Fabiana Quoos; Baldo, Guilherme; de Carvalho, Talita Giacomet; Lagranha, Valeska Lizzi; Giugliani, Roberto; Matte, Ursula

    2010-05-01

    Here, we show the effects of cryopreservation and hypothermic storage upon cell viability and enzyme release in alginate beads containing baby hamster kidney cells overexpressing alpha-L-iduronidase (IDUA), the enzyme deficient in mucopolysaccharidosis type I. In addition, we compared two different concentrations of alginate gel (1% and 1.5%) in respect to enzyme release from the beads and their shape and integrity. Our results indicate that in both alginate concentrations, the enzyme is released in lower amounts compared with nonencapsulated cells. Alginate 1% beads presented increased levels of IDUA release, although this group presented more deformities when compared with alginate 1.5% beads. Importantly, both encapsulated groups presented higher cell viability after long cryopreservation period and hypothermic storage. In addition, alginate 1.5% beads presented higher enzyme release after freezing protocols. Taken together, our findings suggest a benefic effect of alginate upon cell viability and functionality. These results may have important application for treatment of both genetic and nongenetic diseases using microencapsulation-based artificial organs.

  4. Lignocellulosic Fermentation of Wild Grass Employing Recombinant Hydrolytic Enzymes and Fermentative Microbes with Effective Bioethanol Recovery

    PubMed Central

    Das, Saprativ P.; Ghosh, Arabinda; Gupta, Ashutosh; Das, Debasish

    2013-01-01

    Simultaneous saccharification and fermentation (SSF) studies of steam exploded and alkali pretreated different leafy biomass were accomplished by recombinant Clostridium thermocellum hydrolytic enzymes and fermentative microbes for bioethanol production. The recombinant C. thermocellum GH5 cellulase and GH43 hemicellulase genes expressed in Escherichia coli cells were grown in repetitive batch mode, with the aim of enhancing the cell biomass production and enzyme activity. In batch mode, the cell biomass (A600 nm) of E. coli cells and enzyme activities of GH5 cellulase and GH43 hemicellulase were 1.4 and 1.6 with 2.8 and 2.2 U·mg−1, which were augmented to 2.8 and 2.9 with 5.6 and 3.8 U·mg−1 in repetitive batch mode, respectively. Steam exploded wild grass (Achnatherum hymenoides) provided the best ethanol titres as compared to other biomasses. Mixed enzyme (GH5 cellulase, GH43 hemicellulase) mixed culture (Saccharomyces cerevisiae, Candida shehatae) system gave 2-fold higher ethanol titre than single enzyme (GH5 cellulase) single culture (Saccharomyces cerevisiae) system employing 1% (w/v) pretreated substrate. 5% (w/v) substrate gave 11.2 g·L−1 of ethanol at shake flask level which on scaling up to 2 L bioreactor resulted in 23 g·L−1 ethanol. 91.6% (v/v) ethanol was recovered by rotary evaporator with 21.2% purification efficiency. PMID:24089676

  5. A biochemical and physicochemical comparison of two recombinant enzymes used for enzyme replacement therapies of hunter syndrome.

    PubMed

    Chung, Yo Kyung; Sohn, Young Bae; Sohn, Jong Mun; Lee, Jieun; Chang, Mi Sun; Kwun, Younghee; Kim, Chi Hwa; Lee, Jin Young; Yook, Yeon Joo; Ko, Ah-Ra; Jin, Dong-Kyu

    2014-05-01

    Mucopolysaccharidosis II (MPS II, Hunter syndrome; OMIM 309900) is an X-linked lysosomal storage disease caused by a deficiency in the enzyme iduronate-2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs). For enzyme replacement therapy (ERT) of Hunter syndrome, two recombinant enzymes, idursulfase (Elaprase(®), Shire Human Genetic Therapies, Lexington, MA) and idursulfase beta (Hunterase(®), Green Cross Corporation, Yongin, Korea), are currently available in Korea. To compare the biochemical and physicochemical differences between idursulfase and idursulfase beta, we examined the formylglycine (FGly) content, specific enzyme activity, mannose-6-phosphate (M6P) content, sialic acid content, and in vitro cell uptake activity of normal human fibroblasts of these two enzymes.The FGly content, which determines the enzyme activity, of idursulfase beta was significantly higher than that of idursulfase (79.4 ± 0.9 vs. 68.1 ± 2.2 %, P < 0.001). In accordance with the FGly content, the specific enzyme activity of idursulfase beta was significantly higher than that of idursulfase (42.6 ± 1.1 vs. 27.8 ± 0.9 nmol/min/μg protein, P < 0.001). The levels of M6P and sialic acid were not significantly different (2.4 ± 0.1 vs 2.4 ± 0.3 mol/mol protein for M6P and 12.3 ± 0.7 vs. 12.4 ± 0.4 mol/mol protein for sialic acid). However, the cellular uptake activity of the normal human fibroblasts in vitro showed a significant difference (Kuptake, 5.09 ± 0.96 vs. 6.50 ± 1.28 nM protein, P = 0.017).In conclusion, idursulfase beta exhibited significantly higher specific enzyme activity than idursulfase, resulting from higher FGly content. These biochemical differences may be partly attributed to clinical efficacy. However, long-term clinical evaluations of Hunter syndrome patients treated with these two enzymes will be needed to demonstrate the clinical implications of significant difference of the enzyme activity and the FGly content.

  6. Recombinant Sox Enzymes from Paracoccus pantotrophus Degrade Hydrogen Sulfide, a Major Component of Oral Malodor

    PubMed Central

    Ramadhani, Atik; Kawada-Matsuo, Miki; Komatsuzawa, Hitoshi; Oho, Takahiko

    2017-01-01

    Hydrogen sulfide (H2S) is emitted from industrial activities, and several chemotrophs possessing Sox enzymes are used for its removal. Oral malodor is a common issue in the dental field and major malodorous components are volatile sulfur compounds (VSCs), including H2S and methyl mercaptan. Paracoccus pantotrophus is an aerobic, neutrophilic facultatively autotrophic bacterium that possesses sulfur-oxidizing (Sox) enzymes in order to use sulfur compounds as an energy source. In the present study, we cloned the Sox enzymes of P. pantotrophus GB17 and evaluated their VSC-degrading activities for the prevention of oral malodor. Six genes, soxX, soxY, soxZ, soxA, soxB, and soxCD, were amplified from P. pantotrophus GB17. Each fragment was cloned into a vector for the expression of 6×His-tagged fusion proteins in Escherichia coli. Recombinant Sox (rSox) proteins were purified from whole-cell extracts of E. coli using nickel affinity chromatography. The enzyme mixture was investigated for the degradation of VSCs using gas chromatography. Each of the rSox enzymes was purified to apparent homogeneity, as confirmed by SDS-PAGE. The rSox enzyme mixture degraded H2S in dose- and time-dependent manners. All rSox enzymes were necessary for degrading H2S. The H2S-degrading activities of rSox enzymes were stable at 25–80°C, and the optimum pH was 7.0. The amount of H2S produced by periodontopathic bacteria or oral bacteria collected from human subjects decreased after an incubation with rSox enzymes. These results suggest that the combination of rSox enzymes from P. pantotrophus GB17 is useful for the prevention of oral malodor. PMID:28260736

  7. [Dual promoters enhance heterologous enzyme production from bacterial phage based recombinant Bacillus subtilis].

    PubMed

    Liu, Gang; Zhang, Yan; Xing, Miao

    2006-03-01

    The effect of dual promoters on recombinant protein production from bacterial phage based Bacillus subtilis expression system was investigated. Alpha amylase (from Bacillus amyloliquefaciens) and penicillin acylase (from Bacillus megaterium) were selected as the indicating enzymes. Both the promoterless genes and the promoter-bearing genes were isolated through PCR amplification with properly designed primers, and were inserted into plasmid pSG703 that contains the lacZ-cat expression cartridge. The lysogenic B. subtilis (phi105 MU331) was transformed with the resultant recombinant plasmids, and the heterologous genes were thereby integrated into the chromosommal DNA of B. subtilis via homologous recombination. The transformants were designated as B. subtilis AMY1, B. subtilis AMY2, B. subtilis PA1, and B. subtilis PA2, respectively. In the recombinant B. subtilis strains, the inserted sequences were located down stream of a strong phage promoter that could be activated by thermal induction. In B. subtilis AMY1 and B. subtilis PA1, transcription of the heterologous genes was only initiated by the phage promoter after heat shock, whereas in B. subtilis AMY2 and B. subtilis PA2, transcription of the heterologous genes was initiated by dual promoters, the phage promoter and the native promoter. The application of dual promoters increased the productivity of both enzymes, with 133% enhancement for alpha-amylase production and 113% enhancement for penicillin acylase production.

  8. Bifunctional recombinant fusion enzyme between maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase of thermophilic microorganism Metallosphaera hakonensis.

    PubMed

    Seo, Ju-Seok; An, Ju Hee; Cheong, Jong-Joo; Choi, Yang Do; Kim, Chung Ho

    2008-09-01

    MhMTS and MhMTH are trehalose (alpha-D-glucopyranosyl- [1,1]-alpha-D-glucopyranose) biosynthesis genes of the thermophilic microorganism Metallosphaera hakonensis, and encode a maltooligosyltrehalose synthase (MhMTS) and a maltooligosyltrehalose trehalohydrolase (MhMTH), respectively. In this study, the two genes were fused inframe in a recombinant DNA, and expressed in Escherichia coli to produce a bifunctional fusion enzyme, MhMTSH. Similar to the two-step reactions with MhMTS and MhMTH, the fusion enzyme catalyzed the sequential reactions on maltopentaose, maltotriosyltrehalose formation, and following hydrolysis, producing trehalose and maltotriose. Optimum conditions for the fusion enzyme-catalyzed trehalose synthesis were around 70 degrees and pH 5.0-6.0. The MhMTSH fusion enzyme exhibited a high degree of thermostability, retaining 80% of the activity when pre-incubated at 70 degrees for 48 h. The stability was gradually abolished by incubating the fusion enzyme at above 80 degrees . The MhMTSH fusion enzyme was active on various sizes of maltooligosaccharides, extending its substrate specificity to soluble starch, the most abundant natural source of trehalose production.

  9. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  10. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  11. Enzyme therapy for pompe disease with recombinant human alpha-glucosidase from rabbit milk.

    PubMed

    Van den Hout, J M; Reuser, A J; de Klerk, J B; Arts, W F; Smeitink, J A; Van der Ploeg, A T

    2001-04-01

    Pompe disease is a metabolic myopathy caused by deficiency of lysosomal acid alpha-glucosidase. In this report we review the first 36 weeks of a clinical study on the safety and efficacy of enzyme therapy aimed at correcting the deficiency. Four patients with infantile Pompe disease were enrolled. They received recombinant human alpha-glucosidase from transgenic rabbit milk. The product is generally well tolerated and reaches the primary target tissues. Normalization of alpha-glucosidase activity in skeletal muscle was obtained and degradation of PAS-positive material was seen in tissue sections. The clinical condition of all patients improved. The effect on heart was most significant, with an impressive reduction of the left ventricular mass index (LVMI). Motor function improved. The positive preliminary results stimulate continuation and extension of efforts towards the realization of enzyme therapy for Pompe disease.

  12. The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair

    PubMed Central

    Amundsen, Susan K.; Taylor, Andrew F.; Smith, Gerald R.

    2000-01-01

    The RecBCD enzyme is required for homologous recombination and DNA repair in Escherichia coli. The structure and function of RecBCD enzyme is altered on its interaction with the recombination hotspot Chi (5′-GCTGGTGG-3′). It has been hypothesized that the RecD subunit plays a role in Chi-dependent regulation of enzyme activity [Thaler, D. S., Sampson, E., Siddiqi, I., Rosenberg, S. M., Stahl, F. W. & Stahl, M. (1988) in Mechanisms and Consequences of DNA Damage Processing, eds. Friedberg, E. & Hanawalt, P. (Liss, New York), pp. 413–422; Churchill, J. J., Anderson, D. G. & Kowalczykowski, S. C. (1999) Genes Dev. 13, 901–911]. We tested the hypothesis that the RecD subunit inhibits recombination by deleting recD from the nuclease- and recombination-deficient mutant recBD1080ACD. We report here that the resulting strain, recBD1080AC, was proficient for recombination and DNA repair. Recombination proficiency was accompanied by a change in enzyme activity: RecBD1080AC enzyme loaded RecA protein onto DNA during DNA unwinding whereas RecBD1080ACD enzyme did not. Together, these genetic and biochemical results demonstrate that RecA loading by RecBCD enzyme is required for recombination in E. coli cells and suggest that RecD interferes with the enzyme domain required for its loading. A nuclease-dependent signal appears to be required for a change in RecD that allows RecA loading. Because RecA loading is not observed with wild-type RecBCD enzyme until it acts at a Chi site, our observations support the view that RecD inhibits recombination until the enzyme acts at Chi. PMID:10840065

  13. Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells.

    PubMed Central

    Godwin, A R; Bollag, R J; Christie, D M; Liskay, R M

    1994-01-01

    We have derived Chinese hamster ovary (CHO) cell hybrids containing herpes simplex virus thymidine kinase (tk) heteroalleles for the study of spontaneous and restriction enzyme-induced interchromosomal recombination. These lines allowed us to make a direct comparison between spontaneous intrachromosomal and interchromosomal recombination using the same tk heteroalleles at the same genomic insertion site. We find that the frequency of interchromosomal recombination is less by a factor of at least 5000 than that of intrachromosomal recombination. Our results with mammalian cells differ markedly from results with Saccharomyces cerevisiae, with which similar studies typically give only a 10-to 30-fold difference. Next, to inquire into the fate of double-strand breaks at either of the two different Xho I linker insertion mutations, we electroporated PaeR7I enzyme, an isoschizomer of Xho I, into these hybrids. A priori, these breaks can be repaired either by recombination from the homology or by end-joining. Despite a predicted bias against recovering end-joining products in our system, all cells characterized by enzyme-induced resistance to hypoxanthine/aminopterin/thymidine were, in fact, due to nonhomologous recombination or end-joining. These results are in agreement with other studies that used extrachromosomal sequences to examine the relative efficiencies of end-joining and homologous recombination in mammalian cells, but are in sharp contrast to results of analogous studies in S. cerevisiae, wherein only products of homologous events are detected. Images Fig. 2 PMID:7809076

  14. A preliminary neutron diffraction study of rasburicase, a recombinant urate oxidase enzyme, complexed with 8-azaxanthin

    SciTech Connect

    Budayova-Spano, Monika; Bonneté, Françoise; Ferté, Natalie; El Hajji, Mohamed; Meilleur, Flora; Blakeley, Matthew Paul; Castro, Bertrand

    2006-03-01

    Neutron diffraction data of hydrogenated recombinant urate oxidase enzyme (Rasburicase), complexed with a purine-type inhibitor 8-azaxanthin, was collected to 2.1 Å resolution from a crystal grown in D{sub 2}O by careful control and optimization of crystallization conditions via knowledge of the phase diagram. Deuterium atoms were clearly seen in the neutron-scattering density map. Crystallization and preliminary neutron diffraction measurements of rasburicase, a recombinant urate oxidase enzyme expressed by a genetically modified Saccharomyces cerevisiae strain, complexed with a purine-type inhibitor (8-azaxanthin) are reported. Neutron Laue diffraction data were collected to 2.1 Å resolution using the LADI instrument from a crystal (grown in D{sub 2}O) with volume 1.8 mm{sup 3}. The aim of this neutron diffraction study is to determine the protonation states of the inhibitor and residues within the active site. This will lead to improved comprehension of the enzymatic mechanism of this important enzyme, which is used as a protein drug to reduce toxic uric acid accumulation during chemotherapy. This paper illustrates the high quality of the neutron diffraction data collected, which are suitable for high-resolution structural analysis. In comparison with other neutron protein crystallography studies to date in which a hydrogenated protein has been used, the volume of the crystal was relatively small and yet the data still extend to high resolution. Furthermore, urate oxidase has one of the largest primitive unit-cell volumes (space group I222, unit-cell parameters a = 80, b = 96, c = 106 Å) and molecular weights (135 kDa for the homotetramer) so far successfully studied with neutrons.

  15. The metallo-. beta. -lactamases of Bacillus cereus 5/B/6: Expression in Echierichia coli, purification, and characterization of the purified recombinant enzyme

    SciTech Connect

    Shaw, R.W.; Clark, S.D.; Hilliard, N.P.; Harman, J.G. )

    1991-03-11

    The gene for the B. cereus 5/B/6 metallo-{beta}-lactamase was subcloned into the E. coli expression vector pRE-2. The resultant recombinants displayed a low level of enzyme activity. Generation of a site-directed mutant of the {beta}-lactamase gene containing both a Nde I site and an initiator codon allowed us to separate the {beta}-lactamase structural gene from its leader sequence. When only the structural gene was cloned into pRE-2, the B. cereus {beta}-lactamase activity was increased 9.8-fold. Purification of the recombinant enzyme from E. coli by ultracentrifugation, gel filtration, anion and cation exchange chromatography allowed the enzyme to be purified to homogeneity with an overall yield of 87%. The properties of the recombinant enzyme were identical to those of the B. cereus enzyme; e.g., the electrophoretic mobilities of the purified recombinant enzyme and the purified B. cereus enzyme were identical in both native and SDS gel electrophoresis As with the B. cereus enzyme, K{sub m} and V{sub max} for the recombinant enzyme are 0.39 mM and 1,333 units/mg protein, respectively. Likewise, the Co(II)-reconstituted recombinant enzyme has electronic spectra with maxima at 347, 551, 617 and 646 nm and extinction coefficients of 900, 250, 173 and 150 M{sup {minus}1} cm{sup {minus}1}, respectively. This heterologous construct and purification scheme will be used to produce and purify site-directed mutant proteins for use in exploring the reaction mechanisms of B. cereus metallo-{beta}-lactamases.

  16. RecBCD Enzyme "Chi Recognition" Mutants Recognize Chi Recombination Hotspots in the Right DNA Context.

    PubMed

    Amundsen, Susan K; Sharp, Jake W; Smith, Gerald R

    2016-09-01

    RecBCD enzyme is a complex, three-subunit protein machine essential for the major pathway of DNA double-strand break repair and homologous recombination in Escherichia coli Upon encountering a Chi recombination-hotspot during DNA unwinding, RecBCD nicks DNA to produce a single-stranded DNA end onto which it loads RecA protein. Conformational changes that regulate RecBCD's helicase and nuclease activities are induced upon its interaction with Chi, defined historically as 5' GCTGGTGG 3'. Chi is thought to be recognized as single-stranded DNA passing through a tunnel in RecC. To define the Chi recognition-domain in RecC and thus the mechanism of the RecBCD-Chi interaction, we altered by random mutagenesis eight RecC amino acids lining the tunnel. We screened for loss of Chi activity with Chi at one site in bacteriophage λ. The 25 recC mutants analyzed thoroughly had undetectable or strongly reduced Chi-hotspot activity with previously reported Chi sites. Remarkably, most of these mutants had readily detectable, and some nearly wild-type, activity with Chi at newly generated Chi sites. Like wild-type RecBCD, these mutants had Chi activity that responded dramatically (up to fivefold, equivalent to Chi's hotspot activity) to nucleotide changes flanking 5' GCTGGTGG 3'. Thus, these and previously published RecC mutants thought to be Chi-recognition mutants are actually Chi context-dependence mutants. Our results fundamentally alter the view that Chi is a simple 8-bp sequence recognized by the RecC tunnel. We propose that Chi hotspots have dual nucleotide sequence interactions, with both the RecC tunnel and the RecB nuclease domain.

  17. Enzymic, spectroscopic and calorimetric studies of a recombinant dextranase expressed in Pichia pastoris.

    PubMed

    Beldarraín, Alejandro; Acosta, Niuris; Betancourt, Lázaro; González, Luis J; Pons, Tirso

    2003-12-01

    Conformational stability and structural characterization of an rDex (recombinant dextranase) expressed in Pichia pastoris were studied by enzymic assays, fluorescence, CD and DSC (differential scanning calorimetry). We also identified two disulphide bridges (Cys9-Cys14, Cys484-Cys488) and two free Cys residues (Cys336, Cys415) that are not conserved between bacterial and fungal dextranases of GH-49 (glycoside hydrolase family 49) by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS. Enzymic and fluorescence studies revealed that rDex is biological and conformationally stable at acidic pH, with maximum activity at pH 4.5-5.0, while CD spectra indicated a secondary structure basically composed of beta-sheets. rDex loses biological activity at neutral pH without total disruption of its conformation. In addition, rDex preserves its conformation close to 60 degrees C, but it is thermally denatured with appreciable aggregation at temperatures above 75 degrees C. DSC studies always displayed irreversible transitions and a strong dependence on the scan rate. Our combined analysis suggested that the denaturation process of rDex is under kinetic control, which is described reasonably well by the two-state kinetic scheme.

  18. Activation of thiamin diphosphate in enzymes.

    PubMed

    Hübner, G; Tittmann, K; Killenberg-Jabs, M; Schäffner, J; Spinka, M; Neef, H; Kern, D; Kern, G; Schneider, G; Wikner, C; Ghisla, S

    1998-06-29

    Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy. The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated. The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate. The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate.

  19. Determining Enzyme Activity by Radial Diffusion

    ERIC Educational Resources Information Center

    Davis, Bill D.

    1977-01-01

    Discusses advantages of radial diffusion assay in determining presence of enzyme and/or rough approximation of amount of enzyme activities. Procedures are included for the preparation of starch-agar plates, and the application and determination of enzyme. Techniques using plant materials (homogenates, tissues, ungerminated embryos, and seedlings)…

  20. Molecular expression of l-asparaginase gene from Nocardiopsis alba NIOT-VKMA08 in Escherichia coli: A prospective recombinant enzyme for leukaemia chemotherapy.

    PubMed

    Meena, Balakrishnan; Anburajan, Lawrance; Vinithkumar, Nambali Valsalan; Shridhar, Divya; Raghavan, Rangamaran Vijaya; Dharani, Gopal; Kirubagaran, Ramalingam

    2016-09-30

    l-Asparaginase is an antineoplastic agent that selectively reduces the level of l-asparagine in blood and diminishes the proliferation of cancerous cells. Studies were carried out on the cloning and heterologous expression of l-asparaginase biosynthesis gene (ansA) from Nocardiopsis alba NIOT-VKMA08 to achieve the stable inducible system that overproduces the glutaminase-free recombinant l-asparaginase. Overexpression of recombinant l-asparaginase was achieved with an optimized final concentration of 1.5mM of isopropyl-β-d-thiogalactoside (IPTG) and the enzyme was expressed as a soluble protein. The recombinant enzyme was purified using nickel-nitrilotriacetic acid (Ni-NTA) chromatography and the purified enzyme disclosed an elevated level of asparaginase activity (158.1IU/mL). Optimum pH and temperature of the purified l-asparaginase for the hydrolysis of l-asparagine were 8.0 and 37°C and it was very specific for its natural substrate, l-asparagine. Detailed studies were carried out on the kinetics of enzyme reaction, catalytic activity, temperature and ionic strength and the thermostability of the l-asparaginase enzyme. The functional characterisation of the recombinant l-asparaginase was studied through Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), in silico sequence analysis and protein structural modelling. Glutaminase activity was not detected in the recombinant l-asparaginase, which could reduce the probable side effects during leukaemia therapy.

  1. PhAP protease from Pseudoalteromonas haloplanktis TAC125: Gene cloning, recombinant production in E. coli and enzyme characterization

    NASA Astrophysics Data System (ADS)

    de Pascale, D.; Giuliani, M.; De Santi, C.; Bergamasco, N.; Amoresano, A.; Carpentieri, A.; Parrilli, E.; Tutino, M. L.

    2010-08-01

    Cold-adapted proteases have been found to be the dominant activity throughout the cold marine environment, indicating their importance in bacterial acquisition of nitrogen-rich complex organic compounds. However, few extracellular proteases from marine organisms have been characterized so far, and the mechanisms that enable their activity in situ are still largely unknown. Aside from their ecological importance and use as model enzyme for structure/function investigations, cold-active proteolytic enzymes offer great potential for biotechnological applications. Our studies on cold adapted proteases were performed on exo-enzyme produced by the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. By applying a proteomic approach, we identified several proteolytic activities from its culture supernatant. PhAP protease was selected for further investigations. The encoding gene was cloned and the protein was recombinantly produced in E. coli cells. The homogeneous product was biochemically characterised and it turned out that the enzyme is a Zn-dependent aminopeptidase, with an activity dependence from assay temperature typical of psychrophilic enzymes.

  2. Molecular characterization of the acyl-coenzyme A:isopenicillin N acyltransferase gene (penDE) from Penicillium chrysogenum and Aspergillus nidulans and activity of recombinant enzyme in Escherichia coli.

    PubMed Central

    Tobin, M B; Fleming, M D; Skatrud, P L; Miller, J R

    1990-01-01

    The final step in the biosynthesis of beta-lactam antibiotics in Penicillium chrysogenum and Aspergillus nidulans involves removal of the L-alpha-aminoadipyl side chain from isopenicillin N (IPN) and exchange with a nonpolar side chain. The enzyme catalyzing this reaction, acyl-coenzyme A:isopenicillin N acyltransferase (acyltransferase), was purified from P. chrysogenum and A. nidulans. Based on NH2-terminal amino acid sequence information, the acyltransferase gene (penDE) from P. chrysogenum and A. nidulans were cloned. In both organisms, penDE was located immediately downstream from the isopenicillin N synthetase gene (pcbC) and consisted of four exons encoding an enzyme of 357 amino acids (approximately 40 kilodaltons [kDa]). The DNA coding sequences showed approximately 73% identity, while the amino acid sequences were approximately 76% identical. Noncoding DNA regions (including the region between pcbC and penDE) were not conserved. Acyltransferase activity from Escherichia coli producing the 40-kDa protein accepted either 6-aminopenicillanic acid or IPN as the substrate and made a penicillinase-sensitive antibiotic in the presence of phenylacetyl coenzyme A. Therefore, a single gene is responsible for converting IPN to penicillin G. The active form of the enzyme may result from processing of the 40-kDa monomeric precursor to a heterodimer containing subunits of 11 and 29 kDa. Images PMID:2120195

  3. Enzyme Activity Experiments Using a Simple Spectrophotometer

    ERIC Educational Resources Information Center

    Hurlbut, Jeffrey A.; And Others

    1977-01-01

    Experimental procedures for studying enzyme activity using a Spectronic 20 spectrophotometer are described. The experiments demonstrate the effect of pH, temperature, and inhibitors on enzyme activity and allow the determination of Km, Vmax, and Kcat. These procedures are designed for teaching large lower-level biochemistry classes. (MR)

  4. In vitro evaluation of Bacopa monniera extract and individual constituents on human recombinant monoamine oxidase enzymes.

    PubMed

    Singh, Rajbir; Ramakrishna, Rachumallu; Bhateria, Manisha; Bhatta, Rabi Sankar

    2014-09-01

    Bacopa monniera is a traditional Ayurvedic medicinal plant that has been used worldwide for its nootropic action. Chemically standardized extract of B. monniera is now available as over the counter herbal remedy to enhance memory in children and adults. Considering the nootropic action of B. monniera, we evaluated the effect of clinically available B. monniera extract and six of B. monniera constituents (bacoside A3, bacopaside I, bacopaside II, bacosaponin C, bacosine, and bacoside A mixture) on recombinant human monoamine oxidase (MAO) enzymes. The effect of B. monniera extract and individual constituents on human recombinant MAO-A and MAO-B enzymes was evaluated using MAO-Glo(TM) assay kit (Promega Corporation, USA), following the instruction manual. IC50 and mode of inhibition were measured for MAO enzymes. Bacopaside I and bacoside A mixture inhibited the MAO-A and MAO-B enzymes. Bacopaside I exhibited mixed mode of inhibition with IC50 and Ki values of 17.08 ± 1.64 and 42.5 ± 3.53 µg/mL, respectively, for MAO-A enzyme. Bacopaside I is the major constituent of B. monniera, which inhibited the MAO-A enzyme selectively.

  5. Temperature effects on product-quality-related enzymes in batch CHO cell cultures producing recombinant tPA.

    PubMed

    Clark, Kevin J R; Chaplin, Frank W R; Harcum, Sarah W

    2004-01-01

    Culture conditions that affect product quality are important to the successful operation and optimization of bioreactors. Previous studies have demonstrated that enzymes, such as proteases and sialidases, accumulate in batch bioreactors. These enzymes are known to be detrimental to the quality of recombinant glycoproteins. Bioreactor temperature has been used to control cell growth and recombinant protein production rates. However, the effect of culture temperature on the production of proteases and sialidases has not been investigated. In this study, Chinese hamster ovary cells were cultured with a temperature profile that decreased from 37 to 34 degrees C over 8 days and with a constant temperature of 37 degrees C. Analysis of extracellular protease activity indicated that two major proteases were present (50 and 69 kDa). The 50 kDa protease activity decreased similarly with time for both culture conditions. The 69 kDa protease activity increased with time for both culture conditions. The constant-temperature cultures had significantly lower 69 kDa protease levels compared to the ramped-temperature cultures in the early stationary phase. Intracellular sialidase activity was present in both cultures. The intracellular sialidase activity increased dramatically for both culture conditions immediately after the cells were inoculated into fresh medium. The initial peak in intracellular sialidase activity was followed by a first-order decay. The intracellular sialidase activities for the two culture conditions were not significantly different. The production of recombinant tissue type plasminogen activator was not significantly different for the two culture conditions. Thus, the previously hypothesized advantages that lower culture temperatures have reduced protease activity and improved productivity do not appear to be universal.

  6. Population genetic analysis of Helicobacter pylori by multilocus enzyme electrophoresis: extensive allelic diversity and recombinational population structure.

    PubMed Central

    Go, M F; Kapur, V; Graham, D Y; Musser, J M

    1996-01-01

    Genetic diversity and relationships in 74 Helicobacter pylori isolates recovered from patients assigned to distinct clinical categories were estimated by examination of allelic variation in six genes encoding metabolic housekeeping enzymes by multilocus enzyme electrophoresis. Seventy-three distinct allele profiles, representing multilocus chromosomal genotypes, were identified. All six loci were highly polymorphic, with an average of 11.2 alleles per locus. The mean genetic diversity in the sample was 0.735, a value that exceeds the level of diversity recorded in virtually all bacterial species studied by multilocus enzyme electrophoresis. A high frequency of occurrence of null alleles (lack of enzyme activity) was identified and warrants further investigation at the molecular level. Lack of linkage disequilibrium (nonrandom association (of alleles over loci) indicates that horizontal transfer and recombination of metabolic enzyme genes have contributed to the generation of chromosomal diversity in H. pylori. In this sample of isolates, there was no statistically significant association of multilocus enzyme electrophoretic types or cluster of related chromosomal types and disease category. PMID:8682800

  7. Recombination activity of copper in silicon

    NASA Astrophysics Data System (ADS)

    Sachdeva, R.; Istratov, A. A.; Weber, E. R.

    2001-10-01

    The carrier recombination activity of copper in n-type and p-type silicon has been investigated. The minority carrier diffusion length has been found to decrease monotonically with increasing copper concentration in n Si and to exhibit a step-like behavior in p-type silicon at Cu concentrations above a certain critical level. It is suggested that the impact of copper on the minority carrier diffusion length is determined by the formation of copper precipitates. This process is retarded in perfect silicon due to the large lattice mismatch between Cu3Si and the silicon lattice and even more retarded in p Si, due to electrostatic repulsion effects between the positively charged copper precipitates and interstitial copper ions. Comparison of the impact of Cu on minority carrier diffusion length obtained with p-Si samples of different resistivity confirmed the electrostatic model. Studies of the impact of copper on minority carrier diffusion length in samples with internal gettering sites indicated that they provide heterogeneous nucleation sites for Cu precipitation at subcritical Cu concentration. Above a certain threshold of Cu concentration, the bulk recombination activity is dominated by quasihomogeneous formation of Cu precipitates, a process that is not detectably affected by the presence of oxide precipitates.

  8. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme... FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus subtilis. The food additive...

  9. Characterization of Soil Samples of Enzyme Activity

    ERIC Educational Resources Information Center

    Freeland, P. W.

    1977-01-01

    Described are nine enzyme essays for distinguishing soil samples. Colorimetric methods are used to compare enzyme levels in soils from different sites. Each soil tested had its own spectrum of activity. Attention is drawn to applications of this technique in forensic science and in studies of soil fertility. (Author/AJ)

  10. D-ribulose-5-phosphate 3-epimerase: Cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme

    SciTech Connect

    Chen, Y.R.; Hartman, F.C.; Lu, T.Y.S.; Larimer, F.W.

    1998-09-01

    The authors have achieved, to their knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-{alpha}-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.

  11. Heparin and related polysaccharides: Synthesis using recombinant enzymes and metabolic engineering

    PubMed Central

    Suflita, Matthew; Fu, Li; He, Wenqin; Koffas, Mattheos; Linhardt, Robert J.

    2015-01-01

    Glycosaminoglycans are linear anionic polysaccharides that exhibit a number of important biological and pharmacological activities. The two most prominent members of this class of polysaccharides are heparin/heparan sulfate and the chondroitin sulfates (including dermatan sulfate). These polysaccharides, having complex structures and polydispersity, are biosynthesized in the Golgi of most animal cells. The chemical synthesis of these glycosaminoglycans is precluded by their structural complexity. Today, we depend on food animal tissues for their isolation and commercial production. Ton quantities of these glycosaminoglycans are used annually as pharmaceuticals and nutraceuticals. The variability of animal-sourced glycosaminoglycans, their inherent impurities, the limited availability of source tissues, the poor control of these source materials, and their manufacturing processes, suggest a need for new approaches for their production. Over the past decade there have been major efforts in the biotechnological production of these glycosaminoglycans. This mini-review focuses on the use of recombinant enzymes and metabolic engineering for the production of heparin and chondroitin sulfates. PMID:26219501

  12. Visualization of enzyme activities inside earthworm pores

    NASA Astrophysics Data System (ADS)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

    In extremely dynamic microhabitats as bio-pores made by earthworm, the in situ enzyme activities are assumed as a footprint of complex biotic interactions. Our study focused on the effect of earthworm on the enzyme activities inside bio-pores and visualizing the differences between bio-pores and earthworm-free soil by zymography technique (Spohn and Kuzyakov, 2013). For the first time, we aimed at quantitative imaging of enzyme activities in bio-pores. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). After two weeks when bio-pore systems were formed by earthworms, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. However, the differences in activity of cellobiohydrolase and leucine aminopeptidase between bio-pore and bulk soil were less pronounced. This demonstrated an applicability of zymography approach to monitor and to distinguish the in situ activity of hydrolytic enzymes in soil biopores.

  13. How thiamine diphosphate is activated in enzymes.

    PubMed

    Kern, D; Kern, G; Neef, H; Tittmann, K; Killenberg-Jabs, M; Wikner, C; Schneider, G; Hübner, G

    1997-01-03

    The controversial question of how thiamine diphosphate, the biologically active form of vitamin B1, is activated in different enzymes has been addressed. Activation of the coenzyme was studied by measuring thermodynamics and kinetics of deprotonation at the carbon in the 2-position (C2) of thiamine diphosphate in the enzymes pyruvate decarboxylase and transketolase by use of nuclear magnetic resonance spectroscopy, proton/deuterium exchange, coenzyme analogs, and site-specific mutant enzymes. Interaction of a glutamate with the nitrogen in the 1'-position in the pyrimidine ring activated the 4'-amino group to act as an efficient proton acceptor for the C2 proton. The protein component accelerated the deprotonation of the C2 atom by several orders of magnitude, beyond the rate of the overall enzyme reaction. Therefore, the earlier proposed concerted mechanism or stabilization of a C2 carbanion can be excluded.

  14. Water modulation of stratum corneum chymotryptic enzyme activity and desquamation.

    PubMed

    Watkinson, A; Harding, C; Moore, A; Coan, P

    2001-09-01

    Exposure to a dry environment leads to depletion of water from the peripheral stratum corneum layers in a process dependent on the relative humidity (RH) and the intrinsic properties of the tissue. We hypothesized that by modulating the water content of the stratum corneum in the surface layers, RH effects the rate of desquamation by modulating the activity of the desquamatory enzymes, and specifically stratum corneum chymotryptic enzyme (SCCE). Using a novel air interface in vitro desquamatory model, we demonstrated RH-dependent corneocyte release with desquamatory rates decreasing below 80% RH. Application of 10% glycerol or a glycerol-containing moisturizing lotion further increased desquamation, even in humid conditions, demonstrating that water was the rate-limiting factor in the final stages of desquamation. Furthermore, even in humid conditions desquamation was sub-maximal. In situ stratum corneum SCCE activity showed a dependence on RH: activity was significantly higher at 100% than at 44% RH. Further increases in SCCE activity were induced by applying a 10% glycerol solution. Since SCCE, a water-requiring enzyme, must function in the water-depleted outer stratum corneum, we sought to determine whether this enzyme has a tolerance to lowered water activity. Using concentrated sucrose solutions to lower water activity, we analysed the activity of recombinant SCCE and compared it to that of trypsin and chymotrypsin. SCCE activity demonstrated a tolerance to water restriction, and this may be an adaptation to maintain enzyme activity even within the water-depleted stratum corneum intercellular space. Overall these findings support the concept that in the upper stratum corneum, RH modulates desquamation by its effect upon SCCE activity, and possibly other desquamatory hydrolases. In addition, SCCE may be adapted to function in the water-restricted stratum corneum intercellular space.

  15. Normal Modes Expose Active Sites in Enzymes

    PubMed Central

    Glantz-Gashai, Yitav; Samson, Abraham O.

    2016-01-01

    Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes. PMID:28002427

  16. Dielectronic Recombination In Active Galactic Nuclei

    NASA Astrophysics Data System (ADS)

    Lukić, D.; Savin, D. W.; Schnell, M.; Brandau, C.; Schmidt, E.; Schippers, S.; Müller, A.; Lestinsky, M.; Sprenger, F.; Wolf, A.; Altun, Z.; Badnell, N. R.

    2006-05-01

    Recent X-ray satelitte observations of active galactic nuclei point out shortcomings in our understanding of low temperature dielectronic recombination (DR) for iron M- shell ions. In order to resolve this issue and to provide reliable iron M-shell DR data for modeling astrophysical plasmas, we are carrying out a series of laboratory measurements using the heavy-ion Test Storage Ring at the Max- Plank-Institute for Nuclear Physics in Heidelberg, Germany. Storage rings are currently the only laboratory method capable of studying low temperature DR. We use our results to produce experimentally- derived DR rate coefficients. We are also providing our data to atomic theorist to benchmark their DR calculations. Here we will report our recent DR results for selected Fe M-shell ions. At temperatures where these ions are predicted to form in photoionized gas, we find a significant discrepancy between our experimental results and previously recommended DR rate coefficients.

  17. Gene cloning of alpha-methylserine aldolase from Variovorax paradoxus and purification and characterization of the recombinant enzyme.

    PubMed

    Nozaki, Hiroyuki; Kuroda, Shinji; Watanabe, Kunihiko; Yokozeki, Kenzo

    2008-10-01

    The alpha-methylserine aldolase gene from Variovorax paradoxus strains AJ110406, NBRC15149, and NBRC15150 was cloned and expressed in Escherichia coli. Formaldehyde release activity from alpha-methyl-L-serine was detected in the cell-free extract of E.coli expressing the gene from three strains. The recombinant enzyme from V. paradoxus NBRC15150 was purified. The Vmax and Km of the enzyme for the formaldehyde release reaction from alpha-methyl-L-serine were 1.89 micromol min(-1) mg(-1) and 1.2 mM respectively. The enzyme was also capable of catalyzing the synthesis of alpha-methyl-L-serine and alpha-ethyl-L-serine from L-alanine and L-2-aminobutyric acid respectively, accompanied by hydroxymethyl transfer from formaldehyde. The purified enzyme also catalyzed alanine racemization. It contained 1 mole of pyridoxal 5'-phosphate per mol of the enzyme subunit, and exhibited a specific spectral peak at 429 nm. With L-alanine and L-2-aminobutyric acid as substrates, the specific peak, assumed to be a result of the formation of a quinonoid intermediate, increased at 498 nm and 500 nm respectively.

  18. Antimutagenic activity of oxidase enzymes

    SciTech Connect

    Agabeili, R.A.

    1986-11-01

    By means of a cytogenetic analysis of chromosomal aberrations in plant cells (Welsh onion, wheat) it was found that the cofactors nicotinamide adenine phosphate (NAD), nicotinamide adenine dinucleotide phosphate (NADPH), and riboflavin possess antimutagenic activity.

  19. In vitro glucuronidation kinetics of deoxynivalenol by human and animal microsomes and recombinant human UGT enzymes.

    PubMed

    Maul, Ronald; Warth, Benedikt; Schebb, Nils Helge; Krska, Rudolf; Koch, Matthias; Sulyok, Michael

    2015-06-01

    The mycotoxin deoxynivalenol (DON), formed by Fusarium species, is one of the most abundant mycotoxins contaminating food and feed worldwide. Upon ingestion, the majority of the toxin is excreted by humans and animal species as glucuronide conjugate. First in vitro data indicated that DON phase II metabolism is strongly species dependent. However, kinetic data on the in vitro metabolism as well as investigations on the specific enzymes responsible for DON glucuronidation in human are lacking. In the present study, the DON metabolism was investigated using human microsomal fractions and uridine-diphosphoglucuronyltransferases (UGTs) as well as liver microsomes from five animal species. Only two of the twelve tested human recombinant UGTs led to the formation of DON glucuronides with a different regiospecificity. UGT2B4 predominantly catalyzed the formation of DON-15-O-glucuronide (DON-15GlcA), while for UGT2B7 the DON-3-O-glucuronide (DON-3GlcA) metabolite prevailed. For human UGTs, liver, and intestinal microsomes, the glucuronidation activities were low. The estimated apparent intrinsic clearance (Clapp,int) for all human UGT as well as tissue homogenates was <1 mL/min mg protein. For the animal liver microsomes, moderate Clapp,int between 1.5 and 10 mL/min mg protein were calculated for carp, trout, and porcine liver. An elevated glucuronidation activity was detected for rat and bovine liver microsomes leading to Clapp,int between 20 and 80 mL/min mg protein. The obtained in vitro data points out that none of the animal models is suitable for estimating the human DON metabolism with respect to the metabolite pattern and formation rate.

  20. The autolytic activity of the recombinant amidase of Staphylococcus saprophyticus is inhibited by its own recombinant GW repeats.

    PubMed

    Hell, Wolfgang; Reichl, Sylvia; Anders, Agnes; Gatermann, Sören

    2003-10-10

    The Aas (autolysin/adhesin of Staphylococcus saprophyticus) is a multifunctional surface protein containing two enzymatic domains an N-acetyl-muramyl-L-alanine amidase, an endo-beta-N-acetyl-D-glucosaminidase, and two different regions of repetitive sequences, an N-terminal and a C-terminal repetitive domain. The C-terminal repetitive domain is built up by the repeats R1, R2 and R3, which interconnect the putative active centers of the amidase and glucosaminidase. To investigate the influence of the C-terminal repeats and the N-terminal repeats on the amidase activity, the repetitive domains and fragments of them were cloned and expressed in Escherichia coli. The influence of the different fragments on the activity of the recombinant amidase of the Aas, consisting of the active center of the enzyme and repeat R1, was investigated in a turbidimetric microassay. The different fragments derived from the C-terminal repeats inhibited the amidase activity, while the N-terminal repeats did not influence the activity of the enzyme. The inhibiting activity increased with the number of GW repeats the recombinant fragment contained. Thus we conclude, that the C-terminal GW repeats and not the N-terminal repeats are necessary for the cell wall targeting and the autolytic function of the amidase.

  1. The Balance between Recombination Enzymes and Accessory Replicative Helicases in Facilitating Genome Duplication

    PubMed Central

    Syeda, Aisha H.; Atkinson, John; Lloyd, Robert G.; McGlynn, Peter

    2016-01-01

    Accessory replicative helicases aid the primary replicative helicase in duplicating protein-bound DNA, especially transcribed DNA. Recombination enzymes also aid genome duplication by facilitating the repair of DNA lesions via strand exchange and also processing of blocked fork DNA to generate structures onto which the replisome can be reloaded. There is significant interplay between accessory helicases and recombination enzymes in both bacteria and lower eukaryotes but how these replication repair systems interact to ensure efficient genome duplication remains unclear. Here, we demonstrate that the DNA content defects of Escherichia coli cells lacking the strand exchange protein RecA are driven primarily by conflicts between replication and transcription, as is the case in cells lacking the accessory helicase Rep. However, in contrast to Rep, neither RecA nor RecBCD, the helicase/exonuclease that loads RecA onto dsDNA ends, is important for maintaining rapid chromosome duplication. Furthermore, RecA and RecBCD together can sustain viability in the absence of accessory replicative helicases but only when transcriptional barriers to replication are suppressed by an RNA polymerase mutation. Our data indicate that the minimisation of replisome pausing by accessory helicases has a more significant impact on successful completion of chromosome duplication than recombination-directed fork repair. PMID:27483323

  2. Enzyme activity in dialkyl phosphate ionic liquids

    SciTech Connect

    Thomas, M.F.; Dunn, J.; Li, L.-L.; Handley-Pendleton, J. M.; van der lelie, D.; Wishart, J. F.

    2011-12-01

    The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariellavolvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.

  3. Recombinant encapsulated cells overexpressing alpha-L-iduronidase correct enzyme deficiency in human mucopolysaccharidosis type I cells.

    PubMed

    Baldo, Guilherme; Quoos Mayer, Fabiana; Burin, Maira; Carrillo-Farga, Joaquín; Matte, Ursula; Giugliani, Roberto

    2012-01-01

    Mucopolysaccharidosis I (MPS I) is an autosomal recessive lysosomal storage disease due to deficient α-L-iduronidase (IDUA) activity. It results in the accumulation of the glycosaminoglycans (GAGs) heparan and dermatan sulfate and leads to several clinical manifestations. Available treatments are limited in their efficacy to treat some aspects of the disease. Thus, new approaches have been studied for the treatment of MPS I. Here, we tested the ability of recombinant baby hamster kidney cells transfected with human IDUA cDNA in correcting skin fibroblasts from MPS I patients in vitro. Our results showed an increase in IDUA activity in MPS I fibroblasts after 15, 30 and 45 days of coculture with the capsules. Cytological analysis showed a marked reduction in GAG storage within MPS I cells. Enzyme uptake by the fibroblasts was blocked in a dose-dependent manner with mannose-6-phosphate (M6P), indicating that cells use the M6P receptor to internalize the recombinant enzyme. Capsules were effective in correcting MPS I cells even after a 12-month period of cryopreservation. Taken together, our results indicate that cell encapsulation is a potential approach for treatment of MPS I. This approach becomes particularly interesting as a complementary approach, since the capsules could be implanted in sites which current treatments available are not able to reach. Future studies will focus on the efficacy of this approach in vivo.

  4. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) for directed enzyme evolution.

    PubMed

    Gonzalez-Perez, David; Molina-Espeja, Patricia; Garcia-Ruiz, Eva; Alcalde, Miguel

    2014-01-01

    Approaches that depend on directed evolution require reliable methods to generate DNA diversity so that mutant libraries can focus on specific target regions. We took advantage of the high frequency of homologous DNA recombination in Saccharomyces cerevisiae to develop a strategy for domain mutagenesis aimed at introducing and in vivo recombining random mutations in defined segments of DNA. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) is a one-pot random mutagenic method for short protein regions that harnesses the in vivo recombination apparatus of yeast. Using this approach, libraries can be prepared with different mutational loads in DNA segments of less than 30 amino acids so that they can be assembled into the remaining unaltered DNA regions in vivo with high fidelity. As a proof of concept, we present two eukaryotic-ligninolytic enzyme case studies: i) the enhancement of the oxidative stability of a H2O2-sensitive versatile peroxidase by independent evolution of three distinct protein segments (Leu28-Gly57, Leu149-Ala174 and Ile199-Leu268); and ii) the heterologous functional expression of an unspecific peroxygenase by exclusive evolution of its native 43-residue signal sequence.

  5. Mutagenic Organized Recombination Process by Homologous In Vivo Grouping (MORPHING) for Directed Enzyme Evolution

    PubMed Central

    Gonzalez-Perez, David; Molina-Espeja, Patricia; Garcia-Ruiz, Eva; Alcalde, Miguel

    2014-01-01

    Approaches that depend on directed evolution require reliable methods to generate DNA diversity so that mutant libraries can focus on specific target regions. We took advantage of the high frequency of homologous DNA recombination in Saccharomyces cerevisiae to develop a strategy for domain mutagenesis aimed at introducing and in vivo recombining random mutations in defined segments of DNA. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) is a one-pot random mutagenic method for short protein regions that harnesses the in vivo recombination apparatus of yeast. Using this approach, libraries can be prepared with different mutational loads in DNA segments of less than 30 amino acids so that they can be assembled into the remaining unaltered DNA regions in vivo with high fidelity. As a proof of concept, we present two eukaryotic-ligninolytic enzyme case studies: i) the enhancement of the oxidative stability of a H2O2-sensitive versatile peroxidase by independent evolution of three distinct protein segments (Leu28-Gly57, Leu149-Ala174 and Ile199-Leu268); and ii) the heterologous functional expression of an unspecific peroxygenase by exclusive evolution of its native 43-residue signal sequence. PMID:24614282

  6. A sensitive enzyme immunoassay for amygdalin in food extracts using a recombinant antibody.

    PubMed

    Cho, A-Yeon; Shin, Kum-Joo; Chung, Junho; Oh, Sangsuk

    2008-10-01

    Amygdalin (laterile) is a cyanogenic glycoside commonly found in the pits of many fruits and raw nuts. When amygdalin-containing seeds are crushed and moistened, free cyanide is formed. Pits and nuts containing unusually high levels of amygdalin can therefore cause cyanide poisoning, and detection of amygdalin in food extracts can be a life-saving measure. In this study, we generated recombinant antibodies against amygdalin from a phage display of a combinatorial rabbit/human chimeric antibody library and used it in a sensitive competition enzyme immunoassay system to detect amygdalin in extracts of pits and nuts. The detection limit was determined to be 1 x 10(-9) M.

  7. Activity assessment of microbial fibrinolytic enzymes.

    PubMed

    Kotb, Essam

    2013-08-01

    Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.

  8. Effect of Laser Irradiation on Enzyme Activity

    NASA Astrophysics Data System (ADS)

    Murakami, Satoshi; Kashii, Masafumi; Kitano, Hiroshi; Adachi, Hiroaki; Takano, Kazufumi; Matsumura, Hiroyoshi; Inoue, Tsuyoshi; Mori, Yusuke; Doi, Masaaki; Sugamoto, Kazuomi; Yoshikawa, Hideki; Sasaki, Takatomo

    2005-11-01

    We previously developed a protein crystallization technique using a femtosecond laser and protein crystal processing and detaching techniques using a pulsed UV laser. In this study, we examine the effect of laser irradiation on protein integrity. After several kinds of laser were irradiated on part of a solution of glycerol-6-phosphate dehydrogenase from Leuconostoc mesenteroides, we measured the enzyme activity. Femtosecond and deep-UV laser irradiations have little influence on the whole enzyme activity, whereas the enzyme lost its activity upon high-power near-infrared laser irradiation at a wavelength of 1547 nm. These results suggest that suitable laser irradiation has no remarkable destructive influence on protein crystallization or crystal processing.

  9. Dielectronic Recombination In Active Galactic Nuclei

    NASA Technical Reports Server (NTRS)

    Lukic, D. V.; Schnell, M.; Savin, D. W.; Altun, Z.; Badnell, N.; Brandau, C.; Schmidt, E. W.; Mueller, A.; Schippers, S.; Sprenger, F.; Lestinsky, M.; Wolf, A.

    2006-01-01

    XMM-Newton and Chandra observations of active galactic nuclei (AGN) show rich spectra of X-ray absorption lines. These observations have detected a broad unresolved transition array (UTA) between approx. 15-17 A. This is attributed to inner-shell photoexcitation of M-shell iron ions. Modeling these UTA features is currently limited by uncertainties in the low-temperature dielectronic recombination (DR) data for M-shell iron. In order to resolve this issue, and to provide reliable iron M-shell DR data for plasma modeling, we are carrying out a series of laboratory measurements using the heavy-ion Test Storage Ring (TSR) at the Max-Plank-Institute for Nuclear Physics in Heidelberg, Germany. Currently, laboratory measurements of low temperature DR can only be performed at storage rings. We use the DR data obtained at TSR, to calculate rate coefficients for plasma modeling and to benchmark theoretical DR calculations. Here we report our recent experimental results for DR of Fe XIV forming Fe XIII.

  10. Dielectronic Recombination In Active Galactic Nuclei

    NASA Astrophysics Data System (ADS)

    Lukic, D. V.; Schnell, M.; Savin, D. W.; Altun, Z.; Badnell, N.; Brandau, C.; Schmidt, E. W.; Müller, A.; Schippers, S.; Sprenger, F.; Lestinsky, M.; Wolf, A.

    XMM-Newton and Chandra observations of active galactic nuclei (AGN) show rich spectra of X-ray absorption lines. These observations have detected a broad unresolved transition array (UTA) between ˜ 15-17 Å. This is attributed to inner-shell photoexcitation of M-shell iron ions. Modeling these UTA features is currently limited by uncertainties in the low-temperature dielectronic recombination (DR) data for M-shell iron. In order to resolve this issue, and to provide reliable iron M-shell DR data for plasma modeling, we are carrying out a series of laboratory measurements using the heavy-ion Test Storage Ring (TSR) at the Max-Plank-Institute for Nuclear Physics in Heidelberg, Germany. Currently, laboratory measurements of low temperature DR can only be performed at storage rings. We use the DR data obtained at TSR, to calculate rate coefficients for plasma modeling and to benchmark theoretical DR calculations. Here we report our recent experimental results for DR of Fe XIV forming Fe XIII.

  11. Synthesis of the milk oligosaccharide 2'-fucosyllactose using recombinant bacterial enzymes.

    PubMed

    Albermann, C; Piepersberg, W; Wehmeier, U F

    2001-08-23

    The enzymatic synthesis of GDP-beta-L-fucose and its enzymatic transfer reaction using recombinant enzymes from bacterial sources was examined. The GDP-D-mannose 4,6-dehydratase and the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase from Escherichia coli K-12, respectively, were used to catalyse the conversion of GDP-alpha-D-mannose to GDP-beta-L-fucose with 78% yield. For the transfer of the L-fucose to an acceptor, we cloned and overproduced the alpha-(1-->2)-fucosyltransferase (FucT2) protein from Helicobacter pylori. We were able to synthesise 2'-fucosyllactose using the overproduced FucT2 enzyme, enzymatically synthesised GDP-L-fucose and lactose. The isolation of 2'-fucosyllactose was accomplished by anion-exchange chromatography and gel filtration to give 65% yield.

  12. Halophilic enzyme activation induced by salts

    PubMed Central

    Ortega, Gabriel; Laín, Ana; Tadeo, Xavier; López-Méndez, Blanca; Castaño, David; Millet, Oscar

    2011-01-01

    Halophilic archea (halobacteriae) thrive in hypersaline environments, avoiding osmotic shock by increasing the ion concentration of their cytoplasm by up to 3–6 M. To remain folded and active, their constitutive proteins have evolved towards a biased amino acid composition. High salt concentration affects catalytic activity in an enzyme-dependent way and a unified molecular mechanism remains elusive. Here, we have investigated a DNA ligase from Haloferax volcanii (Hv LigN) to show that K+ triggers catalytic activity by preferentially stabilising a specific conformation in the reaction coordinate. Sodium ions, in turn, do not populate such isoform and the enzyme remains inactive in the presence of this co-solute. Our results show that the halophilic amino acid signature enhances the enzyme's thermodynamic stability, with an indirect effect on its catalytic activity. This model has been successfully applied to reengineer Hv LigN into an enzyme that is catalytically active in the presence of NaCl. PMID:22355525

  13. Human recombinant arginase enzyme reduces plasma arginine in mouse models of arginase deficiency.

    PubMed

    Burrage, Lindsay C; Sun, Qin; Elsea, Sarah H; Jiang, Ming-Ming; Nagamani, Sandesh C S; Frankel, Arthur E; Stone, Everett; Alters, Susan E; Johnson, Dale E; Rowlinson, Scott W; Georgiou, George; Lee, Brendan H

    2015-11-15

    Arginase deficiency is caused by deficiency of arginase 1 (ARG1), a urea cycle enzyme that converts arginine to ornithine. Clinical features of arginase deficiency include elevated plasma arginine levels, spastic diplegia, intellectual disability, seizures and growth deficiency. Unlike other urea cycle disorders, recurrent hyperammonemia is typically less severe in this disorder. Normalization of plasma arginine levels is the consensus treatment goal, because elevations of arginine and its metabolites are suspected to contribute to the neurologic features. Using data from patients enrolled in a natural history study conducted by the Urea Cycle Disorders Consortium, we found that 97% of plasma arginine levels in subjects with arginase deficiency were above the normal range despite conventional treatment. Recently, arginine-degrading enzymes have been used to deplete arginine as a therapeutic strategy in cancer. We tested whether one of these enzymes, a pegylated human recombinant arginase 1 (AEB1102), reduces plasma arginine in murine models of arginase deficiency. In neonatal and adult mice with arginase deficiency, AEB1102 reduced the plasma arginine after single and repeated doses. However, survival did not improve likely, because this pegylated enzyme does not enter hepatocytes and does not improve hyperammonemia that accounts for lethality. Although murine models required dosing every 48 h, studies in cynomolgus monkeys indicate that less frequent dosing may be possible in patients. Given that elevated plasma arginine rather than hyperammonemia is the major treatment challenge, we propose that AEB1102 may have therapeutic potential as an arginine-reducing agent in patients with arginase deficiency.

  14. Cloning and expression of a phloretin hydrolase gene from Eubacterium ramulus and characterization of the recombinant enzyme.

    PubMed

    Schoefer, Lilian; Braune, Annett; Blaut, Michael

    2004-10-01

    Phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl)propionic acid during flavonoid degradation in Eubacterium ramulus. The gene encoding the enzyme was cloned by screening a gene library for hydrolase activity. The insert of a clone conferring phloretin hydrolase activity was sequenced. Sequence analysis revealed an open reading frame of 822 bp (phy), a putative promoter region, and a terminating stem-loop structure. The deduced amino acid sequence of phy showed similarities to a putative protein of the 2,4-diacetylphloroglucinol biosynthetic operon from Pseudomonas fluorescens. The phloretin hydrolase was heterologously expressed in Escherichia coli and purified. The molecular mass of the native enzyme was approximately 55 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of phy indicated molecular masses of 30 and 30.8 kDa, respectively, suggesting that the enzyme is a homodimer. The recombinant phloretin hydrolase catalyzed the hydrolysis of phloretin to equimolar amounts of phloroglucinol and 3-(4-hydroxyphenyl)propionic acid. The optimal temperature and pH of the catalyzed reaction mixture were 37 degrees C and 7.0, respectively. The K(m) for phloretin was 13 +/- 3 microM and the k(cat) was 10 +/- 2 s(-1). The enzyme did not transform phloretin-2'-glucoside (phloridzin), neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, or trans-1,3-diphenyl-2,3-epoxy-propan-1-one. The catalytic activity of the phloretin hydrolase was reduced by N-bromosuccinimide, o-phenanthroline, N-ethylmaleimide, and CuCl(2) to 3, 20, 35, and 85%, respectively. Phloroglucinol and 3-(4-hydroxyphenyl)propionic acid reduced the activity to 54 and 70%, respectively.

  15. A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS.

    PubMed

    Li, Xiaofeng; Suhar, Tom; Glass, Lateca; Rajaraman, Ganesh

    2014-03-03

    Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each.

  16. An NMR Study of Enzyme Activity.

    ERIC Educational Resources Information Center

    Peterman, Keith E.; And Others

    1989-01-01

    A laboratory experiment designed as a model for studying enzyme activity with a basic spectrometer is presented. Included are background information, experimental procedures, and a discussion of probable results. Stressed is the value of the use of Nuclear Magnetic Resonance in biochemistry. (CW)

  17. A new study of cell disruption to release recombinant thermostable enzyme from Escherichia coli by thermolysis.

    PubMed

    Ren, Xiaodong; Yu, Dawei; Yu, Lei; Gao, Gui; Han, Siping; Feng, Yan

    2007-05-10

    Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical, chemical or enzymatic disruption technology. In this study, a novel thermolysis method was used to disrupt E. coli cells to release a recombinant thermostable esterase. We found that heat treatment of E. coli was highly effective to destroy the integrity of bacterial cell walls and release the recombinant hyperthermophilic esterase at temperatures above 60 degrees C. The effects of temperature, pH and cell concentration on the efficiency of cell disruption were examined. The most effective temperature for cell disruption was at 80 degrees C. The pH and cell concentration had only minor effect on the release of the hyperthermophilic esterase. In addition, we found that the hyperthermophilic esterase could be purified at the early stage of the thermolysis, which is a major advantage of the thermolysis method. Finally, a comparison between thermolysis and traditional methods for the disruption of cells and the release of the thermostable enzyme was made.

  18. [Effects of Hg on soil enzyme activity].

    PubMed

    Yang, Chun-Lu; Sun, Tie-Heng; He, Wen-Xiang; Chen, Su

    2007-03-01

    With simulation test, this paper studied the effects of Hg on the activities of urease, invertase and neutral phosphotase in four soils. The results showed that Hg inhibited soil urease and invertase activities markedly, but its inhibitory effect differed with test soils. There was a significant logarithmic correlation between the concentration of HgCl2 and the activities of these two enzymes (P < 0.05). In test soils, the ED50 of urease activity was 87.99, 5.47, 24.05 and 19.88 mg x kg(-1), and that of invertase activity was 76.68, 727.49, 236.52 and 316.59 mg x kg(-1), respectively. Urease was more sensitive than invertase to Hg contamination, while organic matter had a protective effect on soil enzymes. Soil neutral phosphatase was not sensitive to Hg contamination, except that it was significantly activated by Hg in the meadow brown soil applied with plenty of organic fertilizer.

  19. Enhanced expression of an endoglucanase in Bacillus subtilis by using the sucrose-inducible sacB promoter and improved properties of the recombinant enzyme.

    PubMed

    Liu, Sen-Lin; Du, Kun

    2012-06-01

    An endoglucanase from Bacillus akibai I-1 was successfully overexpressed in Bacillus subtilis 168 and the expression level of the recombinant enzyme was greatly enhanced by using the sucrose-inducible sacB promoter. The endoglucanase activity in the culture supernatant of recombinant B. subtilis by using itself promoter (HpaII) in plasmid pMA5 was 3U/ml. Interestingly, with the addition of sacB promoter at downstream from the HpaII promoter or the replacement of HpaII promoter by the sacB promoter, the endoglucanase activities reached 62 and 60U/ml, respectively, under the optimal culture conditions. These results demonstrated that the sacB promoter might be more efficient for the expression of the endoglucanase than the HpaII promoter. More interestingly, the purified native enzyme had broad pH stability, good thermostability and resistibility to various metal ions and chelating agents examined, while the recombinant enzyme had improved resistibility to SDS, which was stable in 0.2% (w/v) laundry detergent and thus showed great potential in detergents industry.

  20. Iodothyronine deiodinase enzyme activities in bone.

    PubMed

    Williams, Allan J; Robson, Helen; Kester, Monique H A; van Leeuwen, Johannes P T M; Shalet, Stephen M; Visser, Theo J; Williams, Graham R

    2008-07-01

    Euthyroid status is essential for normal skeletal development and maintenance of the adult skeleton, but the mechanisms which control supply of thyroid hormone to bone cells are poorly understood. Thyroid hormones enter target cells via monocarboxylate transporter-8 (MCT8), which provides a functional link between thyroid hormone uptake and metabolism in the regulation of T3-action but has not been investigated in bone. Most circulating active thyroid hormone (T3) is derived from outer ring deiodination of thyroxine (T4) mediated by the type 1 deiodinase enzyme (D1). The D2 isozyme regulates intra-cellular T3 supply and determines saturation of the nuclear T3-receptor (TR), whereas a third enzyme (D3) inactivates T4 and T3 to prevent hormone availability and reduce TR-saturation. The aim of this study was to determine whether MCT8 is expressed in the skeleton and whether chondrocytes, osteoblasts and osteoclasts express functional deiodinases. Gene expression was analyzed by RT-PCR and D1, D2 and D3 function by sensitive and highly specific determination of enzyme activities. MCT8 mRNA was expressed in chondrocytes, osteoblasts and osteoclasts at all stages of cell differentiation. D1 activity was undetectable in all cell types, D2 activity was only present in mature osteoblasts whereas D3 activity was evident throughout chondrocyte, osteoblast and osteoclast differentiation in primary cell cultures. These data suggest that T3 availability especially during skeletal development may be limited by D3-mediated catabolism rather than by MCT8 mediated cellular uptake or D2-dependent T3 production.

  1. Targeted cellular metabolism for cancer chemotherapy with recombinant arginine-degrading enzymes

    PubMed Central

    Savaraj, Niramol; Feun, Lynn G.

    2010-01-01

    It has been shown that a subset of human cancers, notably, melanoma and hepatocellular carcinoma (HCC) are auxotrophic for arginine (Arg), because they do not express argininosuccinate synthetase (ASS), the rate-limiting enzyme for the biosynthesis of arginine from citrulline. These ASS-negative cancer cells require Arg from extracellular sources for survival. When they are exposed to recombinant Arg-degrading enzymes, e.g. arginine deiminase (ADI) or arginase, they die because of Arg starvation; whereas normal cells which express ASS are able to survive. A pegylated ADI (ADI-PEG20) has been developed for clinical trials for advanced melanoma and HCC; and favorable results have been obtained. ADI-PEG20 treatment induces autophagy in auxotrophic cancer cells leading to cell death. Clinical studies in melanoma patients show that re-expression of ASS is associated with ADI-PEG20 resistance. ADI-PEG20 treatment down-regulates the expression of HIF-1α but up-regulates c-Myc in culture melanoma cells. Induction of ASS by ADI-PEG20 involves positive regulators c-Myc and Sp4 and negative regulator HIF1α. Since both HIF-1α and c-Myc play important roles in cancer cell energy metabolism, together these results suggest that targeted cancer cell metabolism through modulation of HIF-1α and c-Myc expression may improve the efficacy of ADI-PEG20 in treating Arg auxotrophic tumors. PMID:21152246

  2. Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility

    PubMed Central

    Smith, Madison A.; Gonzalez, Jesica; Hussain, Anjum; Oldfield, Rachel N.; Johnston, Kathryn A.; Lopez, Karlo M.

    2016-01-01

    Lysyl oxidase is an important extracellular matrix enzyme that has not been fully characterized due to its low solubility. In order to circumvent the low solubility of this enzyme, three solubility tags (Nus-A, Thioredoxin (Trx), and Glutathione-S-Transferase (GST)) were engineered on the N-terminus of mature lysyl oxidase. Total enzyme yields were determined to be 1.5 mg for the Nus-A tagged enzyme (0.75 mg/L of media), 7.84 mg for the Trx tagged enzyme (3.92 mg/L of media), and 9.33 mg for the GST tagged enzyme (4.67 mg/L of media). Enzymatic activity was calculated to be 0.11 U/mg for the Nus-A tagged enzyme and 0.032 U/mg for the Trx tagged enzyme, and no enzymatic activity was detected for the GST tagged enzyme. All three solubility-tagged forms of the enzyme incorporated copper; however, the GST tagged enzyme appears to bind adventitious copper with greater affinity than the other two forms. The catalytic cofactor, lysyl tyrosyl quinone (LTQ), was determined to be 92% for the Nus-A and Trx tagged lysyl oxidase using the previously reported extinction coefficient of 15.4 mM−1 cm−1. No LTQ was detected for the GST tagged lysyl oxidase. Given these data, it appears that Nus-A is the most suitable tag for obtaining soluble and active recombinant lysyl oxidase from E. coli culture. PMID:26942005

  3. Hydrolysis and transglycosylation activity of a thermostable recombinant beta-glycosidase from Sulfolobus acidocaldarius.

    PubMed

    Park, Ah-Reum; Kim, Hye-Jung; Lee, Jung-Kul; Oh, Deok-Kun

    2010-04-01

    We expressed a putative beta-galactosidase from Sulfolobus acidocaldarius in Escherichia coli and purified the recombinant enzyme using heat treatment and Hi-Trap ion-exchange chromatography. The resultant protein gave a single 57-kDa band by SDS-PAGE and had a specific activity of 58 U/mg. The native enzyme existed as a dimer with a molecular mass of 114 kDa by gel filtration. The maximum activity of this enzyme was observed at pH 5.5 and 90 degrees C. The half-lives of the enzyme at 70, 80, and 90 degrees C were 494, 60, and 0.2 h, respectively. The hydrolytic activity with p-nitrophenyl(pNP) substrates followed the order p-nitrophenyl-beta-D-fucopyranoside > pNP-beta-D-glucopyranoside > pNP-beta-D-galactopyranoside > pNP-beta-D-mannopyranoside > pNP-beta-D-xylopyranoside, but not toward aryl-alpha-glycosides or pNP-beta-L-arabinofuranoside. Thus, the enzyme was actually a beta-glycosidase. The beta-glycosidase exhibited transglycosylation activity with pNP-beta-D-galactopyranoside, pNP-beta-D-glucopyranoside, and pNP-beta-D-fucopyranoside in decreasing order of activity, in the reverse order of its hydrolytic activity. The hydrolytic activity was higher toward cellobiose than toward lactose, but the transglycosylation activity was lower with cellobiose than with lactose.

  4. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... preparation derived from a recombinant Bacillus subtilis. 173.115 Section 173.115 Food and Drugs FOOD AND DRUG... Bacillus subtilis. The food additive alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation, may be... derived from a modified Bacillus subtilis strain that contains the gene coding for α-ALDC from...

  5. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

    PubMed

    Bi, Xiaodong; Liu, Zhen

    2014-12-16

    Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

  6. Saccharification of Lignocelluloses by Carbohydrate Active Enzymes of the White Rot Fungus Dichomitus squalens

    PubMed Central

    Rytioja, Johanna; Hildén, Kristiina; Mäkinen, Susanna; Vehmaanperä, Jari; Hatakka, Annele; Mäkelä, Miia R.

    2015-01-01

    White rot fungus Dichomitus squalens is an efficient lignocellulose degrading basidiomycete and a promising source for new plant cell wall polysaccharides depolymerizing enzymes. In this work, we focused on cellobiohydrolases (CBHs) of D. squalens. The native CBHI fraction of the fungus, consisting three isoenzymes, was purified and it maintained the activity for 60 min at 50°C, and was stable in acidic pH. Due to the lack of enzyme activity assay for detecting only CBHII activity, CBHII of D. squalens was produced recombinantly in an industrially important ascomycete host, Trichoderma reesei. CBH enzymes of D. squalens showed potential in hydrolysis of complex lignocellulose substrates sugar beet pulp and wheat bran, and microcrystalline cellulose, Avicel. Recombinant CBHII (rCel6A) of D. squalens hydrolysed all the studied plant biomasses. Compared to individual activities, synergistic effect between rCel6A and native CBHI fraction of D. squalens was significant in the hydrolysis of Avicel. Furthermore, the addition of laccase to the mixture of CBHI fraction and rCel6A significantly enhanced the amount of released reducing sugars from sugar beet pulp. Especially, synergy between individual enzymes is a crucial factor in the tailor-made enzyme mixtures needed for hydrolysis of different plant biomass feedstocks. Our data supports the importance of oxidoreductases in improved enzyme cocktails for lignocellulose saccharification. PMID:26660105

  7. High-Throughput Analysis of Enzyme Activities

    SciTech Connect

    Lu, Guoxin

    2007-01-01

    High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surface of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different

  8. 76 FR 3150 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-19

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines...: The NIH Guidelines currently require that recombinant DNA experiments designed to create...

  9. 75 FR 42114 - Office of Biotechnology Activities; Recombinant DNA Research: Proposed Action Under the NIH...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-20

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Proposed Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH... transgenic rodents by recombinant DNA technology must be registered with the Institutional...

  10. Angiotensin Converting Enzyme Activity in Alopecia Areata

    PubMed Central

    Namazi, Mohammad Reza; Handjani, Farhad; Eftekhar, Ebrahim; Kalafi, Amir

    2014-01-01

    Background. Alopecia areata (AA) is a chronic inflammatory disease of the hair follicle. The exact pathogenesis of AA remains unknown, although recent studies support a T-cell mediated autoimmune process. On the other hand, some studies have proposed that the renin-angiotensin-aldosterone system (RAAS) may play a role in autoimmunity. Therefore, we assessed serum activity of angiotensin converting enzyme (ACE), a component of this system, in AA. Methods. ACE activity was measured in the sera of 19 patients with AA and 16 healthy control subjects. In addition, the relationship between severity and duration of the disease and ACE activity was evaluated. Results. Serum ACE activity was higher in the patient group (55.81 U/L) compared to the control group (46.41 U/L), but the difference was not statistically significant (P = 0.085). Also, there was no correlation between ACE activity and severity (P = 0.13) and duration of disease (P = 0.25) in the patient group. Conclusion. The increased serum ACE activity found in this study may demonstrate local involvement of the RAAS in the pathogenesis of AA. Assessment of ACE in a study with a larger sample size as well as in tissue samples is recommended in order to further evaluate the possible role of RAAS in AA. PMID:25349723

  11. Detection of contaminating enzymatic activity in plant-derived recombinant biotechnology products.

    PubMed

    Brinson, Robert G; Giulian, Gary G; Kelman, Zvi; Marino, John P

    2014-12-02

    Residual impurities in recombinantly produced protein biologics, such as host cell proteins (HCP), can potentially cause unwanted toxic or immunogenic responses in patients. Additionally, undetected impurities found in recombinant proteins used in cell culture may adversely impact basic research and biotechnology applications. Currently, the enzyme-linked immunosorbent assay (ELISA) is the standard for detection of residual HCP contamination in recombinantly produced biologics. Alternatively, two-dimensional liquid chromatography coupled to mass spectrometry is being developed as a tool for assessing this critical quality attribute. Both of these methods rely on the direct detection of HCPs and some previous knowledge of the contaminant. For contaminating enzymes, the mass level of the impurity may fall below the threshold of detection of these methods and underestimate the true impact. To address this point, here we demonstrate facile detection and characterization of contaminating phytase activity in rice-derived recombinant human serum albumin (rHSA) using a sensitive, label-free nuclear magnetic resonance (NMR) spectroscopy assay. We observed varying degrees of phytase contamination in biotechnology-grade rHSA from various manufacturers by monitoring the degradation of adenosine-5'-triphosphate and myo-inositol-1,2,3,4,5,6-hexakisphosphate by (31)P NMR. The observed lot-to-lot variability may result in irreproducible cell culture results and should be evaluated as a possible critical quality attribute in plant-derived biotherapeutics.

  12. Secretion of active recombinant phytase from soybean cell-suspension cultures.

    PubMed Central

    Li, J; Hegeman, C E; Hanlon, R W; Lacy, G H; Denbow, M D; Grabau, E A

    1997-01-01

    Phytase, an enzyme that degrades the phosphorus storage compound phytate, has the potential to enhance phosphorus availability in animal diets when engineered into soybean (Glycine max) seeds. The phytase gene from Aspergillus niger was inserted into soybean transformation plasmids under control of constitutive and seed-specific promoters, with and without a plant signal sequence. Suspension cultures were used to confirm phytase expression in soybean cells. Phytase mRNA was observed in cultures containing constitutively expressed constructs. Phytase activity was detected in the culture medium from transformants that received constructs containing the plant signal sequence, confirming expectations that the protein would follow the default secretory pathway. Secretion also facilitated characterization of the biochemical properties of recombinant phytase. Soybean-synthesized phytase had a lower molecular mass than did the fungal enzyme. However, deglycosylation of the recombinant and fungal phytase yielded polypeptides of identical molecular mass (49 kD). Temperature and pH optima of the recombinant phytase were indistinguishable from the commercially available fungal phytase. Thermal inactivation studies of the recombinant phytase suggested that the additional protein stability would be required to withstand the elevated temperatures involved in soybean processing. PMID:9232886

  13. Combined effect of improved cell yield and increased specific productivity enhances recombinant enzyme production in genome-reduced Bacillus subtilis strain MGB874.

    PubMed

    Manabe, Kenji; Kageyama, Yasushi; Morimoto, Takuya; Ozawa, Tadahiro; Sawada, Kazuhisa; Endo, Keiji; Tohata, Masatoshi; Ara, Katsutoshi; Ozaki, Katsuya; Ogasawara, Naotake

    2011-12-01

    Genome reduction strategies to create genetically improved cellular biosynthesis machineries for proteins and other products have been pursued by use of a wide range of bacteria. We reported previously that the novel Bacillus subtilis strain MGB874, which was derived from strain 168 and has a total genomic deletion of 874 kb (20.7%), exhibits enhanced production of recombinant enzymes. However, it was not clear how the genomic reduction resulted in elevated enzyme production. Here we report that deletion of the rocDEF-rocR region, which is involved in arginine degradation, contributes to enhanced enzyme production in strain MGB874. Deletion of the rocDEF-rocR region caused drastic changes in glutamate metabolism, leading to improved cell yields with maintenance of enzyme productivity. Notably, the specific enzyme productivity was higher in the reduced-genome strain, with or without the rocDEF-rocR region, than in wild-type strain 168. The high specific productivity in strain MGB874 is likely attributable to the higher expression levels of the target gene resulting from an increased promoter activity and plasmid copy number. Thus, the combined effects of the improved cell yield by deletion of the rocDEF-rocR region and the increased specific productivity by deletion of another gene(s) or the genomic reduction itself enhanced the production of recombinant enzymes in MGB874. Our findings represent a good starting point for the further improvement of B. subtilis reduced-genome strains as cell factories for the production of heterologous enzymes.

  14. Staphylococcus simulans Recombinant Lysostaphin: Production, Purification, and Determination of Antistaphylococcal Activity.

    PubMed

    Boksha, I S; Lavrova, N V; Grishin, A V; Demidenko, A V; Lyashchuk, A M; Galushkina, Z M; Ovchinnikov, R S; Umyarov, A M; Avetisian, L R; Chernukha, M Iu; Shaginian, I A; Lunin, V G; Karyagina, A S

    2016-05-01

    Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by Sigma-Aldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.

  15. Diagnosis of Oropouche Virus Infection Using a Recombinant Nucleocapsid Protein-Based Enzyme Immunoassay

    PubMed Central

    Saeed, Mohammad F.; Nunes, Marcio; Vasconcelos, Pedro F.; Travassos Da Rosa, Amelia P. A.; Watts, Douglas M.; Russell, Kevin; Shope, Robert E.; Tesh, Robert B.; Barrett, Alan D. T.

    2001-01-01

    Oropouche (ORO) virus is an emerging infectious agent that has caused numerous outbreaks of an acute febrile (dengue-like) illness among humans in Brazil, Peru, and Panama. Diagnosis of ORO virus infection is based mainly on serology. Two different antigens, hamster serum antigen (HSA) and Vero cell lysate antigen (VCLA), are currently used in enzyme immunoassays (EIAs) in Brazil and Peru, respectively, to investigate the epidemiology of ORO virus infection. Both antigens involve use of infectious virus, and for this reason their use is restricted. Consequently, the frequency and distribution of ORO virus infection are largely unexplored in other countries of South America. This report describes the use of a bacterially expressed recombinant nucleocapsid (rN) protein of ORO virus in EIAs for the diagnosis of ORO virus infection. The data revealed that the purified rN protein is comparable to the authentic viral N protein in its antigenic characteristics and is highly sensitive and specific in EIAs. Among 183 serum samples tested, a high degree of concordance was found between rN protein-based EIA and HSA- and VCLA-based EIAs for the detection of both ORO virus-specific immunoglobulin M (IgM) and IgG antibodies. The high sensitivity, specificity, and safety of the rN protein-based EIA make it a useful diagnostic technique that can be widely used to detect ORO virus infection in South America. PMID:11427552

  16. The pH optimum of native uracil-DNA glycosylase of Archaeoglobus fulgidus compared to recombinant enzyme indicates adaption to cytosolic pH.

    PubMed

    Knævelsrud, Ingeborg; Kazazic, Sabina; Birkeland, Nils-Kåre; Bjelland, Svein

    2014-01-01

    Uracil-DNA glycosylase of Archaeoglobus fulgidus (Afung) in cell extracts exhibited maximal activity around pH 6.2 as compared to pH 4.8 for the purified recombinant enzyme expressed in Escherichia coli. Native Afung thus seems to be adapted to the intracellular pH of A. fulgidus, determined to be 7.0±0.1. Both recombinant and native Afung exhibited a broad temperature optimum for activity around 80°C, reflecting the A. fulgidus optimal growth temperature of 83°C. Adaption to the neutral conditions in the A. fulgidus cytoplasm might be due to covalent modifications or accessory factors, or due to a different folding when expressed in the native host.

  17. Human recombinant beta-secretase immobilized enzyme reactor for fast hits' selection and characterization from a virtual screening library.

    PubMed

    De Simone, Angela; Mancini, Francesca; Cosconati, Sandro; Marinelli, Luciana; La Pietra, Valeria; Novellino, Ettore; Andrisano, Vincenza

    2013-01-25

    In the present work, a human recombinant BACE1 immobilized enzyme reactor (hrBACE1-IMER) has been applied for the sensitive fast screening of 38 compounds selected through a virtual screening approach. HrBACE1-IMER was inserted into a liquid chromatograph coupled with a fluorescent detector. A fluorogenic peptide substrate (M-2420), containing the β-secretase site of the Swedish mutation of APP, was injected and cleaved in the on-line HPLC-hrBACE1-IMER system, giving rise to the fluorescent product. The compounds of the library were tested for their ability to inhibit BACE1 in the immobilized format and to reduce the area related to the chromatographic peak of the fluorescent enzymatic product. The results were validated in solution by using two different FRET methods. Due to the efficient virtual screening methodology, more than fifty percent of the selected compounds showed a measurable inhibitory activity. One of the most active compound (a bis-indanone derivative) was characterized in terms of IC(50) and K(i) determination on the hrBACE1-IMER. Thus, the hrBACE1-IMER has been confirmed as a valid tool for the throughput screening of different chemical entities with potency lower than 30μM for the fast hits' selection and for mode of action determination.

  18. Glycyl radical activating enzymes: structure, mechanism, and substrate interactions.

    PubMed

    Shisler, Krista A; Broderick, Joan B

    2014-03-15

    The glycyl radical enzyme activating enzymes (GRE-AEs) are a group of enzymes that belong to the radical S-adenosylmethionine (SAM) superfamily and utilize a [4Fe-4S] cluster and SAM to catalyze H-atom abstraction from their substrate proteins. GRE-AEs activate homodimeric proteins known as glycyl radical enzymes (GREs) through the production of a glycyl radical. After activation, these GREs catalyze diverse reactions through the production of their own substrate radicals. The GRE-AE pyruvate formate lyase activating enzyme (PFL-AE) is extensively characterized and has provided insights into the active site structure of radical SAM enzymes including GRE-AEs, illustrating the nature of the interactions with their corresponding substrate GREs and external electron donors. This review will highlight research on PFL-AE and will also discuss a few GREs and their respective activating enzymes.

  19. Recombination activity of interfaces in multicrystalline silicon

    SciTech Connect

    Peshcherova, S. M.; Yakimov, E. B.; Nepomnyashchikh, A. I.; Pavlova, L. A.; Feklisova, O. V.

    2015-06-15

    The electrical activity of grain boundaries in multicrystalline silicon grown from metallurgical silicon by the Bridgman method is investigated by the method of electron-beam induced current. The main tendencies of atypical manifestation of the local electrical activity of Σ3(111) and Σ9(110) special boundaries are revealed. The structural features of the grain boundaries after selective etching and the impurity-distribution characteristics in multicrystalline silicon are determined by the methods of electron backscattering diffraction and electron-probe microanalysis.

  20. Expression and purification of biologically active recombinant human paraoxonase 1 from inclusion bodies of Escherichia coli.

    PubMed

    Bajaj, Priyanka; Tripathy, Rajan K; Aggarwal, Geetika; Pande, Abhay H

    2015-11-01

    Human PON1 (h-PON1) is a Ca(2+)-dependent serum enzyme and can hydrolyze (and inactivate) a wide range of substrates. It is a multifaceted enzyme and exhibit anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial, and organophosphate (OP)-detoxifying properties. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against these conditions in humans. Insufficient hydrolyzing activity of native h-PON1 against desirable substrate affirms the urgent need to develop improved variant(s) of h-PON1 having enhanced activity. Production of recombinant h-PON1 (rh-PON1) using an Escherichia coli expression system is a key to develop such variant(s). However, generation of rh-PON1 using E. coli expression system has been elusive until now because of the aggregation of over-expressed rh-PON1 protein in inactive form as inclusion bodies (IBs) in the bacterial cells. In this study, we have over-expressed rh-PON1(wt) and rh-PON1(H115W;R192K) proteins as IBs in E. coli, and refolded the inactive enzymes present in the IBs to their active form using in vitro refolding. The active enzymes were isolated from the refolding mixture by ion-exchange chromatography. The catalytic properties of the refolded enzymes were similar to their soluble counterparts. Our results show that the pure and the active variant of rh-PON1 enzyme having enhanced hydrolyzing activity can be produced in large quantities using E. coli expression system. This method can be used for the industrial scale production of rh-PON1 enzymes and will aid in developing h-PON1 as a therapeutic candidate.

  1. Temperature and the catalytic activity of enzymes: a fresh understanding.

    PubMed

    Daniel, Roy M; Danson, Michael J

    2013-09-02

    The discovery of an additional step in the progression of an enzyme from the active to inactive state under the influence of temperature has led to a better match with experimental data for all enzymes that follow Michaelis-Menten kinetics, and to an increased understanding of the process. The new model of the process, the Equilibrium Model, describes an additional mechanism by which temperature affects the activity of enzymes, with implications for ecological, metabolic, structural, and applied studies of enzymes.

  2. Detection of Leptospira-Specific Antibodies Using a Recombinant Antigen-Based Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Chen, Hua-Wei; Zhang, Zhiwen; Halsey, Eric S.; Guevara, Carolina; Canal, Enrique; Hall, Eric; Maves, Ryan; Tilley, Drake H.; Kochel, Tadeusz J.; Ching, Wei-Mei

    2013-01-01

    We produced three highly purified recombinant antigens rLipL32, rLipL41, and rLigA-Rep (leptospiral immunoglobulin-like A repeat region) for the detection of Leptospira-specific antibodies in an enzyme-linked immunosorbent assay (ELISA). The performance of these recombinant antigens was evaluated using 121 human sera. Among them, 63 sera were microscopic agglutination test (MAT)-confirmed positive sera from febrile patients in Peru, 22 sera were indigenous MAT-negative febrile patient sera, and 36 sera were from patients with other febrile diseases from Southeast Asia, where leptospirosis is also endemic. Combining the results of immunoglobulin M (IgM) and IgG detection from these three antigens, the overall sensitivity is close to 90% based on the MAT. These results suggest that an ELISA using multiple recombinant antigens may be used as an alternative method for the detection of Leptospira-specific antibodies. PMID:24166046

  3. Prdm9 controls activation of mammalian recombination hotspots.

    PubMed

    Parvanov, Emil D; Petkov, Petko M; Paigen, Kenneth

    2010-02-12

    Mammalian meiotic recombination, which preferentially occurs at specialized sites called hotspots, ensures the orderly segregation of meiotic chromosomes and creates genetic variation among offspring. A locus on mouse chromosome 17, which controls activation of recombination at multiple distant hotspots, has been mapped within a 181-kilobase interval, three of whose genes can be eliminated as candidates. The remaining gene, Prdm9, codes for a zinc finger containing histone H3K4 trimethylase that is expressed in early meiosis and whose deficiency results in sterility in both sexes. Mus musculus exhibits five alleles of Prdm9; human populations exhibit two predominant alleles and multiple minor alleles. The identification of Prdm9 as a protein regulating mammalian recombination hotspots initiates molecular studies of this important biological control system.

  4. Enzyme and root activities in surface-flow constructed wetlands.

    PubMed

    Kong, Ling; Wang, Yu-Bin; Zhao, Li-Na; Chen, Zhang-He

    2009-07-01

    Sixteen small-scale wetlands planted with four plant species were constructed for domestic wastewater purification. The objective of this study was to determine the correlations between contaminant removal and soil enzyme activity, root activity, and growth in the constructed wetlands. The results indicated that correlations between contaminant removal efficiency and enzyme activity varied depending on the contaminants. The removal efficiency of NH4+ was significantly correlated with both urease and protease activity in all wetlands, and the removal of total phosphorus and soluble reactive phosphorus was significantly correlated with phosphatase activity in most wetlands, while the removal of total nitrogen, NO3(-) , and chemical oxygen demand (COD) was significantly correlated with enzyme activity only in a few instances. Correlations between soil enzyme activity and root activity varied among species. Activities of all enzymes were significantly correlated with root activity in Vetiveria zizanioides and Phragmites australis wetlands, but not in Hymenocallis littoralis wetlands. Significant correlations between enzyme activity and root biomass and between enzyme activity and root growth were found mainly in Cyperus flabelliformis wetlands. Root activity was significantly correlated with removal efficiencies of all contaminants except NO3(-) and COD in V. zizanioides wetlands. Enzyme activities and root activity showed single-peak seasonal patterns. Activities of phosphatase, urease, and cellulase were significantly higher in the top layer of the substrate than in the deeper layers, and there were generally no significant differences between the deeper layers (deeper than 15 cm).

  5. Enzymic characterization of two recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis and their effect on root growth and cell wall extension.

    PubMed

    Maris, An; Suslov, Dmitry; Fry, Stephen C; Verbelen, Jean-Pierre; Vissenberg, Kris

    2009-01-01

    Xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes involved in the modification of load-bearing cell wall components. They cleave xyloglucan chains and, often, re-form bonds to the non-reducing ends of available xyloglucan molecules in plant primary cell walls. The enzymic properties and effects on root growth of two Arabidopsis thaliana XTHs belonging to subgroup I/II, that are predominantly expressed in root hairs and in non-elongating zones of the root, were analysed here. AtXTH14 and AtXTH26 were recombinantly produced in Pichia and subsequently purified. Both proteins were found to exhibit xyloglucan endotransglucosylase (XET; EC 2.4.1.207) but not xyloglucan endohydrolase (XEH; EC 3.2.1.151) activity. Their endotransglucosylase activity was at least 70x greater on xyloglucan rather than on mixed-linkage beta-glucan. Differences were found in pH- and temperature-dependence as well as in acceptor-substrate preferences. Furthermore, the specific activity of XET was approximately equal for the two enzymes. Removal of N-linked sugar residues by Endo H treatment reduced XET activity to 60%. Constant-load extensiometry experiments revealed that the enzymes reduce the extension in a model system of heat-inactivated isolated cell walls. When given to growing roots, either of these XTH proteins reduced cell elongation in a concentration-dependent manner and caused abnormal root hair morphology. This is the first time that recombinant and purified XTHs added to growing roots have exhibited a clear effect on cell elongation. It is proposed that these specific XTH isoenzymes play a role in strengthening the side-walls of root-hairs and cell walls in the root differentiation zone after the completion of cell expansion.

  6. Recombinant Human Peptidoglycan Recognition Proteins Reveal Antichlamydial Activity.

    PubMed

    Bobrovsky, Pavel; Manuvera, Valentin; Polina, Nadezhda; Podgorny, Oleg; Prusakov, Kirill; Govorun, Vadim; Lazarev, Vassili

    2016-07-01

    Peptidoglycan recognition proteins (PGLYRPs) are innate immune components that recognize the peptidoglycan and lipopolysaccharides of bacteria and exhibit antibacterial activity. Recently, the obligate intracellular parasite Chlamydia trachomatis was shown to have peptidoglycan. However, the antichlamydial activity of PGLYRPs has not yet been demonstrated. The aim of our study was to test whether PGLYRPs exhibit antibacterial activity against C. trachomatis Thus, we cloned the regions containing the human Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4 genes for subsequent expression in human cell lines. We obtained stable HeLa cell lines that secrete recombinant human PGLYRPs into culture medium. We also generated purified recombinant PGLYRP1, -2, and -4 and confirmed their activities against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. Furthermore, we examined the activities of recombinant PGLYRPs against C. trachomatis and determined their MICs. We also observed a decrease in the infectious ability of chlamydial elementary bodies in the next generation after a single exposure to PGLYRPs. Finally, we demonstrated that PGLYRPs attach to C. trachomatis elementary bodies and activate the expression of the chlamydial two-component stress response system. Thus, PGLYRPs inhibit the development of chlamydial infection.

  7. Recombinant Human Peptidoglycan Recognition Proteins Reveal Antichlamydial Activity

    PubMed Central

    Manuvera, Valentin; Polina, Nadezhda; Podgorny, Oleg; Prusakov, Kirill; Govorun, Vadim; Lazarev, Vassili

    2016-01-01

    Peptidoglycan recognition proteins (PGLYRPs) are innate immune components that recognize the peptidoglycan and lipopolysaccharides of bacteria and exhibit antibacterial activity. Recently, the obligate intracellular parasite Chlamydia trachomatis was shown to have peptidoglycan. However, the antichlamydial activity of PGLYRPs has not yet been demonstrated. The aim of our study was to test whether PGLYRPs exhibit antibacterial activity against C. trachomatis. Thus, we cloned the regions containing the human Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4 genes for subsequent expression in human cell lines. We obtained stable HeLa cell lines that secrete recombinant human PGLYRPs into culture medium. We also generated purified recombinant PGLYRP1, -2, and -4 and confirmed their activities against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. Furthermore, we examined the activities of recombinant PGLYRPs against C. trachomatis and determined their MICs. We also observed a decrease in the infectious ability of chlamydial elementary bodies in the next generation after a single exposure to PGLYRPs. Finally, we demonstrated that PGLYRPs attach to C. trachomatis elementary bodies and activate the expression of the chlamydial two-component stress response system. Thus, PGLYRPs inhibit the development of chlamydial infection. PMID:27160295

  8. Serologic diagnosis of canine and equine borreliosis: use of recombinant antigens in enzyme-linked immunosorbent assays.

    PubMed Central

    Magnarelli, L A; Flavell, R A; Padula, S J; Anderson, J F; Fikrig, E

    1997-01-01

    Serum samples from dogs and equids suspected of having canine or equine borreliosis, respectively, were analyzed in polyvalent enzyme-linked immunosorbent assays (ELISAs) with whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Purified preparations of recombinant antigens included outer surface protein A (OspA), OspB, OspC, OspE, OspF, and p41-G (a fragment of flagellin). Of the 36 dog sera that reacted positively to whole-cell antigen, 32 (88.9%) contained antibodies to one or more recombinant antigens. Reactivities to OspF (88.9% positive) and p41-G (75% positive) were most prevalent. In analyses of 30 equid sera positive in an ELISA with whole cells, 24 (80%) contained antibodies to one or more recombinant antigens. Seropositivities in ELISAs with p41-G (50% positive) and OspF (46.7% positive) were more than twofold greater than in ELISAs with OspA, OspB, or OspC (10 to 20% positive). In parallel tests of eight canine and three equine sera, there was good agreement in results of Western blot (immunoblot) analyses and ELISAs. Although dog and equid sera with antibodies to whole-cell B. burgdorferi frequently reacted positively to one or more recombinant antigens, the inclusion of OspF and p41-G antigens in ELISAs was most useful in the serologic diagnosis of canine and equine borreliosis. PMID:8968901

  9. Development of a keratinase activity assay using recombinant chicken feather keratin substrates

    PubMed Central

    Jin, Hyeon-Su; Park, Seon Yeong; Kim, Kyungmin; Lee, Yong-Jik; Nam, Gae-Won; Kang, Nam Joo; Lee, Dong-Woo

    2017-01-01

    Poultry feathers consist mainly of the protein keratin, which is rich in β-pleated sheets and consequently resistant to proteolysis. Although many keratinases have been identified, the reasons for their substrate specificity towards β-keratin remain unclear due to difficulties in preparing a soluble feather keratin substrate for use in activity assays. In the present study, we overexpressed Gallus gallus chromosomes 2 and 27 β-keratin-encoding genes in Escherichia coli, purified denatured recombinant proteins by Ni2+ affinity chromatography, and refolded by stepwise dialysis to yield soluble keratins. To assess the keratinolytic activity, we compared the proteolytic activity of crude extracts from the feather- degrading bacterium Fervidobacterium islandicum AW-1 with proteinase K, trypsin, and papain using purified recombinant keratin and casein as substrates. All tested proteases showed strong proteolytic activities for casein, whereas only F. islandicum AW-1 crude extracts and proteinase K exhibited pronounced keratinolytic activity for the recombinant keratin. Moreover, LC-MS/MS analysis of keratin hydrolysates allowed us to predict the P1 sites of keratinolytic enzymes in the F. islandicum AW-1 extracts, thereby qualifying and quantifying the extent of keratinolysis. The soluble keratin-based assay has clear therapeutic and industrial potential for the development of a high-throughput screening system for proteases hydrolyzing disease-related protein aggregates, as well as mechanically resilient keratin-based polymers. PMID:28231319

  10. Mg-chelatase of tobacco: identification of a Chl D cDNA sequence encoding a third subunit, analysis of the interaction of the three subunits with the yeast two-hybrid system, and reconstitution of the enzyme activity by co-expression of recombinant CHL D, CHL H and CHL I.

    PubMed

    Papenbrock, J; Gräfe, S; Kruse, E; Hänel, F; Grimm, B

    1997-11-01

    the three subunits of tobacco Mg-chelatase. The reconstitution of the recombinant enzyme activity required additional ATP.

  11. High Inorganic Triphosphatase Activities in Bacteria and Mammalian Cells: Identification of the Enzymes Involved

    PubMed Central

    Lakaye, Bernard; Servais, Anne-Catherine; Scholer, Georges; Fillet, Marianne; Elias, Benjamin; Derochette, Jean-Michel; Crommen, Jacques; Wins, Pierre; Bettendorff, Lucien

    2012-01-01

    Background We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. Methodology/Principal Findings Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPPi) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPPi but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. Conclusions and General Significance We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPPi in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPPi, which could be cytotoxic because of its high affinity for Ca2+, thereby interfering with Ca2+ signaling. PMID:22984449

  12. Detection of recombinant and cellular MALT1 paracaspase activity.

    PubMed

    Nagel, Daniel; Krappmann, Daniel

    2015-01-01

    MALT1 (mucosa-associated lymphoid tissue protein 1) is a key regulator of antigen-induced NF-κB activation in the adaptive immune response. Activation of proteolytic activity of the MALT1 paracaspase was shown to boost the immune response. Additionally, MALT1 proteolytic activity is essential for the survival of MALT1-dependent lymphoma, such as the activated B-cell type (ABC) of diffuse large B-cell lymphoma (DLBCL) or MALT lymphoma. The functional impact of MALT1 paracaspase on T-cell activation and lymphomagenesis suggests that MALT1 is a promising therapeutic target for the treatment of autoimmune diseases and distinct lymphoma entities. To evaluate the requirement of MALT1 in further detail, direct measurement of its activity status is of great importance. We have established a fluorogenic cleavage assay which can be used to measure activity of recombinant and cellular MALT1. Here we describe the basis of the cleavage assay and include a detailed protocol for recombinant production of MALT1 and also the cellular immunoprecipitation of endogenous MALT1 to determine its proteolytic activity.

  13. Spatial distribution of enzyme activities in the rhizosphere

    NASA Astrophysics Data System (ADS)

    Razavi, Bahar S.; Zarebanadkouki, Mohsen; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2015-04-01

    The rhizosphere, the tiny zone of soil surrounding roots, certainly represents one of the most dynamic habitat and interfaces on Earth. Activities of enzymes produced by both plant roots and microbes are the primary biological drivers of organic matter decomposition and nutrient cycling. That is why there is an urgent need in spatially explicit methods for the determination of the rhizosphere extension and enzyme distribution. Recently, zymography as a new technique based on diffusion of enzymes through the 1 mm gel plate for analysis has been introduced (Spohn & Kuzyakov, 2013). We developed the zymography technique to visualize the enzyme activities with a higher spatial resolution. For the first time, we aimed at quantitative imaging of enzyme activities as a function of distance from the root tip and the root surface in the soil. We visualized the two dimensional distribution of the activity of three enzymes: β-glucosidase, phosphatase and leucine amino peptidase in the rhizosphere of maize using fluorogenically labelled substrates. Spatial-resolution of fluorescent images was improved by direct application of a substrate saturated membrane to the soil-root system. The newly-developed direct zymography visualized heterogeneity of enzyme activities along the roots. The activity of all enzymes was the highest at the apical parts of individual roots. Across the roots, the enzyme activities were higher at immediate vicinity of the roots (1.5 mm) and gradually decreased towards the bulk soil. Spatial patterns of enzyme activities as a function of distance from the root surface were enzyme specific, with highest extension for phosphatase. We conclude that improved zymography is promising in situ technique to analyze, visualize and quantify spatial distribution of enzyme activities in the rhizosphere hotspots. References Spohn, M., Kuzyakov, Y., 2013. Phosphorus mineralization can be driven by microbial need for carbon. Soil Biology & Biochemistry 61: 69-75

  14. APS kinase from Arabidopsis thaliana: genomic organization, expression, and kinetic analysis of the recombinant enzyme.

    PubMed

    Lee, S; Leustek, T

    1998-06-09

    The gene encoding 5'-adenylylsulfate (APS) kinase (EC 2.7.1.25) (APK) was cloned from Arabidopsis thaliana. There is a single APK locus in A. thaliana. The coding sequence of the gene is composed of 7 exons, interrupted by 6 introns. A transcriptional initiation site was detected 120 bp 5' of the initiation codon. APK mRNA is slightly more abundant in leaves than in roots of A. thaliana and its level does not change in response to sulfur starvation. The APK protein, synthesized in vitro, is able to enter isolated intact chloroplasts. Recombinant APS kinase shows maximal activity at 10 microM APS with 5 mM ATP, but it is inhibited at APS concentrations above 10 microM. The inhibition is alleviated at higher ATP concentrations. Reciprocal plot analysis showed that the theoretical Vmax is approximately 1.2 mumol min-1 mg-1 at 25 degrees C, pH 8.0; the K(m) values are 3.6 microM APS and 1.8 mM ATP.

  15. Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity

    PubMed Central

    Dolinska, Monika B.; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T.; Brooks, Brian P.; Sergeev, Yuri V.

    2014-01-01

    Background Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. Methodology/Principal Findings The intra-melanosomal domain of human tyrosinase (residues 19–469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. Conclusions/Significance The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure – function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1. PMID:24392141

  16. Refolded Recombinant Human Paraoxonase 1 Variant Exhibits Prophylactic Activity Against Organophosphate Poisoning.

    PubMed

    Bajaj, Priyanka; Tripathy, Rajan K; Aggarwal, Geetika; Datusalia, Ashok K; Sharma, Shyam S; Pande, Abhay H

    2016-09-01

    Organophosphate (OP) compounds are neurotoxic chemicals, and current treatments available for OP-poisoning are considered as unsatisfactory and inadequate. There is an urgent need for the development of more effective treatment(s) for OP-poisoning. Human paraoxonase 1 (h-PON1) is known to hydrolyze a variety of OP-compounds and is a leading candidate for the development of prophylactic and therapeutic agent against OP-poisoning in humans. Non-availability of effective system(s) for the production of recombinant h-PON1 (rh-PON1) makes it hard to produce improved variant(s) of this enzyme and analyze their in vivo efficacy in animal models. Production of recombinant h-PON1 (rh-PON1) using an Escherichia coli expression system is a key to develop variant(s) of h-PON1. Recently, we have developed a procedure to produce active rh-PON1 enzymes by using E. coli expression system. In this study, we have characterized the OP-hydrolyzing properties of refolded rh-PON1(wt) and rh-PON1(H115W;R192K) variant. Our results show that refolded rh-PON1(H115W;R192K) variant exhibit enhanced OP-hydrolyzing activity in in vitro and ex vivo assays and exhibited prophylactic activity in mouse model of OP-poisoning, suggesting that refolded rh-PON1 can be developed as a therapeutic candidate.

  17. Cold-active enzymes studied by comparative molecular dynamics simulation.

    PubMed

    Spiwok, Vojtech; Lipovová, Petra; Skálová, Tereza; Dusková, Jarmila; Dohnálek, Jan; Hasek, Jindrich; Russell, Nicholas J; Králová, Blanka

    2007-04-01

    Enzymes from cold-adapted species are significantly more active at low temperatures, even those close to zero Celsius, but the rationale of this adaptation is complex and relatively poorly understood. It is commonly stated that there is a relationship between the flexibility of an enzyme and its catalytic activity at low temperature. This paper gives the results of a study using molecular dynamics simulations performed for five pairs of enzymes, each pair comprising a cold-active enzyme plus its mesophilic or thermophilic counterpart. The enzyme pairs included alpha-amylase, citrate synthase, malate dehydrogenase, alkaline protease and xylanase. Numerous sites with elevated flexibility were observed in all enzymes; however, differences in flexibilities were not striking. Nevertheless, amino acid residues common in both enzymes of a pair (not present in insertions of a structure alignment) are generally more flexible in the cold-active enzymes. The further application of principle component analysis to the protein dynamics revealed that there are differences in the rate and/or extent of opening and closing of the active sites. The results indicate that protein dynamics play an important role in catalytic processes where structural rearrangements, such as those required for active site access by substrate, are involved. They also support the notion that cold adaptation may have evolved by selective changes in regions of enzyme structure rather than in global change to the whole protein.

  18. Ultrasound in Enzyme Activation and Inactivation

    NASA Astrophysics Data System (ADS)

    Mawson, Raymond; Gamage, Mala; Terefe, Netsanet Shiferaw; Knoerzer, Kai

    As discussed in previous chapters, most effects due to ultrasound arise from cavitation events, in particular, collapsing cavitation bubbles. These collapsing bubbles generate very high localized temperatures and pressure shockwaves along with micro-streaming that is associated with high shear forces. These effects can be used to accelerate the transport of substrates and reaction products to and from enzymes, and to enhance mass transfer in enzyme reactor systems, and thus improve efficiency. However, the high velocity streaming, together with the formation of hydroxy radicals and heat generation during collapsing of bubbles, may also potentially affect the biocatalyst stability, and this can be a limiting factor in combined ultrasound/enzymatic applications. Typically, enzymes can be readily denatured by slight changes in environmental conditions, including temperature, pressure, shear stress, pH and ionic strength.

  19. Microbial Enzyme Activity and Carbon Cycling in Grassland Soil Fractions

    NASA Astrophysics Data System (ADS)

    Allison, S. D.; Jastrow, J. D.

    2004-12-01

    Extracellular enzymes are necessary to degrade complex organic compounds present in soils. Using physical fractionation procedures, we tested whether old soil carbon is spatially isolated from degradative enzymes across a prairie restoration chronosequence in Illinois, USA. We found that carbon-degrading enzymes were abundant in all soil fractions, including macroaggregates, microaggregates, and the clay fraction, which contains carbon with a mean residence time of ~200 years. The activities of two cellulose-degrading enzymes and a chitin-degrading enzyme were 2-10 times greater in organic matter fractions than in bulk soil, consistent with the rapid turnover of these fractions. Polyphenol oxidase activity was 3 times greater in the clay fraction than in the bulk soil, despite very slow carbon turnover in this fraction. Changes in enzyme activity across the restoration chronosequence were small once adjusted for increases in soil carbon concentration, although polyphenol oxidase activity per unit carbon declined by 50% in native prairie versus cultivated soil. These results are consistent with a `two-pool' model of enzyme and carbon turnover in grassland soils. In light organic matter fractions, enzyme production and carbon turnover both occur rapidly. However, in mineral-dominated fractions, both enzymes and their carbon substrates are immobilized on mineral surfaces, leading to slow turnover. Soil carbon accumulation in the clay fraction and across the prairie restoration chronosequence probably reflects increasing physical isolation of enzymes and substrates on the molecular scale, rather than the micron to millimeter scale.

  20. Manganese enzymes with binuclear active sites

    SciTech Connect

    Dismukes, G.C.

    1996-11-01

    The purpose of this article is twofold. First, to review the recent literature dealing with the mechanisms of catalysis by binuclear manganese enzymes. Second, to summarize and illustrate the general principles of catalysis which distinguish binuclear metalloenzymes from monometallic centers. This review covers primarily the published literature from 1991 up to May 1996. A summary of the major structurally characterized dimanganese enzymes is given. These perform various reaction types including several redox reactions, (de)hydrations, isomerizations, (de)phosphorylation, and phosphoryl transfer. 114 refs.

  1. A Simple and Accurate Method for Measuring Enzyme Activity.

    ERIC Educational Resources Information Center

    Yip, Din-Yan

    1997-01-01

    Presents methods commonly used for investigating enzyme activity using catalase and presents a new method for measuring catalase activity that is more reliable and accurate. Provides results that are readily reproduced and quantified. Can also be used for investigations of enzyme properties such as the effects of temperature, pH, inhibitors,…

  2. High-yield expression in Escherichia coli and purification of mouse ubiquitin-activating enzyme E1.

    PubMed

    Carvalho, Andreia F; Pinto, Manuel P; Grou, Cláudia P; Vitorino, Rui; Domingues, Pedro; Yamao, Fumiaki; Sá-Miranda, Clara; Azevedo, Jorge E

    2012-07-01

    Research in the ubiquitin field requires large amounts of ubiquitin-activating enzyme (E1) for in vitro ubiquitination assays. Typically, the mammalian enzyme is either isolated from natural sources or produced recombinantly using baculovirus/insect cell protein expression systems. Escherichia coli is seldom used to produce mammalian E1 probably due to the instability and insolubility of this high-molecular mass protein. In this report, we show that 5-10 mg of histidine-tagged mouse E1 can be easily obtained from a 1 l E. coli culture. A low temperature during the protein induction step was found to be critical to obtain an active enzyme.

  3. Three faces of recombination activating gene 1 (RAG1) mutations.

    PubMed

    Patiroglu, Turkan; Akar, Himmet Haluk; Van Der Burg, Mirjam

    2015-12-01

    Severe combined immune deficiency (SCID) is a group of genetic disorder associated with development of T- and/or B-lymphocytes. Recombination-activating genes (RAG1/2) play a critical role on VDJ recombination process that leads to the production of a broad T-cell receptor (TCR) and B-cell receptor (BCR) repertoire in the development of T and B cells. RAG1/2 genes mutations result in various forms of primary immunodeficiency, ranging from classic SCID to Omenn syndrome (OS) to atypical SCID with such as granuloma formation and autoimmunity. Herein, we reported 4 patients with RAG1 deficiency: classic SCID was seen in two patients who presented with recurrent pneumonia and chronic diarrhoea, and failure to thrive. OS was observed in one patient who presented with chronic diarrhoea, skin rash, recurrent lower respiratory infections, and atypical SCID was seen in one patient who presented with Pyoderma gangrenosum (PG) and had novel RAG1 mutation.

  4. Synergistic action of recombinant accessory hemicellulolytic and pectinolytic enzymes to Trichoderma reesei cellulase on rice straw degradation.

    PubMed

    Laothanachareon, Thanaporn; Bunterngsook, Benjarat; Suwannarangsee, Surisa; Eurwilaichitr, Lily; Champreda, Verawat

    2015-12-01

    Synergism between core cellulases and accessory hydrolytic/non-hydrolytic enzymes is the basis of efficient hydrolysis of lignocelluloses. In this study, the synergistic action of three recombinant accessory enzymes, namely GH62 α-l-arabinofuranosidase (ARA), CE8 pectin esterase (PET), and GH10 endo-1,4-beta-xylanase (XYL) from Aspergillus aculeatus expressed in Pichia pastoris to a commercial Trichoderma reesei cellulase (Accellerase® 1500; ACR) on hydrolysis of alkaline pretreated rice straw was studied using a mixture design approach. Applying the full cubic model, the optimal ratio of quaternary enzyme mixture was predicted to be ACR:ARA:PET:XYL of 0.171:0.079:0.100:0.150, which showed a glucose releasing efficiency of 0.173 gglc/FPU, higher than the binary ACR:XYL mixture (0.122 gglc/FPU) and ACR alone (0.081 gglc/FPU) leading to a 47.3% increase in glucose yield compared with that from ACR at the same cellulase dosage. The result demonstrates the varying degree of synergism of accessory enzymes to cellulases useful for developing tailor-made enzyme systems for bio-industry.

  5. The de-ubiquitylating enzymes USP26 and USP37 regulate homologous recombination by counteracting RAP80

    PubMed Central

    Typas, Dimitris; Luijsterburg, Martijn S.; Wiegant, Wouter W.; Diakatou, Michaela; Helfricht, Angela; Thijssen, Peter E.; van de Broek, Bram; Mullenders, Leon H.; van Attikum, Haico

    2015-01-01

    The faithful repair of DNA double-strand breaks (DSBs) is essential to safeguard genome stability. DSBs elicit a signaling cascade involving the E3 ubiquitin ligases RNF8/RNF168 and the ubiquitin-dependent assembly of the BRCA1-Abraxas-RAP80-MERIT40 complex. The association of BRCA1 with ubiquitin conjugates through RAP80 is known to be inhibitory to DSB repair by homologous recombination (HR). However, the precise regulation of this mechanism remains poorly understood. Through genetic screens we identified USP26 and USP37 as key de-ubiquitylating enzymes (DUBs) that limit the repressive impact of RNF8/RNF168 on HR. Both DUBs are recruited to DSBs where they actively remove RNF168-induced ubiquitin conjugates. Depletion of USP26 or USP37 disrupts the execution of HR and this effect is alleviated by the simultaneous depletion of RAP80. We demonstrate that USP26 and USP37 prevent excessive spreading of RAP80-BRCA1 from DSBs. On the other hand, we also found that USP26 and USP37 promote the efficient association of BRCA1 with PALB2. This suggests that these DUBs limit the ubiquitin-dependent sequestration of BRCA1 via the BRCA1-Abraxas-RAP80-MERIT40 complex, while promoting complex formation and cooperation of BRCA1 with PALB2-BRCA2-RAD51 during HR. These findings reveal a novel ubiquitin-dependent mechanism that regulates distinct BRCA1-containing complexes for efficient repair of DSBs by HR. PMID:26101254

  6. Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs)

    PubMed Central

    Gong, Peijie; Li, Shuxiu; Wang, Yuejin; Zhang, Chaohong

    2016-01-01

    Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the ‘Vitis vinifera cv. Pinot Noir’ and ‘Vitis vinifera cv. Thompson Seedless’ varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs. PMID:27551866

  7. Diffusional correlations among multiple active sites in a single enzyme.

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2014-04-07

    Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

  8. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  9. Enzyme activities along a latitudinal transect in Western Siberia

    NASA Astrophysics Data System (ADS)

    Schnecker, Jörg; Wild, Birgit; Eloy Alves, Ricardo J.; Gentsch, Norman; Gittel, Antje; Knoltsch, Anna; Lashchinskiy, Nikolay; Mikutta, Robert; Takriti, Mounir; Richter, Andreas

    2014-05-01

    Decomposition of soil organic matter (SOM) and thus carbon and nutrient cycling in soils is mediated by the activity of extracellular enzymes. The specific activities of these enzymes and their ratios to each other represent the link between the composition of soil organic matter and the nutrient demand of the microbial community. Depending on the difference between microbial nutrient demand and substrate availability, extracellular enzymes can enhance or slow down different nutrient cycles in the soil. We investigated activities of six extracellular enzymes (cellobiohydrolase, leucine-amino-peptidase, N-acetylglucosaminidase, chitotriosidase, phosphatase and phenoloxidase) in the topsoil organic horizon, topsoil mineral horizon and subsoil horizon in seven ecosystems along a 1,500 km-long North-South transect in Western Siberia. The transect included sites in the southern tundra, northern taiga, middle taiga, southern taiga, forest-steppe (in forested patches as well as in adjacent meadows) and Steppe. We found that enzyme patterns varied stronger with soil depth than between ecosystems. Differences between horizons were mainly based on the increasing ratio of oxidative enzymes to hydrolytic enzymes. Differences between sites were more pronounced in topsoil than in subsoil mineral horizons, but did not reflect the north-south transect and the related gradients in temperature and precipitation. The observed differences between sites in topsoil horizons might therefore result from differences in vegetation rather than climatic factors. The decreasing variability in the enzyme pattern with depth might also indicate that the composition of soil organic matter becomes more similar with soil depth, most likely by an increasing proportion of microbial remains compared to plant derived constituents of SOM. This also indicates, that SOM becomes less divers the more it is processed by soil microorganisms. Our findings highlight the importance of soil depth on enzyme

  10. High-level expression of active recombinant ubiquitin carboxyl-terminal hydrolase of Drosophila melanogaster in Pichia pastoris.

    PubMed

    Jin, Feng-liang; Xu, Xiao-xia; Yu, Xiao-qiang; Ren, Shun-xiang

    2009-06-01

    Ubiquitin carboxyl-terminal hydrolases (UCHs) are implicated in the proteolytic processing of polymeric ubiquitin. The high specificity for the recognition site makes UCHs useful enzymes for in vitro cleavage of ubiquitin fusion proteins. In this work, an active C-terminal His-tagged UCH from Drosophila melanogaster (DmUCH) was produced as a secretory form in a recombinant strain of the methylotrophic yeast Pichia pastoris. The production of recombinant DmUCH by Mut(s) strain was much higher than that by Mut(+) strain, which was confirmed by Western blot analysis. When expression was induced at pH 6.0 in a BMMY/methanol medium, the concentration of recombinant DmUCH reached 210 mg l(-1). With the (His)(6)-tag, the recombinant DmUCH was easily purified by Ni-NTA chromatography and 18 mg pure active DmUCH were obtained from 100ml culture broth supernatant. Ubiquitin-magainin fusion protein was efficiently cleaved by DmUCH, yielding recombinant magainin with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant magainin was purified to homogeneity easily by reversed-phase HPLC. Analysis of the recombinant magainin by ESI-MS showed that the molecular weight of the purified recombinant magainin was 2465 Da, which perfectly matches the mass calculated from the amino acid sequence. The result of mass spectrometry confirmed that the purified His-tagged DmUCH can recognize the ubiquitin-magainin fusion protein and cleave it at the carboxyl terminus of ubiquitin precisely. Our results showed that P. pastoris is a robust system to express the secreted form of DmUCH.

  11. TREATABILITY STUDY BULLETIN: ENZYME-ACTIVATED CELLULOSE TECHNOLOGY - THORNECO, INC

    EPA Science Inventory

    The Enzyme-Activated Cellulose Technology developed by Thorneco, Inc. uses cellulose placed into one or more cylindrical towers to remove metals and organic compounds from an aqueous solution. The cellulose is coated with a proprietary enzyme. Operating parameters that can affe...

  12. Production of 3-hydroxypropionic acid from 3-hydroxypropionaldehyde by recombinant Escherichia coli co-expressing Lactobacillus reuteri propanediol utilization enzymes.

    PubMed

    Sabet-Azad, Ramin; Sardari, Roya R R; Linares-Pastén, Javier A; Hatti-Kaul, Rajni

    2015-03-01

    3-Hydroxypropionic acid (3-HP) is an important platform chemical for the biobased chemical industry. Lactobacillus reuteri produces 3-HP from glycerol via 3-hydroxypropionaldehyde (3-HPA) through a CoA-dependent propanediol utilization (Pdu) pathway. This study was performed to verify and evaluate the pathway comprising propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL), and propionate kinase (PduW) for formation of 3-HP from 3-HPA. The pathway was confirmed using recombinant Escherichia coli co-expressing PduP, PduL and PduW of L. reuteri DSM 20016 and mutants lacking expression of either enzyme. Growing and resting cells of the recombinant strain produced 3-HP with a yield of 0.3mol/mol and 1mol/mol, respectively, from 3-HPA. 3-HP was the sole product with resting cells, while growing cells produced 1,3-propanediol as co-product. 3-HP production from glycerol was achieved with a yield of 0.68mol/mol by feeding recombinant E. coli with 3-HPA produced by L. reuteri and recovered using bisulfite-functionalized resin.

  13. Photoreactivating enzyme activity in the rat tapeworm, Hymenolepis diminuta

    SciTech Connect

    Woodhead, A.D.; Achey, P.M.

    1981-01-01

    There has been considerable speculation about the occurrence of photoreactivating enzyme in different organisms and about its biologic purpose. We have developed a simple, sensitive assay for estimating pyrimidine dimers in DNA which is useful in making a rapid survey for the presence of the enzyme. Using this method, we have found photoreactivating enzyme activity in the tissues of the rat tapeworm, Hymenolepis diminuta. This parasite spends the majority of its life span in the bodies of its definitive or intermediate hosts, but a period is spent externally. We suggest that photoreactivating enzyme may be important in perserving the integrity of embryonic DNA during this free-living stage.

  14. Photoreactivating enzyme activity in the rat tapeworm, Hymenolepis diminuta

    SciTech Connect

    Woodhead, A.D.; Achey, P.M.

    1981-06-01

    There has been considerable speculation about the occurrence of photoreactivating enzyme in different organisms and about its biological purpose. We have developed a simple, sensitive assay for estimating pyrimidine dimers in DNA which is useful in making a rapid survey for the presence of the enzyme. Using this method, we have found photoreactivating enzyme activity in the tissues of the rat tapeworm Hymenolepis diminuta. This parasite spends the majority of its life span in the bodies of its definitive or intermediate hosts, but a period is spent externally. We suggest that photoreactivating enzyme may be important in preserving the integrity of embryonic DNA during this free-living stage.

  15. Real-time analysis of RAG complex activity in V(D)J recombination.

    PubMed

    Zagelbaum, Jennifer; Shimazaki, Noriko; Esguerra, Zitadel Anne; Watanabe, Go; Lieber, Michael R; Rothenberg, Eli

    2016-10-18

    Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS-RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination.

  16. Enzyme activity control by responsive redoxpolymers.

    PubMed

    Nagel, Birgit; Warsinke, Axel; Katterle, Martin

    2007-06-05

    A new thermoresponsive poly-N-isopropylacrylamide (PNIPAM)-ferrocene polymer was synthesized and characterized. PNIPAMFoxy bears additional oxirane groups which were used for attachment by a self-assembly process on a cysteamine-modified gold electrode to create a thin hydrophilic film. The new redox polymer enabled electrical communication between the cofactor pyrrolinoquinoline quinone (PQQ) of soluble glucose dehydrogenase (sGDH) and the electrode for sensitive detection of this enzyme as a prospective protein label. The temperature influence on the redox polymer/enzyme complex was investigated. An inverse temperature response behavior of surface bound PNIPAMFoxy compared to the soluble polymer was found and is discussed in detail. The highest efficiency of mediated electron transfer for the immobilized PNIPAMFoxy with sGDH was observed at 24 degrees C, which was twice as high as that of its soluble counterpart. A steady-state electrooxidation current densitiy of 4.5 microA.cm-2 was observed in the presence of 10 nM sGDH and 5 mM glucose. A detection limit of 0.5 nM of soluble PQQ-sGDH was obtained.

  17. Recombination activating activity of XRCC1 analogous genes in X-ray sensitive and resistant CHO cell lines

    NASA Astrophysics Data System (ADS)

    Golubnitchaya-Labudová, O.; Portele, A.; Vaçata, V.; Lubec, G.; Rink, H.; Höfer, M.

    1997-10-01

    The XRCC1 gene (X-ray repair cross complementing) complements the DNA repair deficiency of the radiation sensitive Chinese hamster ovary (CHO) mutant cell line EM9 but the mechanism of the correction is not elucidated yet. XRCC1 shows substantial homology to the RAG2 gene (recombination activating gene) and we therefore tried to answer question, whether structural similarities (sequence of a putative recombination activating domain, aa 332-362 for XRCC1 and aa 286-316 in RAG2) would reflect similar functions of the homologous, putative recombination activating domain. PCR experiments revealed that no sequence homologous to the structural part of human XRCC1 was present in cDNA of CHO. Differential display demonstrated two putative recombination activating domains in the parental CHO line AA8 and one in the radiosensitive mutant EM9. Southern blot experiments showed the presence of several genes with partial homology to human XRCC1. Recombination studies consisted of expressing amplified target domains within chimeric proteins in recA - bacteria and subsequent detection of recombination events by sequencing the recombinant plasmids. Recombination experiments demonstrated recombination activating activity of all putative recombination activating domains amplified from AA8 and EM9 genomes as reflected by deletions within the insert of the recombinant plasmids. The recombination activating activity of XRCC1 analogues could explain a mechanism responsible for the correction of the DNA repair defect in EM9.

  18. Human xylosyltransferase I: functional and biochemical characterization of cysteine residues required for enzymic activity.

    PubMed

    Müller, Sandra; Schöttler, Manuela; Schön, Sylvia; Prante, Christian; Brinkmann, Thomas; Kuhn, Joachim; Götting, Christian; Kleesiek, Knut

    2005-03-01

    XT-I (xylosyltransferase I) is the initial enzyme in the post-translational biosynthesis of glycosaminoglycan chains in proteoglycans. To gain insight into the structure-function relationship of the enzyme, a soluble active form of human XT-I was expressed in High Five insect cells with an apparent molecular mass of 90 kDa. Analysis of the electrophoretic mobility of the protein under non-reducing and reducing conditions indicated that soluble XT-I does not form homodimers through disulphide bridges. In addition, the role of the cysteine residues was investigated by site-directed mutagenesis combined with chemical modifications of XT-I by N-phenylmaleimide. Replacement of Cys471 or Cys574 with alanine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of soluble recombinant XT-I by forming disulphide bonds. On the other hand, N-phenylmaleimide treatment showed no effect on wild-type XT-I but strongly inactivated the cysteine mutants in a dose-dependant manner, indicating that seven intramolecular disulphide bridges are formed in wild-type XT-I. The inhibitory effect of UDP on the XT-I activity of C561A (Cys561-->Ala) mutant enzyme was significantly reduced compared with all other tested cysteine mutants. In addition, we tested for binding to UDP-agarose beads. The inactive mutants revealed no significantly different nucleotide-binding properties. Our study demonstrates that recombinant XT-I is organized as a monomer with no free thiol groups and strongly suggests that the catalytic activity does not depend on the presence of free thiol groups, furthermore, we identified five cysteine residues which are critical for enzyme activity.

  19. Naturally occurring active N-domain of human angiotensin I-converting enzyme.

    PubMed Central

    Deddish, P A; Wang, J; Michel, B; Morris, P W; Davidson, N O; Skidgel, R A; Erdös, E G

    1994-01-01

    Angiotensin I-converting enzyme (ACE, kininase II) is a single-chain protein containing two active site domains (named N- and C-domains according to position in the chain). ACE is bound to plasma membranes by its C-terminal hydrophobic transmembrane anchor. Ileal fluid, rich in ACE activity, obtained from patients after surgical colectomy was used as the source. Column chromatography, including modified affinity chromatography on lisinopril-Sepharose, yielded homogeneous ACE after only a 45-fold purification. N-terminal sequencing of ileal ACE and partial sequencing of CNBr fragments revealed the presence of an intact N terminus but only a single N-domain active site, ending between residues 443 and 559. Thus, ileal-fluid ACE is a unique enzyme differing from the widely distributed two-domain somatic enzyme or the single C-domain testicular (germinal) ACE. The molecular mass of ileal ACE is 108 kDa and when deglycosylated, the molecular mass is 68 kDa, indicating extensive glycosylation (37% by weight). In agreement with the results reported with recombinant variants of ACE, the ileal enzyme is less Cl(-)-dependent than somatic ACE; release of the C-terminal dipeptide from a peptide substrate was optimal in only 10 mM Cl-. In addition to hydrolyzing at the C-terminal end of peptides, ileal ACE efficiently cleaved the protected N-terminal tripeptide from the luteinizing hormone-releasing hormone and its congener 6-31 times faster, depending on the Cl- concentration, than the C-domain in recombinant testicular ACE. Thus we have isolated an active human ACE consisting of a single N-domain. We suggest that there is a bridge section of about 100 amino acids between the active N- and C-domains of somatic ACE where it may be proteolytically cleaved to liberate the active N-domain. These findings have potential relevance and importance in the therapeutic application of ACE inhibitors. PMID:8052664

  20. Function and biotechnology of extremophilic enzymes in low water activity

    PubMed Central

    2012-01-01

    Enzymes from extremophilic microorganisms usually catalyze chemical reactions in non-standard conditions. Such conditions promote aggregation, precipitation, and denaturation, reducing the activity of most non-extremophilic enzymes, frequently due to the absence of sufficient hydration. Some extremophilic enzymes maintain a tight hydration shell and remain active in solution even when liquid water is limiting, e.g. in the presence of high ionic concentrations, or at cold temperature when water is close to the freezing point. Extremophilic enzymes are able to compete for hydration via alterations especially to their surface through greater surface charges and increased molecular motion. These properties have enabled some extremophilic enzymes to function in the presence of non-aqueous organic solvents, with potential for design of useful catalysts. In this review, we summarize the current state of knowledge of extremophilic enzymes functioning in high salinity and cold temperatures, focusing on their strategy for function at low water activity. We discuss how the understanding of extremophilic enzyme function is leading to the design of a new generation of enzyme catalysts and their applications to biotechnology. PMID:22480329

  1. Sustained gastrointestinal activity of dendronized polymer-enzyme conjugates

    NASA Astrophysics Data System (ADS)

    Fuhrmann, Gregor; Grotzky, Andrea; Lukić, Ružica; Matoori, Simon; Luciani, Paola; Yu, Hao; Zhang, Baozhong; Walde, Peter; Schlüter, A. Dieter; Gauthier, Marc A.; Leroux, Jean-Christophe

    2013-07-01

    Methods to stabilize and retain enzyme activity in the gastrointestinal tract are investigated rarely because of the difficulty of protecting proteins from an environment that has evolved to promote their digestion. Preventing the degradation of enzymes under these conditions, however, is critical for the development of new protein-based oral therapies. Here we show that covalent conjugation to polymers can stabilize orally administered therapeutic enzymes at different locations in the gastrointestinal tract. Architecturally and functionally diverse polymers are used to protect enzymes sterically from inactivation and to promote interactions with mucin on the stomach wall. Using this approach the in vivo activity of enzymes can be sustained for several hours in the stomach and/or in the small intestine. These findings provide new insight and a firm basis for the development of new therapeutic and imaging strategies based on orally administered proteins using a simple and accessible technology.

  2. Generating Functional Recombinant NRPS Enzymes in the Laboratory Setting via Peptidyl Carrier Protein Engineering.

    PubMed

    Owen, Jeremy G; Calcott, Mark J; Robins, Katherine J; Ackerley, David F

    2016-11-17

    Non-ribosomal peptide synthetases (NRPSs) are modular enzymatic assembly lines where substrates and intermediates undergo rounds of transformation catalyzed by adenylation (A), condensation (C), and thioesterase (TE) domains. Central to the NRPS biosynthesis are peptidyl carrier protein (PCP) domains, small, catalytically inactive domains that shuttle substrates and intermediates between the catalytic modules and govern product release from TE domains. There is strong interest in recombination of NRPS systems to generate new chemical entities. However, the intrinsic complexity of these systems has been a major challenge. Here, we employ domain substitution and random mutagenesis to recapitulate NRPS evolution, focusing on PCP domains. Using NRPS model systems that produce two different pigmented molecules, pyoverdine and indigoidine, we found that only evolutionarily specialized recombinant PCP domains could interact effectively with the native TE domain for product release. Overall, we highlight that substituted PCP domains require very minor changes to result in functional NRPSs, and infer that positive selection pressure may improve recombinant NRPS outcomes.

  3. Yeast Recombination Enhancer Is Stimulated by Transcription Activation

    PubMed Central

    Ercan, Sevinc; Reese, Joseph C.; Workman, Jerry L.; Simpson, Robert T.

    2005-01-01

    Saccharomyces cerevisiae mating type switching is a gene conversion event that exhibits donor preference. MATa cells choose HMLα for recombination, and MATα cells choose HMRa. Donor preference is controlled by the recombination enhancer (RE), located between HMLα and MATa on the left arm of chromosome III. A number of a-cell specific noncoding RNAs are transcribed from the RE locus. Mcm1 and Fkh1 regulate RE activity in a cells. Here we show that Mcm1 binding is required for both the transcription of the noncoding RNAs and Fkh1 binding. This requirement can be bypassed by inserting another promoter into the RE. Moreover, the insertion of this promoter increases donor preference and opens the chromatin structure around the conserved domains of RE. Additionally, we determined that the level of Fkh1 binding positively correlates with the level of donor preference. We conclude that the role of Mcm1 in RE is to open chromatin around the conserved domains and activate transcription; this facilitates Fkh1 binding and the level of this binding determines the level of donor preference. PMID:16135790

  4. Combining peptide modeling and capillary electrophoresis-mass spectrometry for characterization of enzymes cleavage patterns: recombinant versus natural bovine pepsin A.

    PubMed

    Simó, Carolina; González, Ramón; Barbas, Coral; Cifuentes, Alejandro

    2005-12-01

    Nowadays there is an increasing number of recombinant enzymes made available to industry. Before replacing the use of natural enzymes with their cognate recombinant counterparts, one important issue to address is their actual equivalence. For a given recombinant proteolytic enzyme, its equivalence can be investigated by comparing its cleavage specificity with that obtained from the natural enzyme. This is mostly done by analyzing the fragments (i.e., peptidic map) attained after enzymatic digestion of a given protein used as substrate. The peptidic maps obtained are typically characterized using separation techniques together with MS and MS/MS systems. However, these procedures are known to be difficult and labor-intensive. In this work, the combined use of a theoretical model that relates electrophoretic behavior of peptides to their sequence together with capillary electrophoresis-mass spectrometry (CE-MS) is proposed to characterize in a very fast and simple way the cleavage specificity of new recombinant enzymes. Namely, the effectiveness of this procedure is demonstrated by analyzing in few minutes the fragments obtained from a protein hydrolysated using recombinant and natural pepsin A. The usefulness of this strategy is further corroborated by CE-MS/MS. The proposed procedure is applicable in many other proteomic studies involving CE-MS of peptides.

  5. Activation volumes of enzymes adsorbed on silica particles.

    PubMed

    Schuabb, Vitor; Czeslik, Claus

    2014-12-30

    The immobilization of enzymes on carrier particles is useful in many biotechnological processes. In this way, enzymes can be separated from the reaction solution by filtering and can be reused in several cycles. On the other hand, there is a series of examples of free enzymes in solution that can be activated by the application of pressure. Thus, a potential loss of enzymatic activity upon immobilization on carrier particles might be compensated by pressure. In this study, we have determined the activation volumes of two enzymes, α-chymotrypsin (α-CT) and horseradish peroxidase (HRP), when they are adsorbed on silica particles and free in solution. The experiments have been carried out using fluorescence assays under pressures up to 2000 bar. In all cases, activation volumes were found to depend on the applied pressure, suggesting different compressions of the enzyme-substrate complex and the transition state. The volume profiles of free and adsorbed HRP are similar. For α-CT, larger activation volumes are found in the adsorbed state. However, up to about 500 bar, the enzymatic reaction of α-CT, which is adsorbed on silica particles, is characterized by a negative activation volume. This observation suggests that application of pressure might indeed be useful to enhance the activity of enzymes on carrier particles.

  6. In vitro excision of adeno-associated virus DNA from recombinant plasmids: Isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences

    SciTech Connect

    Gottlieb, J.; Muzyczka, N.

    1988-06-01

    When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, the authors isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G . C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G . C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.

  7. Characterization of Biosensors Based on Recombinant Glutamate Oxidase: Comparison of Crosslinking Agents in Terms of Enzyme Loading and Efficiency Parameters

    PubMed Central

    Ford, Rochelle; Quinn, Susan J.; O’Neill, Robert D.

    2016-01-01

    Amperometric l-glutamate (Glu) biosensors, based on both wild-type and a recombinant form of l-glutamate oxidase (GluOx), were designed and characterized in terms of enzyme-kinetic, sensitivity and stability parameters in attempts to fabricate a real-time Glu monitoring device suitable for future long-term detection of this amino acid in biological and other complex media. A comparison of the enzyme from these two sources showed that they were similar in terms of biosensor performance. Optimization of the loading of the polycationic stabilization agent, polyethyleneimine (PEI), was established before investigating a range of crosslinking agents under different conditions: glutaraldehyde (GA), polyethylene glycol (PEG), and polyethylene glycol diglycidyl ether (PEGDE). Whereas PEI-free biosensor designs lost most of their meager Glu sensitivity after one or two days, configurations with a 2:5 ratio of dip-evaporation applications of PEI(1%):GluOx(400 U/mL) displayed a 20-fold increase in their initial sensitivity, and a decay half-life extended to 10 days. All the crosslinkers studied had no effect on initial Glu sensitivity, but enhanced biosensor stability, provided the crosslinking procedure was carried out under well-defined conditions. The resulting biosensor design based on the recombinant enzyme deposited on a permselective layer of poly-(ortho-phenylenediamine), PoPD/PEI2/GluOx5/PEGDE, displayed good sensitivity (LOD < 0.2 μM), response time (t90% < 1 s) and stability over a 90-day period, making it an attractive candidate for future long-term monitoring of Glu concentration dynamics in complex media. PMID:27669257

  8. 76 FR 62816 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-11

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines... recombinant DNA research. OBA is also specifying the risk group for several viruses not previously listed...

  9. 75 FR 31795 - Office of Biotechnology Activities; Recombinant DNA Research: Amended Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-04

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA... ] under Section III-A-1 of the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH... the NIH Recombinant DNA Advisory Committee has been deferred at the request of the...

  10. 76 FR 44339 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-25

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines... attenuated strains of bacteria and viruses that are frequently used in recombinant DNA research. OBA is...

  11. 75 FR 69687 - Office of Biotechnology Activities Recombinant DNA Research: Proposed Actions Under the NIH...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-15

    ... of Biotechnology Activities Recombinant DNA Research: Proposed Actions Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) ACTION: Notice of consideration of proposed...- vector system may be certified only after review by the NIH Recombinant DNA Advisory Committee (RAC)...

  12. 76 FR 27653 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-12

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines... Kluyveromyces lactis as a host-vector 1 system has been reviewed by the NIH ] Recombinant DNA Advisory...

  13. 78 FR 66751 - Office of Science Policy, Office of Biotechnology Activities; Recombinant or Synthetic Nucleic...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-06

    ... Activities; Recombinant or Synthetic Nucleic Acid Molecule Research: Action Under the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) AGENCY: NIH, Public... containment for research involving recombinant or synthetic nucleic acid molecules. Section II-A,...

  14. Effect of early feed restriction and enzyme supplementation on digestive enzyme activities in broilers.

    PubMed

    Pinheiro, D F; Cruz, V C; Sartori, J R; Vicentini Paulino, M L M

    2004-09-01

    The effect of feed restriction and enzymatic supplementation on intestinal and pancreatic enzyme activities and weight gain was studied in broiler chickens. Quantitative feed restriction was applied to chickens from 7 to 14 d of age. An enzyme complex mainly consisting of protease and amylase was added to the chicken ration from hatching to the end of the experiment. Birds subjected to feed restriction whose diet was not supplemented showed an increase in sucrase, amylase, and lipase activities immediately after the restriction period. Amylase, lipase, and chymotrypsin activities were higher in chickens subjected to feed restriction and fed a supplemented diet than in those only subjected to feed restriction. Trypsin activity increased after feed restriction and after supplementation, but there was no interaction between these effects. Early feed restriction had no effect on enzyme activity in 42-d-old chickens. Chickens subjected to early restriction and fed the supplemented diet presented higher sucrase, maltase, and lipase activities than nonsupplemented ones (P < 0.05). There was no effect of early feed restriction or diet supplementation on weight gain to 42 d. Percentage weight gain from 14 to 42 d of age was equivalent in feed-restricted and ad libitum fed birds. Feed-restricted broilers fed a supplemented diet showed a higher percentage weight gain than nonsupplemented birds. We conclude that enzymatic supplementation potentiates the effect of feed restriction on digestive enzyme activity and on weight gain.

  15. Compounds from Silicones Alter Enzyme Activity in Curing Barnacle Glue and Model Enzymes

    PubMed Central

    Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H.

    2011-01-01

    Background Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. Methodology/Principal Findings GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Conclusions/Significance Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management. PMID:21379573

  16. Response surface methodology to optimize partition and purification of two recombinant oxidoreductase enzymes, glucose dehydrogenase and d-galactose dehydrogenase in aqueous two-phase systems.

    PubMed

    Shahbaz Mohammadi, Hamid; Mostafavi, Seyede Samaneh; Soleimani, Saeideh; Bozorgian, Sajad; Pooraskari, Maryam; Kianmehr, Anvarsadat

    2015-04-01

    Oxidoreductases are an important family of enzymes that are used in many biotechnological processes. An experimental design was applied to optimize partition and purification of two recombinant oxidoreductases, glucose dehydrogenase (GDH) from Bacillus subtilis and d-galactose dehydrogenase (GalDH) from Pseudomonas fluorescens AK92 in aqueous two-phase systems (ATPS). Response surface methodology (RSM) with a central composite rotatable design (CCRD) was performed to optimize critical factors like polyethylene glycol (PEG) concentration, concentration of salt and pH value. The best partitioning conditions was achieved in an ATPS composed of 12% PEG-6000, 15% K2HPO4 with pH 7.5 at 25°C, which ensured partition coefficient (KE) of 66.6 and 45.7 for GDH and GalDH, respectively. Under these experimental conditions, the activity of GDH and GalDH was 569.5U/ml and 673.7U/ml, respectively. It was found that these enzymes preferentially partitioned into the top PEG-rich phase and appeared as single bands on SDS-PAGE gel. Meanwhile the validity of the response model was confirmed by a good agreement between predicted and experimental results. Collectively, according to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of any enzyme from oxidoreductase family.

  17. ENZYMIC ACTIVITY IN FREEZE DRIED FOODS

    DTIC Science & Technology

    and bananas. Factors studied include, polyphenol oxidase , peroxidase, sucrase, alpha and beta amylase, pectinesterase and ascorbase activity as well...storage of freeze-dried and frozen peas at different moisture was studied. Lipase activity and production of free fatty acid was following during long term

  18. Transcription analysis of pyranose dehydrogenase from the basidiomycete Agaricus bisporus and characterization of the recombinantly expressed enzyme.

    PubMed

    Gonaus, Christoph; Kittl, Roman; Sygmund, Christoph; Haltrich, Dietmar; Peterbauer, Clemens

    2016-03-01

    Agaricus bisporus is a litter degrading basidiomycete commonly found in humic-rich environments. It is used as model organism and cultivated in large scale for food industry. Due to its ecological niche it produces a variety of enzymes for detoxification and degradation of humified plant litter. One of these, pyranose dehydrogenase, is thought to play a role in detoxification and lignocellulose degradation. It is a member of the glucose-methanol-choline family of flavin-dependent enzymes and oxidizes a wide range of sugars with concomitant reduction of electron acceptors like quinones. In this work, transcription of pdh in A. bisporus was investigated with real-time PCR revealing influence of the carbon source on pdh expression levels. The gene was isolated and heterologously expressed in Pichia pastoris. Characterization of the recombinant enzyme showed a higher affinity towards disaccharides compared to other tested pyranose dehydrogenases from related Agariceae. Homology modeling and sequence alignments indicated that two loops of high sequence variability at substrate access site could play an important role in modulating these substrate specificities.

  19. The BCL11A transcription factor directly activates RAG gene expression and V(D)J recombination.

    PubMed

    Lee, Baeck-seung; Dekker, Joseph D; Lee, Bum-kyu; Iyer, Vishwanath R; Sleckman, Barry P; Shaffer, Arthur L; Ippolito, Gregory C; Tucker, Philip W

    2013-05-01

    Recombination-activating gene 1 protein (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining (VDJ) segment recombination, an essential process for antigen receptor expression and lymphocyte development. The transcription factor BCL11A is required for B cell development, but its molecular function(s) in B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds the RAG1 promoter and Erag enhancer to activate RAG1 and RAG2 transcription in pre-B cells. We employed BCL11A overexpression with recombination substrates in a cultured pre-B cell line as well as Cre recombinase-mediated Bcl11a(lox/lox) deletion in explanted murine pre-B cells to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination.

  20. Investigation of enzyme activity by SERRS using poly-functionalised benzotriazole derivatives as enzyme substrates.

    PubMed

    Ingram, Andrew M; Stirling, Kirsten; Faulds, Karen; Moore, Barry D; Graham, Duncan

    2006-08-07

    New methods of measuring biologically relevant concentrations of enzymes are necessary to allow greater understanding of biological systems. We have previously shown that aryl azo benzotriazolyl alkyl esters can act as enzyme substrates, with the progress of the reaction being monitored using SERRS (see Nat. Biotechnol., 2004, 22, 1133, ref. ). This is a wholly novel analytical application of SERRS, and the low detection levels of the technique allow for an ultra-sensitive enzyme assay. Masked enzyme substrates are used that are invisible to SERRS until enzymatic hydrolysis. Turnover of the substrate by the enzyme leads to the release of the surface-seeking dye necessary for SERRS, and intense signals are produced. Here we report an improved synthesis of 2H-benzotriazolyl alkyl esters via nucleophilic substitution of a chloromethyl ester by benzotriazolyl azo dyes, giving up to a ten-fold increase on previously reported yields. Introduction of electron-withdrawing groups to the benzotriazole ring allows control over the SERRS properties of the compounds. This is of great significance in expanding the synthetic flexibility and subsequently the fundamental use of these compounds as ultra-sensitive and selective reporters of enzyme activity.

  1. Enzyme:nanoparticle bioconjugates with two sequential enzymes: stoichiometry and activity of malate dehydrogenase and citrate synthase on Au nanoparticles.

    PubMed

    Keighron, Jacqueline D; Keating, Christine D

    2010-12-21

    We report the synthesis and characterization of bioconjugates in which the enzymes malate dehydrogenase (MDH) and/or citrate synthase (CS) were adsorbed to 30 nm diameter Au nanoparticles. Enzyme:Au stoichiometry and kinetic parameters (specific activity, k(cat), K(M), and activity per particle) were determined for MDH:Au, CS:Au, and three types of dual-activity MDH/CS:Au bioconjugates. For single-activity bioconjugates (MDH:Au and CS:Au), the number of enzyme molecules adsorbed per particle was dependent upon the enzyme concentration in solution, with multilayers forming at high enzyme:Au solution ratios. The specific activity of adsorbed enzyme increased with increasing number adsorbed per particle for CS:Au, but was less sensitive to stoichiometry for MDH:Au. Dual activity bioconjugates were prepared in three ways: (1) by adsorption of MDH followed by CS, (2) by adsorption of CS followed by MDH, and (3) by coadsorption of both enzymes from the same solution. The resulting bioconjugates differed substantially in the number of enzyme molecules adsorbed per particle, the specific activity of the adsorbed enzymes, and also the enzymatic activity per particle. Bioconjugates formed by adding CS to the Au nanoparticles before MDH was added exhibited higher specific activities for both enzymes than those formed by adding the enzymes in the reverse order. These bioconjugates also had 3-fold higher per-particle sequential activity for conversion of malate to citrate, despite substantially fewer copies of both enzymes present.

  2. Identification of putative active site residues of ACAT enzymes.

    PubMed

    Das, Akash; Davis, Matthew A; Rudel, Lawrence L

    2008-08-01

    In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

  3. Inhibition of existing denitrification enzyme activity by chloramphenicol

    USGS Publications Warehouse

    Brooks, M.H.; Smith, R.L.; Macalady, D.L.

    1992-01-01

    Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (>50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log- phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.

  4. Activity of selected hydrolytic enzymes in Allium sativum L. anthers.

    PubMed

    Winiarczyk, Krystyna; Gębura, Joanna

    2016-05-01

    The aim of the study was to determine enzymatic activity in sterile Allium sativum anthers in the final stages of male gametophyte development (the stages of tetrads and free microspores). The analysed enzymes were shown to occur in the form of numerous isoforms. In the tetrad stage, esterase activity was predominant, which was manifested by the greater number of isoforms of the enzyme. In turn, in the microspore stage, higher numbers of isoforms of acid phosphatases and proteases were detected. The development of sterile pollen grains in garlic is associated with a high level of protease and acid phosphatase activity and lower level of esterase activities in the anther locule. Probably this is the first description of the enzymes activity (ACPH, EST, PRO) in the consecutives stages of cell wall formation which is considered to be one of the causes of male sterility in flowering plant.

  5. Enzyme activities in mitochondria isolated from ripening tomato fruit.

    PubMed

    Jeffery, D; Goodenough, P W; Weitzman, P D

    1986-09-01

    Mitochondria were isolated from tomato (Lycopersicon esculentum L.) fruit at the mature green, orange-green and red stages and from fruit artificially suspended in their ripening stage. The specific activities of citrate synthase (EC 4.1.3.7), malate dehydrogenase (EC 1.1.1.37), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and NAD-linked malic enzyme (EC 1.1.1.38) were determined. The specific activities of all these enzymes fell during ipening, although the mitochondria were fully functional as demonstrated by the uptake of oxygen. The fall in activity of mitochondrial malate dehydrogenase was accompanied by a similar fall in the activity of the cytosolic isoenzyme. Percoll-purified mitochondria isolated from mature green fruit remained intact for more than one week and at least one enzyme, citrate synthase, did not exhibit the fall in specific activity found in normal ripening fruit.

  6. Interfacial activation-based molecular bioimprinting of lipolytic enzymes.

    PubMed Central

    Mingarro, I; Abad, C; Braco, L

    1995-01-01

    Interfacial activation-based molecular (bio)-imprinting (IAMI) has been developed to rationally improve the performance of lipolytic enzymes in nonaqueous environments. The strategy combinedly exploits (i) the known dramatic enhancement of the protein conformational rigidity in a water-restricted milieu and (ii) the reported conformational changes associated with the activation of these enzymes at lipid-water interfaces, which basically involves an increased substrate accessibility to the active site and/or an induction of a more competent catalytic machinery. Six model enzymes have been assayed in several model reactions in nonaqueous media. The results, rationalized in light of the present biochemical and structural knowledge, show that the IAMI approach represents a straightforward, versatile method to generate manageable, activated (kinetically trapped) forms of lipolytic enzymes, providing under optimal conditions nonaqueous rate enhancements of up to two orders of magnitude. It is also shown that imprintability of lipolytic enzymes depends not only on the nature of the enzyme but also on the "quality" of the interface used as the template. PMID:7724558

  7. Interfacial activation-based molecular bioimprinting of lipolytic enzymes.

    PubMed

    Mingarro, I; Abad, C; Braco, L

    1995-04-11

    Interfacial activation-based molecular (bio)-imprinting (IAMI) has been developed to rationally improve the performance of lipolytic enzymes in nonaqueous environments. The strategy combinedly exploits (i) the known dramatic enhancement of the protein conformational rigidity in a water-restricted milieu and (ii) the reported conformational changes associated with the activation of these enzymes at lipid-water interfaces, which basically involves an increased substrate accessibility to the active site and/or an induction of a more competent catalytic machinery. Six model enzymes have been assayed in several model reactions in nonaqueous media. The results, rationalized in light of the present biochemical and structural knowledge, show that the IAMI approach represents a straightforward, versatile method to generate manageable, activated (kinetically trapped) forms of lipolytic enzymes, providing under optimal conditions nonaqueous rate enhancements of up to two orders of magnitude. It is also shown that imprintability of lipolytic enzymes depends not only on the nature of the enzyme but also on the "quality" of the interface used as the template.

  8. Chimeric enzymes with improved cellulase activities

    DOEpatents

    Xu, Qi; Baker, John O; Himmel, Michael E

    2015-03-31

    Nucleic acid molecules encoding chimeric cellulase polypeptides that exhibit improved cellulase activities are disclosed herein. The chimeric cellulase polypeptides encoded by these nucleic acids and methods to produce the cellulases are also described, along with methods of using chimeric cellulases for the conversion of cellulose to sugars such as glucose.

  9. Catalytically active nanomaterials: a promising candidate for artificial enzymes.

    PubMed

    Lin, Youhui; Ren, Jinsong; Qu, Xiaogang

    2014-04-15

    Natural enzymes, exquisite biocatalysts mediating every biological process in living organisms, are able to accelerate the rate of chemical reactions up to 10(19) times for specific substrates and reactions. However, the practical application of enzymes is often hampered by their intrinsic drawbacks, such as low operational stability, sensitivity of catalytic activity to environmental conditions, and high costs in preparation and purification. Therefore, the discovery and development of artificial enzymes is highly desired. Recently, the merging of nanotechnology with biology has ignited extensive research efforts for designing functional nanomaterials that exhibit various properties intrinsic to enzymes. As a promising candidate for artificial enzymes, catalytically active nanomaterials (nanozymes) show several advantages over natural enzymes, such as controlled synthesis in low cost, tunability in catalytic activities, as well as high stability against stringent conditions. In this Account, we focus on our recent progress in exploring and constructing such nanoparticulate artificial enzymes, including graphene oxide, graphene-hemin nanocomposites, carbon nanotubes, carbon nanodots, mesoporous silica-encapsulated gold nanoparticles, gold nanoclusters, and nanoceria. According to their structural characteristics, these enzyme mimics are categorized into three classes: carbon-, metal-, and metal-oxide-based nanomaterials. We aim to highlight the important role of catalytic nanomaterials in the fields of biomimetics. First, we provide a practical introduction to the identification of these nanozymes, the source of the enzyme-like activities, and the enhancement of activities via rational design and engineering. Then we briefly describe new or enhanced applications of certain nanozymes in biomedical diagnosis, environmental monitoring, and therapeutics. For instance, we have successfully used these biomimetic catalysts as colorimetric probes for the detection of

  10. Recombinant plasmids for encoding restriction enzymes DpnI and DpnII of streptococcus pneumontae

    DOEpatents

    Lacks, Sanford A.

    1990-01-01

    Chromosomal DNA cassettes containing genes encoding either the DpnI or DpnII restriction endonucleases from Streptococcus pneumoniae are cloned into a streptococcal vector, pLS101. Large amounts of the restriction enzymes are produced by cells containing the multicopy plasmids, pLS202 and pLS207, and their derivatives pLS201, pLS211, pLS217, pLS251 and pLS252.

  11. Recombinant plasmids for encoding restriction enzymes DpnI and DpnII of Streptococcus pneumontae

    DOEpatents

    Lacks, S.A.

    1990-10-02

    Chromosomal DNA cassettes containing genes encoding either the DpnI or DpnII restriction endonucleases from Streptococcus pneumoniae are cloned into a streptococcal vector, pLS101. Large amounts of the restriction enzymes are produced by cells containing the multicopy plasmids, pLS202 and pLS207, and their derivatives pLS201, pLS211, pLS217, pLS251 and pLS252. 9 figs.

  12. Development of a flatfish-specific enzyme-linked immunosorbent assay for Fsh using a recombinant chimeric gonadotropin.

    PubMed

    Chauvigné, François; Verdura, Sara; Mazón, María José; Boj, Mónica; Zanuy, Silvia; Gómez, Ana; Cerdà, Joan

    2015-09-15

    In flatfishes with asynchronous and semicystic spermatogenesis, such as the Senegalese sole (Solea senegalensis), the specific roles of the pituitary gonadotropins during germ cell development, particularly of the follicle-stimulating hormone (Fsh), are still largely unknown in part due to the lack of homologous immunoassays for this hormone. In this study, an enzyme-linked immunosorbent assay (ELISA) for Senegalese sole Fsh was developed by generating a rabbit antiserum against a recombinant chimeric single-chain Fsh molecule (rFsh-C) produced by the yeast Pichia pastoris. The rFsh-C N- and C-termini were formed by the mature sole Fsh β subunit (Fshβ) and the chicken glycoprotein hormone common α subunit (CGA), respectively. Depletion of the antiserum to remove anti-CGA antibodies further enriched the sole Fshβ-specific antibodies, which were used to develop the ELISA using the rFsh-C for the standard curve. The sensitivity of the assay was 10 and 50 pg/ml for Fsh measurement in plasma and pituitary, respectively, and the cross-reactivity with a homologous recombinant single-chain luteinizing hormone was 1%. The standard curve for rFsh-C paralleled those of serially diluted plasma and pituitary extracts of other flatfishes, such as the Atlantic halibut, common sole and turbot. In Senegalese sole males, the highest plasma Fsh levels were found during early spermatogenesis but declined during enhanced spermiation, as found in teleosts with cystic spermatogenesis. In pubertal males, however, the circulating Fsh levels were as high as in adult spermiating fish, but interestingly the Fsh receptor in the developing testis containing only spermatogonia was expressed in Leydig cells but not in the primordial Sertoli cells. These results indicate that a recombinant chimeric Fsh can be used to generate specific antibodies against the Fshβ subunit and to develop a highly sensitive ELISA for Fsh measurements in diverse flatfishes.

  13. Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF, recycling of hydrolytic enzymes and yeast, and recombinant cellulase-producing Aspergillus niger.

    PubMed

    Cavka, Adnan; Alriksson, Björn; Rose, Shaunita H; van Zyl, Willem H; Jönsson, Leif J

    2014-08-01

    Bioethanol and enzymes were produced from fiber sludges through sequential microbial cultivations. After a first simultaneous saccharification and fermentation (SSF) with yeast, the bioethanol concentrations of sulfate and sulfite fiber sludges were 45.6 and 64.7 g/L, respectively. The second SSF, which included fresh fiber sludges and recycled yeast and enzymes from the first SSF, resulted in ethanol concentrations of 38.3 g/L for sulfate fiber sludge and 24.4 g/L for sulfite fiber sludge. Aspergillus niger carrying the endoglucanase-encoding Cel7B gene of Trichoderma reesei was grown in the spent fiber sludge hydrolysates. The cellulase activities obtained with spent hydrolysates of sulfate and sulfite fiber sludges were 2,700 and 2,900 nkat/mL, respectively. The high cellulase activities produced by using stillage and the significant ethanol concentrations produced in the second SSF suggest that onsite enzyme production and recycling of enzyme are realistic concepts that warrant further attention.

  14. Enzyme activation through the utilization of intrinsic dianion binding energy.

    PubMed

    Amyes, T L; Malabanan, M M; Zhai, X; Reyes, A C; Richard, J P

    2016-11-29

    We consider 'the proposition that the intrinsic binding energy that results from the noncovalent interaction of a specific substrate with the active site of the enzyme is considerably larger than is generally believed. An important part of this binding energy may be utilized to provide the driving force for catalysis, so that the observed binding energy represents only what is left over after this utilization' [Jencks,W.P. (1975) Adv. Enzymol. Relat. Areas. Mol. Biol., 43: , 219-410]. The large ~12 kcal/mol intrinsic substrate phosphodianion binding energy for reactions catalyzed by triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase and glycerol-3-phosphate dehydrogenase is divided into 4-6 kcal/mol binding energy that is expressed on the formation of the Michaelis complex in anchoring substrates to the respective enzyme, and 6-8 kcal/mol binding energy that is specifically expressed at the transition state in activating the respective enzymes for catalysis. A structure-based mechanism is described where the dianion binding energy drives a conformational change that activates these enzymes for catalysis. Phosphite dianion plays the active role of holding TIM in a high-energy closed active form, but acts as passive spectator in showing no effect on transition-state structure. The result of studies on mutant enzymes is presented, which support the proposal that the dianion-driven enzyme conformational change plays a role in enhancing the basicity of side chain of E167, the catalytic base, by clamping the base between a pair of hydrophobic side chains. The insight these results provide into the architecture of enzyme active sites and the development of strategies for the de novo design of protein catalysts is discussed.

  15. Distribution and activity of hydrogenase enzymes in subsurface sediments

    NASA Astrophysics Data System (ADS)

    Adhikari, R.; Nickel, J.; Glombitza, C.; Spivack, A. J.; D'Hondt, S. L.; Kallmeyer, J.

    2013-12-01

    Metabolically active microbial communities are present in a wide range of subsurface environments. Techniques like enumeration of microbial cells, activity measurements with radiotracer assays and the analysis of porewater constituents are currently being used to explore the subsurface biosphere, alongside with molecular biological analyses. However, many of these techniques reach their detection limits due to low microbial activity and abundance. Direct measurements of microbial turnover not just face issues of insufficient sensitivity, they only provide information about a single specific process rather than an overall microbial activity. Since hydrogenase enzymes are intracellular and ubiquitous in subsurface microbial communities, the enzyme activity represents a measure of total activity of the entire microbial community. A hydrogenase activity assay could quantify total metabolic activity without having to identify specific processes. This would be a major advantage in subsurface biosphere studies, where several metabolic processes can occur simultaneously. We quantified hydrogenase enzyme activity and distribution in sediment samples from different aquatic subsurface environments (Lake Van, Barents Sea, Equatorial Pacific and Gulf of Mexico) using a tritium-based assay. We found enzyme activity at all sites and depths. Volumetric hydrogenase activity did not show much variability between sites and sampling depths, whereas cell-specific activity ranged from 10-5 to 1 nmol H2 cell-1 d-1. Activity was lowest in sediment layers where nitrate was detected. Higher activity was associated with samples in which sulfate was the predominant electron acceptor. We found highest activity in samples from environments with >10 ppm methane in the pore water. The results show that cell-specific hydrogenase enzyme activity increases with decreasing energy yield of the electron acceptor used. It is not possible to convert volumetric or cell-specific hydrogenase activity into a

  16. Conformation and activity of recombinant human fibroblast interferon-beta.

    PubMed

    Boublik, M; Moschera, J A; Wei, C; Kung, H F

    1990-04-01

    Conformation of highly purified recombinant human fibroblast interferon-beta (rHuIFN-beta) was correlated with its biological activity. The extent of ordered secondary structure was determined by circular dichroic (CD) spectroscopy in various buffer conditions to establish conditions of protein stability and its potential for helix formation. The highest "helicity" (about 50 +/- 5% of alpha-helices) and the highest antiviral activities (4-10 x 10(7) units/mg) were found in 50% ethylene glycol, 1 M NaCl and 0.05 M Na3PO4, pH 7.2 (Buffer I); 80 mM citric acid, 20 mM Na2HPO4, pH 2.9 (Buffer II); and 25 mM NH4OAc, 125 mM NaCl, pH 5.1 (Buffer III). Both helicity and antiviral activity of the IFN-beta decrease in parallel with denaturation by urea, heat, and/or by repeated cycles of freezing and thawing. Low pH (pH 2.9 Buffer II) exhibits a distinct stabilizing effect on the structure and antiviral activity of IFN-beta against heat denaturation.

  17. Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

    PubMed Central

    Barnes, H J; Arlotto, M P; Waterman, M R

    1991-01-01

    When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system. Images PMID:1829523

  18. [Activity of antioxidant enzymes in patients with liver cirrhosis].

    PubMed

    Czeczot, Hanna; Scibior, Dorota; Skrzycki, Michał; Podsiad, Małgorzata

    2006-01-01

    The aim of our studies was the estimation of activities of antioxidant enzymes in patients with liver cirrhosis. We investigated activities of superoxide dismutases (CuZnSOD, MnSOD), catalase (CAT), selenium dependent GSH peroxidase (Se-GSH-Px), selenium independent GSH peroxidase (non-Se-GSH-Px), GSH-S-transferase (GST), GSH reductase (GSHR) and the level ofreduced gutathione (GSH) in cirrhotic and healthy liver tissues. The activities of CuZnSOD, MnSOD, CAT and GSH-dependent enzymes (except GSHR) were found to be lower in cirrhotic tissue compared to healthy liver. Those changes were associated with decrease of GSH level in cirrhotic tissue compared with control liver tissue. Our results show that antioxidant barrier in liver cirrhosis is impaired. It is associated with decrease of glutathione level and changes of activities of antioxidant enzymes (SOD, CAT, GSHPx, GST, GSHR) in liver cirrhosis compared with healthy liver.

  19. Cloning and Expression of Phytase appA Gene from Shigella sp. CD2 in Pichia pastoris and Comparison of Properties with Recombinant Enzyme Expressed in E. coli.

    PubMed

    Pal Roy, Moushree; Mazumdar, Deepika; Dutta, Subhabrata; Saha, Shyama Prasad; Ghosh, Shilpi

    2016-01-01

    The phytase gene appAS was isolated from Shigella sp. CD2 genomic library. The 3.8 kb DNA fragment contained 1299 bp open reading frame encoding 432 amino acid protein (AppAS) with 22 amino acid signal peptide at N-terminal and three sites of N-glycosylation. AppAS contained the active site RHGXRXP and HDTN sequence motifs, which are conserved among histidine acid phosphatases. It showed maximum identity with phytase AppA of Escherichia coli and Citrobacter braakii. The appAS was expressed in Pichia pastoris and E. coli to produce recombinant phytase rAppAP and rAppAE, respectively. Purified glycosylated rAppAP and nonglycosylated rAppAE had specific activity of 967 and 2982 U mg(-1), respectively. Both had pH optima of 5.5 and temperature optima of 60°C. Compared with rAppAE, rAppAP was 13 and 17% less active at pH 3.5 and 7.5 and 11 and 18% less active at temperature 37 and 50°C, respectively; however, it was more active at higher incubation temperatures. Thermotolerance of rAppAP was 33% greater at 60°C and 24% greater at 70°C, when compared with rAppAE. Both the recombinant enzymes showed high specificity to phytate and resistance to trypsin. To our knowledge, this is the first report on cloning and expression of phytase from Shigella sp.

  20. Cloning and Expression of Phytase appA Gene from Shigella sp. CD2 in Pichia pastoris and Comparison of Properties with Recombinant Enzyme Expressed in E. coli

    PubMed Central

    Pal Roy, Moushree; Mazumdar, Deepika; Dutta, Subhabrata; Saha, Shyama Prasad; Ghosh, Shilpi

    2016-01-01

    The phytase gene appAS was isolated from Shigella sp. CD2 genomic library. The 3.8 kb DNA fragment contained 1299 bp open reading frame encoding 432 amino acid protein (AppAS) with 22 amino acid signal peptide at N-terminal and three sites of N-glycosylation. AppAS contained the active site RHGXRXP and HDTN sequence motifs, which are conserved among histidine acid phosphatases. It showed maximum identity with phytase AppA of Escherichia coli and Citrobacter braakii. The appAS was expressed in Pichia pastoris and E. coli to produce recombinant phytase rAppAP and rAppAE, respectively. Purified glycosylated rAppAP and nonglycosylated rAppAE had specific activity of 967 and 2982 U mg-1, respectively. Both had pH optima of 5.5 and temperature optima of 60°C. Compared with rAppAE, rAppAP was 13 and 17% less active at pH 3.5 and 7.5 and 11 and 18% less active at temperature 37 and 50°C, respectively; however, it was more active at higher incubation temperatures. Thermotolerance of rAppAP was 33% greater at 60°C and 24% greater at 70°C, when compared with rAppAE. Both the recombinant enzymes showed high specificity to phytate and resistance to trypsin. To our knowledge, this is the first report on cloning and expression of phytase from Shigella sp. PMID:26808559

  1. Chondroitinase ABC I from Proteus vulgaris: cloning, recombinant expression and active site identification.

    PubMed

    Prabhakar, Vikas; Capila, Ishan; Bosques, Carlos J; Pojasek, Kevin; Sasisekharan, Ram

    2005-02-15

    GalAGs (galactosaminoglycans) are one subset of the GAG (glycosaminoglycan) family of chemically heterogeneous polysaccharides that are involved in a wide range of biological processes. These complex biomacromolecules are believed to be responsible for the inhibition of nerve regeneration following injury to the central nervous system. The enzymic degradation of GAG chains in damaged nervous tissue by cABC I (chondroitinase ABC I), a broad-specificity lyase that degrades GalAGs, promotes neural recovery. In the present paper, we report the subcloning of cABC I from Proteus vulgaris, and discuss a simple methodology for the recombinant expression and purification of this enzyme. The originally expressed cABC I clone resulted in an enzyme with negligible activity against a variety of GalAG substrates. Sequencing of the cABC I clone revealed four point mutations at issue with the electron-density data of the cABC I crystal structure. Site-directed mutagenesis produced a clone with restored GalAG-degrading function. We have characterized this enzyme biochemically, including an analysis of its substrate specificity. By coupling structural inspections of cABC I and an evaluation of sequence homology against other GAG-degrading lyases, a set of amino acids was chosen for further study. Mutagenesis studies of these residues resulted in the first experimental evidence of cABC I's active site. This work will facilitate the structure-function characterization of biomedically relevant GalAGs and further the development of therapeutics for nerve regeneration.

  2. Fluorogenic Peptide Substrate for Quantification of Bacterial Enzyme Activities

    PubMed Central

    Al-Abdullah, Ismail H.; Bagramyan, Karine; Bilbao, Shiela; Qi, Meirigeng; Kalkum, Markus

    2017-01-01

    A novel peptide substrate (A G G P L G P P G P G G) was developed for quantifying the activities of bacterial enzymes using a highly sensitive Fluorescence Resonance Energy Transfer (FRET) based assay. The peptide substrate was cleaved by collagenase class I, II, Liberase MTF C/T, collagenase NB1, and thermolysin/neutral protease, which was significantly enhanced in the presence of CaCl2. However, the activities of these enzymes were significantly decreased in the presence of ZnSO4 or ZnCl2. Collagenase I, II, Liberase MTF C/T, thermolysin/neutral protease share similar cleavage sites, L↓G and P↓G. However, collagenase NB1 cleaves the peptide substrate at G↓P and P↓L, in addition to P↓G. The enzyme activity is pH dependent, within a range of 6.8 to 7.5, but was significantly diminished at pH 8.0. Interestingly, the peptide substrate was not cleaved by endogenous pancreatic protease such as trypsin, chymotrypsin, and elastase. In conclusion, the novel peptide substrate is collagenase, thermolysin/neutral protease specific and can be applied to quantify enzyme activities from different microbes. Furthermore, the assay can be used for fine-tuning reaction mixtures of various agents to enhance the overall activity of a cocktail of multiple enzymes and achieve optimal organ/tissue digestion, while protecting the integrity of the target cells. PMID:28287171

  3. Cancer genes: rare recombinants instead of activated oncogenes (a review).

    PubMed Central

    Duesberg, P H

    1987-01-01

    The 20 known transforming (onc) genes of retroviruses are defined by sequences that are transduced from cellular genes termed protooncogenes or cellular oncogenes. Based on these sequences, viral onc genes have been postulated to be transduced cellular cancer genes, and proto-onc genes have been postulated to be latent cancer genes that can be activated from within the cell to cause virus-negative tumors. The hypothesis is popular because it promises direct access to cellular cancer genes. However, the existence of latent cancer genes presents a paradox, since such genes are clearly undesirable. The hypothesis predicts that viral onc genes and proto-onc genes are isogenic; that expression of proto-onc genes induces tumors; that activated proto-onc genes transform diploid cells upon transfection, like viral onc genes; and that diploid tumors exist. As yet, none of these predictions is confirmed. Instead: Structural comparisons between viral onc genes, essential retroviral genes, and proto-onc genes show that all viral onc genes are indeed new genes, rather than transduced cellular cancer genes. They are recombinants put together from truncated viral and truncated proto-onc genes. Proto-onc genes are frequently expressed in normal cells. To date, not one activated proto-onc gene has been isolated that transforms diploid cells. Above all, no diploid tumors with activated proto-onc genes have been found. Moreover, the probability of spontaneous transformation in vivo is at least 10(9) times lower than predicted from the mechanisms thought to activate proto-onc genes. Therefore, the hypothesis that proto-onc genes are latent cellular oncogenes appears to be an overinterpretation of sequence homology to structural and functional homology with viral onc genes. Here it is proposed that only rare truncations and illegitimate recombinations that alter the germ-line configuration of cellular genes generate viral and possibly cellular cancer genes. The clonal chromosome

  4. Patterns of functional enzyme activity in fungus farming ambrosia beetles

    PubMed Central

    2012-01-01

    Introduction In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. Results We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Conclusion Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose

  5. Chemoproteomic profiling of host and pathogen enzymes active in cholera

    PubMed Central

    Hatzios, Stavroula K.; Hubbard, Troy; Sasabe, Jumpei; Munera, Diana; Clark, Lars; Bachovchin, Daniel A.; Qadri, Firdausi; Ryan, Edward T.; Davis, Brigid M.; Weerapana, Eranthie; Waldor, Matthew K.

    2016-01-01

    Activity-based protein profiling (ABPP) is a chemoproteomic tool for detecting active enzymes in complex biological systems. We used ABPP to identify secreted bacterial and host serine hydrolases that are active in animals infected with the cholera pathogen Vibrio cholerae. Four V. cholerae proteases were consistently active in infected rabbits, and one, VC0157 (renamed IvaP), was also active in human cholera stool. Inactivation of IvaP influenced the activity of other secreted V. cholerae and rabbit enzymes in vivo, while genetic disruption of all four proteases increased the abundance and binding of an intestinal lectin—intelectin—to V. cholerae in infected rabbits. Intelectin also bound to other enteric bacterial pathogens, suggesting it may constitute a previously unrecognized mechanism of bacterial surveillance in the intestine that is inhibited by pathogen-secreted proteases. Our work demonstrates the power of activity-based proteomics to reveal host-pathogen enzymatic dialogue in an animal model of infection. PMID:26900865

  6. Does diet influence salivary enzyme activities in elephant species?

    PubMed

    Boehlke, Carolin; Pötschke, Sandra; Behringer, Verena; Hannig, Christian; Zierau, Oliver

    2017-01-01

    Asian elephants (Elephas maximus) and African elephants (Loxodonta africana) are herbivore generalists; however, Asian elephants might ingest a higher proportion of grasses than Africans. Although some studies have investigated nutrition-specific morphological adaptations of the two species, broader studies on salivary enzymes in both elephant species are lacking. This study focuses on the comparison of salivary enzymes activity profiles in the two elephant species; these enzymes are relevant for protective and digestive functions in humans. We aimed to determine whether salivary amylase (sAA), lysozyme (sLYS), and peroxidase (sPOD) activities have changed in a species-specific pattern during evolutionary separation of the elephant genera. Saliva samples of 14 Asian and eight African elephants were collected in three German zoos. Results show that sAA and sLYS are salivary components of both elephant species in an active conformation. In contrast, little to no sPOD activity was determined in any elephant sample. Furthermore, sAA activity was significantly higher in Asian compared with African elephants. sLYS and sPOD showed no species-specific differences. The time of food provision until sample collection affected only sAA activity. In summary, the results suggest several possible factors modulating the activity of the mammal-typical enzymes, such as sAA, sLYS, and sPOD, e.g., nutrition and sampling procedure, which have to be considered when analyzing differences in saliva composition of animal species.

  7. Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

    SciTech Connect

    Mittal, Seema; Cai, Yufeng; Nalam, Madhavi N.L.; Bolon, Daniel N.A.; Schiffer, Celia A.

    2012-09-11

    Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.

  8. Evolutionary transitions in enzyme activity of ant fungus gardens.

    PubMed

    De Fine Licht, Henrik H; Schiøtt, Morten; Mueller, Ulrich G; Boomsma, Jacobus J

    2010-07-01

    Fungus-growing (attine) ants and their fungal symbionts passed through several evolutionary transitions during their 50 million year old evolutionary history. The basal attine lineages often shifted between two main cultivar clades, whereas the derived higher-attine lineages maintained an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens are targeted primarily toward partial degradation of plant cell walls, reflecting a plesiomorphic state of nondomesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major breakdown of cell walls. The adaptive significance of the lower-attine symbiont shifts remains unclear. One of these shifts was obligate, but digestive advantages remained ambiguous, whereas the other remained facultative despite providing greater digestive efficiency.

  9. Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme

    PubMed Central

    Levati, Elisabetta; Sartini, Sara; Bolchi, Angelo; Ottonello, Simone; Montanini, Barbara

    2016-01-01

    Moonlighting proteins, including metabolic enzymes acting as transcription factors (TF), are present in a variety of organisms but have not been described in higher fungi so far. In a previous genome-wide analysis of the TF repertoire of the plant-symbiotic fungus Tuber melanosporum, we identified various enzymes, including the sulfur-assimilation enzyme phosphoadenosine-phosphosulfate reductase (PAPS-red), as potential transcriptional activators. A functional analysis performed in the yeast Saccharomyces cerevisiae, now demonstrates that a specific variant of this enzyme, PAPS-red A, localizes to the nucleus and is capable of transcriptional activation. TF moonlighting, which is not present in the other enzyme variant (PAPS-red B) encoded by the T. melanosporum genome, relies on a transplantable C-terminal polypeptide containing an alternating hydrophobic/hydrophilic amino acid motif. A similar moonlighting activity was demonstrated for six additional proteins, suggesting that multitasking is a relatively frequent event. PAPS-red A is sulfur-state-responsive and highly expressed, especially in fruitbodies, and likely acts as a recruiter of transcription components involved in S-metabolism gene network activation. PAPS-red B, instead, is expressed at low levels and localizes to a highly methylated and silenced region of the genome, hinting at an evolutionary mechanism based on gene duplication, followed by epigenetic silencing of this non-moonlighting gene variant. PMID:27121330

  10. Inducible trehalase enzyme activity of Morchella conica Persoon mycelium.

    PubMed

    Czövek, Pálma; Király, I

    2011-03-01

    Morchella conica Pers. strains of the study were isolated from fruit bodies collected in ash-mixed forests. At first, the strains were cultured on potato dextrose agar (PDA), then on modified Murashige and Skoog (MS) solid agar media. A normal-growing strain was chosen for the trehalase induction experiments. During the trehalase induction treatment, mycelia were grown in liquid culture containing different concentrations of trehalose. After the induction period of trehalase enzymes, physiological state of the mycelium and the oxidative stress were monitored in the vegetative mycelia by measuring the change of the malondialdehyde content, superoxide dismutase enzyme activity, the fresh and dry weight. The examined Morchella conica strain utilized the trehalose properly. The rising amount of the trehalose triggered the increase of the mycelial trehalase enzyme activity. Our results clearly proved that both neutral and acidic trehalase isoenzyme activity of the Morchella conica mycelium are inducible and are playing important role in the utilization of external trehalose.

  11. A DNA enzyme with N-glycosylase activity

    NASA Technical Reports Server (NTRS)

    Sheppard, T. L.; Ordoukhanian, P.; Joyce, G. F.

    2000-01-01

    In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10(6)-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N-glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.

  12. The 3'-to-5' exonuclease activity of vaccinia virus DNA polymerase is essential and plays a role in promoting virus genetic recombination.

    PubMed

    Gammon, Don B; Evans, David H

    2009-05-01

    Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3'-to-5' proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3' ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDV(r)) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3'-to-5' exonuclease domain of the viral polymerase. Viruses encoding a CDV(r) mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3' ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3'-to-5' exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.

  13. Bioethanol production from leafy biomass of mango (Mangifera indica) involving naturally isolated and recombinant enzymes.

    PubMed

    Das, Saprativ P; Ravindran, Rajeev; Deka, Deepmoni; Jawed, Mohammad; Das, Debasish; Goyal, Arun

    2013-01-01

    The present study describes the usage of dried leafy biomass of mango (Mangifera indica) containing 26.3% (w/w) cellulose, 54.4% (w/w) hemicellulose, and 16.9% (w/w) lignin, as a substrate for bioethanol production from Zymomonas mobilis and Candida shehatae. The substrate was subjected to two different pretreatment strategies, namely, wet oxidation and an organosolv process. An ethanol concentration (1.21 g/L) was obtained with Z. mobilis in a shake-flask simultaneous saccharification and fermentation (SSF) trial using 1% (w/v) wet oxidation pretreated mango leaves along with mixed enzymatic consortium of Bacillus subtilis cellulase and recombinant hemicellulase (GH43), whereas C. shehatae gave a slightly higher (8%) ethanol titer of 1.31 g/L. Employing 1% (w/v) organosolv pretreated mango leaves and using Z. mobilis and C. shehatae separately in the SSF, the ethanol titers of 1.33 g/L and 1.52 g/L, respectively, were obtained. The SSF experiments performed with 5% (w/v) organosolv-pretreated substrate along with C. shehatae as fermentative organism gave a significantly enhanced ethanol titer value of 8.11 g/L using the shake flask and 12.33 g/L at the bioreactor level. From the bioreactor, 94.4% (v/v) ethanol was recovered by rotary evaporator with 21% purification efficiency.

  14. Permanent Genetic Access to Transiently Active Neurons via TRAP: Targeted Recombination in Active Populations

    PubMed Central

    Guenthner, Casey J.; Miyamichi, Kazunari; Yang, Helen H.; Heller, H. Craig; Luo, Liqun

    2013-01-01

    SUMMARY Targeting genetically encoded tools for neural circuit dissection to relevant cellular populations is a major challenge in neurobiology. We developed a new approach, Targeted Recombination in Active Populations (TRAP), to obtain genetic access to neurons that were activated by defined stimuli. This method utilizes mice in which the tamoxifen-dependent recombinase CreERT2 is expressed in an activity-dependent manner from the loci of the immediate early genes Arc and Fos. Active cells that express CreERT2 can undergo recombination only when tamoxifen is present, allowing genetic access to neurons that are active during a time window of less than 12 h. We show that TRAP can selectively provide access to neurons activated by specific somatosensory, visual, and auditory stimuli, and by experience in a novel environment. When combined with tools for labeling, tracing, recording, and manipulating neurons, TRAP offers a powerful new approach for understanding how the brain processes information and generates behavior. PMID:23764283

  15. Cloning of Bacillus licheniformis xylanase gene and characterization of recombinant enzyme

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hemicellulose is a major component of lignocellulose biomass. Complete enzymatic degradation of this substrate requires several different activities, including xylanase. We isolated a strain of Bacillus licheniformis from a hot springs environment that exhibited xylanase activity. A gene encoding...

  16. Evaluation of Babesia bigemina 200 kDa recombinant antigen in enzyme-linked immunosorbent assay.

    PubMed

    Altangerel, Khukhuu; Alhassan, Andy; Iseki, Hiroshi; Sivakumar, Thillaiampalam; Boldbaatar, Damdinsuren; Yokoyama, Naoaki; Igarashi, Ikuo

    2009-07-01

    A truncated fragment of the gene encoding the 200-kDa protein (P200) of Babesia bigemina was cloned into a plasmid vector, pGEX-4 T-1 and expressed in Escherichia coli as a glutathione-S-transferase fused protein. An indirect enzyme-linked immunosorbent assay (ELISA) using the rp200/CT detected specific antibodies in cattle experimentally infected with B. bigemina. Furthermore, the antigen did not cross-react with antibodies to Babesia bovis, a closely related Babesia parasite indicating that rp200/CT is a specific antigen for the diagnosis of B. bigemina infection. Additionally, ELISA using p200/CT and polymerase chain reaction were conducted on serum and corresponding DNA samples obtained from field cattle to evaluate the diagnostic utility of the p200/CT antigen. Results from the current study suggest that p200/CT ELISA is a sensitive and specific method for improved serodiagnosis of B. bigemina infection.

  17. Regulation of eNOS enzyme activity by posttranslational modification.

    PubMed

    Heiss, Elke H; Dirsch, Verena M

    2014-01-01

    The regulation of endothelial NO synthase (eNOS) employs multiple different cellular control mechanisms impinging on level and activity of the enzyme. This review aims at summarizing the current knowledge on the posttranslational modifications of eNOS, including acylation, nitrosylation, phosphorylation, acetylation, glycosylation and glutathionylation. Sites, mediators and impact on enzyme localization and activity of the single modifications will be discussed. Moreover, interdependence, cooperativity and competition between the different posttranslational modifications will be elaborated with special emphasis on the susceptibility of eNOS to metabolic cues.

  18. High throughput sequencing reveals alterations in the recombination signatures with diminishing Spo11 activity.

    PubMed

    Rockmill, Beth; Lefrançois, Philippe; Voelkel-Meiman, Karen; Oke, Ashwini; Roeder, G Shirleen; Fung, Jennifer C

    2013-10-01

    Spo11 is the topoisomerase-like enzyme responsible for the induction of the meiosis-specific double strand breaks (DSBs), which initiates the recombination events responsible for proper chromosome segregation. Nineteen PCR-induced alleles of SPO11 were identified and characterized genetically and cytologically. Recombination, spore viability and synaptonemal complex (SC) formation were decreased to varying extents in these mutants. Arrest by ndt80 restored these events in two severe hypomorphic mutants, suggesting that ndt80-arrested nuclei are capable of extended DSB activity. While crossing-over, spore viability and synaptonemal complex (SC) formation defects correlated, the extent of such defects was not predictive of the level of heteroallelic gene conversions (prototrophs) exhibited by each mutant. High throughput sequencing of tetrads from spo11 hypomorphs revealed that gene conversion tracts associated with COs are significantly longer and gene conversion tracts unassociated with COs are significantly shorter than in wild type. By modeling the extent of these tract changes, we could account for the discrepancy in genetic measurements of prototrophy and crossover association. These findings provide an explanation for the unexpectedly low prototroph levels exhibited by spo11 hypomorphs and have important implications for genetic studies that assume an unbiased recovery of prototrophs, such as measurements of CO homeostasis. Our genetic and physical data support previous observations of DSB-limited meioses, in which COs are disproportionally maintained over NCOs (CO homeostasis).

  19. Tissue Plasminogen Activator Neurotoxicity is Neutralized by Recombinant ADAMTS 13

    PubMed Central

    Fan, Mengchen; Xu, Haochen; Wang, Lixiang; Luo, Haiyu; Zhu, Ximin; Cai, Ping; Wei, Lixiang; Lu, Lu; Cao, Yongliang; Ye, Rong; Fan, Wenying; Zhao, Bing-Qiao

    2016-01-01

    Tissue plasminogen activator (tPA) is an effective treatment for ischemic stroke, but its neurotoxicity is a significant problem. Here we tested the hypothesis that recombinant ADAMTS 13 (rADAMTS 13) would reduce tPA neurotoxicity in a mouse model of stroke. We show that treatment with rADAMTS 13 in combination with tPA significantly reduced infarct volume compared with mice treated with tPA alone 48 hours after stroke. The combination treatment significantly improved neurological deficits compared with mice treated with tPA or vehicle alone. These neuroprotective effects were associated with significant reductions in fibrin deposits in ischemic vessels and less severe cell death in ischemic brain. The effect of rADAMTS13 on tPA neurotoxicity was mimicked by the N-methyl-D-aspartate (NMDA) receptor antagonist M-801, and was abolished by injection of NMDA. Moreover, rADAMTS 13 prevents the neurotoxicity effect of tPA, by blocking its interaction with the NMDA receptor NR2B and the attendant phosphorylation of NR2B and activation of ERK1/2. Finally, the NR2B-specific NMDA receptor antagonist ifenprodil abolished tPA neurotoxicity and rADAMTS 13 treatment had no further beneficial effect. Our data suggest that the combination of rADAMTS 13 and tPA may provide a novel treatment of ischemic stroke by diminishing the neurotoxic effects of exogenous tPA. PMID:27181025

  20. Activation and Stabilization of Olive Recombinant 13-Hydroperoxide Lyase Using Selected Additives.

    PubMed

    Jacopini, Sabrina; Vincenti, Sophie; Mariani, Magali; Brunini-Bronzini de Caraffa, Virginie; Gambotti, Claude; Desjobert, Jean-Marie; Muselli, Alain; Costa, Jean; Tomi, Félix; Berti, Liliane; Maury, Jacques

    2016-12-24

    The stabilization of olive recombinant hydroperoxide lyases (rHPLs) was investigated using selected chemical additives. Two rHPLs were studied: HPL full-length and HPL with its chloroplast transit peptide deleted (matured HPL). Both olive rHPLs are relatively stable at 4 °C, and enzyme activity can be preserved (about 100% of the rHPL activities are maintained) during 5 weeks of storage at -20 or at -80 °C in the presence of glycerol (10%, v/v). Among the additives used in this study, glycine (2.5% w/v), NaCl (0.5 M), and Na2SO4 (0.25 M) provided the highest activation of HPL full-length activity, while the best matured HPL activity was obtained with Na2SO4 (0.25 M) and NaCl (1 M). Although the inactivation rate constants (k) showed that these additives inactivate both rHPLs, their use is still relevant as they strongly increase HPL activity. Results of C6-aldehyde production assays also showed that glycine, NaCl, and Na2SO4 are appropriate additives and that NaCl appears to be the best additive, at least for hexanal production.

  1. Production of recombinant beta-hexosaminidase A, a potential enzyme for replacement therapy for Tay-Sachs and Sandhoff diseases, in the methylotrophic yeast Ogataea minuta.

    PubMed

    Akeboshi, Hiromi; Chiba, Yasunori; Kasahara, Yoshiko; Takashiba, Minako; Takaoka, Yuki; Ohsawa, Mai; Tajima, Youichi; Kawashima, Ikuo; Tsuji, Daisuke; Itoh, Kohji; Sakuraba, Hitoshi; Jigami, Yoshifumi

    2007-08-01

    Human beta-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of alpha- and beta-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using alpha-1,6-mannosyltransferase-deficient (och1Delta) yeast as the host. Genes encoding the alpha- and beta-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (alphaalpha) and HexB (betabeta). A total of 57 mg of beta-hexosaminidase isozymes, of which 13 mg was HexA (alphabeta), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the beta-subunit. The purified HexA was treated with alpha-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 +/- 0.1 and 1.7 +/- 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the beta-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed.

  2. Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities

    PubMed Central

    Harlan, Fiona Karen; Lusk, Jason Scott; Mohr, Breanna Michelle; Guzikowski, Anthony Peter; Batchelor, Robert Hardy; Jiang, Ying

    2016-01-01

    Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson’s Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and

  3. Peptide-Recombinant VP6 Protein Based Enzyme Immunoassay for the Detection of Group A Rotaviruses in Multiple Host Species

    PubMed Central

    Sircar, Subhankar; Saurabh, Sharad; Gulati, Baldev R.; Singh, Neeraj; Singh, Arvind Kumar; Joshi, Vinay G.; Banyai, Krisztian; Dhama, Kuldeep

    2016-01-01

    We developed a novel enzyme immunoassay for the detection of group A rotavirus (RVA) antigen in fecal samples of multiple host species. The assay is based on the detection of conserved VP6 protein using anti-recombinant VP6 antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immunodominant epitopes within VP6 protein) antibodies as detector antibodies. The clinical utility of the assay was evaluated using a panel of 914 diarrhoeic fecal samples from four different host species (bovine, porcine, poultry and human) collected from diverse geographical locations in India. Using VP6- based reverse transcription-polymerase chain reaction (RT-PCR) as the gold standard, we found that the diagnostic sensitivity (DSn) and specificity (DSp) of the new assay was high [bovine (DSn = 94.2% & DSp = 100%); porcine (DSn = 94.6% & DSp = 93.3%); poultry (DSn = 74.2% & DSp = 97.7%) and human (DSn = 82.1% & DSp = 98.7%)]. The concordance with RT-PCR was also high [weighted kappa (k) = 0.831–0.956 at 95% CI = 0.711–1.0] as compared to RNA-polyacrylamide gel electrophoresis (RNA-PAGE). The performance characteristics of the new immunoassay were comparable to those of the two commercially available ELISA kits. Our results suggest that this peptide-recombinant protein based assay may serve as a preliminary assay for epidemiological surveillance of RVA antigen and for evaluation of vaccine effectiveness especially in low and middle income settings. PMID:27391106

  4. Enzyme activities by indicator of quality in organic soil

    NASA Astrophysics Data System (ADS)

    Raigon Jiménez, Mo; Fita, Ana Delores; Rodriguez Burruezo, Adrián

    2016-04-01

    The analytical determination of biochemical parameters, as soil enzyme activities and those related to the microbial biomass is growing importance by biological indicator in soil science studies. The metabolic activity in soil is responsible of important processes such as mineralization and humification of organic matter. These biological reactions will affect other key processes involved with elements like carbon, nitrogen and phosphorus , and all transformations related in soil microbial biomass. The determination of biochemical parameters is useful in studies carried out on organic soil where microbial processes that are key to their conservation can be analyzed through parameters of the metabolic activity of these soils. The main objective of this work is to apply analytical methodologies of enzyme activities in soil collections of different physicochemical characteristics. There have been selective sampling of natural soils, organic farming soils, conventional farming soils and urban soils. The soils have been properly identified conserved at 4 ° C until analysis. The enzyme activities determinations have been: catalase, urease, cellulase, dehydrogenase and alkaline phosphatase, which bring together a representative group of biological transformations that occur in the soil environment. The results indicate that for natural and agronomic soil collections, the values of the enzymatic activities are within the ranges established for forestry and agricultural soils. Organic soils are generally higher level of enzymatic, regardless activity of the enzyme involved. Soil near an urban area, levels of activities have been significantly reduced. The vegetation cover applied to organic soils, results in greater enzymatic activity. So the quality of these soils, defined as the ability to maintain their biological productivity is increased with the use of cover crops, whether or spontaneous species. The practice of cover based on legumes could be used as an ideal choice

  5. Inhibitory Effects of Garcinia cambogia Extract on CYP2B6 Enzyme Activity.

    PubMed

    Yu, Jun Sang; Choi, Min Sun; Park, Jong Suk; Rehman, Shaheed Ur; Nakamura, Katsunori; Yoo, Hye Hyun

    2017-03-13

    This study assessed the inhibitory effects of Garcinia cambogia extract on the cytochrome P450 enzymes in vitro. G. cambogia extract was incubated with cytochrome P450 isozyme-specific substrates in human liver microsomes and recombinant CYP2B6 isozyme, and the formation of the marker metabolites was measured to investigate the inhibitory potential on cytochrome P450 enzyme activities. The results showed that G. cambogia extract has significant inhibitory effects on CYP2B6 activity in a concentration-dependent manner. Furthermore, the inhibition was potentiated following preincubation with NADPH, indicating that G. cambogia extract is a time-dependent inhibitor of CYP2B6. Meanwhile, hydroxycitric acid, the major bioactive ingredient of G. cambogia extract, did not exhibit significant inhibition effects on cytochrome P450 enzyme activities. G. cambogia extract could modulate the pharmacokinetics of CYP2B6 substrate drugs and lead to interactions with those drugs. Therefore, caution may be required with respect to concomitant intake of dietary supplements containing G. cambogia extract with CYP2B6 substrates.

  6. Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of Alcaligenes faecalis. Cofactor assembly and catalytic properties of recombinant enzyme expressed in Paracoccus denitrificans.

    PubMed

    Hothi, Parvinder; Khadra, Khalid Abu; Combe, Jonathan P; Leys, David; Scrutton, Nigel S

    2005-11-01

    The heterologous expression of tryptophan trytophylquinone (TTQ)-dependent aromatic amine dehydrogenase (AADH) has been achieved in Paracoccus denitrificans. The aauBEDA genes and orf-2 from the aromatic amine utilization (aau) gene cluster of Alcaligenes faecalis were placed under the regulatory control of the mauF promoter from P. denitrificans and introduced into P. denitrificans using a broad-host-range vector. The physical, spectroscopic and kinetic properties of the recombinant AADH were indistinguishable from those of the native enzyme isolated from A. faecalis. TTQ biogenesis in recombinant AADH is functional despite the lack of analogues in the cloned aau gene cluster for mauF, mauG, mauL, mauM and mauN that are found in the methylamine utilization (mau) gene cluster of a number of methylotrophic organisms. Steady-state reaction profiles for recombinant AADH as a function of substrate concentration differed between 'fast' (tryptamine) and 'slow' (benzylamine) substrates, owing to a lack of inhibition by benzylamine at high substrate concentrations. A deflated and temperature-dependent kinetic isotope effect indicated that C-H/C-D bond breakage is only partially rate-limiting in steady-state reactions with benzylamine. Stopped-flow studies of the reductive half-reaction of recombinant AADH with benzylamine demonstrated that the KIE is elevated over the value observed in steady-state turnover and is independent of temperature, consistent with (a) previously reported studies with native AADH and (b) breakage of the substrate C-H bond by quantum mechanical tunnelling. The limiting rate constant (k(lim)) for TTQ reduction is controlled by a single ionization with pK(a) value of 6.0, with maximum activity realized in the alkaline region. Two kinetically influential ionizations were identified in plots of k(lim)/K(d) of pK(a) values 7.1 and 9.3, again with the maximum value realized in the alkaline region. The potential origin of these kinetically influential

  7. Structurally unique recombinant Kazal-type proteinase inhibitor retains activity when terminally extended and glycosylated.

    PubMed

    Kludkiewicz, Barbara; Kodrík, Dalibor; Grzelak, Krystyna; Nirmala, Xavier; Sehnal, Frantisek

    2005-10-01

    Recombinant derivatives of the Kazal-type serine proteinase inhibitor GmSPI2 (36 amino acid residues), which is a component of insect silk, were prepared in the expression vector Pichia pastoris. The rhSPI2 had a C-terminal hexahistidine tag attached to the GmSPI2 sequence, rtSPI2 was extended with GluAlaAla at the N-terminus, and rfSPI2 included this N-terminal extension and a C-terminal tail of 22 residues (myc epitope and hexahistidine). A portion of the secreted rfSI2 was O-glycosylated with a trimannosyl or hexamannosyl. The native inhibitor was active slightly on trypsin and highly on subtilisin and proteinase K. The extended C-terminus in rhSPI2 and rfSPI2 enhanced activity on the two latter enzymes and rendered rfSPI2 active on elastase and pronase, but abolished the inhibition of trypsin. The glycosylation of rfSPI2 reduced its inhibitory activity to a level comparable with the native inhibitor. The rtSPI2 with tripeptide extension at the N-terminus and no C-terminal modification was clearly less active than the native inhibitor. None of the tested compounds inhibited alpha-chymotrypsin and the non-serine proteinases.

  8. Variation in Soil Enzyme Activities in a Temperate Agroforestry Watershed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Integration of agroforestry and grass buffers into row crop watersheds improves overall environmental quality, including soil quality. The objective of this study was to examine management and landscape effects on soil carbon, soil nitrogen, microbial diversity, enzyme activity, and DNA concentrati...

  9. Molecular characterization of a cold-active recombinant xylanase from Flavobacterium johnsoniae and its applicability in xylan hydrolysis

    PubMed Central

    Chen, Shicheng; Kaufman, Michael G.; Miazgowicz, Kerri L.; Bagdasarian, Michael; Walker, Edward D.

    2014-01-01

    A novel xylanase gene, xyn10A, was cloned from Flavobacterium johsoniae, overexpressed in a flavobacterial expression system, the recombinant enzyme purified by Ni-affinity chromatography, and enzyme structure and activity analyzed. Xyn10A was found to be a modular xylanase with an Fn3 accessory domain on its N-terminal and a catalytic region on the C-terminal. The optimum pH and temperature for Xyn10A was 8.0 and 30° C, but Xyn10A retained 50% activity at 4°C, indicating that Xyn10A is a cold-active xylanase. A Fn3-deletion xylanase had relative activity ca. 3.6-fold lower than the wild-type, indicating that Fn3 promotes xylanase activity. The Fn3 region also contributed to stability of the enzyme at elevated temperatures. However, Fn3 did not bind this xylanase to insoluble substrates. The enzyme hydrolyzed xylo-oligosaccharides into xylobiose, and xylose with xylobiose as the main product, confirming that Xyn10A is a strict endo-β-1,4-xylanase. Xyn10A also hydrolyzed birchwood and beechwood xylan to yield mainly xylose, xylobiose and xylotriose. PMID:23196234

  10. Chemoprotective activity of boldine: modulation of drug-metabolizing enzymes.

    PubMed

    Kubínová, R; Machala, M; Minksová, K; Neca, J; Suchý, V

    2001-03-01

    Possible chemoprotective effects of the naturally occurring alkaloid boldine, a major alkaloid of boldo (Peumus boldus Mol.) leaves and bark, including in vitro modulations of drug-metabolizing enzymes in mouse hepatoma Hepa-1 cell line and mouse hepatic microsomes, were investigated. Boldine manifested inhibition activity on hepatic microsomal CYP1A-dependent 7-ethoxyresorufin O-deethylase and CYP3A-dependent testosterone 6 beta-hydroxylase activities and stimulated glutathione S-transferase activity in Hepa-1 cells. In addition to the known antioxidant activity, boldine could decrease the metabolic activation of other xenobiotics including chemical mutagens.

  11. [Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

    PubMed

    Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

    2015-09-01

    Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

  12. Giardia, Entamoeba, and Trichomonas enzymes activate metronidazole (nitroreductases) and inactivate metronidazole (nitroimidazole reductases).

    PubMed

    Pal, Dibyarupa; Banerjee, Sulagna; Cui, Jike; Schwartz, Aaron; Ghosh, Sudip K; Samuelson, John

    2009-02-01

    Infections with Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis, which cause diarrhea, dysentery, and vaginitis, respectively, are each treated with metronidazole. Here we show that Giardia, Entamoeba, and Trichomonas have oxygen-insensitive nitroreductase (ntr) genes which are homologous to those genes that have nonsense mutations in metronidazole-resistant Helicobacter pylori isolates. Entamoeba and Trichomonas also have nim genes which are homologous to those genes expressed in metronidazole-resistant Bacteroides fragilis isolates. Recombinant Giardia, Entamoeba, and Trichomonas nitroreductases used NADH rather than the NADPH used by Helicobacter, and two recombinant Entamoeba nitroreductases increased the metronidazole sensitivity of transformed Escherichia coli strains. Conversely, the recombinant nitroimidazole reductases (NIMs) of Entamoeba and Trichmonas conferred very strong metronidazole resistance to transformed bacteria. The Ehntr1 gene of the genome project HM-1:IMSS strain of Entamoeba histolytica had a nonsense mutation, and the same nonsense mutation was present in 3 of 22 clinical isolates of Entamoeba. While ntr and nim mRNAs were variably expressed by cultured Entamoeba and Trichomonas isolates, there was no relationship to metronidazole sensitivity. We conclude that microaerophilic protists have bacterium-like enzymes capable of activating metronidazole (nitroreductases) and inactivating metronidazole (NIMs). While Entamoeba and Trichomonas displayed some of the changes (nonsense mutations and gene overexpression) associated with metronidazole resistance in bacteria, these changes did not confer metronidazole resistance to the microaerophilic protists examined here.

  13. Carotenoid-cleavage activities of crude enzymes from Pandanous amryllifolius.

    PubMed

    Ningrum, Andriati; Schreiner, Matthias

    2014-11-01

    Carotenoid degradation products, known as norisoprenoids, are aroma-impact compounds in several plants. Pandan wangi is a common name of the shrub Pandanus amaryllifolius. The genus name 'Pandanus' is derived from the Indonesian name of the tree, pandan. In Indonesia, the leaves from the plant are used for several purposes, e.g., as natural colorants and flavor, and as traditional treatments. The aim of this study was to determine the cleavage of β-carotene and β-apo-8'-carotenal by carotenoid-cleavage enzymes isolated from pandan leaves, to investigate dependencies of the enzymatic activities on temperature and pH, to determine the enzymatic reaction products by using Headspace Solid Phase Microextraction Gas Chromatography/Mass Spectrophotometry (HS-SPME GC/MS), and to investigate the influence of heat treatment and addition of crude enzyme on formation of norisoprenoids. Crude enzymes from pandan leaves showed higher activity against β-carotene than β-apo-8'-carotenal. The optimum temperature of crude enzymes was 70°, while the optimum pH value was 6. We identified β-ionone as the major volatile reaction product from the incubations of two different carotenoid substrates, β-carotene and β-apo-8'-carotenal. Several treatments, e.g., heat treatment and addition of crude enzymes in pandan leaves contributed to the norisoprenoid content. Our findings revealed that the crude enzymes from pandan leaves with carotenoid-cleavage activity might provide a potential application, especially for biocatalysis, in natural-flavor industry.

  14. Inhibition of the recBCD-dependent activation of Chi recombinational hot spots in SOS-induced cells of Escherichia coli.

    PubMed Central

    Rinken, R; Wackernagel, W

    1992-01-01

    Nucleotide sequences called Chi (5'-GCTGGTGG-3') enhance homologous recombination near their location by the RecBCD enzyme in Escherichia coli (Chi activation). A partial inhibition of Chi activation measured in lambda red gam mutant crosses was observed after treatment of wild-type cells with DNA-damaging agents including UV, mitomycin, and nalidixic acid. Inhibition of Chi activation was not accompanied by an overall decrease of recombination. A lexA3 mutation which blocks induction of the SOS system prevented the inhibition of Chi activation, indicating that an SOS function could be responsible for the inhibition. Overproduction of the RecD subunit of the RecBCD enzyme from a multicopy plasmid carrying the recD gene prevented the induced inhibition of Chi activation, whereas overproduction of RecB or RecC subunits did not. It is proposed that in SOS-induced cells the RecBCD enzyme is modified into a Chi-independent recombination enzyme, with the RecD subunit being the regulatory switch key. PMID:1310498

  15. Recombinant microorganisms for increased production of organic acids

    DOEpatents

    Yi, Jian; Kleff, Susanne; Guettler, Michael V

    2013-04-30

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  16. Recombinant microorganisms for increased production of organic acids

    DOEpatents

    Yi, Jian [East Lansing, MI; Kleff, Susanne [East Lansing, MI; Guettler, Michael V [Holt, MI

    2012-02-21

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  17. Transaldolase B of Escherichia coli K-12: cloning of its gene, talB, and characterization of the enzyme from recombinant strains.

    PubMed Central

    Sprenger, G A; Schörken, U; Sprenger, G; Sahm, H

    1995-01-01

    A previously recognized open reading frame (T. Yura, H. Mori, H. Nagai, T. Nagata, A. Ishihama, N. Fujita, K. Isono, K. Mizobuchi, and A. Nakata, Nucleic Acids Res. 20:3305-3308) from the 0.2-min region of the Escherichia coli K-12 chromosome is shown to encode a functional transaldolase activity. After cloning of the gene onto high-copy-number vectors, transaldolase B (D-sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate dihydroxyacetone transferase; EC 2.2.1.2) was overexpressed up to 12.7 U mg of protein-1 compared with less than 0.1 U mg of protein-1 in wild-type homogenates. The enzyme was purified from recombinant E. coli K-12 cells by successive ammonium sulfate precipitations (45 to 80% and subsequently 55 to 70%) and two anion-exchange chromatography steps (Q-Sepharose FF, Fractogel EMD-DEAE tentacle column; yield, 130 mg of protein from 12 g of cell wet weight) and afforded an apparently homogeneous protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a subunit size of 35,000 +/- 1,000 Da. As the enzyme had a molecular mass of 70,000 Da by gel filtration, transaldolase B is likely to form a homodimer. N-terminal amino acid sequencing of the protein verified its identity with the product of the cloned gene talB. The specific activity of the purified enzyme determined at 30 degrees C with the substrates fructose-6-phosphate (donor of C3 compound) and erythrose-4-phosphate (acceptor) at an optimal pH (50 mM glycylglycine [pH 8.5]) was 60 U mg-1.Km values for the substrates fructose-6-phosphate and erythrose-4-phosphate were determined at 1,200 and 90 microM, respectively. Kinetic constants for the other two physiological reactants, D,L-glyceraldehyde 3-phosphate (Km, 38 microM; relative activity [V(rel)], 8%) and sedoheptulose-7-phosphate (K(m), 285 microM; V(rel), 5%) were also determined. Fructose acted as a C(3) donor at a high apparent K(m) (>/=M) and with a V(rel) of 12%. The enzyme was inhibited by Tris-HCl, phosphate, or

  18. Expression of recombinant organophosphorus hydrolase in the original producer of the enzyme, Sphingobium fuliginis ATCC 27551.

    PubMed

    Nakayama, Kosuke; Ohmori, Takeshi; Ishikawa, Satoshi; Iwata, Natsumi; Seto, Yasuo; Kawahara, Kazuyoshi

    2016-05-01

    The plasmid encoding His-tagged organophosphorus hydrolase (OPH) cloned from Sphingobium fuliginis was modified to be transferred back to this bacterium. The replication function of S. amiense plasmid was inserted at downstream of OPH gene, and S. fuliginis was transformed with this plasmid. The transformant produced larger amount of active OPH with His-tag than E. coli.

  19. Chronic administration of recombinant IL-6 upregulates lipogenic enzyme expression and aggravates high-fat-diet-induced steatosis in IL-6-deficient mice.

    PubMed

    Vida, Margarita; Gavito, Ana Luisa; Pavón, Francisco Javier; Bautista, Dolores; Serrano, Antonia; Suarez, Juan; Arrabal, Sergio; Decara, Juan; Romero-Cuevas, Miguel; Rodríguez de Fonseca, Fernando; Baixeras, Elena

    2015-07-01

    Interleukin-6 (IL-6) has emerged as an important mediator of fatty acid metabolism with paradoxical effects in the liver. Administration of IL-6 has been reported to confer protection against steatosis, but plasma and tissue IL-6 concentrations are elevated in chronic liver diseases, including fatty liver diseases associated with obesity and alcoholic ingestion. In this study, we further investigated the role of IL-6 on steatosis induced through a high-fat diet (HFD) in wild-type (WT) and IL-6-deficient (IL-6(-/-)) mice. Additionally, HFD-fed IL-6(-/-) mice were also chronically treated with recombinant IL-6 (rIL-6). Obesity in WT mice fed a HFD associated with elevated serum IL-6 levels, fatty liver, upregulation of carnitine palmitoyltransferase 1 (CPT1) and signal transducer and activator of transcription-3 (STAT3), increased AMP kinase phosphorylation (p-AMPK), and downregulation of the hepatic lipogenic enzymes fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1). The HFD-fed IL-6(-/-) mice showed severe steatosis, no changes in CPT1 levels or AMPK activity, no increase in STAT3 amounts, inactivated STAT3, and marked downregulation of the expression of acetyl-CoA carboxylase (ACCα/β), FAS and SCD1. The IL-6 chronic replacement in HFD-fed IL-6 -/-: mice restored hepatic STAT3 and AMPK activation but also increased the expression of the lipogenic enzymes ACCα/β, FAS and SCD1. Furthermore, rIL-6 administration was associated with aggravated steatosis and elevated fat content in the liver. We conclude that, in the context of HFD-induced obesity, the administration of rIL-6 might contribute to the aggravation of fatty liver disease through increasing lipogenesis.

  20. Chronic administration of recombinant IL-6 upregulates lipogenic enzyme expression and aggravates high-fat-diet-induced steatosis in IL-6-deficient mice

    PubMed Central

    Vida, Margarita; Gavito, Ana Luisa; Pavón, Francisco Javier; Bautista, Dolores; Serrano, Antonia; Suarez, Juan; Arrabal, Sergio; Decara, Juan; Romero-Cuevas, Miguel; Rodríguez de Fonseca, Fernando; Baixeras, Elena

    2015-01-01

    ABSTRACT Interleukin-6 (IL-6) has emerged as an important mediator of fatty acid metabolism with paradoxical effects in the liver. Administration of IL-6 has been reported to confer protection against steatosis, but plasma and tissue IL-6 concentrations are elevated in chronic liver diseases, including fatty liver diseases associated with obesity and alcoholic ingestion. In this study, we further investigated the role of IL-6 on steatosis induced through a high-fat diet (HFD) in wild-type (WT) and IL-6-deficient (IL-6−/−) mice. Additionally, HFD-fed IL-6−/− mice were also chronically treated with recombinant IL-6 (rIL-6). Obesity in WT mice fed a HFD associated with elevated serum IL-6 levels, fatty liver, upregulation of carnitine palmitoyltransferase 1 (CPT1) and signal transducer and activator of transcription-3 (STAT3), increased AMP kinase phosphorylation (p-AMPK), and downregulation of the hepatic lipogenic enzymes fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1). The HFD-fed IL-6−/− mice showed severe steatosis, no changes in CPT1 levels or AMPK activity, no increase in STAT3 amounts, inactivated STAT3, and marked downregulation of the expression of acetyl-CoA carboxylase (ACCα/β), FAS and SCD1. The IL-6 chronic replacement in HFD-fed IL-6−/− mice restored hepatic STAT3 and AMPK activation but also increased the expression of the lipogenic enzymes ACCα/β, FAS and SCD1. Furthermore, rIL-6 administration was associated with aggravated steatosis and elevated fat content in the liver. We conclude that, in the context of HFD-induced obesity, the administration of rIL-6 might contribute to the aggravation of fatty liver disease through increasing lipogenesis. PMID:26035386

  1. Myelostimulatory activity of recombinant human interleukin-2 in mice

    SciTech Connect

    Talmadge, J.E.; Schneider, M.; Keller, J.; Ruscetti, F.; Longo, D.; Pennington, R.; Bowersox, O.; Tribble, H.

    1989-05-01

    In a series of studies designed to extend our understanding of interleukin-2 (IL-2) and to study the effect of biologic response modifiers on bone marrow, we observed that administering recombinant human (rH) IL-2 to normal mice resulted in an increase in the frequency of colony-forming units-culture (CFU-C) in bone marrow. In addition, rH IL-2 was able to accelerate host recovery from cyclophosphamide (CTX)- or radiation-induced bone marrow depression and peripheral blood leukopenia. Not only can rH IL-2 accelerate, in a dose-dependent manner, the return of bone marrow, peripheral blood cellularity, and CFU-C frequency to normal levels following cytoreduction by CTX or irradiation, but it also significantly increases CFU-C frequency to greater than normal levels. Furthermore, rH IL-2 can significantly prolong survival of animals receiving a lethal dose of irradiation or CTX. Thus, multiple mechanisms are responsible for the synergistic therapeutic activity associated with rH IL-2 and CTX. rH IL-2 does not act only as an immunomodulatory agent in the presence or absence of suppressor T cells, but also accelerates host recovery from cytoreductive agents, resulting in decreased leukopenia and perhaps resistances to secondary infection. Thus, rH IL-2 plus chemotherapy may increase therapeutic activity against neoplastic disease, not only by adding immune stimulation to the direct antitumor effect of the drug but also by allowing delivery of higher, more effective doses of chemotherapy.

  2. The active site structure and mechanism of phosphoenolpyruvate utilizing enzymes

    SciTech Connect

    Cheng, K.C.

    1989-01-01

    Arginine specific reagents showed irreversible inhibition of avian liver mitochondrial phosphoenolpyruvate carboxykinase. Potent protection against modification was elicited by CO{sub 2} or CO{sub 2} in the presence of other substrates. Labeling of enzyme with (7-{sup 14}C) phenylglyoxal showed that 1 or 2 arginines are involved in CO{sub 2} binding and activation. Peptide map studies showed this active site arginine residues is located at position 289. Histidine specific reagents showed pseudo first order inhibition of avian mitochondrial phosphoenolpyruvate carboxykinase activity. The best protection against modification was elicited by IDP or IDP and Mn{sup +2}. One histidine residue is at or near the phosphoenolpyruvate binding site as demonstrated in the increased absorbance at 240 nm and proton relaxation rate studies. Circular dichroism studies reveal that enzyme structure was perturbed by diethylpyrocarbonate modification. Metal binding studies suggest that this enzyme has only one metal binding site. The putative binding sites from several GTP and phosphoenolpyruvate utilizing enzymes are observed in P-enolpyruvate carboxykinase from different species.

  3. High-throughput screening to estimate single or multiple enzymes involved in drug metabolism: microtitre plate assay using a combination of recombinant CYP2D6 and human liver microsomes.

    PubMed

    Yamamoto, T; Suzuki, A; Kohno, Y

    2003-08-01

    1. The purpose of this study was to estimate readily involvement of single or multiple enzymes in the metabolism of a drug through inhibitory assessment. Inhibitory effects of various compounds on CYP2D6 activity assayed by formation of fluorescent metabolite from 3-[2-(N,N-diethyl-N-methyl-ammonium)ethyl]-7-methoxy-4-methyl-coumarin (AMMC) were assessed using microtitre plate (MTP) assays with a combination of recombinant CYP2D6 and human liver microsomes (HLM). 2. Among various compounds studied, antipsychotic drugs extensively inhibited recombinant CYP2D6 activity and the IC50 values were generally lower than those of antidepressants and antiarrhythmic drugs. 3. After pre-incubation, the IC50 values of mianserin, chlorpromadine, risperidone, thioridazine, alprenolol, propafenone and dextromethorphan increased but the values of timolol, S-metoprolol and propranolol substantially decreased compared with those in case of co-incubation. 4. The IC50 values of typical substrates of CYP2D6 (bufuralol and dextromethorphan at lower substrate concentration) in inhibition studies using HLM, were similar to those in the case of recombinant CYP2D6, but the values of the compounds that are metabolized by multiple CYP forms (perphenazine and chlorpromazine) in HLM were much larger. 5. If the ratio (HLM/rCYP ratio) of IC50 values between HLM and recombinant CYP2D6 exceeds approximately 2, it suggests that other CYP forms in addition to CYP2D6 might be involved in the metabolism of the test compounds. From the advantage such as speed, high throughput and ease of the technique, the MTP assay using a combination of the recombinant CYP2D6 and HLM is useful to estimate the involvement of single or multiple enzymes in the metabolism of drugs at the stage of drug discovery.

  4. Quantitative determination of recombinant bovine somatotropin in commercial shrimp feed using a competitive enzyme-linked immunosorbent assay.

    PubMed

    Munro, James L; Boon, Virginia A

    2010-02-10

    Recombinant bovine somatotropin (rbST), also known as growth hormone, is used to enhance production and development of animals within the agriculture and aquaculture industries. Its use is controversial because of its potential effects on human and animal health. To screen for rbST in shrimp feed, a competitive enzyme-linked immunosorbent assay (ELISA) with an inhibition step was developed. Sample and rbST antibody (rabbit anti-rbST) were incubated at room temperature for 30 min. Subsequently, this competitive reaction was transferred to a microplate coated with rbST, using goat antirabbit IgG linked with horseradish peroxidise as the secondary antibody. Substrates for peroxidise were added, and the absorbance at 410 nm was determined. The applicability of the method was assessed using rbST extracted from "spiked" shrimp feed samples. The assay was reproducible and linear with R(2) values greater than 0.98 over the standard curve range of 20-500 microg/g. The intra- and interday precisions expressed as relative standard deviations were 3.4 and 5.3%, respectively. The mean recovery from 15 spiked feed samples was 105%. This assay will be a valuable tool for quantitative detection of rbST by both governments and commercial companies and can be modified for other types of feed.

  5. Evaluation of a recombinant rhoptry protein 2 enzyme-linked immunoassay for the diagnosis of toxoplasmosis acquired during pregnancy

    PubMed Central

    Capobiango, Jaqueline Dario; Pagliari, Sthefany; Pasquali, Aline Kuhn Sbruzzi; Nino, Beatriz; Ferreira, Fernanda Pinto; Monica, Thaís Cabral; Tschurtschenthaler, Nely Norder; Navarro, Italmar Teodorico; Garcia, João Luis; Mitsuka-Breganó, Regina; Reiche, Edna Maria Vissoci

    2015-01-01

    The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detectToxoplasma gondii ROP2-specific IgG in samples from pregnant women. The study included 236 samples that were divided into groups according to serological screening profiles for toxoplasmosis: unexposed (n = 65), probable acute infection (n = 48), possible acute infection (n = 58) and exposed to the parasite (n = 65). When an indirect immunofluorescence assay forT. gondii-specific IgG was considered as a reference test, the ELISA-rROP2 had a sensitivity of 61.8%, specificity of 62.8%, predictive positive value of 76.6% and predictive negative value of 45.4% (p = 0.0002). The ELISA-rROP2 reacted with 62.5% of the samples from pregnant women with probable acute infection and 40% of the samples from pregnant women with previous exposure (p = 0.0180). Seropositivity was observed in 50/57 (87.7%) pregnant women with possible infection. The results underscored that T. gondii rROP2 is recognised by specific IgG antibodies in both the acute and chronic phases of toxoplasmosis acquired during pregnancy. However, the sensitivity of the ELISA-rROP2 was higher in the pregnant women with probable and possible acute infections and IgM reactivity. PMID:26517651

  6. Evaluation of a recombinant rhoptry protein 2 enzyme-linked immunoassay for the diagnosis of toxoplasmosis acquired during pregnancy.

    PubMed

    Capobiango, Jaqueline Dario; Pagliari, Sthefany; Pasquali, Aline Kuhn Sbruzzi; Nino, Beatriz; Ferreira, Fernanda Pinto; Monica, Thaís Cabral; Tschurtschenthaler, Nely Norder; Navarro, Italmar Teodorico; Garcia, João Luis; Mitsuka-Breganó, Regina; Reiche, Edna Maria Vissoci

    2015-09-01

    The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detect Toxoplasma gondii ROP2-specific IgG in samples from pregnant women. The study included 236 samples that were divided into groups according to serological screening profiles for toxoplasmosis: unexposed (n = 65), probable acute infection (n = 48), possible acute infection (n = 58) and exposed to the parasite (n = 65). When an indirect immunofluorescence assay forT. gondii-specific IgG was considered as a reference test, the ELISA-rROP2 had a sensitivity of 61.8%, specificity of 62.8%, predictive positive value of 76.6% and predictive negative value of 45.4% (p = 0.0002). The ELISA-rROP2 reacted with 62.5% of the samples from pregnant women with probable acute infection and 40% of the samples from pregnant women with previous exposure (p = 0.0180). Seropositivity was observed in 50/57 (87.7%) pregnant women with possible infection. The results underscored that T. gondii rROP2 is recognised by specific IgG antibodies in both the acute and chronic phases of toxoplasmosis acquired during pregnancy. However, the sensitivity of the ELISA-rROP2 was higher in the pregnant women with probable and possible acute infections and IgM reactivity.

  7. Comparison of a recombinant-antigen enzyme immunoassay with Treponema pallidum hemagglutination test for serological confirmation of syphilis.

    PubMed

    Rodríguez, Islay; Alvarez, Elvio L; Fernández, Carmen; Miranda, Alina

    2002-04-01

    A recombinant-antigen enzyme immunoassay (EIA), BioSCREEN anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38), secondary (n = 10), early latent (n = 28) and congenital syphilis (n = 2), patients with leptospirosis ( n= 8), infectious mononucleosis (n = 7), hepatitis (n = 9), diabetes mellitus (n = 11), rheumatoid arthritis (n = 13), leprosy (n = 11), tuberculosis (n = 9), HIV/Aids ( n= 12), systemic lupus erythematosus (n = 4), rheumatic fever (n = 3), old-persons (n = 9), pregnant women (n = 29) and blood donors (n = 164). The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.

  8. Micropollutant degradation via extracted native enzymes from activated sludge.

    PubMed

    Krah, Daniel; Ghattas, Ann-Kathrin; Wick, Arne; Bröder, Kathrin; Ternes, Thomas A

    2016-05-15

    A procedure was developed to assess the biodegradation of micropollutants in cell-free lysates produced from activated sludge of a municipal wastewater treatment plant (WWTP). This proof-of-principle provides the basis for further investigations of micropollutant biodegradation via native enzymes in a solution of reduced complexity, facilitating downstream protein analysis. Differently produced lysates, containing a variety of native enzymes, showed significant enzymatic activities of acid phosphatase, β-galactosidase and β-glucuronidase in conventional colorimetric enzyme assays, whereas heat-deactivated controls did not. To determine the enzymatic activity towards micropollutants, 20 compounds were spiked to the cell-free lysates under aerobic conditions and were monitored via LC-ESI-MS/MS. The micropollutants were selected to span a wide range of different biodegradabilities in conventional activated sludge treatment via distinct primary degradation reactions. Of the 20 spiked micropollutants, 18 could be degraded by intact sludge under assay conditions, while six showed reproducible degradation in the lysates compared to the heat-deactivated negative controls: acetaminophen, N-acetyl-sulfamethoxazole (acetyl-SMX), atenolol, bezafibrate, erythromycin and 10,11-dihydro-10-hydroxycarbamazepine (10-OH-CBZ). The primary biotransformation of the first four compounds can be attributed to amide hydrolysis. However, the observed biotransformations in the lysates were differently influenced by experimental parameters such as sludge pre-treatment and the addition of ammonium sulfate or peptidase inhibitors, suggesting that different hydrolase enzymes were involved in the primary degradation, among them possibly peptidases. Furthermore, the transformation of 10-OH-CBZ to 9-CA-ADIN was caused by a biologically-mediated oxidation, which indicates that in addition to hydrolases further enzyme classes (probably oxidoreductases) are present in the native lysates. Although the

  9. Chi hotspots trigger a conformational change in the helicase-like domain of AddAB to activate homologous recombination

    PubMed Central

    Gilhooly, Neville S.; Carrasco, Carolina; Gollnick, Benjamin; Wilkinson, Martin; Wigley, Dale B.; Moreno-Herrero, Fernando; Dillingham, Mark S.

    2016-01-01

    In bacteria, the repair of double-stranded DNA breaks is modulated by Chi sequences. These are recognised by helicase-nuclease complexes that process DNA ends for homologous recombination. Chi activates recombination by changing the biochemical properties of the helicase-nuclease, transforming it from a destructive exonuclease into a recombination-promoting repair enzyme. This transition is thought to be controlled by the Chi-dependent opening of a molecular latch, which enables part of the DNA substrate to evade degradation beyond Chi. Here, we show that disruption of the latch improves Chi recognition efficiency and stabilizes the interaction of AddAB with Chi, even in mutants that are impaired for Chi binding. Chi recognition elicits a structural change in AddAB that maps to a region of AddB which resembles a helicase domain, and which harbours both the Chi recognition locus and the latch. Mutation of the latch potentiates the change and moderately reduces the duration of a translocation pause at Chi. However, this mutant displays properties of Chi-modified AddAB even in the complete absence of bona fide hotspot sequences. The results are used to develop a model for AddAB regulation in which allosteric communication between Chi binding and latch opening ensures quality control during recombination hotspot recognition. PMID:26762979

  10. Microbial Community Structure and Enzyme Activities in Semiarid Agricultural Soils

    NASA Astrophysics Data System (ADS)

    Acosta-Martinez, V. A.; Zobeck, T. M.; Gill, T. E.; Kennedy, A. C.

    2002-12-01

    The effect of agricultural management practices on the microbial community structure and enzyme activities of semiarid soils of different textures in the Southern High Plains of Texas were investigated. The soils (sandy clay loam, fine sandy loam and loam) were under continuous cotton (Gossypium hirsutum L.) or in rotations with peanut (Arachis hypogaea L.), sorghum (Sorghum bicolor L.) or wheat (Triticum aestivum L.), and had different water management (irrigated or dryland) and tillage (conservation or conventional). Microbial community structure was investigated using fatty acid methyl ester (FAME) analysis by gas chromatography and enzyme activities, involved in C, N, P and S cycling of soils, were measured (mg product released per kg soil per h). The activities of b-glucosidase, b-glucosaminidase, alkaline phosphatase, and arylsulfatase were significantly (P<0.05) increased in soils under cotton rotated with sorghum or wheat, and due to conservation tillage in comparison to continuous cotton under conventional tillage. Principal component analysis showed FAME profiles of these soils separated distinctly along PC1 (20 %) and PC2 (13 %) due to their differences in soil texture and management. No significant differences were detected in FAME profiles due to management practices for the same soils in this sampling period. Enzyme activities provide early indications of the benefits in microbial populations and activities and soil organic matter under crop rotations and conservation tillage in comparison to the typical practices in semiarid regions of continuous cotton and conventional tillage.

  11. Novel Feruloyl Esterase from Lactobacillus fermentum NRRL B-1932 and Analysis of the Recombinant Enzyme Produced in Escherichia coli

    PubMed Central

    Bischoff, Kenneth M.; Anderson, Amber M.; Rich, Joseph O.

    2016-01-01

    ABSTRACT A total of 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity using agar plates containing ethyl ferulate as the sole carbon source, and Lactobacillus fermentum NRRL B-1932 demonstrated the strongest FE activity among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate. FE activities were monitored using high-performance liquid chromatography with an acetonitrile-trifluoroacetic acid gradient. To produce sufficient purified FE from L. fermentum strain NRRL B-1932 (LfFE), the cDNA encoding LfFE (Lffae) was amplified and cloned by using available closely related genome sequences and overexpressed in Escherichia coli. A 29.6-kDa LfFE protein was detected from the protein extract of E. coli BL21(pLysS) carrying pET28bLffae upon IPTG (isopropyl-β-d-thiogalactopyranoside) induction. The recombinant LfFE containing a polyhistidine tag was purified by nickel-nitrilotriacetic acid affinity resin. The purified LfFE showed strong activities against several artificial substrates, including p-nitrophenyl acetate and 4-methylumbelliferyl p-trimethylammoniocinnamate chloride. The optimum pH and temperature of the recombinant LfFE were around 6.5 and 37°C, respectively, as determined using either crude or purified recombinant LfFE. This study will be essential for the production of the LfFE in E. coli on a larger scale that could not be readily achieved by L. fermentum fermentation. IMPORTANCE The production of feruloyl esterase (FE) from Lactobacillus fermentum NRRL B-1932 reported in this study will have immense potential commercial applications not only in biofuel production but also in pharmaceutical, polymer, oleo chemical, cosmetic additive, and detergent industries, as well as human health-related applications, including food flavoring, functional foods, probiotic agents, preventive medicine, and animal feed. Given the essential role FE plays in the production of hydroxycinnamic acids and ferulic acid

  12. Functionally diverse biotin-dependent enzymes with oxaloacetate decarboxylase activity.

    PubMed

    Lietzan, Adam D; St Maurice, Martin

    2014-02-15

    Biotin-dependent enzymes catalyze carboxylation, decarboxylation and transcarboxylation reactions that participate in the primary metabolism of a wide range of organisms. In all cases, the overall reaction proceeds via two half reactions that take place in physically distinct active sites. In the first half-reaction, a carboxyl group is transferred to the 1-N' of a covalently tethered biotin cofactor. The tethered carboxybiotin intermediate subsequently translocates to a second active site where the carboxyl group is either transferred to an acceptor substrate or, in some bacteria and archaea, is decarboxylated to biotin and CO2 in order to power the export of sodium ions from the cytoplasm. A homologous carboxyltransferase domain is found in three enzymes that catalyze diverse overall reactions: carbon fixation by pyruvate carboxylase, decarboxylation and sodium transport by the biotin-dependent oxaloacetate decarboxylase complex, and transcarboxylation by transcarboxylase from Propionibacterium shermanii. Over the past several years, structural data have emerged which have greatly advanced the mechanistic description of these enzymes. This review assembles a uniform description of the carboxyltransferase domain structure and catalytic mechanism from recent studies of pyruvate carboxylase, oxaloacetate decarboxylase and transcarboxylase, three enzymes that utilize an analogous carboxyltransferase domain to catalyze the biotin-dependent decarboxylation of oxaloacetate.

  13. A generic rate law for surface-active enzymes.

    PubMed

    Kartal, Onder; Ebenhöh, Oliver

    2013-09-02

    Many biochemical reactions are confined to interfaces, such as membranes or cell walls. Despite their importance, no canonical rate laws describing the kinetics of surface-active enzymes exist. Combining the approach chosen by Michaelis and Menten 100 years ago with concepts from surface chemical physics, we here present an approach to derive generic rate laws of enzymatic processes at surfaces. We illustrate this by a simple reversible conversion on a surface to stress key differences to the classical case in solution. The available area function, a concept from surface physics which enters the rate law, covers different models of adsorption and presents a unifying perspective on saturation effects and competition between enzymes. A remarkable implication is the direct dependence of the rate of a given enzyme on all other enzymatic species able to bind at the surface. The generic approach highlights general principles of the kinetics of surface-active enzymes and allows to build consistent mathematical models of more complex pathways involving reactions at interfaces.

  14. Extracellular enzyme activity and biogeochemical cycling in restored prairies

    NASA Astrophysics Data System (ADS)

    Lynch, L.; Hernandez, D.; Schade, J. D.

    2011-12-01

    Winter microbial activity in mid-latitude prairie ecosystems is thermally sensitive and significantly influenced by snow depth. Snow insulates the soil column facilitating microbial processing of complex organic substrates. Previous studies in forests and tundra ecosystems suggest patterns of substrate utilization and limitation are seasonal; above freezing, soil microbes access fresh litter inputs and sugar exudates from plant roots, while under frozen condition they recycle nutrients incorporated in microbial biomass. In order to liberate nutrients required for carbon degradation, soil microbes invest energy in the production of extracellular enzymes that cleave monomers from polymer bonds. The inverse relationship between relative enzyme abundance and substrate availability makes enzyme assays a useful proxy to assess changes in resources over time. Our objective in this study was to assess patterns in microbial biomass, nutrient availability, and extracellular enzyme activity in four snow exclosure sites over a seven-month period. Over the past three years, we have maintained a snow removal experiment on two restored prairies in central Minnesota. In each prairie, snow was continuously removed annually from two 4 x 4 m plots by shoveling after each snow event. Extractable C, N and P, and microbial C, N and P in soil samples were measured in samples collected from these snow removal plots, as well as in adjacent unmanipulated prairie control plots. Pools of C, N, and P were estimated using standard extraction protocols, and microbial pools were estimated using chloroform fumigation direct extraction (CFDE). We conducted fluorometric extracellular enzyme assays (EEA) to assess how the degradation potential of cellulose (cellobiohydrolase, CBH), protein (leucine aminopeptidase, LAP), and phosphate esters (phosphatase, PHOS) changed seasonally. Microbial C and N declined between October and June, while microbial P declined during the fall and winter, but increased

  15. Antioxidant enzyme activities in maize plants colonized with Piriformospora indica.

    PubMed

    Kumar, Manoj; Yadav, Vikas; Tuteja, Narendra; Johri, Atul Kumar

    2009-03-01

    The bioprotection performance of Piriformospora indica against the root parasite Fusarium verticillioides was studied. We found that maize plants first grown with F. verticillioides and at day 10 inoculated with P. indica showed improvements in biomass, and root length and number as compared with plants grown with F. verticillioides alone. To validate our finding that inoculation with P. indica suppresses colonization by F. verticillioides, we performed PCR analyses using P. indica- and F. verticillioides-specific primers. Our results showed that inoculation with P. indica suppresses further colonization by F. verticillioides. We hypothesized that as the colonization by P. indica increases, the presence of/colonization by F. verticillioides decreases. In roots, catalase (CAT), glutathione reductase (GR), glutathione S-transferase (GST) and superoxide dismutase (SOD) activities were found to be higher in F. verticillioides-colonized plants than in non-colonized plants. Increased activity of antioxidant enzymes minimizes the chances of oxidative burst (excessive production of reactive oxygen species), and therefore F. verticillioides might be protected from the oxidative defence system during colonization. We also observed decreased antioxidant enzyme activities in plants first inoculated with F. verticillioides and at day 10 inoculated with P. indica as compared with plants inoculated with F. verticillioides alone. These decreased antioxidant enzyme activities due to the presence of P. indica help the plant to overcome the disease load of F. verticillioides. We propose that P. indica can be used as a bioprotection agent against the root parasite F. verticillioides.

  16. Extracellular enzyme activity in a willow sewage treatment system.

    PubMed

    Brzezinska, Maria Swiontek; Lalke-Porczyk, Elżbieta; Kalwasińska, Agnieszka

    2012-12-01

    This paper presents the results of studies on the activity of extra-cellular enzymes in soil-willow vegetation filter soil which is used in the post-treatment of household sewage in an onsite wastewater treatment system located in central Poland. Wastewater is discharged from the detached house by gravity into the onsite wastewater treatment system. It flows through a connecting pipe into a single-chamber septic tank and is directed by the connecting pipe to a control well to be further channelled in the soil-willow filter by means of a subsurface leaching system. Soil samples for the studies were collected from two depths of 5 cm and 1 m from three plots: close to the wastewater inflow, at mid-length of the plot and close to its terminal part. Soil samples were collected from May to October 2009. The activity of the extra-cellular enzymes was assayed by the fluorometric method using 4-methylumbelliferyl and 7-amido-4-methylcoumarin substrate. The ranking of potential activity of the assayed enzymes was the same at 5 cm and 1 m soil depths, i.e. esterase > phosphmomoesterase > leucine-aminopeptidase > β-glucosidase > α-glucosidase. The highest values of enzymatic activity were recorded in the surface layer of the soil at the wastewater inflow and decreased with increasing distance from that point.

  17. Enzyme-like activities of algal polysaccharide - cerium complexes

    NASA Astrophysics Data System (ADS)

    Wang, Dongfeng; Sun, Jipeng; Du, Dehong; Ye, Shen; Wang, Changhong; Zhou, Xiaoling; Xue, Changhu

    2005-01-01

    Water-soluble algal polysaccharides (APS) (alginic acid, fucoidan and laminaran) possess many pharmacological activities. The results of this study showed that the APS-Ce4+ complexes have some enzyme-like activities. Fucoidan and its complex with Ce4+ have activities similar to those of SOD. The activities of laminaran, alginic acid and their complexes are not measurable. The APS do not show measurable activities in the digestion of plasmid DNA. In contrast, the APS - Ce4+ complexes show these measurable activities under the comparable condition when APS bind Ce4+ and form homogenous solutions. The laminaran - Ce4+ complex shows the most obvious activity in the digestion of plasmid DNA, pNPP and chloropy-rifos under neutral conditions.

  18. Activity-Based Screening of Metagenomic Libraries for Hydrogenase Enzymes.

    PubMed

    Adam, Nicole; Perner, Mirjam

    2017-01-01

    Here we outline how to identify hydrogenase enzymes from metagenomic libraries through an activity-based screening approach. A metagenomic fosmid library is constructed in E. coli and the fosmids are transferred into a hydrogenase deletion mutant of Shewanella oneidensis (ΔhyaB) via triparental mating. If a fosmid exhibits hydrogen uptake activity, S. oneidensis' phenotype is restored and hydrogenase activity is indicated by a color change of the medium from yellow to colorless. This new method enables screening of 48 metagenomic fosmid clones in parallel.

  19. Digestive enzymes activity in larvae of Cameraria ohridella (Lepidoptera: Gracillariidae).

    PubMed

    Stygar, Dominika; Dolezych, Bogdan; Nakonieczny, Mirosław; Migula, Pawel; Michalczyk, Katarzyna; Zaak, Maria

    2010-10-01

    This article presents the activity of carbohydratases and proteases in the midgut of Cameraria ohridella larvae--an oligophagous pest whose preferred feeding is horse chestnuts leaves. Optimal media pH of the assayed enzymes were similar to those of other Lepidopterans. Relatively high amylase activity, as well as maltase and sucrase activities, indicates that starch and sucrose are the main digested saccharides. Trehalase activity was similar to that described in other Lepidopterans. Activities of glycosidases were significantly lower than those of disaccharidases what suggests that neither cellulose nor glycosides are important for C. ohridella. Trypsin is the main endoprotease of this pest. Like in other leaf-eaters carboxypeptidase activity was higher than that of aminopeptidase. The activity of the majority of examined enzymes increased in the following successive pest generations, which could be explained by the decreased nutritional value of older leaves. Probably this phenomenon in hydrolases activity in Cameraria is a nonspecific mechanism present at this stage of co-evolution of the horse chestnut and its pest.

  20. [Lysosomal enzyme activity in white blood cells in leukemias].

    PubMed

    Rybakova, L P; Kharchenko, M F

    1996-01-01

    Total enzyme activity of acidic hydrolases and total neutral proteinase were compared in the post-nuclear fraction of leukocytes from healthy subjects and leukemia patients. The levels of acidic phosphotase and neutral proteinase in lymphoid cells of healthy donors were 11 and 7 times lower than those in myeloid cells, respectively. Patients suffering chronic myeloid leukemia revealed enhanced levels of beta-glucuronidase and neutral proteinases whereas B-chronic lymphoid leukemia involved acidic hydrolase concentrations lower than normal. As chronic myeloid leukemia advanced, neutral proteinase activity dropped dramatically (2.5 times); an aggressive course of B-chronic lymphoid leukemia was accompanied by a 3-fold decrease in acidic hydrolase level. The results may be used as indirect evidence of differences in the role of lysosomal enzymes in the mechanism of protein processing involved in myeloid and lymphoid proliferative pathologies.

  1. Activity of anandamide (AEA) metabolic enzymes in rat placental bed.

    PubMed

    Fonseca, B M; Battista, N; Correia-da-Silva, G; Rapino, C; Maccarrone, M; Teixeira, N A

    2014-11-01

    Endocannabinoids are endogenous lipid mediators, with anandamide (AEA) being the first member identified. It is now widely accepted that AEA influences early pregnancy events and its levels, which primarily depend on its synthesis by an N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and degradation by a fatty acid amide hydrolase (FAAH), must be tightly regulated. Previous studies demonstrated that AEA levels require in situ regulation of these respective metabolic enzymes, and thus, any disturbance in AEA levels may impact maternal remodeling processes occurring during placental development. In this study, the activities of the AEA-metabolic enzymes that result in the establishment of proper local AEA levels during rat gestation were examined. Here, we demonstrate that during placentation NAPE-PLD and FAAH activities change in a temporal manner. Our findings suggest that NAPE-PLD and FAAH create the appropriate AEA levels required for tissue remodeling in the placental bed, a process essential to pregnancy maintenance.

  2. A DNA enzyme with Mg(2+)-Dependent RNA Phosphoesterase Activity

    NASA Technical Reports Server (NTRS)

    Breaker, Ronald R.; Joyce, Gerald F.

    1995-01-01

    Previously we demonstrated that DNA can act as an enzyme in the Pb(2+)-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of approx. 10(exp -4)/ min in the presence of 1 mM Pb(OAc)2 at pH 7.0 and 23 C. The Mg(2+) - dependent reaction is more difficult, with an uncatalyzed rate of approx. 10(exp -7)/ min under comparable conditions. Mg(2+) - dependent cleavage has special relevance to biology because it is compatible with intracellular conditions. Using in vitro selection, we sought to develop a family of phosphoester-cleaving DNA enzymes that operate in the presence of various divalent metals, focusing particularly on the Mg(2+) - dependent reaction. Results: We generated a population of greater than 10(exp 13) DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phosphoester in the presence of 1 mM Mg(2+), Mn(2+), Zn(2+) or Pb(2+). Examination of individual clones from the Mg(2+) lineage after the sixth round revealed a catalytic motif comprised of a three-stem junction.This motif was partially randomized and subjected to seven additional rounds of selective amplification, yielding catalysts with a rate of 0.01/ min. The optimized DNA catalyst was divided into separate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover conditions. Conclusions: We have generated a Mg(2+) - dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate approx. 10(exp 5) - fold greater than that of the uncatalyzed reaction. This activity is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.

  3. An enzyme activity capable of endotransglycosylation of heteroxylan polysaccharides is present in plant primary cell walls.

    PubMed

    Johnston, Sarah L; Prakash, Roneel; Chen, Nancy J; Kumagai, Monto H; Turano, Helen M; Cooney, Janine M; Atkinson, Ross G; Paull, Robert E; Cheetamun, Roshan; Bacic, Antony; Brummell, David A; Schröder, Roswitha

    2013-01-01

    Heteroxylans in the plant cell wall have been proposed to have a role analogous to that of xyloglucans or heteromannans, forming growth-restraining networks by interlocking cellulose microfibrils. A xylan endotransglycosylase has been identified that can transglycosylate heteroxylan polysaccharides in the presence of xylan-derived oligosaccharides. High activity was detected in ripe fruit of papaya (Carica papaya), but activity was also found in a range of other fruits, imbibed seeds and rapidly growing seedlings of cereals. Xylan endotransglycosylase from ripe papaya fruit used a range of heteroxylans, such as wheat arabinoxylan, birchwood glucuronoxylan and various heteroxylans from dicotyledonous primary cell walls purified from tomato and papaya fruit, as donor molecules. As acceptor molecules, the enzyme preferentially used xylopentaitol over xylohexaitol or shorter-length acceptors. Xylan endotransglycosylase was active over a broad pH range and could perform transglycosylation reactions up to 55 °C. Xylan endotransglycosylase activity was purified from ripe papaya fruit by ultrafiltration and cation exchange chromatography. Highest endotransglycosylase activity was identified in fractions that also contained high xylan hydrolase activity and correlated with the presence of the endoxylanase CpaEXY1. Recombinant CpaEXY1 protein transiently over-expressed in Nicotiana benthamiana leaves showed both endoxylanase and xylan endotransglycosylase activities in vitro, suggesting that CpaEXY1 is a single enzyme with dual activity in planta. Purified native CpaEXY1 showed two- to fourfold higher endoxylanase than endotransglycosylase activity, suggesting that CpaEXY1 may act primarily as a hydrolase. We propose that xylan endotransglycosylase activity (like xyloglucan and mannan endotransglycosylase activities) could be involved in remodelling or re-arrangement of heteroxylans of the cellulose-non-cellulosic cell wall framework.

  4. Induction of homologous recombination between sequence repeats by the activation induced cytidine deaminase (AID) protein.

    PubMed

    Buerstedde, Jean-Marie; Lowndes, Noel; Schatz, David G

    2014-07-08

    The activation induced cytidine deaminase (AID) protein is known to initiate somatic hypermutation, gene conversion or switch recombination by cytidine deamination within the immunoglobulin loci. Using chromosomally integrated fluorescence reporter transgenes, we demonstrate a new recombinogenic activity of AID leading to intra- and intergenic deletions via homologous recombination of sequence repeats. Repeat recombination occurs at high frequencies even when the homologous sequences are hundreds of bases away from the positions of AID-mediated cytidine deamination, suggesting DNA end resection before strand invasion. Analysis of recombinants between homeologous repeats yielded evidence for heteroduplex formation and preferential migration of the Holliday junctions to the boundaries of sequence homology. These findings broaden the target and off-target mutagenic potential of AID and establish a novel system to study induced homologous recombination in vertebrate cells.DOI: http://dx.doi.org/10.7554/eLife.03110.001.

  5. Sample storage for soil enzyme activity and bacterial community profiles.

    PubMed

    Wallenius, K; Rita, H; Simpanen, S; Mikkonen, A; Niemi, R M

    2010-04-01

    Storage of samples is often an unavoidable step in environmental data collection, since available analytical capacity seldom permits immediate processing of large sample sets needed for representative data. In microbiological soil studies, sample pretreatments may have a strong influence on measurement results, and thus careful consideration is required in the selection of storage conditions. The aim of this study was to investigate the suitability of prolonged (up to 16 weeks) frozen or air-dried storage for divergent soil materials. The samples selected to this study were mineral soil (clay loam) from an agricultural field, humus from a pine forest and compost from a municipal sewage sludge composting field. The measured microbiological parameters included functional profiling with ten different hydrolysing enzyme activities determined by artificial fluorogenic substrates, and structural profiling with bacterial 16S rDNA community fingerprints by amplicon length heterogeneity analysis (LH-PCR). Storage of samples affected the observed fluorescence intensity of the enzyme assay's fluorophor standards dissolved in soil suspension. The impact was highly dependent on the soil matrix and storage method, making it important to use separate standardisation for each combination of matrix type, storage method and time. Freezing proved to be a better storage method than air-drying for all the matrices and enzyme activities studied. The effect of freezing on the enzyme activities was small (<20%) in clay loam and forest humus and moderate (generally 20-30%) in compost. The most dramatic decreases (>50%) in activity were observed in compost after air-drying. The bacterial LH-PCR community fingerprints were unaffected by frozen storage in all matrices. The effect of storage treatments was tested using a new statistical method based on showing similarity rather than difference of results.

  6. In vivo enzyme activity in inborn errors of metabolism

    SciTech Connect

    Thompson, G.N.; Walter, J.H.; Leonard, J.V.; Halliday, D. )

    1990-08-01

    Low-dose continuous infusions of (2H5)phenylalanine, (1-13C)propionate, and (1-13C)leucine were used to quantitate phenylalanine hydroxylation in phenylketonuria (PKU, four subjects), propionate oxidation in methylmalonic acidaemia (MMA, four subjects), and propionic acidaemia (PA, four subjects) and leucine oxidation in maple syrup urine disease (MSUD, four subjects). In vivo enzyme activity in PKU, MMA, and PA subjects was similar to or in excess of that in adult controls (range of phenylalanine hydroxylation in PKU, 3.7 to 6.5 mumol/kg/h, control 3.2 to 7.9, n = 7; propionate oxidation in MMA, 15.2 to 64.8 mumol/kg/h, and in PA, 11.1 to 36.0, control 5.1 to 19.0, n = 5). By contrast, in vivo leucine oxidation was undetectable in three of the four MSUD subjects (less than 0.5 mumol/kg/h) and negligible in the remaining subject (2 mumol/kg/h, control 10.4 to 15.7, n = 6). These results suggest that significant substrate removal can be achieved in some inborn metabolic errors either through stimulation of residual enzyme activity in defective enzyme systems or by activation of alternate metabolic pathways. Both possibilities almost certainly depend on gross elevation of substrate concentrations. By contrast, only minimal in vivo oxidation of leucine appears possible in MSUD.

  7. Enzymatic vitreolysis with recombinant tissue plasminogen activator for vitreomacular traction

    PubMed Central

    Raczyńska, Dorota; Lipowski, Paweł; Zorena, Katarzyna; Skorek, Andrzej; Glasner, Paulina

    2015-01-01

    Aims The aim of our research was to gain data about the efficacy of intravitreal injections of a recombinant tissue plasminogen activator (rTPA) in dissolving vitreoretinal tractions (VRTs). Materials and methods The study group consisted of patients of our Ophthalmology Clinic who had received an injection of rTPA (TPA Group) for an existent vitreomacular traction confirmed by optical coherence tomography and stereoscopic examinations. The control group consisted of patients who had declined treatment despite the existence of a vitreomacular traction confirmed by the same diagnostic methods. Each group consisted of 30 people (30 eyes). The observation period was 6 months. Conclusion In both groups some of the VRTs had dissolved. In the TPA group the traction dissolved in 10 patients (33.33%) and in the control group only in 5 (16.67%). It is also important to point out that the mean baseline membrane thickness was higher in the TPA group than in the control group. Observing patients in both groups we noticed that the dissolution of vitreoretinal membrane occurred most frequently in those cases where the membrane was thin. In the TPA group, the mean membrane thickness after 6 months decreased considerably. At the same time, no significant change in the membrane thickness could be observed in the control group. Observation of the retinal thickness allows us to draw the following conclusion: in the TPA group, the retinal thickness in the macular area (edema) had decreased over the study period, whereas in the control group it had increased. In those cases where the traction had dissolved, the edema of the retina decreased by the end of the 6-month period in both groups. In the TPA group, the dissolution of the membrane occurred most often within 3 months from the primary injection. Based on statistics, we can confirm that in the control group there was a decrease in visual acuity during the 6 months of the study period. At the same time, visual acuity in the TPA

  8. The Diagnosis of Human Fascioliasis by Enzyme-Linked Immunosorbent Assay (ELISA) Using Recombinant Cathepsin L Protease

    PubMed Central

    Gonzales Santana, Bibiana; Vasquez Camargo, Fabio; Parkinson, Michael

    2013-01-01

    Background Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis. Methodology/Principal findings Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases. Conclusions/Significance A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this

  9. Babesia gibsoni: Serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP.

    PubMed

    Goo, Youn-Kyoung; Jia, Honglin; Aboge, G Oluga; Terkawi, M Alaa; Kuriki, Ken; Nakamura, Chinatsu; Kumagai, Akiko; Zhou, Jinlin; Lee, Eung-goo; Nishikawa, Yoshifumi; Igarashi, Ikuo; Fujisaki, Kozo; Xuan, Xuenan

    2008-04-01

    The thrombospondin-related adhesive protein of Babesia gibsoni (BgTRAP) is known as an immunodominant antigen and is, therefore, considered as a candidate for the development of a diagnostic reagent for canine babesiosis. The recombinant BgTRAP (rBgTRAP) expressed in Escherichia coli was tested in an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to B. gibsoni in dogs. The ELISA with rBgTRAP clearly differentiated between B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. The sera collected from dogs experimentally infected with closely related parasites, B. canis canis, B. canis vogeli, B. canis rossi, and Neospora caninum, showed no cross-reactivity by the ELISA with rBgTRAP. A total of 107 blood samples collected from dogs that had been diagnosed as having babesiosis at veterinary hospitals in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA and PCR. Ninety-six (89.7%) and 89 (83.2%) of the tested samples were positive by the ELISA and PCR, respectively, while 11 (10.3%) and 4 (3.7%) were ELISA+/PCR- and ELISA-/PCR+, respectively. In addition, the sensitivity of the ELISA with rBgTRAP was much higher than that of previously established ELISAs with rBgP50, rBgSA1, and rBgP32. These results indicate that the rBgTRAP is the most promising diagnostic antigen for the detection of an antibody to B. gibsoni in dogs and that the combined ELISA/PCR approach could provide the most reliable diagnosis for clinical sites.

  10. Enzyme-linked immunosorbent assay employing a recombinant antigen for detection of protective antibody against swine erysipelas.

    PubMed

    Imada, Yumiko; Mori, Yasuyuki; Daizoh, Masaji; Kudoh, Kazuma; Sakano, Tetsuya

    2003-11-01

    The specificities and sensitivities of five recombinant proteins of the surface protective antigen (SpaA) of Erysipelothrix rhusiopathiae were examined by indirect enzyme-linked immunosorbent assay (ELISA) with the aim of developing a reliable serological test for the detection of protective antibody against E. rhusiopathiae. Fully mature protein and the N-terminal 416 amino acids (SpaA416) showed sufficient antigenicities, and further examination was done with SpaA416 because of its higher yield. The antibody titers of pigs experimentally immunized with commercial live vaccine and two types of inactivated vaccines clearly increased after immunization, and all pigs were completely protected against challenge with virulent strains. On the other hand, the antibody titers of nonimmunized control pigs remained very low until they were challenged, and all showed severe symptoms or subsequently died. Interference with the production of antibody against live vaccine by maternal antibody or porcine respiratory and reproductive syndrome virus infection 1 week after vaccination was also clearly detected. Because the ELISA titer correlated well with the protection results, the specificity and sensitivity of the ELISA were further evaluated with sera collected from pigs reared on 1 farm on which animals had acute septicemia, 2 farms on which the animals were infected or free from infection, and 10 farms on which the animals were vaccinated with live vaccine, among others. The ELISA titers clearly revealed the conditions of the herds. These results indicate that the SpaA416 ELISA is an effective method not only for evaluating pigs for the presence of protective antibody levels resulting from vaccination or maternal antibody but also for detecting antibody produced by natural infection. This test has important potential for the effective control of swine erysipelas.

  11. Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes.

    PubMed

    Xu, Hao; Majmudar, Jaimeen D; Davda, Dahvid; Ghanakota, Phani; Kim, Ki H; Carlson, Heather A; Showalter, Hollis D; Martin, Brent R; Amidon, Gordon L

    2015-09-08

    Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating preclinical animal models and potential drug-drug interactions. Despite significant adoption of prodrug methodologies for enhanced pharmacokinetics, functional annotation of prodrug activating enzymes is laborious and often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase enzyme family is broadly reactive with reporter-linked fluorophosphonates, which have shown to provide a mechanism-based covalent labeling strategy to assay the activation state and active site occupancy of cellular serine amidases, esterases, and thioesterases. Here we describe a modified ABPP approach using direct substrate competition to identify activating enzymes for an ethyl ester prodrug, the influenza neuraminidase inhibitor oseltamivir. Substrate-competitive ABPP analysis identified carboxylesterase 1 (CES1) as an oseltamivir-activating enzyme in intestinal cell homogenates. Saturating concentrations of oseltamivir lead to a four-fold reduction in the observed rate constant for CES1 inactivation by fluorophosphonates. WWL50, a reported carbamate inhibitor of mouse CES1, blocked oseltamivir hydrolysis activity in human cell homogenates, confirming CES1 is the primary prodrug activating enzyme for oseltamivir in human liver and intestinal cell lines. The related carbamate inhibitor WWL79 inhibited mouse but not human CES1, providing a series of probes for analyzing prodrug activation mechanisms in different preclinical models. Overall, we present a substrate-competitive activity-based profiling approach for broadly surveying candidate prodrug hydrolyzing enzymes and outline the kinetic parameters for activating enzyme discovery, ester prodrug design, and

  12. Characterization of the native and denatured herceptin by enzyme linked immunosorbent assay and quartz crystal microbalance using a high-affinity single chain fragment variable recombinant antibody.

    PubMed

    Shang, Yuqin; Mernaugh, Ray; Zeng, Xiangqun

    2012-10-02

    Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain a single chain fragment variable (scFv, designated 2B4) to a linear synthetic peptide representing Herceptin's heavy chain CDR3. Enzyme linked immunosorbent assays (ELISAs) and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35-220.5 nM) dynamic range. Herceptin denatures and forms significant amounts of aggregates when heated. UV-vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 10(13) M(-2). The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize nonspecific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of the use of QCM to characterize human therapeutic antibodies in samples are also discussed.

  13. Effect of alginate microencapsulation on the catalytic efficiency and in vitro enzyme-prodrug therapeutic efficacy of cytosine deaminase and of recombinant E. coli expressing cytosine deaminase.

    PubMed

    Funaro, Michael G; Nemani, Krishnamurthy V; Chen, Zhihang; Bhujwalla, Zaver M; Griswold, Karl E; Gimi, Barjor

    2016-02-01

    Cytosine deaminase (CD) catalyses the enzymatic conversion of the non-toxic prodrug 5-fluorocytosine (5-FC) to the potent chemotherapeutic form, 5-fluorouracil (5-FU). Intratumoral delivery of CD localises chemotherapy dose while reducing systemic toxicity. Encapsulation in biocompatible microcapsules immunoisolates CD and protects it from degradation. We report on the effect of alginate encapsulation on the catalytic and functional activity of isolated CD and recombinant E. coli engineered to express CD (E. coli(CD)). Alginate microcapsules containing either CD or Escherichia coli(CD) were prepared using ionotropic gelation. Conversion of 5-FC to 5-FU was quantitated in unencapsulated and encapsulated CD/E. coli(CD) using spectrophotometry, with a slower rate of conversion observed following encapsulation. Both encapsulated CD/5-FC and E. coli(CD)/5-FC resulted in cell kill and reduced proliferation of 9 L rat glioma cells, which was comparable to direct 5-FU treatment. Our results show that encapsulation preserves the therapeutic potential of CD and E. coli(CD) is equally effective for enzyme-prodrug therapy.

  14. Endoplasmic reticulum localization and activity of maize auxin biosynthetic enzymes.

    PubMed

    Kriechbaumer, Verena; Seo, Hyesu; Park, Woong June; Hawes, Chris

    2015-09-01

    Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied. Active research over >60 years has shed light on many of the molecular mechanisms of its action including transport, perception, signal transduction, and a variety of biosynthetic pathways in various species, tissues, and developmental stages. The complexity and redundancy of the auxin biosynthetic network and enzymes involved raises the question of how such a system, producing such a potent agent as auxin, can be appropriately controlled at all. Here it is shown that maize auxin biosynthesis takes place in microsomal as well as cytosolic cellular fractions from maize seedlings. Most interestingly, a set of enzymes shown to be involved in auxin biosynthesis via their activity and/or mutant phenotypes and catalysing adjacent steps in YUCCA-dependent biosynthesis are localized to the endoplasmic reticulum (ER). Positioning of auxin biosynthetic enzymes at the ER could be necessary to bring auxin biosynthesis in closer proximity to ER-localized factors for transport, conjugation, and signalling, and allow for an additional level of regulation by subcellular compartmentation of auxin action. Furthermore, it might provide a link to ethylene action and be a factor in hormonal cross-talk as all five ethylene receptors are ER localized.

  15. Extracellular enzyme activities and nutrient availability during artificial groundwater recharge.

    PubMed

    Kolehmainen, Reija E; Korpela, Jaana P; Münster, Uwe; Puhakka, Jaakko A; Tuovinen, Olli H

    2009-02-01

    Natural organic matter (NOM) removal is the main objective of artificial groundwater recharge (AGR) for drinking water production and biodegradation plays a substantial role in this process. This study focused on the biodegradation of NOM and nutrient availability for microorganisms in AGR by the determination of extracellular enzyme activities (EEAs) and nutrient concentrations along a flow path in an AGR aquifer (Tuusula Water Works, Finland). Natural groundwater in the same area but outside the influence of recharge was used as a reference. Determination of the specific alpha-d-glucosidase (alpha-Glu), beta-d-glucosidase (beta-Glu), phosphomonoesterase (PME), leucine aminopeptidase (LAP) and acetate esterase (AEST) activities by fluorogenic model substrates revealed major increases in the enzymatic hydrolysis rates in the aquifer within a 10m distance from the basin. The changes in the EEAs along the flow path occurred simultaneously with decreases in nutrient concentrations. The results support the assumption that the synthesis of extracellular enzymes in aquatic environments is up and down regulated by nutrient availability. The EEAs in the basin sediment and pore water samples (down to 10cm) were in the same order of magnitude as in the basin water, suggesting similar nutritional conditions. Phosphorus was likely to be the limiting nutrient at this particular AGR site. Furthermore, the extracellular enzymes functioned in a synergistic and cooperative way.

  16. Identification of a recombinant inulin fructotransferase (difructose dianhydride III forming) from Arthrobacter sp. 161MFSha2.1 with high specific activity and remarkable thermostability.

    PubMed

    Wang, Xiao; Yu, Shuhuai; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2015-04-08

    Difructose dianhydride III (DFA III) is a functional carbohydrate produced from inulin by inulin fructotransferase (IFTase, EC 4.2.2.18). In this work, an IFTase gene from Arthrobacter sp. 161MFSha2.1 was cloned and expressed in Escherachia coli. The recombinant enzyme was purified by metal affinity chromatography. It showed significant inulin hydrolysis activity, and the produced main product from inulin was determined as DFA III by nuclear magnetic resonance analysis. The molecular mass of the purified protein was calculated to be 43 and 125 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, suggesting the native enzyme might be a homotrimer. The recombinant enzyme showed maximal activity as 2391 units/mg at pH 6.5 and 55 °C. It displayed the highest thermostability among previously reported IFTases (DFA III forming) and was stable up to 80 °C for 4 h of incubation. The smallest substrate was determined as nystose. The conversion ratio of inulin to DFA III reached 81% when 100 g/L inulin was catalyzed by 80 nM recombinant enzyme for 20 min at pH 6.5 and 55 °C. All of these data indicated that the IFTase (DFA III forming) from Arthrobacter sp. 161MFSha2.1 had great potential for industrial DFA III production.

  17. Permanent genetic access to transiently active neurons via TRAP: targeted recombination in active populations.

    PubMed

    Guenthner, Casey J; Miyamichi, Kazunari; Yang, Helen H; Heller, H Craig; Luo, Liqun

    2013-06-05

    Targeting genetically encoded tools for neural circuit dissection to relevant cellular populations is a major challenge in neurobiology. We developed an approach, targeted recombination in active populations (TRAP), to obtain genetic access to neurons that were activated by defined stimuli. This method utilizes mice in which the tamoxifen-dependent recombinase CreER(T2) is expressed in an activity-dependent manner from the loci of the immediate early genes Arc and Fos. Active cells that express CreER(T2) can only undergo recombination when tamoxifen is present, allowing genetic access to neurons that are active during a time window of less than 12 hr. We show that TRAP can provide selective access to neurons activated by specific somatosensory, visual, and auditory stimuli and by experience in a novel environment. When combined with tools for labeling, tracing, recording, and manipulating neurons, TRAP offers a powerful approach for understanding how the brain processes information and generates behavior.

  18. Mycobacterium tuberculosis Lipolytic Enzymes as Potential Biomarkers for the Diagnosis of Active Tuberculosis

    PubMed Central

    Brust, Belinda; Lecoufle, Mélanie; Tuaillon, Edouard; Dedieu, Luc; Canaan, Stéphane; Valverde, Viviane; Kremer, Laurent

    2011-01-01

    Background New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB. Methods Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. Results A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. Conclusion These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high

  19. Direct Ionic Regulation of the Activity of Myo-Inositol Biosynthesis Enzymes in Mozambique Tilapia.

    PubMed

    Villarreal, Fernando D; Kültz, Dietmar

    2015-01-01

    Myo-inositol (Ins) is a major compatible osmolyte in many cells, including those of Mozambique tilapia (Oreochromis mossambicus). Ins biosynthesis is highly up-regulated in tilapia and other euryhaline fish exposed to hyperosmotic stress. In this study, enzymatic regulation of two enzymes of Ins biosynthesis, Ins phosphate synthase (MIPS) and inositol monophosphatase (IMPase), by direct ionic effects is analyzed. Specific MIPS and IMPase isoforms from Mozambique tilapia (MIPS-160 and IMPase 1) were selected based on experimental, phylogenetic, and structural evidence supporting their role for Ins biosynthesis during hyperosmotic stress. Recombinant tilapia IMPase 1 and MIPS-160 activity was assayed in vitro at ionic conditions that mimic changes in the intracellular milieu during hyperosmotic stress. The in vitro activities of MIPS-160 and IMPase 1 are highest at alkaline pH of 8.8. IMPase 1 catalytic efficiency is strongly increased during hyperosmolality (particularly for the substrate D-Ins-3-phosphate, Ins-3P), mainly as a result of [Na+] elevation. Furthermore, the substrate-specificity of IMPase 1 towards D-Ins-1-phosphate (Ins-1P) is lower than towards Ins-3P. Because MIPS catalysis results in Ins-3P this results represents additional evidence for IMPase 1 being the isoform that mediates Ins biosynthesis in tilapia. Our data collectively demonstrate that the Ins biosynthesis enzymes are activated under ionic conditions that cells are exposed to during hypertonicity, resulting in Ins accumulation, which, in turn, results in restoration of intracellular ion homeostasis. We propose that the unique and direct ionic regulation of the activities of Ins biosynthesis enzymes represents an efficient biochemical feedback loop for regulation of intracellular physiological ion homeostasis during hyperosmotic stress.

  20. RecBC enzyme activity is required for far-UV induced respiration shutoff in Escherichia coli K12.

    PubMed

    Swenson, P A; Norton, I L

    1986-01-01

    Shutoff of respiration is one of a number of recA+ lexA+ dependent (SOS) responses caused by far ultraviolet (245 nm) radiation (UV) damage of DNA in Escherichia coli cells. Thus far no rec/lex response has been shown to require the recB recC gene product, the RecBC enzyme. We report in this paper that UV-induced respiration shutoff did not occur in either of these radiation-sensitive derivatives of K12 strain AB1157 nor in the recB recC double mutant. The sbcB gene product is exonuclease I and it has been reported that the triple mutant strain recB recC sbcB has near normal recombination efficiency and resistance to UV. The sbcB strain shut off its respiration after UV but the triple mutant did not show UV-induced respiration shutoff; the shutoff and death responses were uncoupled. We concluded that respiration shutoff requires RecBC enzyme activity. The RecBC enzyme has ATP-dependent double-strand exonuclease activity, helicase activity and several other activities. We tested a recBC+ (double dagger) mutant strain (recC 1010) that had normal recombination efficiency and resistance to UV but which possessed no ATP-dependent double-strand exonuclease activity. This strain did not shut off its respiration. The presence or absence of other RecBC enzyme activities in this mutant is not known. These results support the hypothesis that ATP-dependent double-strand exonuclease activity is necessary for UV-induced respiration shutoff.

  1. Protoplast fusion enhances lignocellulolytic enzyme activities in Trichoderma reesei.

    PubMed

    Cui, Yu-xiao; Liu, Jia-jing; Liu, Yan; Cheng, Qi-yue; Yu, Qun; Chen, Xin; Ren, Xiao-dong

    2014-12-01

    Protoplast fusion was used to obtain a higher production of lignocellulolytic enzymes with protoplast fusion in Trichoderma reesei. The fusant strain T. reesei JL6 was obtained from protoplast fusion from T. reesei strains QM9414, MCG77, and Rut C-30. Filter paper activity of T. reesei JL6 increased by 18% compared with that of Rut C-30. β-Glucosidase, hemicellulase and pectinase activities of T. reesei JL6 were also higher. The former activity was 0.39 Uml(-1), while those of QM9414, MCG77, and Rut C-30 were 0.13, 0.11, and 0.16 Uml(-1), respectively. Pectinase and hemicellulase activities of JL6 were 5.4 and 15.6 Uml(-1), respectively, which were slightly higher than those of the parents. The effects of corn stover and wheat bran carbon sources on the cellulase production and growth curve of T. reesei JL6 were also investigated.

  2. An Extended Polyanion Activation Surface in Insulin Degrading Enzyme

    PubMed Central

    Song, Eun Suk; Ozbil, Mehmet; Zhang, Tingting; Sheetz, Michael; Lee, David; Tran, Danny; Li, Sheng; Prabhakar, Rajeev; Hersh, Louis B.; Rodgers, David W.

    2015-01-01

    Insulin degrading enzyme (IDE) is believed to be the major enzyme that metabolizes insulin and has been implicated in the degradation of a number of other bioactive peptides, including amyloid beta peptide (Aβ), glucagon, amylin, and atrial natriuretic peptide. IDE is activated toward some substrates by both peptides and polyanions/anions, possibly representing an important control mechanism and a potential therapeutic target. A binding site for the polyanion ATP has previously been defined crystallographically, but mutagenesis studies suggest that other polyanion binding modes likely exist on the same extended surface that forms one wall of the substrate-binding chamber. Here we use a computational approach to define three potential ATP binding sites and mutagenesis and kinetic studies to confirm the relevance of these sites. Mutations were made at four positively charged residues (Arg 429, Arg 431, Arg 847, Lys 898) within the polyanion-binding region, converting them to polar or hydrophobic residues. We find that mutations in all three ATP binding sites strongly decrease the degree of activation by ATP and can lower basal activity and cooperativity. Computational analysis suggests conformational changes that result from polyanion binding as well as from mutating residues involved in polyanion binding. These findings indicate the presence of multiple polyanion binding modes and suggest the anion-binding surface plays an important conformational role in controlling IDE activity. PMID:26186535

  3. Purified recombinant hypothetical protein coded by open reading frame Rv1885c of Mycobacterium tuberculosis exhibits a monofunctional AroQ class of periplasmic chorismate mutase activity.

    PubMed

    Prakash, Prachee; Aruna, Bandi; Sardesai, Abhijit A; Hasnain, Seyed E

    2005-05-20

    Naturally occurring variants of the enzyme chorismate mutase are known to exist that exhibit diversity in enzyme structure, regulatory properties, and association with other proteins. Chorismate mutase was not annotated in the initial genome sequence of Mycobacterium tuberculosis (Mtb) because of low sequence similarity between known chorismate mutases. Recombinant protein coded by open reading frame Rv1885c of Mtb exhibited chorismate mutase activity in vitro. Biochemical and biophysical characterization of the recombinant protein suggests its resemblance to the AroQ class of chorismate mutases, prototype examples of which include the Escherichia coli and yeast chorismate mutases. We also demonstrate that unlike the corresponding proteins of E. coli, Mtb chorismate mutase does not have any associated prephenate dehydratase or dehydrogenase activity, indicating its monofunctional nature. The Rv1885c-encoded chorismate mutase showed allosteric regulation by pathway-specific as well as cross-pathway-specific ligands, as evident from proteolytic cleavage protection and enzyme assays. The predicted N-terminal signal sequence of Mtb chorismate mutase was capable of functioning as one in E. coli, suggesting that Mtb chorismate mutase belongs to the AroQ class of chorismate mutases. It was evident that Rv1885c may not be the only enzyme with chorismate mutase enzyme function within Mtb, based on our observation of the presence of chorismate mutase activity displayed by another hypothetical protein coded by open reading frame Rv0948c, a novel instance of the existence of two monofunctional chorismate mutases ever reported in any pathogenic bacterium.

  4. Tissue enzyme activities in the loggerhead sea turtle (Caretta caretta).

    PubMed

    Anderson, Eric T; Socha, Victoria L; Gardner, Jennifer; Byrd, Lynne; Manire, Charles A

    2013-03-01

    The loggerhead sea turtle, Caretta caretta, one of the seven species of threatened or endangered sea turtles worldwide, is one of the most commonly encountered marine turtles off the eastern coast of the United States and Gulf of Mexico. Although biochemical reference ranges have been evaluated for several species of sea turtles, tissue specificity of the commonly used plasma enzymes is lacking. This study evaluated the tissue specificity of eight enzymes, including amylase, lipase, creatine kinase (CK), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), in 30 tissues from five stranded loggerhead sea turtles with no evidence of infectious disease. Amylase and lipase showed the greatest tissue specificity, with activity found only in pancreatic samples. Creatine kinase had high levels present in skeletal and cardiac muscle, and moderate levels in central nervous system and gastrointestinal samples. Gamma-glutamyl transferase was found in kidney samples, but only in very low levels. Creatine kinase, ALP, AST, and LDH were found in all tissues evaluated and ALT was found in most, indicating low tissue specificity for these enzymes in the loggerhead.

  5. Charge Shielding of PIP2 by Cations Regulates Enzyme Activity of Phospholipase C

    PubMed Central

    Seo, Jong Bae; Jung, Seung-Ryoung; Huang, Weigang; Zhang, Qisheng; Koh, Duk-Su

    2015-01-01

    Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane by phospholipase C (PLC) generates two critical second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. For the enzymatic reaction, PIP2 binds to positively charged amino acids in the pleckstrin homology domain of PLC. Here we tested the hypothesis that positively charged divalent and multivalent cations accumulate around the negatively charged PIP2, a process called electrostatic charge shielding, and therefore inhibit electrostatic PIP2-PLC interaction. This charge shielding of PIP2 was measured quantitatively with an in vitro enzyme assay using WH-15, a PIP2 analog, and various recombinant PLC proteins (β1, γ1, and δ1). Reduction of PLC activity by divalent cations, polyamines, and neomycin was well described by a theoretical model considering accumulation of cations around PIP2 via their electrostatic interaction and chemical binding. Finally, the charge shielding of PIP2 was also observed in live cells. Perfusion of the cations into cells via patch clamp pipette reduced PIP2 hydrolysis by PLC as triggered by M1 muscarinic receptors with a potency order of Mg2+ < spermine4+ < neomycin6+. Accumulation of divalent cations into cells through divalent-permeable TRPM7 channel had the same effect. Altogether our results suggest that Mg2+ and polyamines modulate the activity of PLCs by controlling the amount of free PIP2 available for the enzymes and that highly charged biomolecules can be inactivated by counterions electrostatically. PMID:26658739

  6. Charge Shielding of PIP2 by Cations Regulates Enzyme Activity of Phospholipase C.

    PubMed

    Seo, Jong Bae; Jung, Seung-Ryoung; Huang, Weigang; Zhang, Qisheng; Koh, Duk-Su

    2015-01-01

    Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane by phospholipase C (PLC) generates two critical second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. For the enzymatic reaction, PIP2 binds to positively charged amino acids in the pleckstrin homology domain of PLC. Here we tested the hypothesis that positively charged divalent and multivalent cations accumulate around the negatively charged PIP2, a process called electrostatic charge shielding, and therefore inhibit electrostatic PIP2-PLC interaction. This charge shielding of PIP2 was measured quantitatively with an in vitro enzyme assay using WH-15, a PIP2 analog, and various recombinant PLC proteins (β1, γ1, and δ1). Reduction of PLC activity by divalent cations, polyamines, and neomycin was well described by a theoretical model considering accumulation of cations around PIP2 via their electrostatic interaction and chemical binding. Finally, the charge shielding of PIP2 was also observed in live cells. Perfusion of the cations into cells via patch clamp pipette reduced PIP2 hydrolysis by PLC as triggered by M1 muscarinic receptors with a potency order of Mg2+ < spermine4+ < neomycin6+. Accumulation of divalent cations into cells through divalent-permeable TRPM7 channel had the same effect. Altogether our results suggest that Mg2+ and polyamines modulate the activity of PLCs by controlling the amount of free PIP2 available for the enzymes and that highly charged biomolecules can be inactivated by counterions electrostatically.

  7. Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification

    PubMed Central

    Leelavathi, Sadhu; Doria, Enrico; Reddy, Vanga Siva; Cella, Rino

    2015-01-01

    Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2 increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry. PMID:26137472

  8. Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification.

    PubMed

    Longoni, Paolo; Leelavathi, Sadhu; Doria, Enrico; Reddy, Vanga Siva; Cella, Rino

    2015-01-01

    Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2 increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry.

  9. Global Profiling of Carbohydrate Active Enzymes in Human Gut Microbiome

    PubMed Central

    Mande, Sharmila S.

    2015-01-01

    Motivation Carbohydrate Active enzyme (CAZyme) families, encoded by human gut microflora, play a crucial role in breakdown of complex dietary carbohydrates into components that can be absorbed by our intestinal epithelium. Since nutritional wellbeing of an individual is dependent on the nutrient harvesting capability of the gut microbiome, it is important to understand how CAZyme repertoire in the gut is influenced by factors like age, geography and food habits. Results This study reports a comprehensive in-silico analysis of CAZyme profiles in the gut microbiomes of 448 individuals belonging to different geographies, using similarity searches of the corresponding gut metagenomic contigs against the carbohydrate active enzymes database. The study identifies a core group of 89 CAZyme families that are present across 85% of the gut microbiomes. The study detects several geography/age-specific trends in gut CAZyme repertoires of the individuals. Notably, a group of CAZymes having a positive correlation with BMI has been identified. Further this group of BMI-associated CAZymes is observed to be specifically abundant in the Firmicutes phyla. One of the major findings from this study is identification of three distinct groups of individuals, referred to as 'CAZotypes', having similar CAZyme profiles. Distinct taxonomic drivers for these CAZotypes as well as the probable dietary basis for such trends have also been elucidated. The results of this study provide a global view of CAZyme profiles across individuals of various geographies and age-groups. These results re-iterate the need of a more precise understanding of the role of carbohydrate active enzymes in human nutrition. PMID:26544883

  10. Constitutive cellulase production from glucose using the recombinant Trichoderma reesei strain overexpressing an artificial transcription activator.

    PubMed

    Zhang, Xiaoyue; Li, Yonghao; Zhao, Xinqing; Bai, Fengwu

    2017-01-01

    The high cost of cellulase production presents biggest challenge in biomass deconstruction. Cellulase production by Trichoderma reesei using low cost carbon source is of great interest. In this study, an artificial transcription activator containing the Cre1 binding domain linked to the Xyr1 effector and binding domains was designed and constitutively overexpressed in T. reesei RUT C30. The recombinant strain T. reesei zxy-2 displayed constitutive cellulase production using glucose as a sole carbon source, and the production titer was 12.75-fold of that observed with T. reesei RUT C30 in shake flask culture. Moreover, FPase and xylanase titers of 2.63 and 108.72IU/mL, respectively, were achieved using glucose as sole carbon source within 48h in a 7-L fermenter by batch fermentation using T. reesei zxy-2. The crude enzyme obtained was used to hydrolyze alkali pretreated corn stover, and a high glucose yield of 99.18% was achieved.

  11. Application of capillary enzyme micro-reactor in enzyme activity and inhibitors studies of glucose-6-phosphate dehydrogenase.

    PubMed

    Camara, Mohamed Amara; Tian, Miaomiao; Guo, Liping; Yang, Li

    2015-05-15

    In this study, we present an on-line measurement of enzyme activity and inhibition of Glucose-6-phosphate dehydrogenase (G6PDH) enzyme using capillary electrophoresis based immobilized enzyme micro-reactor (CE-based IMER). The IMER was prepared using a two-step protocol based on electrostatic assembly. The micro-reactor exhibited good stability and reproducibility for on-line assay of G6PDH enzyme. Both the activity as well as the inhibition of the G6PDH enzyme by six inhibitors, including three metals (Cu(2+), Pb(2+), Cd(2+)), vancomycin, urea and KMnO4, were investigated using on-line assay of the CE-based IMERs. The enzyme activity and inhibition kinetic constants were measured using the IMERs which were found to be consistent with those using traditional off-line enzyme assays. The kinetic mechanism of each inhibitor was also determined. The present study demonstrates the feasibility of using CE-based IMERs for rapid and efficient on-line assay of G6PDH, an important enzyme in the pentosephosphate pathway of human metabolism.

  12. Protein stability and enzyme activity at extreme biological temperatures.

    PubMed

    Feller, Georges

    2010-08-18

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.

  13. Serum angiotensin converting enzyme activity in chronic beryllium disease.

    PubMed

    Newman, L S; Orton, R; Kreiss, K

    1992-07-01

    Serum angiotensin converting enzyme (SACE) activity is used as a marker of sarcoidosis activity and severity, but in chronic beryllium disease (CBD) the studies of SACE give conflicting results. We examined SACE activity in 23 CBD patients, five patients with beryllium sensitization, and 25 beryllium-exposed control subjects. CBD patients underwent complete clinical evaluation, including physical examination, pulmonary function testing, exercise physiology testing, chest radiography, and bronchoscopy with bronchoalveolar lavage and biopsy. CBD SACE activity was systematically compared with these clinical markers of disease severity. Of CBD patients, 22% had elevated SACE activity. The test did not discriminate CBD patients from those in the beryllium-sensitized or beryllium-exposed groups. However, SACE activity in CBD correlated with the extent of pulmonary granulomatous inflammation as reflected by the symptom of breathlessness, the number of white cells in bronchoalveolar lavage (r = 0.44), the number of lavage lymphocytes (r = 0.58), the lavage lymphocyte percentage (r = 0.55), and the profusion of small opacities on chest radiograph (r = 0.41). The test-retest reliability of the assay was high (r = 0.84), as was the agreement between fresh and -70 degrees C frozen sera (r = 0.93). We conclude that SACE activity levels may reflect the extent of pulmonary granulomatous inflammation in CBD but that the test does not help discriminate disease from nondisease.

  14. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a...

  15. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a...

  16. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a...

  17. Engineering Enzymes in Energy Crops: Conditionally Activated Enzymes Expressed in Cellulosic Energy Crops

    SciTech Connect

    2010-01-15

    Broad Funding Opportunity Announcement Project: Enzymes are required to break plant biomass down into the fermentable sugars that are used to create biofuel. Currently, costly enzymes must be added to the biofuel production process. Engineering crops to already contain these enzymes will reduce costs and produce biomass that is more easily digested. In fact, enzyme costs alone account for $0.50-$0.75/gallon of the cost of a biomass-derived biofuel like ethanol. Agrivida is genetically engineering plants to contain high concentrations of enzymes that break down cell walls. These enzymes can be “switched on” after harvest so they won’t damage the plant while it’s growing.

  18. A Thermostable Bilirubin-Oxidizing Enzyme from Activated Sludge Isolated by a Metagenomic Approach

    PubMed Central

    Kimura, Nobutada; Kamagata, Yoichi

    2016-01-01

    A gene coding for a multicopper oxidase (BopA) was identified through the screening of a metagenomic library constructed from wastewater treatment activated sludge. The recombinant BopA protein produced in Escherichia coli exhibited oxidation activity toward 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in the presence of copper, suggesting that BopA is laccase. A bioinformatic analysis of the bopA gene sequence indicated that it has a phylogenetically bacterial origin, possibly derived from a bacterium within the phylum Deinococcus-Thermus. Purified BopA exhibited maximum activity at pH 7.5 with bilirubin as its substrate and was found to be active over a markedly broad pH range from 6 to 11. It also showed notable thermostability; its activity remained intact even after a heat treatment at 90°C for 60 min. This enzyme is a thermostable-bilirubin oxidase that exhibits markedly higher thermostability than that previously reported for laccases. PMID:27885197

  19. Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins.

    PubMed

    Tang, Jin-Bao; Tang, Ying; Yang, Hong-Ming

    2015-02-15

    Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag-BirA system. Through the high streptavidin (SA)-biotin interaction, the divalent biotinylated APs were clustered in the SA-biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ-AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for the ZZ protein.

  20. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, Eric E.; Roessler, Paul G.

    1999-01-01

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities.

  1. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, E.E.; Roessler, P.G.

    1999-07-27

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities. 8 figs.

  2. Characterization and chillproofing activity of two enzymes from Streptomyces species.

    PubMed

    Etok, C A; Eka, O U

    1996-01-01

    Two enzymes, amylase and protease of Streptomyces species were purified by a combination of ion exchange chromatography and gel filtration and characterized. The amylase had an exoaction on starch yielding maltose as a major end product and was identified as beta-amylase. The purified amylase had a molecular weight of 48,000 and was maximally active at 35 degrees C and at pH 6.0. On the other hand, protease had a molecular weight of 21,000 and was most active at pH 10.0 and at a temperature of 30 degrees C. The Km or MICHAELIS constant of amylase for maize starch was 0.333 mg/ml while that of protease for casein was 2.5 mg/ml. The feasibility of using the purified protease for various industrial application especially in the chillproofing of beer is discussed.

  3. A bifunctional enzyme from Rhodococcus erythropolis exhibiting secondary alcohol dehydrogenase-catalase activities.

    PubMed

    Martinez-Rojas, Enriqueta; Kurt, Tutku; Schmidt, Udo; Meyer, Vera; Garbe, Leif-Alexander

    2014-11-01

    Alcohol dehydrogenases have long been recognized as potential biocatalyst for production of chiral fine and bulk chemicals. They are relevant for industry in enantiospecific production of chiral compounds. In this study, we identified and purified a nicotinamide adenine dinucleotide (NAD)-dependent secondary alcohol dehydrogenase (SdcA) from Rhodococcus erythropolis oxidizing γ-lactols into γ-lactones. SdcA showed broad substrate specificity on γ-lactols; secondary aliphatic alcohols with 8 and 10 carbon atoms were also substrates and oxidized with (2S)-stereospecificity. The enzyme exhibited moderate stability with a half-life of 5 h at 40 °C and 20 days at 4 °C. Mass spectrometric identification revealed high sequence coverage of SdcA amino acid sequence to a highly conserved catalase from R. erythropolis. The corresponding encoding gene was isolated from genomic DNA and subsequently overexpressed in Escherichia coli BL21 DE3 cells. In addition, the recombinant SdcA was purified and characterized in order to confirm that the secondary alcohol dehydrogenase and catalase activity correspond to the same enzyme.

  4. Evolution of an Antibiotic Resistance Enzyme Constrained by Stability and Activity Trade-offs

    SciTech Connect

    Wang, Xiaojun; Minasov, George; Shoichet, Brian K.

    2010-03-08

    Pressured by antibiotic use, resistance enzymes have been evolving new activities. Does such evolution have a cost? To investigate this question at the molecular level, clinically isolated mutants of the {beta}-lactamase TEM-1 were studied. When purified, mutant enzymes had increased activity against cephalosporin antibiotics but lost both thermodynamic stability and kinetic activity against their ancestral targets, penicillins. The X-ray crystallographic structures of three mutant enzymes were determined. These structures suggest that activity gain and stability loss is related to an enlarged active site cavity in the mutant enzymes. In several clinically isolated mutant enzymes, a secondary substitution is observed far from the active site (Met182 {yields} Thr). This substitution had little effect on enzyme activity but restored stability lost by substitutions near the active site. This regained stability conferred an advantage in vivo. This pattern of stability loss and restoration may be common in the evolution of new enzyme activity.

  5. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes.

    PubMed

    Chu, Wen-Ting; Wang, Jin

    2016-06-14

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the "hot-spot" within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design.

  6. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes

    NASA Astrophysics Data System (ADS)

    Chu, Wen-Ting; Wang, Jin

    2016-06-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the “hot-spot” within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design.

  7. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes

    PubMed Central

    Chu, Wen-Ting; Wang, Jin

    2016-01-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the “hot-spot” within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design. PMID:27298067

  8. Dynamically achieved active site precision in enzyme catalysis.

    PubMed

    Klinman, Judith P

    2015-02-17

    CONSPECTUS: The grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes' enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme-substrate interactions and beyond. This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C-H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed.

  9. Comparative insecticide susceptibility and detoxification enzyme activities among pestiferous blattodea.

    PubMed

    Valles, S M; Koehler, P G; Brenner, R J

    1999-11-01

    Topical bioassays using propoxur, chlorpyrifos, and lambda-cyhalothrin were conducted on eight cockroach species. Based on lethal dose values, the relative toxicities of the insecticide classes were generally pyrethroid > carbamate > organophosphorous. Lambda-Cyhalothrin and propoxur were more toxic toward the Blattidae as compared with the Blattellidae. The order of lambda-cyhalothrin toxicity was Periplaneta americana > Periplaneta brunnea = Periplaneta australasiae = Periplaneta fuliginosa = Blatta orientalis > Blattella asahinai = Blattella germanica > Blattella vaga. The order of propoxur toxicity was B. orientalis > P. americana > P. brunnea = P. australasiae > B. asahinai > P. fuliginosa = B. germanica > B. vaga. The order of chlorpyrifos toxicity was P. americana > B. asahinai = B. vaga > B. orientalis = P. australasiae = P. brunnea > B. germanica = P. fuliginosa. Detoxification enzyme activities for each species also were measured and compared with insecticide toxicity. Propoxur LD50 was significantly (P = 0.01; r = 0.81) correlated with glutathione S-transferase activity. Lambda-Cyhalothrin LD50 correlated with methoxyresorufin O-demethylase activity (P = 0.01; r = 0.81), carboxylesterase activity (P = 0.03; r = - 0.75), general esterase activity (P = 0.02; r = - 0.79), and cockroach weight (P = 0.01; r = -0.95).

  10. Enzyme-amplified protein micorarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies

    SciTech Connect

    Varnum, Susan M.; Warner, Marvin G.; Dockendorff, Brian P.; Anheier, Norman C.; Lou, Jianlong; Marks, James D.; Smith, Leonard A.; Feldhaus, Michael J.; Grate, Jay W.; Bruckner-Lea, Cindy J.

    2006-06-16

    With the use of high-affinity recombinant monoclonal antibodies against the receptor binding domain of botulinum neurotoxin A (BoNT/A), two separate immunoassay platforms were developed for either the sensitive or the rapid detection of BoNT/A. An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of BoNT in buffer and clinical fluids. This assay has the sensitivity to detect BoNT in diverse samples down to 14 fM (1.4 pg/mL). Using the recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. While the ELISA microarray assay, because of its sensitivity, offers an alternative to the mouse bioassay, the renewable surface assay has potential as a rapid screening assay for the analysis of complex environmental samples.

  11. County-Scale Spatial Distribution of Soil Enzyme Activities and Enzyme Activity Indices in Agricultural Land: Implications for Soil Quality Assessment

    PubMed Central

    Xie, Baoni; Wang, Junxing; He, Wenxiang; Wang, Xudong; Wei, Gehong

    2014-01-01

    Here the spatial distribution of soil enzymatic properties in agricultural land was evaluated on a county-wide (567 km2) scale in Changwu, Shaanxi Province, China. The spatial variations in activities of five hydrolytic enzymes were examined using geostatistical methods. The relationships between soil enzyme activities and other soil properties were evaluated using both an integrated total enzyme activity index (TEI) and the geometric mean of enzyme activities (GME). At the county scale, soil invertase, phosphatase, and catalase activities were moderately spatially correlated, whereas urease and dehydrogenase activities were weakly spatially correlated. Correlation analysis showed that both TEI and GME were better correlated with selected soil physicochemical properties than single enzyme activities. Multivariate regression analysis showed that soil OM content had the strongest positive effect while soil pH had a negative effect on the two enzyme activity indices. In addition, total phosphorous content had a positive effect on TEI and GME in orchard soils, whereas alkali-hydrolyzable nitrogen and available potassium contents, respectively, had negative and positive effects on these two enzyme indices in cropland soils. The results indicate that land use changes strongly affect soil enzyme activities in agricultural land, where TEI provides a sensitive biological indicator for soil quality. PMID:25610908

  12. Expression of enzymatically active, recombinant barley alpha-glucosidase in yeast and immunological detection of alpha-glucosidase from seed tissue.

    PubMed

    Tibbot, B K; Henson, C A; Skadsen, R W

    1998-10-01

    An alpha-glucosidase cDNA clone derived from barley aleurone tissue was expressed in Pichia pastoris and Escherichia coli. The gene was fused with the N-terminal region of the Saccharomyces cerevisiae alpha-factor secretory peptide and placed under control of the Pichia AOX1 promoter in the vector pPIC9. Enzymatically active, recombinant alpha-glucosidase was synthesized and secreted from the yeast upon induction with methanol. The enzyme hydrolyzed maltose > trehalose > nigerose > isomaltose. Maltase activity occurred over the pH range 3.5-6.3 with an optimum at pH 4.3, classifying the enzyme as an acid alpha-glucosidase. The enzyme had a Km of 1.88 mM and Vmax of 0.054 micromol/min on maltose. The recombinant alpha-glucosidase expressed in E. coli was used to generate polyclonal antibodies. The antibodies detected 101 and 95 kDa forms of barley alpha-glucosidase early in seed germination. Their levels declined sharply later in germination, as an 81 kDa alpha-glucosidase became prominent. Synthesis of these proteins also occurred in isolated aleurones after treatment with gibberellin, and this was accompanied by a 14-fold increase in alpha-glucosidase enzyme activity.

  13. Heparin/heparan sulfate 6-O-sulfatase from Flavobacterium heparinum: integrated structural and biochemical investigation of enzyme active site and substrate specificity.

    PubMed

    Myette, James R; Soundararajan, Venkataramanan; Shriver, Zachary; Raman, Rahul; Sasisekharan, Ram

    2009-12-11

    Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

  14. Study on optimization of process parameters for enhancing the multi-hydrolytic enzyme activity in garbage enzyme produced from preconsumer organic waste.

    PubMed

    Arun, C; Sivashanmugam, P

    2017-02-01

    The garbage enzymes produced from preconsumer organic waste containing multi hydrolytic enzyme activity which helps to solubilize the waste activated sludge. The continuous production of garbage enzyme and its scaling up process need a globe optimized condition. In present study the effect of fruit peel composition and sonication time on enzyme activity were investigated. Garbage enzyme produced from 6g pineapple peels: 4g citrus peels pre-treated with ultrasound for 20min shows higher hydrolytic enzymes activity. Simultaneously statistical optimization tools were used to model garbage enzyme production with higher activity of amylase, lipase and protease. The maximum activity of amylase, lipase and protease were predicted to be 56.409, 44.039, 74.990U/ml respectively at optimal conditions (pH (6), temperature (37°C), agitation (218 RPM) and fermentation duration (3days)). These optimized conditions can be successfully used for large scale production of garbage enzyme with higher hydrolytic enzyme activity.

  15. Characterization of non-oxidative transaldolase and transketolase enzymes in the pentose phosphate pathway with regard to xylose utilization by recombinant Saccharomyces cerevisiae.

    PubMed

    Matsushika, Akinori; Goshima, Tetsuya; Fujii, Tatsuya; Inoue, Hiroyuki; Sawayama, Shigeki; Yano, Shinichi

    2012-06-10

    The activity of transaldolase and transketolase, key enzymes in the non-oxidative pentose phosphate pathway, is rate-limiting for xylose utilization in recombinant Saccharomyces cerevisiae. Overexpression of TAL1 and TKL1, the major transaldolase and transketolase genes, increases the flux from the pentose phosphate pathway into the glycolytic pathway. However, the functional roles of NQM1 and TKL2, the secondary transaldolase and transketolase genes, especially in xylose utilization, remain unclear. This study focused on characterization of NQM1 and TKL2, together with TAL1 and TKL1, regarding their roles in xylose utilization and fermentation. Knockout or overexpression of these four genes on the phenotype of xylose-utilizing S. cerevisiae strains was also examined. Transcriptional analysis indicated that the expression of TAL1, NQM1, and TKL1 was up-regulated in the presence of xylose. A significant decrease in both growth on xylose and xylose-fermenting ability in tal1Δ and tkl1Δ mutants confirmed that TAL1 and TKL1 are essential for xylose assimilation and fermentation. Gene disruption analysis using a tkl1Δ mutant revealed that TKL1 is also required for utilization of glucose. Growth on xylose and xylose-fermenting ability were slightly influenced by deletion of NQM1 or TKL2 when xylose was used as the sole carbon source. Moreover, the rate of xylose consumption and ethanol production was slightly impaired in TKL1- and TKL2-overexpressing strains. NQM1 and TKL2 may thus play a physiological role via an effect on the non-oxidative pentose phosphate pathway in the xylose metabolic pathway, although their roles in xylose utilization and fermentation are less important than those of TAL1 and TKL1.

  16. Angiotensin-converting enzyme inhibitory activity in Mexican Fresco cheese.

    PubMed

    Torres-Llanez, M J; González-Córdova, A F; Hernandez-Mendoza, A; Garcia, H S; Vallejo-Cordoba, B

    2011-08-01

    The objective of this study was to evaluate if Mexican Fresco cheese manufactured with specific lactic acid bacteria (LAB) presented angiotensin I-converting enzyme inhibitory (ACEI) activity. Water-soluble extracts (3 kDa) obtained from Mexican Fresco cheese prepared with specific LAB (Lactococcus, Lactobacillus, Enterococcus, and mixtures: Lactococcus-Lactobacillus and Lactococcus-Enterococcus) were evaluated for ACEI activity. Specific peptide fractions with high ACEI were analyzed using reverse phase-HPLC coupled to mass spectrometry for determination of amino acid sequence. Cheese containing Enterococcus faecium or a Lactococcus lactis ssp. lactis-Enterococcus faecium mixture showed the largest number of fractions with ACEI activity and the lowest half-maximal inhibitory concentration (IC(50); <10 μg/mL). Various ACEI peptides derived from β-casein [(f(193-205), f(193-207), and f(193-209)] and α(S1)-casein [f(1-15), f(1-22), f(14-23), and f(24-34)] were found. The Mexican Fresco cheese manufactured with specific LAB strains produced peptides with potential antihypertensive activity.

  17. Effects of Fertilization on Tomato Growth and Soil Enzyme Activity

    NASA Astrophysics Data System (ADS)

    Mu, Zhen; Hu, Xue-Feng; Cheng, Chang; Luo, Zhi-qing

    2015-04-01

    To study the effects of different fertilizer applications on soil enzyme activity, tomato plant growth and tomato yield and quality, a field experiment on tomato cultivation was carried out in the suburb of Shanghai. Three fertilizer treatments, chemical fertilizer (CF) (N, 260 g/kg; P, 25.71g/kg; K, 83.00g/kg), rapeseed cake manure (CM) (N, 37.4 g/kg; P, 9.0 g/kg; K, 8.46 g/kg), crop-leaf fermenting manure (FM) (N, 23.67 g/kg; P, 6.39 g/kg; K 44.32 g/kg), and a control without using any fertilizers (CK), were designed. The total amounts of fertilizer application to each plot for the CF, CM, FM and CK were 0.6 kg, 1.35 kg, 3.75 kg and 0 kg, respectively, 50% of which were applied as base fertilizer, and another 50% were applied after the first fruit picking as top dressing. Each experimental plot was 9 m2 (1 m × 9 m) in area. Each treatment was replicated for three times. No any pesticides and herbicides were applied during the entire period of tomato growth to prevent their disturbance to soil microbial activities. Soil enzyme activities at each plot were constantly tested during the growing period; the tomato fruit quality was also constantly analyzed and the tomato yield was calculated after the final harvesting. The results were as follows: (1) Urease activity in the soils treated with the CF, CM and FM increased quickly after applying base fertilizer. That with the CF reached the highest level. Sucrase activity was inhibited by the CF and CM to some extent, which was 32.4% and 11.2% lower than that with the CK, respectively; while that with the FM was 15.7% higher than that with the CK. Likewise, catalase activity with the CF increased by 12.3% - 28.6%; that with the CM increased by 87.8% - 95.1%; that with the FM increased by 86.4% - 93.0%. Phosphatase activity with the CF increased rapidly and reached a maximum 44 days after base fertilizer application, and then declined quickly. In comparison, that with the CM and FM increased slowly and reached a maximum

  18. Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

    NASA Astrophysics Data System (ADS)

    Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; Fakhrullin, Rawil F.

    2015-05-01

    Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (A549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment.

  19. Endothelin-converting enzyme 2 differentially regulates opioid receptor activity

    PubMed Central

    Gupta, A; Fujita, W; Gomes, I; Bobeck, E; Devi, L A

    2015-01-01

    BACKGROUND AND PURPOSE Opioid receptor function is modulated by post-activation events such as receptor endocytosis, recycling and/or degradation. While it is generally understood that the peptide ligand gets co-endocytosed with the receptor, relatively few studies have investigated the role of the endocytosed peptide and peptide processing enzymes in regulating receptor function. In this study, we focused on endothelin-converting enzyme 2 (ECE2), a member of the neprilysin family of metallopeptidases that exhibits an acidic pH optimum, localizes to an intracellular compartment and selectively processes neuropeptides including opioid peptides in vitro, and examined its role in modulating μ receptor recycling and resensitization. EXPERIMENTAL APPROACH The effect of ECE2 inhibition on hydrolysis of the endocytosed peptide was examined using thin-layer chromatography and on μ opioid receptor trafficking using either elisa or microscopy. The effect of ECE2 inhibition on receptor signalling was measured using a cAMP assay and, in vivo, on antinociception induced by intrathecally administered opioids by the tail-flick assay. KEY RESULTS The highly selective ECE2 inhibitor, S136492, significantly impaired μ receptor recycling and signalling by only those ligands that are ECE2 substrates and this was seen both in heterologous cells and in cells endogenously co-expressing μ receptors with ECE2. We also found that ECE2 inhibition attenuated antinociception mediated only by opioid peptides that are ECE2 substrates. CONCLUSIONS AND IMPLICATIONS These results suggest that ECE2, by selectively processing endogenous opioid peptides in the endocytic compartment, plays a role in modulating opioid receptor activity. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24990314

  20. Cerebrospinal fluid enzymes in acute brain injury. 1. Dynamics of changes in CSF enzyme activity after acute experimental brain injury.

    PubMed Central

    Maas, A I

    1977-01-01

    Changes in CSF enzyme activity were studied after brain trauma for their prognostic value. Raised values of CPK and HBDH were demonstrated in the CSF of patients with severe brain injuries. Standardised cold lesions of the brain were induced in cats. The activities of the enzymes CPK, HBDH, LDH, GOT, GPT, and pseudocholinesterase were studied at half hour intervals in the cerebrospinal fluid and at hourly intervals in the serum. A statistically highly significant increase of all enzymes studied developed in the CSF. The greatest changes occurred within four hours of freezing. Large increases could occur in half an hour. Isoenzyme studies demonstrated that CPK and LDH were of cerebral origin. No consistently significant changes could be shown in the serum enzyme activity. It is concluded that after brain injuries, enzymes are released into the extracellular fluid of the brain and transported to the CSF. The limited value of a single enzyme estimation is emphasised. The results described seem to provide indirect evidence for transependymal flow of extracellular fluid in brain oedema. Images PMID:915509

  1. Detection, quantification, and glycotyping of prion protein in specifically activated enzyme-linked immunosorbent assay plates.

    PubMed

    Triantaphyllidou, I E; Sklaviadis, T; Vynios, D H

    2006-12-15

    The conversion of a normal glycoprotein, prion protein (PrP(C)), to its abnormal protease-resistant isoform (PrP(Sc)) seems to be one of the main factors underlying the pathogenesis of spongiform encephalopathies. There are many studies indicating that PrP interacts with glycosaminoglycans, and we exploited this interaction to develop a sensitive solid phase assay for detection of both PrP forms. Glycosaminoglycans, such as chondroitin sulfate and heparin, were immobilized by their negative charge to enzyme-linked immunosorbent assay (ELISA) plate wells activated by glutaraldehyde and spermine. PrP in the samples examined (recombinant PrP or tissue homogenate) was allowed to interact with glycans. The interaction of recombinant PrP was more efficient against immobilized chondroitin sulfate of type A, and a linear correlation with concentration was demonstrated. From this curve, the concentration of each one of the PrP isoforms in biological samples can be determined. In addition, and taking into account that glycosylation of prion protein is species specific, we used similarly activated ELISA plate wells to determine different PrP glycoforms. A monoclonal antibody against PrP was immobilized, and PrP present in the samples (brain homogenates) was bound and visualized by various lectins. The most interesting outcome of the study is the differential binding of ricinus communis agglutinin I to the normal and scrapie brain homogenates. Dattura stramonium lectin and wheat germ agglutinin seem to bind almost equally to both samples, and all three have an increased sensitivity to PrP(Sc) after proteinase K digestion.

  2. Synthesis and characterization of biologically active recombinant elk and horse FSH.

    PubMed

    Fachal, María Victoria; Furlan, Mike; Clark, Rena; Card, Claire E; Chedrese, P Jorge

    2010-02-01

    The objective of this investigation was to clone and express the elk and horse common alpha-subunit and FSH beta-subunit cDNAs, and to produce recombinant FSH from both species in vitro. The RNAs extracted from elk and horse pituitary glands were reverse-transcribed and amplified by polymerase chain reaction. The cDNAs corresponding to both subunits of elk and horse were cloned into the expression vector pBudCE4.1 and transfected into CRL-9096 cells. Expression of both genes was determined in the transfected cells by Northern and Western blot analysis. Recombinant elk and horse FSH secreted in culture media were characterized by an in vitro bioassay and RIA. When the recombinant products were assessed as activity over mass of FSH measured by RIA, the horse product was 5.6 times more potent than the elk product. The recombinant products injected to immature female Wistar rats stimulated ovarian growth. The results suggest that the products obtained correspond to recombinant versions of the native elk and horse FSH. The availability of these recombinant products may aid in the development of more predictable and efficient techniques of ovarian stimulation in cervids, equids, and other species as well.

  3. Development of radiometric assays for quantification of enzyme activities of the key enzymes of thyroid hormones metabolism.

    PubMed

    Pavelka, S

    2014-01-01

    We newly elaborated and adapted several radiometric enzyme assays for the determination of activities of the key enzymes engaged in the biosynthesis (thyroid peroxidase, TPO) and metabolic transformations (conjugating enzymes and iodothyronine deiodinases, IDs) of thyroid hormones (THs) in the thyroid gland and in peripheral tissues, especially in white adipose tissue (WAT). We also elaborated novel, reliable radiometric methods for extremely sensitive determination of enzyme activities of IDs of types 1, 2 and 3 in microsomal fractions of different rat and human tissues, as well as in homogenates of cultured mammalian cells. The use of optimized TLC separation of radioactive products from the unconsumed substrates and film-less autoradiography of radiochromatograms, taking advantage of storage phosphor screens, enabled us to determine IDs enzyme activities as low as 10(-18) katals. In studies of the interaction of fluoxetine (Fluox) with the metabolism of THs, we applied adapted radiometric enzyme assays for iodothyronine sulfotransferases (ST) and uridine 5'-diphospho-glucuronyltransferase (UDP-GT). Fluox is the most frequently used representative of a new group of non-tricyclic antidepressant drugs--selective serotonin re-uptake inhibitors. We used the elaborated assays for quantification the effects of Fluox and for the assessment of the degree of potential induction of rat liver ST and/or UDP-GT enzyme activities by Fluox alone or in combination with T(3). Furthermore, we studied possible changes in IDs activities in murine adipose tissue under the conditions that promoted either tissue hypertrophy (obesogenic treatment) or involution (caloric restriction), and in response to leptin, using our newly developed radiometric enzyme assays for IDs. Our results suggest that deiodinase D1 has a functional role in WAT, with D1 possibly being involved in the control of adipose tissue metabolism and/or accumulation of the tissue. Significant positive correlation between

  4. Cadmium(II)-stimulated enzyme activation of Arabidopsis thaliana phytochelatin synthase 1.

    PubMed

    Ogawa, Shinya; Yoshidomi, Takahiro; Yoshimura, Etsuro

    2011-01-01

    Phytochelatin (PC), a class of heavy metal-binding peptides, is synthesized from the tripeptide glutathione (GSH) and/or previously synthesized PC in a reaction mediated by PC synthase (PCS). In the present study, the PC production rate catalyzed by recombinant Arabidopsis PCS1 (rAtPCS1) in the presence of a constant free Cd(II) level increased steadily and the kinetic parameters were approximated using a substituted-enzyme mechanism in which GSH and bis(glutathionato)cadmium acted as co-substrates. In contrast, the PC production rate as a function of GSH concentration at a constant total Cd(II) concentration reached a maximum, which shifted toward higher GSH concentrations as the concentration of Cd(II) was increased. These observations are consistent with the suggestion that rAtPCS1 possesses a Cd(II) binding site where Cd(II) binds to activate the enzyme. The affinity constant, optimized using a one-site mathematical model, successfully simulated the experimental data for the assay system using lower concentrations of Cd(II) (5 or 10 μM) but not for the assay using higher concentrations (50 or 500 μM), where a sigmoidal increase in PCS activity was evident. Furthermore, the PCS activity determined at a constant GSH concentration as a function of Cd(II) concentration also reached a maximum. These findings demonstrate that rAtPCS1 also possesses a second Cd(II) binding site where Cd(II) binds to induce an inhibitory effect. A two-site mathematical model was applied successfully to account for the observed phenomena, supporting the suggestion that rAtPCS1 possesses two Cd(II) binding sites.

  5. Microbial enzyme activities of peatland soils in south central Alaska lowlands

    EPA Science Inventory

    Microbial enzyme activities related to carbon and nutrient acquisition were measured on Alaskan peatland soils as indicators of nutrient limitation and biochemical sustainability. Peat decomposition is mediated by microorganisms and enzymes that in turn are limited by various ph...

  6. Influence of dietary nutritional composition on caterpillar salivary enzyme activity.

    PubMed

    Babic, Branislav; Poisson, Alexandre; Darwish, Shireef; Lacasse, Jean; Merkx-Jacques, Magali; Despland, Emma; Bede, Jacqueline C

    2008-01-01

    Caterpillars are faced with nutritional challenges when feeding on plants. In addition to harmful secondary metabolites and protein- and water-limitations, tissues may be carbohydrate-rich which may attenuate optimal caterpillar performance. Therefore, caterpillars have multiple strategies to cope with surplus carbohydrates. In this study, we raise the possibility of a pre-ingestive mechanism to metabolically deal with excess dietary sugars. Many Noctuid caterpillars secrete the labial salivary enzyme glucose oxidase (GOX), which oxidizes glucose to hydrogen peroxide and gluconate, a nutritionally unavailable carbohydrate to the insect. Beet armyworm, Spodoptera exigua, larvae were restricted to diets varying in protein to digestible carbohydrate (P:C) ratio (42p:21c; 33p:30c; 21p:42c) and total nutrient concentration (42% and 63%). High mortality and longer developmental time were observed when caterpillars were reared on the C-biased, P-poor diet (21p:42c). As the carbohydrate content of the diet increased, caterpillars egested excess glucose and a diet-dependent difference in assimilated carbohydrates and pupal biomass was not observed, even though caterpillars restricted to the C-biased diet (21p:42c) accumulated greater pupal lipid reserves. Larval labial salivary GOX activity was also diet-dependent and gluconate, the product of GOX activity, was detected in the frass. Unexpectedly, GOX activity was strongly and positively correlated with dietary protein content.

  7. Flavonoid inhibition of aromatase enzyme activity in human preadipocytes.

    PubMed

    Campbell, D R; Kurzer, M S

    1993-09-01

    Eleven flavonoid compounds were compared with aminoglutethimide (AG), a pharmaceutical aromatase inhibitor, for their abilities to inhibit aromatase enzyme activity in a human preadipocyte cell culture system. Flavonoids exerting no effect on aromatase activity were catechin, daidzein, equol, genistein, beta-naphthoflavone (BNF), quercetin and rutin. The synthetic flavonoid, alpha-naphthoflavone (ANF), was the most potent aromatase inhibitor, with an I50 value of 0.5 microM. Three naturally-occurring flavonoids, chrysin, flavone, and genistein 4'-methyl ether (Biochanin A) showed I50 values of 4.6, 68, and 113 microM, respectively, while AG showed an I50 value of 7.4 microM. Kinetic analyses showed that both AG and the flavonoids acted as competitive inhibitors of aromatase. The Ki values, indicating the effectiveness of inhibition, were 0.2, 2.4, 2.4, 22, and 49 microM, for ANF, AG, chrysin, flavone, and Biochanin A, respectively. Chrysin, the most potent of the naturally-occurring flavonoids, was similar in potency and effectiveness to AG, a pharmaceutical aromatase inhibitor used clinically in cases of estrogen-dependent carcinoma. These data suggest that flavonoid inhibition of peripheral aromatase activity may contribute to the observed cancer-preventive hormonal effects of plant-based diets.

  8. Low recombination activity of R region located at both ends of the HIV-1 genome.

    PubMed

    Urbanowicz, Anna; Kurzyńska-Kokorniak, Anna; Jankowska, Anna; Alejska, Magdalena; Figlerowicz, Marek

    2012-01-01

    Although two strand transfer events are indispensable for the synthesis of double-stranded DNA and establishing HIV-1 infection, the molecular basis of these phenomena is still unclear. The first obligatory template switching event occurs just at the beginning of the virus replication cycle and involves two copies of the 97-nucleotide long R region, located one each at the both ends of the HIV-1 genome (HIV-1 R). Thus, one can expect that the molecular mechanism of this process is similar to the mechanism of homologous recombination which operates in RNA viruses. To verify the above-mentioned hypothesis, we attempted to assess the recombination activity of HIV-1 R. To this end, we tested in vitro, how effectively it induces template switching by HIV-1 RT in comparison with another well-characterized sequence supporting frequent homologous crossovers in an unrelated virus (R region derived from Brome mosaic virus--BMV R). We also examined if the RNA sequences neighboring HIV-1 R influence its recombination activity. Finally, we tested if HIV-1 R could cause BMV polymerase complex to switch between RNA templates in vivo. Overall, our results have revealed a relatively low recombination activity of HIV-1 R as compared to BMV R. This observation suggests that different factors modulate the efficiency of the first obligatory strand transfer in HIV-1 and the homology-driven recombination in RNA viruses.

  9. Site-specific immobilization of recombinant antibody fragments through material-binding peptides for the sensitive detection of antigens in enzyme immunoassays.

    PubMed

    Kumada, Yoichi

    2014-11-01

    The immobilization of an antibody is one of the key technologies that are used to enhance the sensitivity and efficiency of the detection of target molecules in immunodiagnosis and immunoseparation. Recombinant antibody fragments such as VHH, scFv and Fabs produced by microorganisms are the next generation of ligand antibodies as an alternative to conventional whole Abs due to a smaller size and the possibility of site-directed immobilization with uniform orientation and higher antigen-binding activity in the adsorptive state. For the achievement of site-directed immobilization, affinity peptides for a certain ligand molecule or solid support must be introduced to the recombinant antibody fragments. In this mini-review, immobilization technologies for the whole antibodies (whole Abs) and recombinant antibody fragments onto the surfaces of plastics are introduced. In particular, the focus here is on immobilization technologies of recombinant antibody fragments utilizing affinity peptide tags, which possesses strong binding affinity towards the ligand molecules. Furthermore, I introduced the material-binding peptides that are capable of direct recognition of the target materials. Preparation and immobilization strategies for recombinant antibody fragments linked to material-binding peptides (polystyrene-binding peptides (PS-tags) and poly (methyl methacrylate)-binding peptide (PMMA-tag)) are the focus here, and are based on the enhancement of sensitivity and a reduction in the production costs of ligand antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

  10. Expression of feline recombinant interferon-gamma in baculovirus and demonstration of biological activity.

    PubMed

    Argyle, D J; Harris, M; Lawrence, C; McBride, K; Barron, R; McGillivray, C; Onions, D E

    1998-07-08

    We have previously reported the cloning of the coding sequence for feline-specific interferon-gamma. Here, we describe the expression of this sequence in a baculovirus system and demonstrate the biological activity of the recombinant protein. The coding sequence for feline interferon was directionally cloned into the baculovirus transfer vector pAcCL29-1. Transfer vector and linearized wild-type AcMNPV (BacPAK6) were used to co-transfect Sf9 cells by calcium phosphate coprecipitation. Subsequently, wild-type and recombinant viruses were separated by plaque assay. Recombinant plaques were expanded and a master stock of virus is produced. Production of biologically active interferon-gamma from infected Sf9 cells was demonstrated using a standard cytopathic effect reduction assay, utilising vesicular stomatitis virus (VSV), and an MHC class II induction assay.

  11. Phosphate-Modified Nucleotides for Monitoring Enzyme Activity.

    PubMed

    Ermert, Susanne; Marx, Andreas; Hacker, Stephan M

    2017-04-01

    Nucleotides modified at the terminal phosphate position have been proven to be interesting entities to study the activity of a variety of different protein classes. In this chapter, we present various types of modifications that were attached as reporter molecules to the phosphate chain of nucleotides and briefly describe the chemical reactions that are frequently used to synthesize them. Furthermore, we discuss a variety of applications of these molecules. Kinase activity, for instance, was studied by transfer of a phosphate modified with a reporter group to the target proteins. This allows not only studying the activity of kinases, but also identifying their target proteins. Moreover, kinases can also be directly labeled with a reporter at a conserved lysine using acyl-phosphate probes. Another important application for phosphate-modified nucleotides is the study of RNA and DNA polymerases. In this context, single-molecule sequencing is made possible using detection in zero-mode waveguides, nanopores or by a Förster resonance energy transfer (FRET)-based mechanism between the polymerase and a fluorophore-labeled nucleotide. Additionally, fluorogenic nucleotides that utilize an intramolecular interaction between a fluorophore and the nucleobase or an intramolecular FRET effect have been successfully developed to study a variety of different enzymes. Finally, also some novel techniques applying electron paramagnetic resonance (EPR)-based detection of nucleotide cleavage or the detection of the cleavage of fluorophosphates are discussed. Taken together, nucleotides modified at the terminal phosphate position have been applied to study the activity of a large diversity of proteins and are valuable tools to enhance the knowledge of biological systems.

  12. Structural snapshots of Xer recombination reveal activation by synaptic complex remodeling and DNA bending

    PubMed Central

    Bebel, Aleksandra; Karaca, Ezgi; Kumar, Banushree; Stark, W Marshall; Barabas, Orsolya

    2016-01-01

    Bacterial Xer site-specific recombinases play an essential genome maintenance role by unlinking chromosome multimers, but their mechanism of action has remained structurally uncharacterized. Here, we present two high-resolution structures of Helicobacter pylori XerH with its recombination site DNA difH, representing pre-cleavage and post-cleavage synaptic intermediates in the recombination pathway. The structures reveal that activation of DNA strand cleavage and rejoining involves large conformational changes and DNA bending, suggesting how interaction with the cell division protein FtsK may license recombination at the septum. Together with biochemical and in vivo analysis, our structures also reveal how a small sequence asymmetry in difH defines protein conformation in the synaptic complex and orchestrates the order of DNA strand exchanges. Our results provide insights into the catalytic mechanism of Xer recombination and a model for regulation of recombination activity during cell division. DOI: http://dx.doi.org/10.7554/eLife.19706.001 PMID:28009253

  13. Correlation Among Soil Enzyme Activities, Root Enzyme Activities, and Contaminant Removal in Two-Stage In Situ Constructed Wetlands Purifying Domestic Wastewater.

    PubMed

    Ni, Lixiao; Xu, Jiajun; Chu, Xianglin; Li, Shiyin; Wang, Peifang; Li, Yiping; Li, Yong; Zhu, Liang; Wang, Chao

    2016-07-01

    Two-stage in situ wetlands (two vertical flow constructed wetlands in parallel and a horizontal flow constructed wetland) were constructed for studying domestic wastewater purification and the correlations between contaminant removal and plant and soil enzyme activities. Results indicated the removal efficiency of NH4 (+) and NO3 (-) were significantly correlated with both urease and protease activity, and the removal of total phosphorus was significantly correlated with phosphatase activity. Chemical oxygen demand removal was not correlated with enzyme activity in constructed wetlands. Plant root enzyme (urease, phosphatase, protease and cellulose) activity correlation was apparent with all contaminant removal in the two vertical flow constructed wetlands. However, the correlation between the plant root enzyme activity and contaminant removal was poor in horizontal flow constructed wetlands. Results indicated that plant roots clearly played a role in the removal of contaminants.

  14. Ultrasonic Monitoring of Enzyme Catalysis; Enzyme Activity in Formulations for Lactose-Intolerant Infants.

    PubMed

    Altas, Margarida C; Kudryashov, Evgeny; Buckin, Vitaly

    2016-05-03

    The paper introduces ultrasonic technology for real-time, nondestructive, precision monitoring of enzyme-catalyzed reactions in solutions and in complex opaque media. The capabilities of the technology are examined in a comprehensive analysis of the effects of a variety of diverse factors on the performance of enzyme β-galactosidase in formulations for reduction of levels of lactose in infant milks. These formulations are added to infant's milk bottles prior to feeding to overcome the frequently observed intolerance to lactose (a milk sugar), a serious issue in healthy development of infants. The results highlight important impediments in the development of these formulations and also illustrate the capability of the described ultrasonic tools in the assessment of the performance of enzymes in complex reaction media and in various environmental conditions.

  15. Genetically engineered immunomodulatory Streptococcus thermophilus strains producing antioxidant enzymes exhibit enhanced anti-inflammatory activities.

    PubMed

    Del Carmen, Silvina; de Moreno de LeBlanc, Alejandra; Martin, Rebeca; Chain, Florian; Langella, Philippe; Bermúdez-Humarán, Luis G; LeBlanc, Jean Guy

    2014-02-01

    The aims of this study were to develop strains of lactic acid bacteria (LAB) having both immunomodulatory and antioxidant properties and to evaluate their anti-inflammatory effects both in vitro, in different cellular models, and in vivo, in a mouse model of colitis. Different Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were cocultured with primary cultures of mononuclear cells. Analysis of the pro- and anti-inflammatory cytokines secreted by these cells after coincubation with candidate bacteria revealed that L. delbrueckii subsp. bulgaricus CRL 864 and S. thermophilus CRL 807 display the highest anti-inflammatory profiles in vitro. Moreover, these results were confirmed in vivo by the determination of the cytokine profiles in large intestine samples of mice fed with these strains. S. thermophilus CRL 807 was then transformed with two different plasmids harboring the genes encoding catalase (CAT) or superoxide dismutase (SOD) antioxidant enzymes, and the anti-inflammatory effects of recombinant streptococci were evaluated in a mouse model of colitis induced by trinitrobenzenesulfonic acid (TNBS). Our results showed a decrease in weight loss, lower liver microbial translocation, lower macroscopic and microscopic damage scores, and modulation of the cytokine production in the large intestines of mice treated with either CAT- or SOD-producing streptococci compared to those in mice treated with the wild-type strain or control mice without any treatment. Furthermore, the greatest anti-inflammatory activity was observed in mice receiving a mixture of both CAT- and SOD-producing streptococci. The addition of L. delbrueckii subsp. bulgaricus CRL 864 to this mixture did not improve their beneficial effects. These findings show that genetically engineering a candidate bacterium (e.g., S. thermophilus CRL 807) with intrinsic immunomodulatory properties by introducing a gene expressing an antioxidant enzyme enhances its anti

  16. Genetically Engineered Immunomodulatory Streptococcus thermophilus Strains Producing Antioxidant Enzymes Exhibit Enhanced Anti-Inflammatory Activities

    PubMed Central

    del Carmen, Silvina; de Moreno de LeBlanc, Alejandra; Martin, Rebeca; Chain, Florian; Langella, Philippe; Bermúdez-Humarán, Luis G.

    2014-01-01

    The aims of this study were to develop strains of lactic acid bacteria (LAB) having both immunomodulatory and antioxidant properties and to evaluate their anti-inflammatory effects both in vitro, in different cellular models, and in vivo, in a mouse model of colitis. Different Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were cocultured with primary cultures of mononuclear cells. Analysis of the pro- and anti-inflammatory cytokines secreted by these cells after coincubation with candidate bacteria revealed that L. delbrueckii subsp. bulgaricus CRL 864 and S. thermophilus CRL 807 display the highest anti-inflammatory profiles in vitro. Moreover, these results were confirmed in vivo by the determination of the cytokine profiles in large intestine samples of mice fed with these strains. S. thermophilus CRL 807 was then transformed with two different plasmids harboring the genes encoding catalase (CAT) or superoxide dismutase (SOD) antioxidant enzymes, and the anti-inflammatory effects of recombinant streptococci were evaluated in a mouse model of colitis induced by trinitrobenzenesulfonic acid (TNBS). Our results showed a decrease in weight loss, lower liver microbial translocation, lower macroscopic and microscopic damage scores, and modulation of the cytokine production in the large intestines of mice treated with either CAT- or SOD-producing streptococci compared to those in mice treated with the wild-type strain or control mice without any treatment. Furthermore, the greatest anti-inflammatory activity was observed in mice receiving a mixture of both CAT- and SOD-producing streptococci. The addition of L. delbrueckii subsp. bulgaricus CRL 864 to this mixture did not improve their beneficial effects. These findings show that genetically engineering a candidate bacterium (e.g., S. thermophilus CRL 807) with intrinsic immunomodulatory properties by introducing a gene expressing an antioxidant enzyme enhances its anti

  17. Expression and Enzyme Activity Detection of a Sepiapterin Reductase Gene from Musca domestica Larva.

    PubMed

    Tang, Yan; Pei, Zhihua; Liu, Lei; Wang, Dongfang; Kong, Lingcong; Liu, Shuming; Jiang, Xiuyun; Gao, Yunhang; Ma, Hongxia

    2017-02-01

    Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases and nitric oxide synthase. Sepiapterin reductase (SPR) catalyzes the final steps of BH4 biosynthesis. Studies on SPR from several insects and other organisms have been reported. However, thus far, enzyme activity of SPR in Musca domestica is kept unknown. In this study, 186 differentially expressed genes including SPR gene from Musca domestica (MDSPR) were screened in subtractive cDNA library. The MDSPR gene was cloned, and the recombinant MDSPI16 protein was expressed as a 51-kDa protein in soluble form. The MDSPR exhibited strong activity to the substrate sepiapterin (SP). The values of Vmax and Km of the MDSPR for SP were 6.83 μM/min and 23.48 μM, and the optimum temperature and pH of MDSPR were 50 °C and 4.0, respectively. This study provides new hypotheses and methods for the production of BH4 using insect-derived SPR.

  18. Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology.

    PubMed

    Collins, Kathleen; Nilsen, Timothy W

    2013-08-01

    Current investigation of RNA transcriptomes relies heavily on the use of retroviral reverse transcriptases. It is well known that these enzymes have many limitations because of their intrinsic properties. This commentary highlights the recent biochemical characterization of a new family of reverse transcriptases, those encoded by group II intron retrohoming elements. The novel properties of these enzymes endow them with the potential to revolutionize how we approach RNA analyses.

  19. Low-scale expression and purification of an active putative iduronate 2-sulfate sulfatase-Like enzyme from Escherichia coli K12.

    PubMed

    Morales-Álvarez, Edwin David; Rivera-Hoyos, Claudia Marcela; Baena-Moncada, Angélica María; Landázuri, Patricia; Poutou-Piñales, Raúl A; Sáenz-Suárez, Homero; Barrera, Luis A; Echeverri-Peña, Olga Y

    2013-04-01

    The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78.13 to 94.35 ng/ml and the specific activity varied from 34.20 to 25.97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold, compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like) emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization, future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli.

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

    PubMed

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

    2015-09-01

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

  1. Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities.

    PubMed

    Meneely, Kathleen M; Luo, Qianyi; Lamb, Audrey L

    2013-11-01

    The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the "interchange" hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, "permute" hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities.

  2. Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities

    PubMed Central

    Meneely, Kathleen M.; Luo, Qianyi; Lamb, Audrey L.

    2013-01-01

    The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the “interchange” hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, “permute” hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities. PMID:24055536

  3. Suppression of human cytochrome P450 aromatase activity by monoclonal and recombinant antibody fragments and identification of a stable antigenic complex.

    PubMed

    Lala, Puloma; Higashiyama, Tadayoshi; Erman, Mary; Griswold, Jennifer; Wagner, Traci; Osawa, Yoshio; Ghosh, Debashis

    2004-03-01

    Human cytochrome P450 aromatase (P450arom) is responsible for biosynthesis of estrogens from androgens. Monoclonal antibody MAb3-2C2 to P450arom specifically binds to a conformational epitope and suppresses the enzyme activity in a dose-dependent manner. The crystal structure of the Fab fragment of MAb3-2C2 has been used to engineer a recombinant single chain antibody fragment (scFv) and a homodimeric variable domain of the light chain (VL(2)). These recombinant antibody fragments have been expressed in Escherichia coli and purified. Here, we show that the recombinant scFv suppresses P450arom activity with an IC(50) value similar to that of natural MAb3-2C2 F(ab')(2). The recombinant VL(2) also exhibits dose-dependent suppression of the P450arom activity, but at a reduced level, demonstrating that the homodimer is unable to fully mimic the complementarity determining region (CDR) of a variable heavy chain (VH)-VL heterodimer. We prepare and purify a stable complex of P450arom with MAb3-2C2 F(ab')(2) and show that the complex migrates and precipitates as a single molecular assembly. Efforts to crystallize P450arom for structure-function studies have yielded small single crystals. Our results suggest that formation of stable complexes with fragments of the monoclonal antibody could provide an alternative method for crystallization of P450arom.

  4. Structure-activity relationship of a recombinant hybrid Manganese superoxide dismutase of Staphylococcus saprophyticus/S. equorum.

    PubMed

    Retnoningrum, Debbie S; Arumsari, Sekar; Artarini, Anita; Ismaya, Wangsa T

    2017-05-01

    Recombinant hybrid Manganese superoxide dismutase from Staphyloccus saphropyticus/S. equorum (rMnSODSeq) exhibits stability at high temperatures. The enzyme occurs as a dimer that dissociates around 52°C prior to unfolding of the monomer around 64°C, demonstrating contribution of the dimeric form to stability. Here, structure - activity relationship of rMnSODSeq was evaluated on the basis of its activity and stability in the presence of inhibitors, NaCl, denaturants, detergents, reducing agents, and at different pH values. The activity was evaluated at both 37°C and 52°C, which the latter is the temperature for dissociation of the dimer. Dimer to monomer transition coincided with significant decrease in residual activity at 52°C. However, the activity assay results at 52°C and 37°C suggest spontaneous re-association of the monomer into dimer. Intriguingly, various new species with melting temperature (TM) values other than those of the dimer or monomer were observed. These species displayed medium to comparable level of residual activities to the native at 37°C. This report suggests that dimer to monomer transition may be not the only explanation for activity loss or decrease.

  5. Expression and activity of recombinant proaerolysin derived from Aeromonas hydrophila cultured from diseased channel catfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proaerolysin-coding gene was cloned from the genomic DNA of A. hydrophila and heterologously expressed in E. coli. The purified recombinant proaerolysin was inactive and could be activated by treatment with proteases, furin and trypsin, and extra-cellular proteins (ECPs, the cell-free supernatant of...

  6. Enzyme catalysis: C-H activation is a Reiske business

    NASA Astrophysics Data System (ADS)

    Bruner, Steven D.

    2011-05-01

    Enzymes that selectively oxidize unactivated C-H bonds are capable of constructing complex molecules with high efficiency. A new member of this enzyme family is RedG, a Reiske-type oxygenase that catalyses chemically challenging cyclizations in the biosynthesis of prodiginine natural products.

  7. Engineering a hyper-catalytic enzyme by photo-activated conformation modulation

    SciTech Connect

    Agarwal, Pratul K

    2012-01-01

    Enzyme engineering for improved catalysis has wide implications. We describe a novel chemical modification of Candida antarctica lipase B that allows modulation of the enzyme conformation to promote catalysis. Computational modeling was used to identify dynamical enzyme regions that impact the catalytic mechanism. Surface loop regions located distal to active site but showing dynamical coupling to the reaction were connected by a chemical bridge between Lys136 and Pro192, containing a derivative of azobenzene. The conformational modulation of the enzyme was achieved using two sources of light that alternated the azobenzene moiety in cis and trans conformations. Computational model predicted that mechanical energy from the conformational fluctuations facilitate the reaction in the active-site. The results were consistent with predictions as the activity of the engineered enzyme was found to be enhanced with photoactivation. Preliminary estimations indicate that the engineered enzyme achieved 8-52 fold better catalytic activity than the unmodulated enzyme.

  8. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion.

    PubMed

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W; Liu, Yan; Walter, Nils G; Yan, Hao

    2016-02-10

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

  9. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

    PubMed Central

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

    2016-01-01

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology. PMID:26861509

  10. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

    NASA Astrophysics Data System (ADS)

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

    2016-02-01

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

  11. Substrate supply, fine roots, and temperature control proteolytic enzyme activity in temperate forest soils.

    PubMed

    Brzostek, Edward R; Finzi, Adrien C

    2011-04-01

    Temperature and substrate availability constrain the activity of the extracellular enzymes that decompose and release nutrients from soil organic matter (SOM). Proteolytic enzymes are the primary class of enzymes involved in the depolymerization of nitrogen (N) from proteinaceous components of SOM, and their activity affects the rate of N cycling in forest soils. The objectives of this study were to determine whether and how temperature and substrate availability affect the activity of proteolytic enzymes in temperate forest soils, and whether the activity of proteolytic enzymes and other enzymes involved in the acquisition of N (i.e., chitinolytic and ligninolytic enzymes) differs between trees species that form associations with either ectomycorrhizal or arbuscular mycorrhizal fungi. Temperature limitation of proteolytic enzyme activity was observed only early in the growing season when soil temperatures in the field were near 4 degrees C. Substrate limitation to proteolytic activity persisted well into the growing season. Ligninolytic enzyme activity was higher in soils dominated by ectomycorrhizal associated tree species. In contrast, the activity of proteolytic and chitinolytic enzymes did not differ, but there were differences between mycorrhizal association in the control of roots on enzyme activity. Roots of ectomycorrhizal species but not those of arbuscular mycorrhizal species exerted significant control over proteolytic, chitinolytic, and ligninolytic enzyme activity; the absence of ectomycorrhizal fine roots reduced the activity of all three enzymes. These results suggest that climate warming in the absence of increases in substrate availability may have a modest effect on soil-N cycling, and that global changes that alter belowground carbon allocation by trees are likely to have a larger effect on nitrogen cycling in stands dominated by ectomycorrhizal fungi.

  12. Controlled exogenous enzyme imbibition and activation in whole chickpea seed enzyme reactor (SER).

    PubMed

    Kliger, Eynav; Fischer, Lutz; Lutz-Wahl, Sabine; Saguy, I Sam

    2011-05-01

    Chickpeas are of excellent quality (protein, vitamins, minerals, unsaturated fatty acids) and very low in phytoestrogen, making them a potentially promising source for vegetarian-based infant formula (VBIF). However, their high starch and fiber concentration could hinder their utilization for infants. To overcome this natural shortcoming, a solid-state "enzymation" (SSE) process was developed in which imbibition of exogenous enzyme facilitates hydrolysis within the intact chickpea seed. The process was termed seed enzyme reactor (SER). Liquid imbibition data of dry chickpeas during soaking were fitted with the Weibull distribution model. The derived Weibull shape parameter, β, value (0.77 ± 0.11) indicated that the imbibition mechanism followed Fickian diffusion. Imbibition occurred through the coat and external layers. The process was tested using green fluorescent protein (GFP) as an exogenous marker, and involved soaking, thermal treatment, peeling, microwave partial drying, rehydration in enzyme solution, and SSE at an adjusted pH, time, and temperature. Amylases, or a combination of amylases and cellulases, resulted in significant carbohydrate hydrolysis (23% and 47% of the available starch, respectively). In addition, chickpea initial raffinose and stachyose concentration was significantly reduced (91% and 92%, respectively). The process could serve as a proof of concept, requiring additional development and optimization to become a full industrial application.

  13. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies.

    PubMed

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2014-02-20

    Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

  14. Effect of copper on the recombination activity of extended defects in silicon

    SciTech Connect

    Feklisova, O. V. Yakimov, E. B.

    2015-06-15

    The effect of copper atoms introduced by high-temperature diffusion on the recombination properties of dislocations and dislocation trails in p-type single-crystal silicon is studied by the electron-beam-induced current technique. It is shown that, in contrast to dislocations, dislocation trails exhibit an increase in recombination activity after the introduction of copper. Bright contrast appearance in the vicinity of dislocation trails is detected after the diffusion of copper and quenching of the samples. The contrast depends on the defect density in these trails.

  15. Evidence for reduced charge recombination in carbon nanotube/perovskite-based active layers

    NASA Astrophysics Data System (ADS)

    Bag, Monojit; Renna, Lawrence A.; Jeong, Seung Pyo; Han, Xu; Cutting, Christie L.; Maroudas, Dimitrios; Venkataraman, D.

    2016-10-01

    Using impedance spectroscopy and computation, we show that incorporation of multi-walled carbon nanotubes (MWCNTs) in the bulk of the active layer of perovskite-based solar cells reduces charge recombination and increases the open circuit voltage. An ∼87% reduction in recombination was achieved when MWCNTs were introduced in the planar-heterostructure perovskite solar cell containing mixed counterions. The open circuit voltage (Voc) of perovskite/MWCNTs devices was increased by 70 mV, while the short circuit current density (Jsc) and fill factor (FF) remained unchanged.

  16. Engineering of Recombinant Poplar Deoxy-D-Xylulose-5-Phosphate Synthase (PtDXS) by Site-Directed Mutagenesis Improves Its Activity

    PubMed Central

    Banerjee, Aparajita; Preiser, Alyssa L.

    2016-01-01

    Deoxyxylulose 5-phosphate synthase (DXS), a thiamine diphosphate (ThDP) dependent enzyme, plays a regulatory role in the methylerythritol 4-phosphate (MEP) pathway. Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the end products of this pathway, inhibit DXS by competing with ThDP. Feedback inhibition of DXS by IDP and DMADP constitutes a significant metabolic regulation of this pathway. The aim of this work was to experimentally test the effect of key residues of recombinant poplar DXS (PtDXS) in binding both ThDP and IDP. This work also described the engineering of PtDXS to improve the enzymatic activity by reducing its inhibition by IDP and DMADP. We have designed and tested modifications of PtDXS in an attempt to reduce inhibition by IDP. This could possibly be valuable by removing a feedback that limits the usefulness of the MEP pathway in biotechnological applications. Both ThDP and IDP use similar interactions for binding at the active site of the enzyme, however, ThDP being a larger molecule has more anchoring sites at the active site of the enzyme as compared to the inhibitors. A predicted enzyme structure was examined to find ligand-enzyme interactions, which are relatively more important for inhibitor-enzyme binding than ThDP-enzyme binding, followed by their modifications so that the binding of the inhibitors can be selectively affected compared to ThDP. Two alanine residues important for binding ThDP and the inhibitors were mutated to glycine. In two of the cases, both the IDP inhibition and the overall activity were increased. In another case, both the IDP inhibition and the overall activity were reduced. This provides proof of concept that it is possible to reduce the feedback from IDP on DXS activity. PMID:27548482

  17. Engineering of Recombinant Poplar Deoxy-D-Xylulose-5-Phosphate Synthase (PtDXS) by Site-Directed Mutagenesis Improves Its Activity.

    PubMed

    Banerjee, Aparajita; Preiser, Alyssa L; Sharkey, Thomas D

    2016-01-01

    Deoxyxylulose 5-phosphate synthase (DXS), a thiamine diphosphate (ThDP) dependent enzyme, plays a regulatory role in the methylerythritol 4-phosphate (MEP) pathway. Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the end products of this pathway, inhibit DXS by competing with ThDP. Feedback inhibition of DXS by IDP and DMADP constitutes a significant metabolic regulation of this pathway. The aim of this work was to experimentally test the effect of key residues of recombinant poplar DXS (PtDXS) in binding both ThDP and IDP. This work also described the engineering of PtDXS to improve the enzymatic activity by reducing its inhibition by IDP and DMADP. We have designed and tested modifications of PtDXS in an attempt to reduce inhibition by IDP. This could possibly be valuable by removing a feedback that limits the usefulness of the MEP pathway in biotechnological applications. Both ThDP and IDP use similar interactions for binding at the active site of the enzyme, however, ThDP being a larger molecule has more anchoring sites at the active site of the enzyme as compared to the inhibitors. A predicted enzyme structure was examined to find ligand-enzyme interactions, which are relatively more important for inhibitor-enzyme binding than ThDP-enzyme binding, followed by their modifications so that the binding of the inhibitors can be selectively affected compared to ThDP. Two alanine residues important for binding ThDP and the inhibitors were mutated to glycine. In two of the cases, both the IDP inhibition and the overall activity were increased. In another case, both the IDP inhibition and the overall activity were reduced. This provides proof of concept that it is possible to reduce the feedback from IDP on DXS activity.

  18. Molluscan mobile elements similar to the vertebrate recombination-activating genes

    PubMed Central

    Panchin, Yuri; Moroz, Leonid L.

    2009-01-01

    Animal genomes contain ~20,000 genes. Additionally millions of genes for antigen receptors are generated in cells of the immune system from the sets of separate gene segments by a mechanism known as the V(D)J somatic recombination. The components of the V(D)J recombination system, Recombination-Activating Gene proteins (RAG1 and RAG2) and recombination signal sequence (RSS), are thought to have “entered” the vertebrate genome as a hypothetical “RAG transposon”. Recently discovered mobile elements have terminal inverted repeats (TIRs) similar to RSS and may encode proteins with a different degree of similarity to RAG1. We describe a novel N-RAG-TP transposon identified from the sea slug Aplysia californica that encodes a protein similar to the N-terminal part of RAG1 in vertebrates. This refines the “RAG transposon” hypothesis and allows us to propose a scenario for V(D)J recombination machinery evolution from a relic transposon related to the existing mobile elements N-RAG-TP, Chapaev and Transib. PMID:18313399

  19. Anti-angiogenesis and anti-tumor activity of recombinant anginex

    SciTech Connect

    Brandwijk, Ricardo J.M.G.E.; Dings, Ruud P.M.; Linden, Edith van der; Mayo, Kevin H.; Thijssen, Victor L.J.L.; Griffioen, Arjan W. . E-mail: aw.griffioen@path.unimaas.nl

    2006-10-27

    Anginex, a synthetic 33-mer angiostatic peptide, specifically inhibits vascular endothelial cell proliferation and migration along with induction of apoptosis in endothelial cells. Here we report on the in vivo characterization of recombinant anginex and use of the artificial anginex gene for gene therapy approaches. Tumor growth of human MA148 ovarian carcinoma in athymic mice was inhibited by 80% when treated with recombinant anginex. Histological analysis of the tumors showed an approximate 2.5-fold reduction of microvessel density, suggesting that angiogenesis inhibition is the cause of the anti-tumor effect. Furthermore, there was a significant correlation between the gene expression patterns of 16 angiogenesis-related factors after treatment with both recombinant and synthetic anginex. To validate the applicability of the anginex gene for gene therapy, stable transfectants of murine B16F10 melanoma cells expressing recombinant anginex were made. Supernatants of these cells inhibited endothelial cell proliferation in vitro. Furthermore, after subcutaneous injection of these cells in C57BL/6 mice, an extensive delay in tumor growth was observed. These data show that the artificial anginex gene can be used to produce a recombinant protein with similar activity as its synthetic counterpart and that the gene can be applied in gene therapy approaches for cancer treatment.

  20. Submillimeter recombination lines in dust-obscured starbursts and active galactic nuclei

    SciTech Connect

    Scoville, N.; Murchikova, L.

    2013-12-10

    We examine the use of submillimeter (submm) recombination lines of H, He, and He{sup +} to probe the extreme ultraviolet (EUV) luminosity of starbursts (SBs) and active galactic nuclei (AGNs). We find that the submm recombination lines of H, He, and He{sup +} are in fact extremely reliable and quantitative probes of the EUV continuum at 13.6 eV to above 54.6 eV. At submm wavelengths, the recombination lines originate from low energy levels (n = 20-50). The maser amplification, which poses significant problems for quantitative interpretation of the higher n, radio frequency recombination lines, is insignificant. Lastly, at submm wavelengths, the dust extinction is minimal. The submm line luminosities are therefore directly proportional to the emission measures (EM{sub ION} = n{sub e} × n {sub ion} × volume) of their ionized regions. We also find that the expected line fluxes are detectable with ALMA and can be imaged at ∼0.''1 resolution in low redshift ultraluminous infrared galaxies. Imaging of the H I lines will provide accurate spatial and kinematic mapping of the star formation distribution in low-z IR-luminous galaxies, and the relative fluxes of the H I and He II recombination lines will strongly constrain the relative contributions of SBs and AGNs to the luminosity. The H I lines should also provide an avenue to constraining the submm dust extinction curve.

  1. Chaperone-like activities of {alpha}-synuclein: {alpha}-Synuclein assists enzyme activities of esterases

    SciTech Connect

    Ahn, Misun; Kim, SeungBum; Kang, Mira; Ryu, Yeonwoo . E-mail: ywryu@ajou.ac.kr; Doohun Kim, T. . E-mail: doohunkim@ajou.ac.kr

    2006-08-11

    {alpha}-Synuclein, a major constituent of Lewy bodies (LBs), has been implicated to play a critical role in the pathogenesis of Parkinson's disease (PD), although the physiological function of {alpha}-synuclein has not yet been known. Here we have shown that {alpha}-synuclein, which has no well-defined secondary or tertiary structure, can protect the enzyme activity of microbial esterases against stress conditions such as heat, pH, and organic solvents. In particular, the flexibility of {alpha}-synuclein and its C-terminal region seems to be important for complex formation, but the structural integrity of the C-terminal region may not be required for stabilization of enzyme activity. In addition, atomic force microscopy (AFM) and in vivo enzyme assays showed highly specific interactions of esterases with {alpha}-synuclein. Our results indicate that {alpha}-synuclein not only protects the enzyme activity of microbial esterases in vitro, but also can stabilize the active conformation of microbial esterases in vivo.

  2. High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity

    PubMed Central

    Iglesias-Figueroa, Blanca; Valdiviezo-Godina, Norberto; Siqueiros-Cendón, Tania; Sinagawa-García, Sugey; Arévalo-Gallegos, Sigifredo; Rascón-Cruz, Quintín

    2016-01-01

    In this study, bovine lactoferrin (bLf), an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf) in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin) demonstrated antibacterial activity against Escherichia coli (E. coli) BL21DE3, Staphylococcus aureus (S. aureus) FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa) ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly. PMID:27294912

  3. Metabolism of the major Echinacea alkylamide N-isobutyldodeca-2E,4E,8Z,10Z-tetraenamide by human recombinant cytochrome P450 enzymes and human liver microsomes.

    PubMed

    Toselli, F; Matthias, A; Bone, K M; Gillam, E M J; Lehmann, R P

    2010-08-01

    Echinacea preparations are used for the treatment and prevention of upper respiratory tract infections. The phytochemicals believed responsible for the immunomodulatory properties are the alkylamides found in ethanolic extracts, with one of the most abundant being the N-isobutyldodeca-2E,4E,8Z,10Z-tetraenamide (1). In this study, we evaluated the human cytochrome P450 enzymes involved in the metabolism of this alkylamide using recombinant P450s, human liver microsomes and pure synthetic compound. Epoxidation, N-dealkylation and hydroxylation products were detected, with different relative amounts produced by recombinant P450s and microsomes. The major forms showing activity toward the metabolism of 1 were CYP1A1, CYP1A2 (both producing the same epoxide and N-dealkylation product), CYP2A13 (producing two epoxides), and CYP2D6 (producing two epoxides and an hydroxylated metabolite). Several other forms showed less activity. In incubations with human liver microsomes and selective inhibitors, CYP2E1 was found to be principally responsible for producing the dominant, hydroxylation product, whereas CYP2C9 was the principal source of the epoxides and CYP1A2 was responsible for the dealkylation product. In summary, in this study the relative impacts of the main human xenobiotic-metabolizing cytochrome P450s on the metabolism of a major Echinacea alkylamide have been established and the metabolites formed have been identified.

  4. Enzyme activities in plasma, kidney, liver, and muscle of five avian species

    USGS Publications Warehouse

    Franson, J.C.; Murray, H.C.; Bunck, C.

    1985-01-01

    Activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatine phosphokinase (CPK), and lactate dehydrogenase (LDH) were determined in plasma, kidney, liver, and muscle from five species of captive birds. Few differences occurred in plasma activities between sexes but considerable differences occurred between species. All five enzymes were detected in each of the tissues sampled. Relative enzyme activities in liver, kidney, and muscle were similar for each species. CPK activity was much higher in muscle than in liver or kidney and, of the five enzymes studied, may be the best indicator of muscle damage. Most of the other enzymes were more evenly distributed among the three tissues, and no organ-specific enzyme could be identified for liver or kidney. Because of interspecific variations in plasma enzyme activities, it is important to establish baseline values for each species to ensure accurate interpretation of results.

  5. Production of bioactive γ-glutamyl transpeptidase in Escherichia coli using SUMO fusion partner and application of the recombinant enzyme to L-theanine synthesis.

    PubMed

    Wang, Qi; Min, Cui; Zhu, Fenfen; Xin, Yinqiang; Zhang, Shuangquan; Luo, Lan; Yin, Zhimin

    2011-05-01

    The amino acid L-theanine (γ-glutamylethylamide) has potential important applications in the food and pharmaceutical industries and increased demand for this compound is expected. It is the major "umami" (good taste) component of tea and its favorable physiological effects on mammals have been reported. An enzymatic method for the synthesis of L-theanine involving recombinant Escherichia coli γ-glutamyltranspeptidase (GGT) has been developed. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Escherichia coli γ-GGT. In order to obtain γ-GGT with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9 (consisting of glycerol and inorganic salts) and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel-nitrilotriacetic acid (Ni-NTA) resin chromatography with a yield of 115 mg per liter fermentation culture. After the SUMO/γ-GGT fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni-NTA column. Finally, about 62 mg recombinant γ-GGT was obtained from 1 l fermentation culture with no less than 95% purity. The recombinant γ-GGT showed great transpeptidase activity, with 1500 U of purified recombinant γ-GGT in a 1-l reaction system, a biosynthesis yield of 41 g of L-theanine was detected by paper chromatography or high pressure liquid chromatography (HPLC). Thus, the application of SUMO technology to the expression and purification of γ-GGT potentially could be employed for the industrial production of L-theanine.

  6. CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems.

    PubMed

    Wu, Zhexue; Lee, Doohyun; Joo, Jeongmin; Shin, Jung-Hoon; Kang, Wonku; Oh, Sangtaek; Lee, Do Yup; Lee, Su-Jun; Yea, Sung Su; Lee, Hye Suk; Lee, Taeho; Liu, Kwang-Hyeon

    2013-11-01

    Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.

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

    PubMed

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

    2017-04-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2017-01-01

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

  9. Characterization and biological activities of recombinant human plasminogen kringle 1-3 produced in Escherichia coli.

    PubMed

    You, Weon-Kyoo; So, Seung-Ho; Sohn, Young-Doug; Lee, Hyosil; Park, Doo-Hong; Chung, Soo-Il; Chung, Kwang-Hoe

    2004-07-01

    Angiogenesis, the formation of new capillaries from preexisting blood vessels, is involved in many pathological conditions, for example, tumorigenesis, diabetic retinopathy, and rheumatoid arthritis. Angiostatin, which contains the kringle 1-4 domains of plasminogen, is known to be a potent inhibitor of angiogenesis and a strong suppressor of various solid tumors. In this study, we expressed recombinant protein containing the kringle 1-3 domains of human plasminogen in Escherichia coli and investigated its biological activities. The protein was successfully refolded from inclusion bodies and purified at a 30% overall yield, as a single peak by HPLC. The purified recombinant protein had biochemical properties that were similar to those of the native form, which included molecular size, lysine-binding capacity, and immunoreactivity with a specific antibody. The recombinant protein was also found to strongly inhibit the proliferation of bovine capillary endothelial cells in vitro, and the formation of new capillaries on chick embryos. In addition, it suppressed the growth of primary Lewis lung carcinoma and B16 melanoma in an in vivo mouse model. Our findings suggest that the recombinant kringle 1-3 domains in a prokaryote expression system have anti-angiogenic activities, which may be useful in clinical and basic research in the field of angiogenesis.

  10. Kinetic study of an enzymic cycling system coupled to an enzymic step: determination of alkaline phosphatase activity.

    PubMed Central

    Valero, E; Varón, R; García-Carmona, F

    1995-01-01

    A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response. PMID:7619054

  11. Annexation of a high-activity enzyme in a synthetic three-enzyme complex greatly decreases the degree of substrate channeling.

    PubMed

    You, Chun; Zhang, Y-H Percival

    2014-06-20

    The self-assembled three-enzyme complex containing triosephosphate isomerase (TIM), aldolase (ALD), and fructose 1,6-biphosphatase (FBP) was constructed via a mini-scaffoldin containing three different cohesins and the three dockerin-containing enzymes. This enzyme complex exhibited 1 order of magnitude higher initial reaction rates than the mixture of noncomplexed three enzymes. In this enzyme cascade reactions, the reaction mediated by ALD was the rate-limiting step. To understand the in-depth role of the rate-limiting enzyme ALD in influencing the substrate channeling effect of synthetic enzyme complexes, low-activity ALD from Thermotoga maritima was replaced with a similar-size ALD isolated from Thermus thermophilus, where the latter had more than 5 times specific activity of the former. The synthetic three-enzyme complexes annexed with either low-activity or high-activity ALDs exhibited higher initial reaction rates than the mixtures of the two-enzyme complex (TIM-FBP) and the nonbound low-activity or high activity ALD at the same enzyme concentration. It was also found that the annexation of more high-activity ALD in the synthetic enzyme complexes drastically decreased the degree of substrate channeling from 7.5 to 1.5. These results suggested that the degree of substrate channeling in synthetic enzyme complexes depended on the enzyme choice. This study implied that the construction of synthetic enzyme enzymes in synthetic cascade pathways could be a very important tool to accrelerate rate-limiting steps controlled by low-activity enzymes.

  12. An inhibition enzyme immuno assay exploring recombinant invariant surface glycoprotein and monoclonal antibodies for surveillance of surra in animals.

    PubMed

    Rudramurthy, G R; Sengupta, P P; Ligi, M; Rahman, H

    2017-02-20

    The present study is aimed at the development of inhibition ELISA (I-ELISA) exploring monoclonal antibodies (MAbs) and recombinant invariant surface glycoprotein. The extracellular domain (ED) of invariant surface glycoprotein (ISG-75) from Trypanosoma evasni has been heterologously expressed in Pichia pastoris (X-33). The recombinant ISG-75 (rISG-75ED) was characterized by immunoblot and ELISA, followed by the production of MAbs against rISG-75ED. The MAbs were characterized by immunoblot and then explored in the development of I-ELISA for the detection of surra. The diagnostic potential of the developed test has been evaluated using 1192 field sera sample including cattle, buffalo, donkey, horse and camel. The statistical analysis of the data showed optimum combination of diagnostic sensitivity and specificity at 98.8% and 99.2% respectively, with cut-off percentage inhibition (PI) value of >45. The Cohen's kappa coefficient of agreement was found to be 0.98. Hence, the diagnostic test developed in the present study can be exploited as a potential and reliable tool in the serodiagnosis and surveillance of surra in animals.

  13. Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity

    PubMed Central

    Funk, Michael A.; Judd, Evan T.; Marsh, E. Neil G.; Elliott, Sean J.; Drennan, Catherine L.

    2014-01-01

    Anaerobic degradation of the environmental pollutant toluene is initiated by the glycyl radical enzyme benzylsuccinate synthase (BSS), which catalyzes the radical addition of toluene to fumarate, forming benzylsuccinate. We have determined crystal structures of the catalytic α-subunit of BSS with its accessory subunits β and γ, which both bind a [4Fe-4S] cluster and are essential for BSS activity in vivo. We find that BSSα has the common glycyl radical enzyme fold, a 10-stranded β/α-barrel that surrounds the glycyl radical cofactor and active site. Both accessory subunits β and γ display folds related to high potential iron–sulfur proteins but differ substantially from each other in how they interact with the α-subunit. BSSγ binds distally to the active site, burying a hydrophobic region of BSSα, whereas BSSβ binds to a hydrophilic surface of BSSα that is proximal to the active site. To further investigate the function of BSSβ, we determined the structure of a BSSαγ complex. Remarkably, we find that the barrel partially opens, allowing the C-terminal region of BSSα that houses the glycyl radical to shift within the barrel toward an exit pathway. The structural changes that we observe in the BSSαγ complex center around the crucial glycyl radical domain, thus suggesting a role for BSSβ in modulating the conformational dynamics required for enzyme activity. Accompanying proteolysis experiments support these structural observations. PMID:24982148

  14. Identification of active sites in amidase: Evolutionary relationship between amide bond- and peptide bond-cleaving enzymes

    PubMed Central

    Kobayashi, Michihiko; Fujiwara, Yoshie; Goda, Masahiko; Komeda, Hidenobu; Shimizu, Sakayu

    1997-01-01

    Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes. Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism. For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification. With regard to the presumptive active site residue Cys203, a Cys203 → Ala mutant enzyme still retained 11.5% of the original specific activity. In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the amidase. Glutamic acid substitution of Asp191 reduced the specific activity of the mutant enzyme to 1.33% of the wild-type activity. Furthermore, Asp191 → Asn substitution as well as Ser195 → Ala substitution completely abolished the specific activity. It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all amidases, the real active site residues are Asp191 and Ser195 rather than Cys203. Inasmuch as an amide bond (CO-NH2) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various amidases were compared with the active site sequences of various types of proteases. It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two. PMID:9342349

  15. Revealing a Novel Otubain-Like Enzyme from Leishmania infantum with Deubiquitinating Activity toward K48-Linked Substrate

    PubMed Central

    Azevedo, Clênia S.; Guido, Bruna C.; Pereira, Jhonata L.; Nolasco, Diego O.; Corrêa, Rafael; Magalhães, Kelly G.; Motta, Flávia N.; Santana, Jaime M.; Grellier, Philippe; Bastos, Izabela M. D.

    2017-01-01

    Deubiquitinating enzymes (DUBs) play an important role in regulating a variety of eukaryotic processes. In this context, exploring the role of deubiquitination in Leishmania infantum could be a promising alternative to search new therapeutic targets for leishmaniasis. Here we present the first characterization of a DUB from L. infantum, otubain (OtuLi), and its localization within parasite. The recombinant OtuLi (rOtuLi) showed improved activity on lysine 48 (K48)-linked over K63-linked tetra-ubiquitin (Ub) and site-directed mutations on amino acids close to the catalytic site (F82) or involved in Ub interaction (L265 and F182) caused structural changes as shown by molecular dynamics, resulting in a reduction or loss of enzyme activity, respectively. Furthermore, rOtuLi stimulates lipid droplet biogenesis (an inflammatory marker) and induces IL-6 and TNF-α secretion in peritoneal macrophages, both proinflammatory cytokines. Our findings suggest that OtuLi is a cytoplasmic enzyme with K48-linked substrate specificity that could play a part in proinflammatory response in stimulated murine macrophages. PMID:28386537

  16. Guanidinylated Neomycin Mediates Heparan Sulfate–dependent Transport of Active Enzymes to Lysosomes

    PubMed Central

    Sarrazin, Stéphane; Wilson, Beth; Sly, William S; Tor, Yitzhak; Esko, Jeffrey D

    2010-01-01

    Guanidinylated neomycin (GNeo) can transport bioactive, high molecular weight cargo into the interior of cells in a process that depends on cell surface heparan sulfate proteoglycans. In this report, we show that GNeo-modified quantum dots bind to cell surface heparan sulfate, undergo endocytosis and eventually reach the lysosomal compartment. An N-hydroxysuccinimide activated ester of GNeo (GNeo-NHS) was prepared and conjugated to two lysosomal enzymes, β--glucuronidase (GUS) and α--iduronidase. Conjugation did not interfere with enzyme activity and enabled binding of the enzymes to heparin-Sepharose and heparan sulfate on primary human fibroblasts. Cells lacking the corresponding lysosomal enzyme took up sufficient amounts of the conjugated enzymes to restore normal turnover of glycosaminoglycans. The high capacity of proteoglycan-mediated uptake suggests that this method of delivery might be used for enzyme replacement or introduction of foreign enzymes into cells. PMID:20442709

  17. Integrated lipase production and in situ biodiesel synthesis in a recombinant Pichia pastoris yeast: an efficient dual biocatalytic system composed of cell free enzymes and whole cell catalysts

    PubMed Central

    2014-01-01

    Background Lipase-catalyzed biotransformation of acylglycerides or fatty acids into biodiesel via immobilized enzymes or whole cell catalysts has been considered as one of the most promising methods to produce renewable and environmentally friendly alternative liquid fuels, thus being extensively studied so far. In all previously pursued approaches, however, lipase enzymes are prepared in an independent process separated from enzymatic biodiesel production, which would unavoidably increase the cost and energy consumption during industrial manufacture of this cost-sensitive energy product. Therefore, there is an urgent need to develop novel cost-effective biocatalysts and biocatalytic processes with genuine industrial feasibility. Result Inspired by the consolidated bioprocessing of lignocellulose to generate bioethanol, an integrated process with coupled lipase production and in situ biodiesel synthesis in a recombinant P. pastoris yeast was developed in this study. The novel and efficient dual biocatalytic system based on Thermomyces lanuginosus lipase took advantage of both cell free enzymes and whole cell catalysts. The extracellular and intracellular lipases of growing yeast cells were simultaneously utilized to produce biodiesel from waste cooking oils in situ and in one pot. This integrated system effectively achieved 58% and 72% biodiesel yield via concurrent esterified-transesterified methanolysis and stepwise hydrolysis-esterification at 3:1 molar ratio between methanol and waste cooking oils, respectively. Further increasing the molar ratio of methanol to waste cooking oils to 6:1 led to an 87% biodiesel yield using the stepwise strategy. Both water tolerance and methanol tolerance of this novel system were found to be significantly improved compared to previous non-integrated biodiesel production processes using separately prepared immobilized enzymes or whole cell catalysts. Conclusion We have proposed a new concept of integrated biodiesel production

  18. Copper-induced changes in tissue enzyme activity in a freshwater mussel.

    PubMed

    Rajalakshmi, S; Mohandas, A

    2005-09-01

    Changes in enzyme activity levels are of great diagnostic value. Lysosomal membrane is often the target of injury by xenobiotics, resulting in destabilization. Variations in the activity of acid phosphatase (ACP) a marker enzyme, in gills and hepatopancreas of the freshwater mussel Lamellidens corrianus (Lea) exposed to different concentrations of copper for 24, 120, and 168 h are discussed. The aim was to determine if the metal caused any variation in enzyme activity in the two tissues studied and, if so, whether the length of exposure had any influence on enzyme activity. ACP activity was determined as described in Sigma Technical Bulletin No. 104 and expressed as micromoles of p-nitrophenol liberated per milligram of protein per hour. Both concentration of the metal and length of exposure were found to influence enzyme activity. Higher concentrations of metals are assumed to induce stress proteins like metallothioneins.

  19. Development and comparative evaluation of a plate enzyme-linked immunosorbent assay based on recombinant outer membrane antigens Omp28 and Omp31 for diagnosis of human brucellosis.

    PubMed

    Tiwari, Sapana; Kumar, Ashu; Thavaselvam, Duraipandian; Mangalgi, Smita; Rathod, Vedika; Prakash, Archana; Barua, Anita; Arora, Sonia; Sathyaseelan, Kannusamy

    2013-08-01

    Brucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and

  20. Diagnosing Human Anisakiasis: Recombinant Ani s 1 and Ani s 7 Allergens versus the UniCAP 100 Fluorescence Enzyme Immunoassay ▿

    PubMed Central

    Anadón, A. M.; Rodríguez, E.; Gárate, M. T.; Cuéllar, C.; Romarís, F.; Chivato, T.; Rodero, M.; González-Díaz, H.; Ubeira, F. M.

    2010-01-01

    Commercially available serological methods for serodiagnosis of human anisakiasis either are poorly specific or do not include some of the most relevant Anisakis allergens. The use of selected recombinant allergens may improve serodiagnosis. To compare the diagnostic and clinical values of enzyme-linked immunosorbent assay (ELISA) methods based on Ani s 1 and Ani s 7 recombinant allergens and of the UniCAP 100 fluorescence enzyme immunoassay (CAP FEIA) system, we tested sera from 495 allergic and 25 non-food-related allergic patients. The decay in specific IgE antibodies in serum was also investigated in 15 positive patients over a period of 6 to 38 months. Considering sera that tested positive by either Ani s 1 or Ani s 7 ELISA, the CAP FEIA classified 25% of sera as falsely positive, mainly in the group of patients with the lowest levels of anti-Anisakis IgE antibodies, and 1.28% of positive sera as falsely negative. Considering allergens individually, the overall sensitivities of Ani s 7 ELISA and Ani s 1 ELISA were 94% and 61%, respectively. The results also showed that anti-Anisakis IgE antibodies can be detected in serum for longer with Ani s 1 ELISA than with Ani s 7 ELISA and CAP FEIA (P < 0.01). Our findings suggest that ELISA methods with Ani s 7 and Ani s 1 allergens as targets of IgE antibodies are currently the best option for serodiagnosis of human anisakiasis, combining specificity and sensitivity. The different persistence of anti-Ani s 1 and anti-Ani s 7 antibodies in serum may help clinicians to distinguish between recent and old Anisakis infections. PMID:20107002

  1. Spinach Thylakoid Polyphenol Oxidase : ISOLATION, ACTIVATION, AND PROPERTIES OF THE NATIVE CHLOROPLAST ENZYME.

    PubMed

    Golbeck, J H; Cammarata, K V

    1981-05-01

    Polyphenol oxidase activity (E.C. 1.14.18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. The higher molecular weight enzyme is the predominant form in freshly isolated preparations but on aging or further purification, the amount of lower molecular weight enzyme increases at the expense of the higher.Sonication releases polyphenol oxidase from the membrane largely in the latent state. C(18) fatty acids, especially linolenic acid, are potent activators of the enzymic activity. In the absence of added fatty acids, the isolated enzyme spontaneously, but slowly, activates with time.Purified polyphenol oxidase utilizes o-diphenols as substrates and shows no detectable levels of monophenol or p-diphenol oxidase activities. The K(m) values for 3,4-dihydroxyphenylalanine and O(2) are 6.5 and 0.065 millimolar, respectively. Suitable substrates include chlorogenic acid, catechol, caffeic acid, pyrogallol, and dopamine; however, the enzyme is substrate-inhibited by the last four at concentrations near their K(m) A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.

  2. Molecular architectures and functions of radical enzymes and their (re)activating proteins.

    PubMed

    Shibata, Naoki; Toraya, Tetsuo

    2015-10-01

    Certain proteins utilize the high reactivity of radicals for catalysing chemically challenging reactions. These proteins contain or form a radical and therefore named 'radical enzymes'. Radicals are introduced by enzymes themselves or by (re)activating proteins called (re)activases. The X-ray structures of radical enzymes and their (re)activases revealed some structural features of these molecular apparatuses which solved common enigmas of radical enzymes—i.e. how the enzymes form or introduce radicals at the active sites, how they use the high reactivity of radicals for catalysis, how they suppress undesired side reactions of highly reactive radicals and how they are (re)activated when inactivated by extinction of radicals. This review highlights molecular architectures of radical B12 enzymes, radical SAM enzymes, tyrosyl radical enzymes, glycyl radical enzymes and their (re)activating proteins that support their functions. For generalization, comparisons of the recently reported structures of radical enzymes with those of canonical radical enzymes are summarized here.

  3. A fluorescence-based coupling reaction for monitoring the activity of recombinant human NAD synthetase.

    PubMed

    Bembenek, Michael E; Kuhn, Eric; Mallender, William D; Pullen, Lester; Li, Ping; Parsons, Thomas

    2005-10-01

    NAD synthetase is responsible for the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. This reaction provides a biosynthetic route of the coenzyme and, thus, a source of cellular reducing equivalents. Alterations in the oxidative reductive potential of the cell have been implicated as a contributing factor in many disease states. Thus, this enzyme represents a new class of potential drug targets, and, hence, our efforts were focused upon developing a robust assay for utilization in a high throughput screen. Toward that end, we describe a coupled enzyme assay format for the measurement of recombinant human NAD synthetase by employing lactate dehydrogenase in a cycling/amplification reaction linked ultimately to the fluorescence generation of resorufin from resazurin via diaphorase. We present kinetics of the reaction of NAD synthetase in the coupled assay format, optimization conditions, and inhibition of the reaction by gossypol [1,1',6,6',7,7'-hexahydroxy-3,3'-dimethyl-5,5'-bis(1-methylethyl)-[2,2'- binaphthalene]-8,8'-dicarboxaldehyde] and illustrate the robustness of the assay by demonstrating 384-well microtiter plate uniformity statistics. Collectively, our results show that the assay method is both robust and well suited for this class of enzymes involved in the NAD+ biosynthetic pathway.

  4. Safety update on the use of recombinant activated factor VII in approved indications.

    PubMed

    Neufeld, Ellis J; Négrier, Claude; Arkhammar, Per; Benchikh el Fegoun, Soraya; Simonsen, Mette Duelund; Rosholm, Anders; Seremetis, Stephanie

    2015-06-01

    This updated safety review summarises the large body of safety data available on the use of recombinant activated factor VII (rFVIIa) in approved indications: haemophilia with inhibitors, congenital factor VII (FVII) deficiency, acquired haemophilia and Glanzmann's thrombasthenia. Accumulated data up to 31 December 2013 from clinical trials as well as post-marketing data (registries, literature reports and spontaneous reports) were included. Overall, rFVIIa has shown a consistently favourable safety profile, with no unexpected safety concerns, in all approved indications. No confirmed cases of neutralising antibodies against rFVIIa have been reported in patients with congenital haemophilia, acquired haemophilia or Glanzmann's thrombasthenia. The favourable safety profile of rFVIIa can be attributed to the recombinant nature of rFVIIa and its localised mechanism of action at the site of vascular injury. Recombinant FVIIa activates factor X directly on the surface of activated platelets, which are present only at the site of injury, meaning that systemic activation of coagulation is avoided and the risk of thrombotic events (TEs) thus reduced. Nonetheless, close monitoring for signs and symptoms of TE is warranted in all patients treated with any pro-haemostatic agent, including rFVIIa, especially the elderly and any other patients with concomitant conditions and/or predisposing risk factors to thrombosis.

  5. Changes in the spectrum and rates of extracellular enzyme activities in seawater following aggregate formation

    NASA Astrophysics Data System (ADS)

    Ziervogel, K.; Steen, A. D.; Arnosti, C.

    2010-03-01

    Marine snow aggregates are heavily colonized by heterotrophic microorganisms that express high levels of hydrolytic activities, making aggregates hotspots for carbon remineralization in the ocean. To assess how aggregate formation influences the ability of seawater microbial communities to access organic carbon, we compared hydrolysis rates of six polysaccharides in coastal seawater after aggregates had been formed (via incubation on a roller table) with hydrolysis rates in seawater from the same site that had not incubated on a roller table (referred to as whole seawater). Hydrolysis rates in the aggregates themselves were up to three orders of magnitude higher on a volume basis than in whole seawater. The enhancement of enzyme activity in aggregates relative to whole seawater differed by substrate, suggesting that the enhancement was under cellular control, rather than due to factors such as lysis or grazing. A comparison of hydrolysis rates in whole seawater with those in aggregate-free seawater, i.e. the fraction of water from the roller bottles that did not contain aggregates, demonstrated a nuanced microbial response to aggregate formation. Activities of laminarinase and xylanase enzymes in aggregate-free seawater were higher than in whole seawater, while activities of chondroitin, fucoidan, and arabinogalactan hydrolyzing enzymes were lower than in whole seawater. These data suggest that aggregate formation enhanced production of laminarinase and xylanase enzymes, and the enhancement also affected the surrounding seawater. Decreased activities of chondroitin, fucoidan, and arabinoglactan-hydrolyzing enzymes in aggregate-free seawaters relative to whole seawater are likely due to shifts in enzyme production by the aggregate-associated community, coupled with the effects of enzyme degradation. Enhanced activities of laminarin- and xylan-hydrolyzing enzymes in aggregate-free seawater were due at least in part to cell-free enzymes. Measurements of enzyme

  6. Recombination-activating gene 1 and 2 (RAG1 and RAG2) in flounder (Paralichthys olivaceus).

    PubMed

    Wang, Xianlei; Tan, Xungang; Zhang, Pei-Jun; Zhang, Yuqing; Xu, Peng

    2014-12-01

    During the development of B and T lymphocytes, Ig and TCR variable region genes are assembled from germline V, D, and J gene segments by a site-specific recombination reaction known as V(D)J recombination. The process of somatic V(D)J recombination, mediated by the recombination-activating gene (RAG) products, is the most significant characteristic of adaptive immunity in jawed vertebrates. Flounder (Paralichthys olivaceus) RAG1 and RAG2 were isolated by Genome Walker and RT-PCR, and their expression patterns were analysed by RT-PCR and in situ hybridization on sections. RAG1 spans over 7.0 kb, containing 4 exons and 3 introns, and the full-length ORF is 3207 bp, encoding a peptide of 1068 amino acids. The first exon lies in the 5'-UTR, which is an alternative exon. RAG2 full-length ORF is 1062 bp, encodes a peptide of 533 amino acids, and lacks introns in the coding region. In 6-month old flounders, the expression of RAG1 and RAG2 was essentially restricted to the pronephros (head kidney) and mesonephros (truck kidney). Additionally, both of them were mainly expressed in the thymus. These results revealed that the thymus and kidney most likely serve as the primary lymphoid tissues in the flounder.

  7. Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses

    PubMed Central

    2010-01-01

    Background Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricutural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL), and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat), were constructed. and their insecticidal properties analysed against Spodoptera frugiperda larvae. Results Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. Conclusions Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs. PMID:20587066

  8. Acute ischemic stroke after cardiac catheterization: the protamine low-dose recombinant tissue plasminogen activator pathway.

    PubMed

    Guevara, Carlos; Quijada, Alonso; Rosas, Carolina; Bulatova, Katya; Lara, Hugo; Nieto, Elena; Morales, Marcelo

    2016-05-20

    Intravenous thrombolysis is the preferred treatment for acute ischemic stroke; however, it remains unestablished in the area of cardiac catheterization. We report three patients with acute ischemic stroke after cardiac catheterization. After reversing the anticoagulant effect of unfractionated heparin with protamine, all of the patients were successfully off-label thrombolyzed with reduced doses of intravenous recombinant tissue plasminogen activator (0.6 mg/kg). This dose was preferred to reduce the risk of symptomatic cerebral or systemic bleeding. The sequential pathway of protamine recombinant tissue plasminogen activator at reduced doses may be safer for reducing intracranial or systemic bleeding events, whereas remaining efficacious for the treatment of acute ischemic stroke after cardiac catheterization.

  9. Regulation of Enzyme Activities in Drosophila: Genetic Variation Affecting Induction of Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenases in Larvae

    PubMed Central

    Cochrane, Bruce J.; Lucchesi, John C.; Laurie-Ahlberg, C. C.

    1983-01-01

    The genetic basis of modulation by dietary sucrose of the enzyme activities glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities in third instar larvae of Drosophila melanogaster was investigated, using isogenic lines derived from wild populations. Considerable genetically determined variation in response was detected among lines that differed only in their third chromosome constitution. Comparison of crossreacting material between a responding and a nonresponding line showed that the G6PD activity variation is due to changes in G6PD protein level. These differences in responses are localized in the fat body, with 300 m m sucrose in the diet resulting in a sixfold stimulation of G6PD activity and a fourfold one of 6PGD in the line showing the strongest response. In this tissue, the responses of the two enzymes are closely correlated with one another. Using recombinant lines, we obtained data that suggested the existence of more than one gene on chromosome III involved in the regulation of G6PD in the fat body, and at least one of these genes affects the level of 6PGD as well. PMID:6416921

  10. Queuine promotes antioxidant defence system by activating cellular antioxidant enzyme activities in cancer.

    PubMed

    Pathak, Chandramani; Jaiswal, Yogesh K; Vinayak, Manjula

    2008-04-01

    Constant generation of Reactive oxygen species (ROS) during normal cellular metabolism of an organism is generally balanced by similar rate of consumption by antioxidants. Imbalance between ROS production and antioxidant defense results in increased level of ROS causing oxidative stress which leads to promotion of malignancy. Queuine is a hyper modified base analogue of guanine, found at first anti-codon position of Q- family of tRNAs. These tRNAs are completely modified with respect to queuosine in terminally differentiated somatic cells, however hypomodification of Q-tRNAs is close association with cell proliferation. Q-tRNA modification is essential for normal development, differentiation and cellular functions. Queuine is a nutrient factor to eukaryotes. It is found to promote cellular antioxidant defense system and inhibit tumorigenesis. The activities of antioxidant enzymes like catalase, SOD, glutathione peroxidase and glutathione reductase are found to be low in Dalton's lymphoma ascites transplanted (DLAT) mouse liver compared to normal. However, exogenous administration of queuine to DLAT mouse improves the activities of antioxidant enzymes. The results suggest that queuine promotes antioxidant defense system by increasing antioxidant enzyme activities and in turn inhibits oxidative stress and tumorigenesis.

  11. Effects of triazole fungicides on androgenic disruption and CYP3A4 enzyme activity.

    PubMed

    Lv, Xuan; Pan, Liumeng; Wang, Jiaying; Lu, Liping; Yan, Weilin; Zhu, Yanye; Xu, Yiwen; Guo, Ming; Zhuang, Shulin

    2017-03-01

    Triazole fungicides are widely used as broad-spectrum fungicides, non-steroidal antiestrogens and for various industrial applications. Their residues have been frequently detected in multiple environmental and human matrices. The increasingly reported toxicity incidents have led triazole fungicides as emerging contaminants of environmental and public health concern. However, whether triazole fungicides behave as endocrine disruptors by directly mimicking environmental androgens/antiandrogens or exerting potential androgenic disruption indirectly through the inhibition of cytochrome P450 (CYP450) enzyme activity is yet an unresolved question. We herein evaluated five commonly used triazole fungicides including bitertanol, hexaconazole, penconazole, tebuconazole and uniconazole for the androgenic and anti-androgenic activity using two-hybrid recombinant human androgen receptor (AR) yeast bioassay and comparatively evaluated their effects on enzymatic activity of CYP3A4 by P450-Glo™ CYP3A4 bioassay. All five fungicides showed moderate anti-androgenic activity toward human AR with the IC50 ranging from 9.34 μM to 79.85 μM. The anti-androgenic activity remained no significant change after the metabolism mediated by human liver microsomes. These fungicides significantly inhibited the activity of CYP3A4 at the environmental relevant concentrations and the potency ranks as tebuconazole > uniconazole > hexaconazole > penconazole > bitertanol with the corresponding IC50 of 0.81 μM, 0.93 μM, 1.27 μM, 2.22 μM, and 2.74 μM, respectively. We found that their anti-androgenic activity and the inhibition potency toward CYP3A4 inhibition was significantly correlated (R(2) between 0.83 and 0.97, p < 0.001). Our results indicated that the risk assessment of triazole pesticides and structurally similar chemicals should fully consider potential androgenic disrupting effects and the influences on the activity of CYP450s.

  12. Recombination-active defects in silicon ribbon and polycrystalline solar cells

    NASA Technical Reports Server (NTRS)

    Cheng, L. J.

    1984-01-01

    This paper reports results from a study of recombination-active structural defects in silicon ribbon and polycrystalline solar cells using the electron beam induced current (EBIC) technique in a scanning electron microscope. It is demonstrated that low temperature EBIC measurements can reveal a range of defects that are not observable at room temperature, including slip dislocations in silicon dendritic web ribbons as well as decorated twin boundaries and dislocation complexes in cast polycrystalline silicon solar cell materials.

  13. Effect of recombinant erythropoietin on functional activity of cultured human cells.

    PubMed

    Emel'yanova, E A; Kosykh, A V; Sukhanov, Yu V; Vorotelyak, E A; Vasil'ev, A V

    2012-08-01

    We studied the effect of recombinant human erythropoietin on functional activity of skin cells in vitro. It was found that erythropoietin stimulated proliferation of mesenchymal and epithelial cells and effectively protected epidermal HaCaT cells from apoptosis. Insignificant effect of erythropoietin on contraction of collagen gel by mesenchymal cells was revealed. These findings suggest that erythropoietin can be a promising component of wound-healing preparations.

  14. Kinetic Parameters and Cytotoxic Activity of Recombinant Methionine γ-Lyase from Clostridium tetani, Clostridium sporogenes, Porphyromonas gingivalis and Citrobacter freundii

    PubMed Central

    Morozova, E. A.; Kulikova, V. V.; Yashin, D. V.; Anufrieva, N. V.; Anisimova, N. Y.; Revtovich, S. V.; Kotlov, M. I.; Belyi, Y. F.; Pokrovsky, V. S.; Demidkina, T. V.

    2013-01-01

    The steady-state kinetic parameters of pyridoxal 5’-phosphate-dependent recombinant methionine γ -lyase from three pathogenic bacteria, Clostridium tetani, Clostridium sporogenes, and Porphyromonas gingivalis, were determined in β- and γ-elimination reactions. The enzyme from C. sporogenes is characterized by the highest catalytic efficiency in the γ-elimination reaction of L-methionine. It was demonstrated that the enzyme from these three sources exists as a tetramer. The N-terminal poly-histidine fragment of three recombinant enzymes influences their catalytic activity and facilitates the aggregation of monomers to yield dimeric forms under denaturing conditions. The cytotoxicity of methionine γ-lyase from C. sporogenes and C. tetani in comparison with Citrobacter freundii was evaluated using K562, PC-3, LnCap, MCF7, SKOV-3, and L5178y tumor cell lines. K562 (IC50=0.4–1.3 U/ml), PC-3 (IC50=0.1–0.4 U/ml), and MCF7 (IC50=0.04–3.2 U/ml) turned out to be the most sensitive cell lines. PMID:24303205

  15. Kinetic Parameters and Cytotoxic Activity of Recombinant Methionine γ-Lyase from Clostridium tetani, Clostridium sporogenes, Porphyromonas gingivalis and Citrobacter freundii.

    PubMed

    Morozova, E A; Kulikova, V V; Yashin, D V; Anufrieva, N V; Anisimova, N Y; Revtovich, S V; Kotlov, M I; Belyi, Y F; Pokrovsky, V S; Demidkina, T V

    2013-07-01

    The steady-state kinetic parameters of pyridoxal 5'-phosphate-dependent recombinant methionine γ -lyase from three pathogenic bacteria, Clostridium tetani, Clostridium sporogenes, and Porphyromonas gingivalis, were determined in β- and γ-elimination reactions. The enzyme from C. sporogenes is characterized by the highest catalytic efficiency in the γ-elimination reaction of L-methionine. It was demonstrated that the enzyme from these three sources exists as a tetramer. The N-terminal poly-histidine fragment of three recombinant enzymes influences their catalytic activity and facilitates the aggregation of monomers to yield dimeric forms under denaturing conditions. The cytotoxicity of methionine γ-lyase from C. sporogenes and C. tetani in comparison with Citrobacter freundii was evaluated using K562, PC-3, LnCap, MCF7, SKOV-3, and L5178y tumor cell lines. K562 (IC50=0.4-1.3 U/ml), PC-3 (IC50=0.1-0.4 U/ml), and MCF7 (IC50=0.04-3.2 U/ml) turned out to be the most sensitive cell lines.

  16. Expression and purification of the recombinant subunits of toluene/o-xylene monooxygenase and reconstitution of the active complex.

    PubMed

    Cafaro, Valeria; Scognamiglio, Roberta; Viggiani, Ambra; Izzo, Viviana; Passaro, Irene; Notomista, Eugenio; Piaz, Fabrizio Dal; Amoresano, Angela; Casbarra, Annarita; Pucci, Piero; Di Donato, Alberto

    2002-11-01

    This paper describes the cloning of the genes coding for each component of the complex of toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization. Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined. The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA)2, endowed with hydroxylase activity. Tomo F component is an NADH oxidoreductase. The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one [2Fe-2S] cluster, and exhibits a typical flavodoxin absorption spectrum. Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence. We have shown that this depends on minor errors in the gene sequence that we have corrected. C component is a Rieske-type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one [2Fe-2S] cluster. The cluster is very sensitive to oxygen damage. Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p-cresol into 4-methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified. Finally, experimental evidence is reported which strongly support a model for the electron transfer. Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H subcomplex, and eventually to molecular oxygen.

  17. Microaerobic conversion of xylose to ethanol in recombinant Saccharomyces cerevisiae SX6(MUT) expressing cofactor-balanced xylose metabolic enzymes and deficient in ALD6.

    PubMed

    Jo, Sung-Eun; Seong, Yeong-Je; Lee, Hyun-Soo; Lee, Soo Min; Kim, Soo-Jung; Park, Kyungmoon; Park, Yong-Cheol

    2016-06-10

    Xylose is a major monosugar in cellulosic biomass and should be utilized for cost-effective ethanol production. In this study, xylose-converting ability of recombinant Saccharomyces cerevisiae SX6(MUT) expressing NADH-preferring xylose reductase mutant (R276H) and other xylose-metabolic enzymes, and deficient in aldehyde dehydrogenase 6 (Ald6p) were characterized at microaerobic conditions using various sugar mixtures. The reduction of air supply from 0.5vvm to 0.1vvm increased specific ethanol production rate by 75% and did not affect specific xylose consumption rate. In batch fermentations using various concentrations of xylose (50-104g/L), higher xylose concentration enhanced xylose consumption rate and ethanol productivity but reduced ethanol yield, owing to the accumulation of xylitol and glycerol from xylose. SX6(MUT) consumed monosugars in pitch pine hydrolysates and produced 23.1g/L ethanol from 58.7g/L sugars with 0.39g/g ethanol yield, which was 14% higher than the host strain of S. cerevisiae D452-2 without the xylose assimilating enzymes. In conclusion, S. cerevisiae SX6(MUT) was characterized to possess high xylose-consuming ability in microaerobic conditions and a potential for ethanol production from cellulosic biomass.

  18. Spatial distribution of enzyme activities along the root and in the rhizosphere of different plants

    NASA Astrophysics Data System (ADS)

    Razavi, Bahar S.; Zarebanadkouki, Mohsen; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2015-04-01

    Extracellular enzymes are important for decomposition of many biological macromolecules abundant in soil such as cellulose, hemicelluloses and proteins. Activities of enzymes produced by both plant roots and microbes are the primary biological drivers of organic matter decomposition and nutrient cycling. So far acquisition of in situ data about local activity of different enzymes in soil has been challenged. That is why there is an urgent need in spatially explicit methods such as 2-D zymography to determine the variation of enzymes along the roots in different plants. Here, we developed further the zymography technique in order to quantitatively visualize the enzyme activities (Spohn and Kuzyakov, 2013), with a better spatial resolution We grew Maize (Zea mays L.) and Lentil (Lens culinaris) in rhizoboxes under optimum conditions for 21 days to study spatial distribution of enzyme activity in soil and along roots. We visualized the 2D distribution of the activity of three enzymes:β-glucosidase, leucine amino peptidase and phosphatase, using fluorogenically labelled substrates. Spatial resolution of fluorescent images was improved by direct application of a substrate saturated membrane to the soil-root system. The newly-developed direct zymography shows different pattern of spatial distribution of enzyme activity along roots and soil of different plants. We observed a uniform distribution of enzyme activities along the root system of Lentil. However, root system of Maize demonstrated inhomogeneity of enzyme activities. The apical part of an individual root (root tip) in maize showed the highest activity. The activity of all enzymes was the highest at vicinity of the roots and it decreased towards the bulk soil. Spatial patterns of enzyme activities as a function of distance from the root surface were enzyme specific, with highest extension for phosphatase. We conclude that improved zymography is promising in situ technique to analyze, visualize and quantify

  19. A new versatile microarray-based method for high throughput screening of carbohydrate-active enzymes.

    PubMed

    Vidal-Melgosa, Silvia; Pedersen, Henriette L; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G T

    2015-04-03

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths.

  20. A New Versatile Microarray-based Method for High Throughput Screening of Carbohydrate-active Enzymes*

    PubMed Central

    Vidal-Melgosa, Silvia; Pedersen, Henriette L.; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B.; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G. T.

    2015-01-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. PMID:25657012

  1. Pivotal trial with plant cell-expressed recombinant glucocerebrosidase, taliglucerase alfa, a novel enzyme replacement therapy for Gaucher disease.

    PubMed

    Zimran, Ari; Brill-Almon, Einat; Chertkoff, Raul; Petakov, Milan; Blanco-Favela, Francisco; Muñoz, Eduardo Terreros; Solorio-Meza, Sergio E; Amato, Dominick; Duran, Gloria; Giona, Fiorina; Heitner, Rene; Rosenbaum, Hanna; Giraldo, Pilar; Mehta, Atul; Park, Glen; Phillips, Mici; Elstein, Deborah; Altarescu, Gheona; Szleifer, Mali; Hashmueli, Sharon; Aviezer, David

    2011-11-24

    Taliglucerase alfa (Protalix Biotherapeutics, Carmiel, Israel) is a novel plant cell-derived recombinant human β-glucocerebrosidase for Gaucher disease. A phase 3, double-blind, randomized, parallel-group, comparison-dose (30 vs 60 U/kg body weight/infusion) multinational clinical trial was undertaken. Institutional review board approvals were received. A 9-month, 20-infusion trial used inclusion/exclusion criteria in treatment-naive adult patients with splenomegaly and thrombocytopenia. Safety end points were drug-related adverse events: Ab formation and hypersensitivity reactions. Primary efficacy end point was reduction in splenic volume measured by magnetic resonance imaging. Secondary end points were: changes in hemoglobin, hepatic volume, and platelet counts. Exploratory parameters included biomarkers and bone imaging. Twenty-nine patients (11 centers) completed the protocol. There were no serious adverse events; drug-related adverse events were mild/moderate and transient. Two patients (6%) developed non-neutralizing IgG Abs; 2 other patients (6%) developed hypersensitivity reactions. Statistically significant spleen reduction was achieved at 9 months: 26.9% (95% confidence interval [CI]: -31.9, -21.8) in the 30-unit dose group and 38.0% (95% CI: -43.4, -32.8) in the 60-unit dose group (both P < .0001); and in all secondary efficacy end point measures, except platelet counts at the lower dose. These results support safety and efficacy of taliglucerase alfa for Gaucher disease.

  2. Enzyme-linked immunosorbent assay for the detection of canine Leptospira antibodies using recombinant OmpL1 protein.

    PubMed

    Okuda, Masaru; Sakai, Yoshiko; Matsuuchi, Megumi; Oikawa, Tatsuo; Watanabe, Malaika; Itamoto, Kazuhito; Iwata, Hiroyuki; Kano, Rui; Hasegawa, Atsuhiko; Onishi, Takafumi; Inokuma, Hisashi

    2005-03-01

    OmpL1 is a 31-kDa outer membrane protein characterized in 1993 and known to be expressed only in pathogenic Leptospira spp. Recombinant OmpL1 (GST-rOmpL1) was expressed for use as an ELISA antigen for the detection of anti-Leptospira antibodies. In immunoblot analysis, the protein reacted with sera of dogs infected with three different serotypes of Leptospira interrogans, while did not react with sera of dogs both uninfected negative controls and infected with Borrelia burgdorferi, which is closely related to Leptospira spp. Moreover, in ELISA using GST-rOmpL1, the optical density (O.D.) values from the positive controls were very high (1.125 +/- 0.549). In contrast, the O.D. values from clinically healthy dogs and dogs with diseases other than leptospirosis were very low (0.109 +/- 0.046 and 0.089 +/- 0.046, respectively). These data suggest that the detection of anti-Leptospira antibodies by ELISA using the GST-rOmpL1 protein can be applied for diagnosis of canine leptospirosis.

  3. Soybean PM2 protein (LEA3) confers the tolerance of Escherichia coli and stabilization of enzyme activity under diverse stresses.

    PubMed

    Liu, Yun; Zheng, Yizhi; Zhang, Yuqin; Wang, Weimao; Li, Ranhui

    2010-05-01

    Late embryogenesis abundant (LEA) proteins are closely associated with the tolerance of diverse stresses in organisms. To elucidate the function of group 3 LEA proteins, the soybean PM2 protein (LEA3) was expressed in E. coli and the protective function of the PM2 protein was assayed both in vivo and in vitro. The results of a spot assay and survival ratio demonstrated that the expression of the PM2 protein conferred the tolerance to the E. coli recombinant for different temperature conditions (4, -20 or 50 degrees C) or high-salinity stresses (120 mmol/l MgCl(2) or 120 mmol/l CaCl(2)). In addition, it was demonstrated that the in vitro addition of the PM2 protein could prevent the lactate dehydrogenase (LDH) inactivation normally induced by freeze-thaw. In the 62 degrees C condition, the PM2 protein (1:5 mass ratio to LDH) effectively prevented the LDH thermo-denaturation by acting synergistically with trehalose (62.5 microg/ml), although the PM2 protein alone at this concentration showed little protective effect on LDH activity. Furthermore, the results showed that the PM2 protein could partially prevent the thermo-denaturation of the bacterial proteome after boiling for 2 min. Based on these results, we propose that the PM2 protein itself, or together with trehalose, conferred the tolerance to the E. coli recombinant against diverse stresses by protecting proteins and enzyme activity under low- or high- temperature conditions.

  4. New hydroxamate inhibitors of neurotensin-degrading enzymes. Synthesis and enzyme active-site recognition.

    PubMed

    Bourdel, E; Doulut, S; Jarretou, G; Labbe-Jullie, C; Fehrentz, J A; Doumbia, O; Kitabgi, P; Martinez, J

    1996-08-01

    Selective and mixed inhibitors of the three zinc metallopeptidases that degrade neurotensin (NT), e.g. endopeptidase 24-16 (EC 3.4.24.16), endopeptidase 24-11 (EC 3.4.24.11 or neutral endopeptidase, NEP) and endopeptidase 24-15 (EC 3.4.24.15), and leucine-aminopeptidase (type IV-S), that degrades the NT-related peptides, Neuromedin N (NN), are of great interest. On the structural basis of compound JMV 390-1 (N-[3-[(hydroxyamino)carbonyl]-1-oxo-2(R)-benzylpropyl]-L- isoleucyl-L-leucine), which was a full inhibitor of the major NT degrading enzymes, several hydroxamate inhibitors corresponding to the general formula HONHCO-CH2-CH(CH2-C6H5)CO-X-Y-OH (with X-Y = dipeptide) have been synthesized. Compound 7a (X-Y = Ile-Ala) was nearly 40-times more potent in inhibiting EC 24-16 than NEP and more than 800-times more potent than EC 24-15, with an IC50 (12 nM) almost equivalent to that of compound JMV 390-1. Therefore, this compound is an interesting selective inhibitor of EC 24-16, and should be an interesting probe to explore the physiological involvement of EC 24-16 in the metabolism of neurotensin.

  5. An active triple-catalytic hybrid enzyme engineered by linking cyclo-oxygenase isoform-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protector.

    PubMed

    Ruan, Ke-He; So, Shui-Ping; Cervantes, Vanessa; Wu, Hanjing; Wijaya, Cori; Jentzen, Rebecca R

    2008-12-01

    It remains a challenge to achieve the stable and long-term expression (in human cell lines) of a previously engineered hybrid enzyme [triple-catalytic (Trip-cat) enzyme-2; Ruan KH, Deng H & So SP (2006) Biochemistry45, 14003-14011], which links cyclo-oxygenase isoform-2 (COX-2) to prostacyclin (PGI(2)) synthase (PGIS) for the direct conversion of arachidonic acid into PGI(2) through the enzyme's Trip-cat functions. The stable upregulation of the biosynthesis of the vascular protector, PGI(2), in cells is an ideal model for the prevention and treatment of thromboxane A(2) (TXA(2))-mediated thrombosis and vasoconstriction, both of which cause stroke, myocardial infarction, and hypertension. Here, we report another case of engineering of the Trip-cat enzyme, in which human cyclo-oxygenase isoform-1, which has a different C-terminal sequence from COX-2, was linked to PGI(2) synthase and called Trip-cat enzyme-1. Transient expression of recombinant Trip-cat enzyme-1 in HEK293 cells led to 3-5-fold higher expression capacity and better PGI(2)-synthesizing activity as compared to that of the previously engineered Trip-cat enzyme-2. Furthermore, an HEK293 cell line that can stably express the active new Trip-cat enzyme-1 and constantly synthesize the bioactive PGI(2) was established by a screening approach. In addition, the stable HEK293 cell line, with constant production of PGI(2), revealed strong antiplatelet aggregation properties through its unique dual functions (increasing PGI(2) production while decreasing TXA(2) production) in TXA(2) synthase-rich plasma. This study has optimized engineering of the active Trip-cat enzyme, allowing it to become the first to stably upregulate PGI(2) biosynthesis in a human cell line, which provides a basis for developing a PGI(2)-producing therapeutic cell line for use against vascular diseases.

  6. Immunoadjuvant activities of a recombinant chicken IL-12 in chickens vaccinated with Newcastle disease virus recombinant HN protein.

    PubMed

    Su, Bor Sheu; Yin, Hsien Sheng; Chiu, Hua Hsien; Hung, Li Hsiang; Huang, Ji Ping; Shien, Jui Hung; Lee, Long Huw

    2011-08-05

    Recombinant fowlpox virus (rFPV/HN) expressing Newcastle disease virus (NDV) HN gene and rFPV/HN/chIL-12 co-expressing chicken IL-12 (chIL-12) and HN (rHN/chIL-12) genes have been characterized. rHN/chIL-12 or rchIL-12, expressed by our previous construct rFPV/chIL-12, co-administered with rHN was assessed for adjuvant activities of chIL-12. Chickens were vaccinated with various amounts of rHN/chIL-12 mixed with mineral oil (MO), intramuscularly. Levels of hemagglutination-inhibition (HI) antibody production depended on the concentration of the injected rHN or rHN/chIL-12. The lower HI antibody titers were obtained in chicken groups rHN/chIL-12/7-rHN/chIL-12/9, receiving 60ng rHN/8ng chIL-12 with MO, 30ng rHN/4ng chIL-12 with MO or 15ng rHN/2ng chIL-12 with MO, respectively, compared to those in chicken groups rHN/7-rHN/9, receiving rHN with MO alone. However, chickens in group rHN/chIL-12/7 or rHN/chIL-12/8 and rHN with MO alone showed the same effective protection. Chicken group rHN/chIL-12/9 was even more protective than that in group rHN/9. When rchIL-12 was co-injected with 15ng rHN plus MO, chickens produced low levels of HI antibody titers; while higher levels of IFN-γ production and an effective protection rate (83%) were obtained. On the other hand, low levels of IFN-γ production and low protection response (50%) were obtained in chickens injected with rHN with MO alone. Taken together, when the concentration of rHN decreased to certain levels, rchIL-12 reduced HI antibody production. The increase in the induction of IFN-γ production might suggest the enhancement of the cell-mediated immunity which conferred the protection from the NDV challenge.

  7. Recombinant production of Epstein-Barr virus BZLF1 trans-activator and characterization of its DNA-binding specificity.

    PubMed

    Lim, Chun Shen; Goh, Siang Ling; Krishnan, Gopala; Ng, Ching Ching

    2014-03-01

    This paper describes the recombinant production of a biologically active Epstein-Barr virus BZLF1 trans-activator, i.e., Z-encoded broadly reactive activator (ZEBRA), that recognized specific DNA motifs. We used auto-induction for histidine-tagged BZLF1 expression in Escherichia coli and immobilized cobalt affinity membrane chromatography for protein purification under native conditions. We obtained the purified BZLF1 at a yield of 5.4mg per gram of wet weight cells at 75% purity, in which 27% of the recombinant BZLF1 remained biologically active. The recombinant BZLF1 bound to oligonucleotides containing ZEBRA response elements, either AP-1 or ZIIIB, but not a ZIIIB mutant. The recombinant BZLF1 showed a specific DNA-binding activity which could be useful for functional studies.

  8. Changes in the spectrum and rates of extracellular enzyme activities in seawater following aggregate formation

    NASA Astrophysics Data System (ADS)

    Ziervogel, K.; Steen, A. D.; Arnosti, C.

    2009-12-01

    Marine snow aggregates are heavily colonized by heterotrophic microorganisms that express high levels of hydrolytic activities, making aggregates hotspots for carbon remineralization in the ocean. To assess how aggregate formation influences the ability of seawater microbial communities to access organic carbon, we compared hydrolysis rates of six polysaccharides in coastal seawater after aggregates had been formed (via incubation on a roller table) with hydrolysis rates in seawater from the same site that had not incubated on a roller table (referred to as whole seawater). Hydrolysis rates in the aggregates themselves were up to three orders of magnitude higher on a volume basis than in whole seawater. The enhancement of enzyme activity in aggregates relative to whole seawater differed by substrate, suggesting that the enhancement was under cellular control, rather than due to factors such as lysis or grazing. A comparison of hydrolysis rates in whole seawater with those in aggregate-free seawater, i.e. the fraction of water from the roller bottles that did not contain aggregates, demonstrated a nuanced microbial response to aggregate formation. Activities of laminarinase and xylanase enzymes in aggregate-free seawater were higher than in whole seawater, while activities of chondroitin, fucoidan, and arabinogalactan hydrolyzing enzymes were lower than in whole seawater. These data suggest that aggregate formation enhanced production of laminarinase and xylanase enzymes, and the enhancement also affected the surrounding seawater. Decreased activities of chondroitin, fucoidan, and arabinoglactan-hydrolyzing enzymes in aggregate-free seawater relative to whole seawater are likely due to shifts in enzyme production by the aggregate-associated community, coupled with the effects of enzyme degradation. Enhanced activities of laminarin- and xylan-hydrolyzing enzymes in aggregate-free seawater were due at least in part to cell-free enzymes. Measurements of enzyme lifetime

  9. The characterization of the endoglucanase Cel12A from Gloeophyllum trabeum reveals an enzyme highly active on β-glucan.

    PubMed

    Miotto, Lis Schwartz; de Rezende, Camila Alves; Bernardes, Amanda; Serpa, Viviane Isabel; Tsang, Adrian; Polikarpov, Igor

    2014-01-01

    The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50 °C on β-glucan. Under these conditions specific activity was 239.2 ± 9.1 U mg(-1) and the half-life of the enzyme was 84.6 ± 3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the Km was 3.2 ± 0.5 mg mL(-1) and the Vmax was 0.41 ± 0.02 µmol min(-1). Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.

  10. Secretion of an articular cartilage proteoglycan-degrading enzyme activity by murine T lymphocytes in vitro.

    PubMed Central

    Kammer, G M; Sapolsky, A I; Malemud, C J

    1985-01-01

    Destruction of articular cartilage is the hallmark of inflammatory arthritides. Enzymes elaborated by mononuclear cells infiltrating the synovium mediate, in part, the degradation of the cartilage extracellular matrix. Since mononuclear cells are the dominant cell type found in chronic inflammatory synovitis, we investigated whether interaction of immune mononuclear cells with antigen initiated the synthesis and secretion of a proteoglycan-degrading enzyme activity. Proteoglycan-degrading enzyme activity was monitored by the capacity of murine spleen cell conditioned medium to release [3H]serine/35SO4 incorporated into rabbit cartilage proteoglycan monomer fraction (A1D1), and by the relative change in specific viscosity of bovine nasal cartilage proteoglycan monomer. The results demonstrated that both virgin and immune mononuclear cells spontaneously generated proteoglycan-degrading enzyme activity and that cellular activation and proliferation induced by the antigen keyhole limpet hemocyanin or the mitogen phytohemagglutinin was not required. Kinetic studies demonstrated stable release of the enzyme activity over 72 h. Cell separation studies showed that T lymphocytes, a thymoma line, and macrophages separately produced proteoglycan-degrading enzyme activity. The enzyme activity has been partially characterized and appears to belong to a class of neutral pH metal-dependent proteinases. These observations, the first to demonstrate that T lymphocytes secrete an enzyme capable of degrading cartilage proteoglycan, raise the possibility that this enzyme activity contributes to cartilage extracellular matrix destruction in vivo. Moreover, these data support the conclusion that production of this enzyme by T lymphocytes is independent of an antigen-specific stimulus. PMID:3897284

  11. Inhibition of enzyme activity of Rhipicephalus (Boophilus) microplus triosephosphate isomerase and BME26 cell growth by monoclonal antibodies.

    PubMed

    Saramago, Luiz; Franceschi, Mariana; Logullo, Carlos; Masuda, Aoi; Vaz, Itabajara da Silva; Farias, Sandra Estrazulas; Moraes, Jorge

    2012-10-12

    In the present work, we produced two monoclonal antibodies (BrBm37 and BrBm38) and tested their action against the triosephosphate isomerase of Rhipicephalus (Boophilus) microplus (RmTIM). These antibodies recognize epitopes on both the native and recombinant forms of the protein. rRmTIM inhibition  by BrBm37 was up to 85% whereas that of BrBrm38 was 98%, depending on the antibody-enzyme ratio. RmTIM activity was lower in ovarian, gut, and fat body tissue extracts treated with BrBm37 or BrBm38 mAbs. The proliferation of the embryonic tick cell line (BME26) was inhibited by BrBm37 and BrBm38 mAbs. In summary, the results reveal that it is possible to interfere with the RmTIM function using antibodies, even in intact cells.

  12. Inhibition of Enzyme Activity of Rhipicephalus (Boophilus) microplus Triosephosphate Isomerase and BME26 Cell Growth by Monoclonal Antibodies

    PubMed Central

    Saramago, Luiz; Franceschi, Mariana; Logullo, Carlos; Masuda, Aoi; Vaz, Itabajara da Silva; Farias, Sandra Estrazulas; Moraes, Jorge

    2012-01-01

    In the present work, we produced two monoclonal antibodies (BrBm37 and BrBm38) and tested their action against the triosephosphate isomerase of Rhipicephalus (Boophilus) microplus (RmTIM). These antibodies recognize epitopes on both the native and recombinant forms of the protein. rRmTIM inhibition by BrBm37 was up to 85% whereas that of BrBrm38 was 98%, depending on the antibody-enzyme ratio. RmTIM activity was lower in ovarian, gut, and fat body tissue extracts treated with BrBm37 or BrBm38 mAbs. The proliferation of the embryonic tick cell line (BME26) was inhibited by BrBm37 and BrBm38 mAbs. In summary, the results reveal that it is possible to interfere with the RmTIM function using antibodies, even in intact cells. PMID:23202941

  13. Expression and purification of active recombinant equine lysozyme in Escherichia coli.

    PubMed

    Casaite, Vida; Bruzyte, Simona; Bukauskas, Virginijus; Setkus, Arunas; Morozova-Roche, Ludmilla A; Meskys, Rolandas

    2009-11-01

    Equine lysozyme (EL) is a calcium (Ca)-binding lysozyme and is an intermediary link between non-Ca-binding C-type lysozyme and alpha-lactalbumin. The feature of lysozymes to assemble into the fibrils has recently gained considerable attention for the investigation of the functional properties of these proteins. To study the structural and functional properties of EL, a synthetic gene was cloned and EL was overexpressed in Escherichia coli as a fused protein. The His-tagged recombinant EL was accumulated as inclusion bodies. Up to 50 mg/l of the recombinant EL could be achieved after purification by Ni(2+) affinity chromatography, refolding in the presence of arginine, CM-Sepharose column purification following TEV protease cleavage. The purified protein was functionally active, as determined by the lysozyme activity, proving the proper folding of protein. The purified lysozyme was used for the oligomerisation studies. The protein formed amyloid fibrils during incubation in acidic pH and elevated temperature. The recombinant EL forms two types of fibrils: ring shaped and linear, similar to the native EL.

  14. Histidine tag fusion increases expression levels of active recombinant amelogenin in Escherichia coli.

    PubMed

    Svensson, Johan; Andersson, Christer; Reseland, Janne E; Lyngstadaas, Petter; Bülow, Leif

    2006-07-01

    Amelogenin is a dental enamel matrix protein involved in formation of dental enamel. In this study, we have expressed two different recombinant murine amelogenins in Escherichia coli: the untagged rM179, and the histidine tagged rp(H)M180, identical to rM179 except that it carries the additional N-terminal sequence MRGSHHHHHHGS. The effects of the histidine tag on expression levels, and on growth properties of the amelogenin expressing cells were studied. Purification of a crude protein extract containing rp(H)M180 was also carried out using IMAC and reverse-phase HPLC. The results of this study showed clearly that both growth properties and amelogenin expression levels were improved for E. coli cells expressing the histidine tagged amelogenin rp(H)M180, compared to cells expressing the untagged amelogenin rM179. The positive effect of the histidine tag on amelogenin expression is proposed to be due to the hydrophilic nature of the histidine tag, generating a more hydrophilic amelogenin, which is more compatible with the host cell. Human osteoblasts treated with the purified rp(H)M180 showed increased levels of secreted osteocalcin, compared to untreated cells. This response was similar to cells treated with enamel matrix derivate, mainly composed by amelogenin, suggesting that the recombinant protein is biologically active. Thus, the histidine tag favors expression and purification of biologically active recombinant amelogenin.

  15. Single administration of recombinant IL‐6 restores the gene expression of lipogenic enzymes in liver of fasting IL‐6‐deficient mice

    PubMed Central

    Gavito, AL; Cabello, R; Suarez, J; Serrano, A; Pavón, F J; Vida, M; Romero, M; Pardo, V; Bautista, D; Arrabal, S; Decara, J; Cuesta, AL; Valverde, A M; Rodríguez de Fonseca, F

    2016-01-01

    Background and Purpose Lipogenesis is intimately controlled by hormones and cytokines as well as nutritional conditions. IL‐6 participates in the regulation of fatty acid metabolism in the liver. We investigated the role of IL‐6 in mediating fasting/re‐feeding changes in the expression of hepatic lipogenic enzymes. Experimental Approach Gene and protein expression of lipogenic enzymes were examined in livers of wild‐type (WT) and IL‐6‐deficient (IL‐6−/−) mice during fasting and re‐feeding conditions. Effects of exogenous IL‐6 administration on gene expression of these enzymes were evaluated in vivo. The involvement of STAT3 in mediating these IL‐6 responses was investigated by using siRNA in human HepG2 cells. Key Results During feeding, the up‐regulation in the hepatic expression of lipogenic genes presented similar time kinetics in WT and IL‐6−/− mice. During fasting, expression of lipogenic genes decreased gradually over time in both strains, although the initial drop was more marked in IL‐6−/− mice. Protein levels of hepatic lipogenic enzymes were lower in IL‐6−/− than in WT mice at the end of the fasting period. In WT, circulating IL‐6 levels paralleled gene expression of hepatic lipogenic enzymes. IL‐6 administration in vivo and in vitro showed that IL‐6‐mediated signalling was associated with the up‐regulation of hepatic lipogenic enzyme genes. Moreover, silencing STAT3 in HepG2 cells attenuated IL‐6 mediated up‐regulation of lipogenic gene transcription levels. Conclusions and Implications IL‐6 sustains levels of hepatic lipogenic enzymes during fasting through activation of STAT3. Our findings indicate that clinical use of STAT3‐associated signalling cytokines, particularly against steatosis, should be undertaken with caution. PMID:26750868

  16. High-throughput fluorometric measurement of potential soil extracellular enzyme activities.

    PubMed

    Bell, Colin W; Fricks, Barbara E; Rocca, Jennifer D; Steinweg, Jessica M; McMahon, Shawna K; Wallenstein, Matthew D

    2013-11-15

    Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer's particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample). Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil

  17. Extremely thermostable L(+)-lactate dehydrogenase from Thermotoga maritima: cloning, characterization, and crystallization of the recombinant enzyme in its tetrameric and octameric state.

    PubMed Central

    Ostendorp, R.; Auerbach, G.; Jaenicke, R.

    1996-01-01

    L(+)-lactate dehydrogenase (LDH; E.C.1.1.1.27) from the hyperthermophilic bacterium Thermotoga maritima has been shown to represent the most stable LDH isolated so far (Wrba A, Jaenicke R, Huber R, Stetter KO, 1990, Eur J Biochem 188:195-201). In order to obtain the enzyme in amounts sufficient for physical characterization, and to analyze the molecular basis of its intrinsic stability, the gene was cloned and expressed functionally in Escherichia coli. Growth of the cells and purification of the enzyme were performed aerobically at 26 degrees C, i.e., ca. 60 degrees below the optimal growth temperature of Thermotoga. Two enzyme species with LDH activity were purified to homogeneity. Crystals of the enzyme obtained at 4 degrees C show satisfactory diffraction suitable for X-ray analysis up to a resolution of 2.8 A. As shown by gel-permeation chromatography, chemical crosslinking, light scattering, analytical ultracentrifugation, and electron microscopy, the two LDH species represent homotetramers and homooctamers (i.e., dimers of tetramers), with a common subunit molecular mass of 35 kDa. The spectroscopic characteristics (UV absorption, fluorescence emission, near- and far-UV CD) of the two species are indistinguishable. The calculated alpha-helix content is 45%, in accordance with the result of homology modeling. Compared to the tetrameric enzyme, the octamer exhibits reduced specific activity, whereas KM is unalatered. The extreme intrinsic stability of the protein is reflected by its unaltered catalytic activity over 4 h at 85 degrees C; irreversible thermal denaturation becomes significant at approximately 95 degrees C. The anomalous resistance toward chemical denaturation using guanidinium chloride and urea confirms this observation. Both the high optimal temperature and the pH optimum of the catalytic activity correspond to the growth conditions of T. maritima in its natural habitat. PMID:8732758

  18. Catalytic activity of metallic nanoisland coatings. The influence of size effects on the recombination properties

    NASA Astrophysics Data System (ADS)

    Tomilina, O. A.; Berzhansky, V. N.; Tomilin, S. V.; Shaposhnikov, A. N.

    2016-08-01

    The results of investigations of the quantum-size effects influence on selective properties of heterogeneous nanocatalysts are presents. As etalon exothermic reaction was used the reaction of atomic hydrogen recombination. The nanostructured Pd and Pt films on Teflon substrate were used as a samples of heterogeneous nanocatalysts. It was shown that for nanoparticles with various sizes the catalytic activity has the periodic dependence. It has been found that for certain sizes of nanoparticles their catalytic activity is less than that of Teflon substrate.

  19. Specific activities of poetam preparation (superlow-doses of antibodies to erythropoietin) and recombinant erythropoietin.

    PubMed

    Dygai, A M; Zhdanov, V V; Udut, E V; Simanina, E V; Gur'yantseva, L A; Khrichkova, T Yu; Epshtein, O I; Sergeeva, S A

    2006-09-01

    We compared the capacity of superlow-dose of antibodies to erythropoietin (Poetam) and recombinant erythropoietin (Recormon) to stimulate the recovery of adriamycin-suppressed erythropoiesis in mice. Both preparations exhibited high erythron activation capacity and considerably increased the content of erythrocytes and reticulocytes in the peripheral blood and content of erythrokaryocytes and erythroid precursors in the hemopoietic tissue of experimental animals. The effect of Recormon manifested immediately after injection, while the effect of Poetam was somewhat delayed, but more lasting (due to activation of host erythropoietin system).

  20. Retaining and recovering enzyme activity during degradation of TCE by methanotrophs

    SciTech Connect

    Palumbo, A.V.; Strong-Gunderson, J.M.; Carroll, S.

    1997-12-31

    To determine if compounds added during trichloroethylene (TCE) degradation could reduce the loss of enzyme activity or increase enzyme recovery, different compounds serving as energy and carbon sources, pH buffers, or free radical scavengers were tested. Formate and formic acid (reducing power and a carbon source), as well as ascorbic acid and citric acid (free radical scavengers) were added during TCE degradation at a concentration of 2 mM. A saturated solution of calcium carbonate was also tested to address pH concerns. In the presence of formate and methane, only calcium carbonate and formic acid had a beneficial effect on enzyme recovery. The calcium carbonate and formic acid both reduced the loss of enzyme activity and resulted in the highest levels of enzyme activity after recovery. 19 refs., 3 figs.

  1. Construction of Recombinant Pichia pastoris Carrying a Constitutive AvBD9 Gene and Analysis of Its Activity.

    PubMed

    Tu, Jian; Qi, Kezong; Xue, Ting; Wei, Haiting; Zhang, Yongzheng; Wu, Yanli; Zhou, Xiuhong; Lv, Xiaolong

    2015-12-28

    Avian beta-defensin 9 (AvBD9) is a small cationic peptide consisting of 41 amino acids that plays a crucial rule in innate immunity and acquired immunity in chickens. Owing to its wide antibacterial spectrum, lack of a residue, and failure to induce bacterial drug resistance, AvBD9 is expected to become a substitute for conventional antibiotics in the livestock and poultry industries. Using the preferred codon of Pichia pastoris, the mature AvBD9 peptide was designed and synthesized, based on the sequence from GenBank. The P. pastoris constitutive expression vector pGHKα was used to construct a pGHKα-AvBD9 recombinant plasmid. Restriction enzyme digestion was performed using SacI and BglII to remove the ampicillin resistance gene, and the plasmid was electrotransformed into P. pastoris GS115. High-expression strains with G418 resistance were screened, and the culture supernatant was analyzed by Tricine-SDS-PAGE and western blot assay to identify target bands of about 6 kDa. A concentrate of the supernatant containing AvBD9 was used for determination of antimicrobial activity. The supernatant concentrate was effective against Escherichia coli, Salmonella paratyphi, Salmonella pullorum, Pseudomonas aeruginosa, Enterococcus faecalis, and Enterobacter cloacae. The fermentation product of P. pastoris carrying the recombinant AvBD9 plasmid was adjusted to 1.0 × 10(8) CFU/ml and added to the drinking water of white feather broilers at different concentrations. The daily average weight gain and immune organ indices in broilers older than 7 days were significantly improved by the AvBD9 treatment.

  2. The Enterococcus hirae Mur-2 enzyme displays N-acetylglucosaminidase activity

    PubMed Central

    Eckert, Catherine; Magnet, Sophie; Mesnage, Stéphane

    2007-01-01

    Enterococcus hirae produces two autolytic enzymes named Mur-1 and Mur-2, both previously described as N-acetylmuramidases. We used tandem mass spectrometry to show that Mur-2 in fact displays N-acetylglucosaminidase activity. This result reveals that Mur-2 and its counterparts studied to date, which are members of glycosyl hydrolase family 73 from the CAZy (Carbohydrate-Active enZyme) database, display the same catalytic activity. PMID:17258207

  3. Kinetic characterization of recombinant Bacillus coagulans FDP-activated l-lactate dehydrogenase expressed in Escherichia coli and its substrate specificity.

    PubMed

    Jiang, Ting; Xu, Yanbing; Sun, Xiucheng; Zheng, Zhaojuan; Ouyang, Jia

    2014-03-01

    Bacillus coagulans is a homofermentative, acid-tolerant and thermophilic sporogenic lactic acid bacterium, which is capable of producing high yields of optically pure lactic acid. The l-(+)-lactate dehydrogenase (l-LDH) from B. coagulans is considered as an ideal biocatalyst for industrial production. In this study, the gene ldhL encoding a thermostable l-LDH was amplified from B. coagulans NL01 genomic DNA and successfully expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was partially purified and its enzymatic properties were characterized. Sequence analysis demonstrated that the l-LDH was a fructose 1,6-diphosphate-activated NAD-dependent lactate dehydrogenase (l-nLDH). Its molecular weight was approximately 34-36kDa. The Km and Vmax values of the purified l-nLDH for pyruvate were 1.91±0.28mM and 2613.57±6.43μmol(minmg)(-1), respectively. The biochemical properties of l-nLDH showed that the specific activity were up to 2323.29U/mg with optimum temperature of 55°C and pH of 6.5 in the pyruvate reduction and 351.01U/mg with temperature of 55°C and pH of 11.5 in the lactate oxidation. The enzyme also showed some activity in the absence of FDP, with a pH optimum of 4.0. Compared to other lactic acid bacterial l-nLDHs, the enzyme was found to be relatively stable at 50°C. Ca(2+), Ba(2+), Mg(2+) and Mn(2+) ions had activated effects on the enzyme activity, and the enzyme was greatly inhibited by Ni(2+) ion. Besides these, l-nLDH showed the higher specificity towards pyruvate esters, such as methyl pyruvate and ethyl pyruvate.

  4. Expression of catalytically active recombinant Helicobacter pylori urease at wild-type levels in Escherichia coli.

    PubMed Central

    Hu, L T; Mobley, H L

    1993-01-01

    The genes encoding Helicobacter pylori urease, a nickel metalloenzyme, have been cloned and expressed in Escherichia coli. Enzymatic activity, however, has been very weak compared with that in clinical isolates of H. pylori. Conditions under which near wild-type urease activity was achieved were developed. E. coli. SE5000 containing recombinant H. pylori urease genes was grown in minimal medium containing no amino acids, NiCl2 was added to 0.75 microM, and structural genes ureA and ureB (pHP902) were overexpressed in trans to the complete urease gene cluster (pHP808). Under these conditions, E. coli SE5000 pHP808/pHP902) expressed a urease activity up to 87 mumol of urea per min per mg of protein (87 U/mg of protein), a level approaching that of wild-type H. pylori UMAB41 (100 U/mg of protein), from which the genes were cloned. Poor catalytic activity of recombinant clones grown in Luria broth or M9 medium containing 0.5% Casamino Acids was due to chelation of nickel ions by medium components, particularly histidine and cysteine. In cultures containing these amino acids, 63Ni2+ was prevented from being transported into cells and was not incorporated into urease protein. As a consequence, M9 minimal medium cultures containing histidine or cysteine produced only 0.05 and 0.9%, respectively, of active urease produced by control cultures containing no amino acids. We conclude that recombinant H. pylori urease is optimally expressed when Ni2+ transport is not inhibited and when sufficient synthesis of urease subunits UreA and UreB is provided. Images PMID:8500893

  5. Enzymatic activity and motility of recombinant Arabidopsis myosin XI, MYA1.

    PubMed

    Hachikubo, You; Ito, Kohji; Schiefelbein, John; Manstein, Dietmar J; Yamamoto, Keiichi

    2007-06-01

    We expressed recombinant Arabidopsis myosin XI (MYA1), in which the motor domain of MYA1 was connected to an artificial lever arm composed of triple helical repeats of Dictyostelium alpha-actinin, in order to understand its motor activity and intracellular function. The V(max) and K(actin) of the actin-activated Mg(2+) ATPase activity of the recombinant MYA1 were 50.7 Pi head(-1) s(-1) and 30.2 microM, respectively, at 25 degrees C. The recombinant MYA1 could translocate actin filament at the maximum velocity of 1.8 microm s(-1) at 25 degrees C in the in vitro motility assay. The value corresponded to a motility of 3.2 microm s(-1) for native MYA1 if we consider the difference in the lever arm length, and this value was very close to the velocity of cytoplasmic streaming in Arabidopsis hypocotyl epidermal cells. The extent of inhibition by ADP of the motility of MYA1 was similar to that of the well-known processive motor, myosin V, suggesting that MYA1 is a processive motor. The dissociation rate of the actin-MYA1-ADP complex induced by ATP (73.5 s(-1)) and the V(max) value of the actin-activated Mg(2+) ATPase activity revealed that MYA1 stays in the actin-bound state for about 70% of its mechanochemical cycle time. This high ratio of actin-bound states is also a characteristic of processive motors. Our results strongly suggest that MYA1 is a processive motor and involved in vesicle transport and/or cytoplasmic streaming.

  6. Measuring potential denitrification enzyme activity rates using the membrane inlet mass spectrometer

    EPA Science Inventory

    The denitrification enzyme activity (DEA) assay, provides a quantitative assessment of the multi enzyme, biological process of reactive nitrogen removal via the reduction of N03 to N2. Measured in soil, usually under non limiting carbon and nitrate concentrations, this short ter...

  7. Temperature-responsive enzyme-polymer nanoconjugates with enhanced catalytic activities in organic media.

    PubMed

    Zhu, Jingying; Zhang, Yifei; Lu, Diannan; Zare, Richard N; Ge, Jun; Liu, Zheng

    2013-07-11

    A general approach for preparing enzyme-polymer nanoconjugates that respond to temperature in organic media is presented. These nanoconjugates readily dissolve in organic solvents for homogenous catalysis at 40 °C and showed greatly enhanced apparent catalytic activities. The recovery of the soluble enzyme-polymer nanoconjugates is accomplished by temperature-induced precipitation.

  8. Reconciling apparent variability in effects of biochar amendment on soil enzyme activities by assay optimization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We studied the effects of a biochar made from switchgrass on four soil enzymes (ß- glucosidase, ß-N-acetylglucosaminidase, lipase, and leucine aminopeptidase) to determine if biochar would consistently modify soil biological activities. Inconsistent results from enzyme assays of char-amended soils s...

  9. Quantitation of Lipase Activity from a Bee: An Introductory Enzyme Experiment.

    ERIC Educational Resources Information Center

    Farley, Kathleen A.; Jones, Marjorie A.

    1989-01-01

    This four-hour experiment uses a bee as a source of the enzyme which is reacted with a radioactive substrate to determine the specific activity of the enzyme. Uses thin layer chromatography, visible spectrophotometry, and liquid scintillation spectrometry (if not available a Geiger-Muller counter can be substituted). (MVL)

  10. Mechanism of allopurinol-mediated increase in enzyme activity in man

    PubMed Central

    Beardmore, Thomas D.; Cashman, Jay S.; Kelley, William N.

    1972-01-01

    Allopurinol therapy in man interferes with pyrimidine biosynthesis de novo by inhibition of one or both of the two enzymes, orotate phosphoribosyltransferase (OPRT) and orotidylic decarboxylase (ODC), responsible for the conversion of orotic acid to uridine-5′-monophosphate. Inhibition of this pathway in vivo is followed in 1-3 wk by an increase in the activity of both of these enzymes in erythrocytes and of ODC in circulating leukocytes. This drug-mediated increase in enzyme activity in erythrocytes could not be attributed to enzyme stabilization or induction in vivo but appeared to be due to enzymeactivation.” “Activation” of the OPRT enzyme was directly demonstrated in erythrocytes studied in vitro after incubation with oxipurinol, and to a lesser extent, with allopurinol. No evidence for “activation” of the ODC enzyme was demonstrated in vitro. This response to allopurinol therapy provides an excellent model for examining the mechanism of increased enzyme activity in response to drug administration. PMID:5032526

  11. Genome-level and biochemical diversity of the acyl-activating enzyme superfamily in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In higher plants, the superfamily of carboxyl-CoA ligases and related proteins, collectively called acyl activating enzymes (AAEs), has evolved to provide enzymes for many pathways of primary and secondary metabolism and for the conjugation of hormones to amino acids. Across the superfamily there is...

  12. Enzyme activity in terrestrial soil in relation to exploration of the Martian surface

    NASA Technical Reports Server (NTRS)

    Mclaren, A. D.

    1974-01-01

    Sensitive tests for the detection of extracellular enzyme activity in Martian soil was investigated using simulated Martian soil. Enzyme action at solid-liquid water interfaces and at low humidity were studied, and a kinetic scheme was devised and tested based on the growth of microorganisms and the oxidation of ammonium nitrite.

  13. Sediment Microbial Enzyme Activity as an Indicator of Nutrient Limitation in Great Lakes Coastal Wetlands

    EPA Science Inventory

    This study, the first to link microbial enzyme activities to regional-scale anthropogenic stressors, suggests that microbial enzyme regulation of carbon and nutrient dynamics may be sensitive indicators of nutrient dynamics in aquatic ecosystems, but further work is needed to elu...

  14. Illustrating the Effect of pH on Enzyme Activity Using Gibbs Energy Profiles

    ERIC Educational Resources Information Center

    Bearne, Stephen L.

    2014-01-01

    Gibbs energy profiles provide students with a visual representation of the energy changes that occur during enzyme catalysis, making such profiles useful as teaching and learning tools. Traditional kinetic topics, such as the effect of pH on enzyme activity, are often not discussed in terms of Gibbs energy profiles. Herein, the symbolism of Gibbs…

  15. Activity of an enzyme immobilized on superparamagnetic particles in a rotational magnetic field

    SciTech Connect

    Mizuki, Toru; Watanabe, Noriyuki; Nagaoka, Yutaka; Fukushima, Tadamasa; Morimoto, Hisao; Usami, Ron; Maekawa, Toru

    2010-03-19

    We immobilize {alpha}-amylase extracted from Bacillus Iicheniformis on the surfaces of superparamagnetic particles and investigate the effect of a rotational magnetic field on the enzyme's activity. We find that the activity of the enzyme molecules immobilized on superparamagnetic particles increases in the rotational magnetic field and reaches maximum at a certain frequency. We clarify the effect of the cluster structures formed by the superparamagnetic particles on the activity. Enzyme reactions are enhanced even in a tiny volume of solution using the present method, which is very important for the development of efficient micro reactors and micro total analysis systems ({mu}-TAS).

  16. Modification of thiamine pyrophosphate dependent enzyme activity by oxythiamine in Saccharomyces cerevisiae cells.

    PubMed

    Tylicki, Adam; Czerniecki, Jan; Dobrzyn, Pawel; Matanowska, Agnieszka; Olechno, Anna; Strumilo, Slawomir

    2005-10-01

    Oxythiamine is an antivitamin derivative of thiamine that after phosphorylation to oxythiamine pyro phosphate can bind to the active centres of thiamine-dependent enzymes. In the present study, the effect of oxythiamine on the viability of Saccharomyces cerevisiae and the activity of thiamine pyrophosphate dependent enzymes in yeast cells has been investigated. We observed a decrease in pyruvate decarboxylase specific activity on both a control and an oxythiamine medium after the first 6 h of culture. The cytosolic enzymes transketolase and pyruvate decarboxylase decreased their specific activity in the presence of oxythiamine but only during the beginning of the cultivation. However, after 12 h of cultivation, oxythiamine-treated cells showed higher specific activity of cytosolic enzymes. More over, it was established by SDS-PAGE that the high specific activity of pyruvate decarboxylase was followed by an increase in the amount of the enzyme protein. In contrast, the mitochondrial enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, were inhibited by oxythiamine during the entire experiment. Our results suggest that the observed strong decrease in growth rate and viability of yeast on medium with oxythiamine may be due to stronger inhibition of mitochondrial pyruvate dehydrogenase than of cytosolic enzymes.

  17. Antioxidative capacity and enzyme activity in Haematococcus pluvialis cells exposed to superoxide free radicals

    NASA Astrophysics Data System (ADS)

    Liu, Jianguo; Zhang, Xiaoli; Sun, Yanhong; Lin, Wei

    2010-01-01

    The antioxidative capacity of astaxanthin and enzyme activity of reactive oxygen eliminating enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were studied in three cell types of Haematococcus pluvialis exposed to high concentrations of a superoxide anion radical (O{2/-}). The results show that defensive enzymes and astaxanthin-related mechanisms were both active in H. pluvialis during exposure to reactive oxygen species (ROS) such as O{2/-}. Astaxanthin reacted with ROS much faster than did the protective enzymes, and had the strongest antioxidative capacity to protect against lipid peroxidation. The defensive mechanisms varied significantly between the three cell types and were related to the level of astaxanthin that had accumulated in those cells. Astaxanthin-enriched red cells had the strongest antioxidative capacity, followed by brown cells, and astaxanthin-deficient green cells. Although there was no significant increase in expression of protective enzymes, the malondialdehyde (MDA) content in red cells was sustained at a low level because of the antioxidative effect of astaxanthin, which quenched O{2/-} before the protective enzymes could act. In green cells, astaxanthin is very low or absent; therefore, scavenging of ROS is inevitably reliant on antioxidative enzymes. Accordingly, in green cells, these enzymes play the leading role in scavenging ROS, and the expression of these enzymes is rapidly increased to reduce excessive ROS. However, because ROS were constantly increased in this study, the enhance enzyme activity in the green cells was not able to repair the ROS damage, leading to elevated MDA content. Of the four defensive enzymes measured in astaxanthin-deficient green cells, SOD eliminates O{2/-}, POD eliminates H2O2, which is a by-product of SOD activity, and APX and CAT are then initiated to scavenge excessive ROS.

  18. Enzyme-linked immunosorbent assay using recombinant SAG1 antigen to detect Toxoplasma gondii-specific immunoglobulin G antibodies in human sera and saliva.

    PubMed

    Chahed Bel-Ochi, Nouha; Bouratbine, Aïda; Mousli, Mohamed

    2013-04-01

    Serologic detection of Toxoplasma gondii IgG antibodies is widely accepted as a means to determine immune status and susceptibility to Toxoplasma infection during pregnancy. However, current commercial kits present some drawbacks, such as a requirement for whole-parasite antigen preparation or interassay variability. To address these problems, the purpose of this study was to produce a whole sequence of the recombinant T. gondii SAG1 antigen (rSAG1) to assess its diagnostic performance in Toxoplasma IgG screening and to explore a saliva-based method as a noninvasive alternative to serum-based testing. rSAG1 was expressed in recombinant bacteria as inclusion bodies, purified through one-step affinity chromatography, and refolded in native form by dialysis. A large amount was obtained, and the specific antigen immunoreactivity was confirmed by immunoblotting. Two rSAG1-based enzyme-linked immunosorbent assays (ELISAs) applied to paired serum and saliva samples were designed. The rSAG1-based ELISA evaluation consisted of testing intrinsic sensitivity and specificity of 49 serum samples from patients immune to toxoplasmosis and 42 serum samples from nonimmune controls identified by routinely used kits. To assess agreement between serum-based and saliva-based tests, the positive percent agreement (PPA) and negative percent agreement (NPA) between the 2 tests were estimated. The rSAG1 serum-based ELISA detected specific IgG with 100% sensitivity and specificity. The PPA and NPA between the serum-based and saliva-based tests varied according to the selected optical density threshold in saliva. Thus, for a selected cutoff of 0.14, the PPA was 100% and the NPA was 88.1%, whereas for a selected cutoff of 0.29, the PPA was 67.3% and the NPA was 100%.

  19. Enhancement of 2,3-butanediol production from Jerusalem artichoke tuber extract by a recombinant Bacillus sp. strain BRC1 with increased inulinase activity.

    PubMed

    Park, Jang Min; Oh, Baek-Rock; Kang, In Yeong; Heo, Sun-Yeon; Seo, Jeong-Woo; Park, Seung-Moon; Hong, Won-Kyung; Kim, Chul Ho

    2017-03-18

    A Bacillus sp. strain named BRC1 is capable of producing 2,3-butanediol (2,3-BD) using hydrolysates of the Jerusalem artichoke tuber (JAT), a rich source of the fructose polymer inulin. To enhance 2,3-BD production, we undertook an extensive analysis of the Bacillus sp. BRC1 genome, identifying a putative gene (sacC) encoding a fructan hydrolysis enzyme and characterizing the activity of the resulting recombinant protein expressed in and purified from Escherichia coli. Introduction of the sacC gene into Bacillus sp. BRC1 using an expression vector increased enzymatic activity more than twofold. Consistent with this increased enzyme expression, 2,3-BD production from JAT was also increased from 3.98 to 8.10 g L(-1). Fed-batch fermentation of the recombinant strain produced a maximal level of 2,3-BD production of 28.6 g L(-1), showing a high theoretical yield of 92.3%.

  20. [Effect of space flight on the Kosmos-1129 biosatellite on enzyme activity of the rat liver].

    PubMed

    Nemeth, S; Tigranian, R A

    1983-01-01

    After the 18.5 day Cosmos-1129 flight the activity of 7 glucocorticoid-stimulated enzymes of the rat liver was measured. Immediately postflight the activity of tyrosine aminotransferase, tryptophan pyrolase and serine dehydrogenase increased. These enzymes rapidly (within several hours) react to increased glucocorticoids. The activity of aspartate and alanine aminotransferases also increased. These enzymes require many days of a continuous effect of glucocorticoids. The glycogen concentration in the rat liver also grew. At R + 6 the activity of tryptophan pyrolase and serine dehydrogenase decreased and that of the other enzymes returned to normal. The immobilization stress applied postflight led to an increased activity of tyrosine aminotransferase and tryptophan pyrolase. This study gives evidence that after space flight rats are in an acute stress state, evidently, produced by the biosatellite recovery.

  1. Experimental strategy to discover microbes with gluten-degrading enzyme activities

    NASA Astrophysics Data System (ADS)

    Helmerhorst, Eva J.; Wei, Guoxian

    2014-06-01

    Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement.

  2. Array-on-Array Strategy For Activity-Based Enzyme Profiling.

    PubMed

    Sieow, Brendan Fu-Long; Uttamchandani, Mahesh

    2017-01-01

    We describe a novel array on array strategy intended to enhance the throughput of enzymatic activity screening using microarrays. This strategy consists of spotting a first array with large droplets of enzymes with varying concentrations and subsequently spotting a second array with small droplets of fluorogenic substrate on top of the enzyme array. By varying the array on array spotting patterns of different classes of enzyme (e.g., proteases, phosphatases, kinases) and their corresponding fluorogenic substrates, we have the unprecedented ability for testing enzymes and mixed samples in a multiplexed fashion within a single microarray slide. This new approach enables rapid enzyme characterization building upon a one enzyme on one slide droplet-based screening concept previously established.

  3. Experimental Strategy to Discover Microbes with Gluten-degrading Enzyme Activities

    PubMed Central

    Helmerhorst, Eva J.; Wei, Guoxian

    2015-01-01

    Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement. PMID:26113763

  4. Lectin-directed enzyme activated prodrug therapy (LEAPT): Synthesis and evaluation of rhamnose-capped prodrugs.

    PubMed

    Garnier, Philippe; Wang, Xiang-Tao; Robinson, Mark A; van Kasteren, Sander; Perkins, Alan C; Frier, Malcolm; Fairbanks, Antony J; Davis, Benjamin G

    2010-12-01

    The lectin-directed enzyme activated prodrug therapy (LEAPT) bipartite drug delivery system utilizes glycosylated enzyme, localized according to its sugar pattern, and capped prodrugs released by that enzyme. In this way, the sugar coat of a synthetic enzyme determines the site of release of a given drug. Here, prodrugs of doxorubicin and 5-fluorouracil capped by the nonmammalian l-rhamnosyl sugar unit have been efficiently synthesized and evaluated for use in the LEAPT system. Both are stable in blood, released by synthetically d-galactosylated rhamnosidase enzyme, and do not inhibit the uptake of the synthetic enzyme to its liver target. These results are consistent with their proposed mode of action and efficacy in models of liver cancer, and confirm modular flexibility in the drugs that may be used in LEAPT.

  5. Aerobic and anaerobic bioprocessing of activated sludge: floc disintegration by enzymes.

    PubMed

    Ayol, Azize; Filibeli, Ayse; Sir, Diclehan; Kuzyaka, Ersan

    2008-11-01

    Hydrolytic enzymes such as glucosidases, lipases, and proteases have an imperative function at the hydrolysis stage of complex organic structures in the degradation of biodegradable particulate organic matter. As a key factor, extracellular polymeric substances (EPS) control the extracellular hydrolytic enzymes in this degradation mechanism. A flocculated matrix of EPS bridging with bacteria holds back the dewaterability properties of the bioprocessed sludges. Disruption of the flocculated matrix leads to improved solubilization of sludge solids by attacking the hydrolytic enzymes to polymeric substances forming enzyme-substrate complexes. To determine the floc disintegration mechanisms by enzymes during aerobic and anaerobic bioprocessing of sludges, experimental data obtained from three aerobic digesters and three anaerobic digesters were evaluated. As part of a broader project examining the overall fate and effects of hydrolytic enzymes in biological sludge stabilization, this paper compares the performances of aerobic and anaerobic reactors used in this study and reports significant improvements in enzymatic treatment of activated sludge.

  6. Activity modulation of microbial enzymes by llama (Lama glama) heavy-chain polyclonal antibodies during in vivo immune responses.

    PubMed

    Ferrari, A; Weill, F S; Paz, M L; Cela, E M; González Maglio, D H; Leoni, J

    2012-03-01

    Since they were first described in 1993, it was found that recombinant variable fragments (rVHHs) of heavy-chain antibodies (HCAbs) from Camelidae have unusual biophysical properties, as well as a special ability to interact with epitopes that are cryptic for conventional Abs. It has been assumed that in vivo raised polyclonal HCAbs (pHCAbs) should behave in a similar manner than rVHHs; however, this assumption has not been tested sufficiently. Furthermore, our own preliminary work on a single serum sample from a llama immunized with a β-lactamase, has suggested that pHCAbs have no special ability to down-modulate catalytic activity. In this work, we further explored the interaction of pHCAbs from four llamas raised against two microbial enzymes and analyzed it within a short and a long immunization plan. The relative contribution of pHCAbs to serum titer was found to be low compared with that of the most abundant conventional subisotype (IgG(1)), during the whole immunization schedule. Furthermore, pHCAbs not only failed to inhibit the enzymes, but also activated one of them. Altogether, these results suggest that raising high titer inhibitory HCAbs is not a straightforward strategy - neither as a biotechnological strategy nor in the biological context of an immune response against infection - as raising inhibitory rVHHs.

  7. Discovery of an Active RAG Transposon Illuminates the Origins of V(D)J Recombination.

    PubMed

    Huang, Shengfeng; Tao, Xin; Yuan, Shaochun; Zhang, Yuhang; Li, Peiyi; Beilinson, Helen A; Zhang, Ya; Yu, Wenjuan; Pontarotti, Pierre; Escriva, Hector; Le Petillon, Yann; Liu, Xiaolong; Chen, Shangwu; Schatz, David G; Xu, Anlong

    2016-06-30

    Co-option of RAG1 and RAG2 for antigen receptor gene assembly by V(D)J recombination was a crucial event in the evolution of jawed vertebrate adaptive immunity. RAG1/2 are proposed to have arisen from a transposable element, but definitive evidence for this is lacking. Here, we report the discovery of ProtoRAG, a DNA transposon family from lancelets, the most basal extant chordates. A typical ProtoRAG is flanked by 5-bp target site duplications and a pair of terminal inverted repeats (TIRs) resembling V(D)J recombination signal sequences. Between the TIRs reside tail-to-tail-oriented, intron-containing RAG1-like and RAG2-like genes. We demonstrate that ProtoRAG was recently active in the lancelet germline and that the lancelet RAG1/2-like proteins can mediate TIR-dependent transposon excision, host DNA recombination, transposition, and low-efficiency TIR rejoining using reaction mechanisms similar to those used by vertebrate RAGs. We propose that ProtoRAG represents a molecular "living fossil" of the long-sought RAG transposon.

  8. Cre-dependent DNA recombination activates a STING-dependent innate immune response

    PubMed Central

    Pépin, Geneviève; Ferrand, Jonathan; Höning, Klara; Jayasekara, W. Samantha N.; Cain, Jason E.; Behlke, Mark A.; Gough, Daniel J.; G. Williams, Bryan R.; Hornung, Veit; Gantier, Michael P.

    2016-01-01

    Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell–cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies. PMID:27166376

  9. Heterologous Expression of MeLEA3: A 10 kDa Late Embryogenesis Abundant Protein of Cassava, Confers Tolerance to Abiotic Stress in Escherichia coli with Recombinant Protein Showing In Vitro Chaperone Activity.

    PubMed

    Barros, Nicolle L F; da Silva, Diehgo T; Marques, Deyvid N; de Brito, Fabiano M; dos Reis, Savio P; de Souza, Claudia R B

    2015-01-01

    Late embryogenesis abundant (LEA) proteins are small molecular weight proteins involved in acquisition of tolerance to drought, salinity, high temperature, cold, and freezing stress in many plants. Previous studies revealed a cDNA sequence coding for a 10 kDa atypical LEA protein, named MeLEA3, predicted to be located into mitochondria with potential role in salt stress response of cassava (Manihot esculenta Crantz). Here we aimed to produce the recombinant MeLEA3 protein by heterologous expression in Escherichia coli and evaluate the tolerance of bacteria expressing this protein under abiotic stress. Our result revealed that the recombinant MeLEA3 protein conferred a protective function against heat and salt stress in bacterial cells. Also, the recombinant MeLEA3 protein showed in vitro chaperone activity by protection of NdeI restriction enzyme activity under heat stress.

  10. Microbial dynamics and enzyme activities in tropical Andosols depending on land use and nutrient inputs

    NASA Astrophysics Data System (ADS)

    Mganga, Kevin; Razavi, Bahar; Kuzyakov, Yakov

    2015-04-01

    Microbial decomposition of soil organic matter is mediated by enzymes and is a key source of terrestrial CO2 emissions. Microbial and enzyme activities are necessary to understand soil biochemical functioning and identify changes in soil quality. However, little is known about land use and nutrients availability effects on enzyme activities and microbial processes, especially in tropical soils of Africa. This study was conducted to examine how microbial and enzyme activities differ between different land uses and nutrient availability. As Andosols of Mt. Kilimanjaro are limited by nutrient concentrations, we hypothesize that N and P additions will stimulate enzyme activity. N and P were added to soil samples (0-20 cm) representing common land use types in East Africa: (1) savannah, (2) maize fields, (3) lower montane forest, (4) coffee plantation, (5) grasslands and (6) traditional Chagga homegardens. Total CO2 efflux from soil, microbial biomass and activities of β-glucosidase, cellobiohydrolase, chitinase and phosphatase involved in C, N and P cycling, respectively was monitored for 60 days. Total CO2 production, microbial biomass and enzyme activities varied in the order forest soils > grassland soils > arable soils. Increased β-glucosidase and cellobiohydrolase activities after N addition of grassland soils suggest that microorganisms increased N uptake and utilization to produce C-acquiring enzymes. Low N concentration in all soils inhibited chitinase activity. Depending on land use, N and P addition had an inhibitory or neutral effect on phosphatase activity. We attribute this to the high P retention of Andosols and low impact of N and P on the labile P fractions. Enhanced CO2 production after P addition suggests that increased P availability could stimulate soil organic matter biodegradation in Andosols. In conclusion, land use and nutrients influenced soil enzyme activities and microbial dynamics and demonstrated the decline in soil quality after landuse

  11. Investigations on the activation of recombinant microbial pro-transglutaminase: in contrast to proteinase K, dispase removes the histidine-tag.

    PubMed

    Sommer, Christian; Hertel, Thomas C; Schmelzer, Christian E H; Pietzsch, Markus

    2012-02-01

    In order to produce recombinant microbial transglutaminase (rMTG) which is free of the activating protease, dispase was used to activate the pro-rMTG followed by immobilized metal affinity chromatography (IMAC). As shown by MALDI-MS, the dispase does not only cleave the pro-sequence, but unfortunately also cleaves within the C-terminal histidine-tag. Hence, the active rMTG cannot properly bind to the IMAC material. As an alternative, proteinase K was investigated. This protease was successfully applied for the activation of purified pro-rMTG either as free or immobilized enzyme and the free enzyme was also applicable directly in the crude cell extract of E. coli. Thus, it enables a simple two-step activation/purification procedure resulting in protease-free and almost pure transglutaminase preparations. The protocol has been successfully applied to both, wild-type transglutaminase of Streptomyces mobaraensis as well as to the highly active variant S2P. Proteinase K activates the pro-rMTG without unwanted degradation of the histidine-tag. It turned out to be very important to inhibit proteinase K activity, e.g., by PMSF, prior to protein separation by SDS-PAGE.

  12. Activation of a Covalent Enzyme-Substrate Bond by Noncovalent Interaction with an Effector

    PubMed Central

    Malhotra, O. P.; Bernhard, Sidney A.

    1973-01-01

    The absorption spectrum of an activesite specific chromophoric acyl enzyme, sturgeon β-(2-furyl)-acryloyl-glyceraldehyde-3-phosphate dehydrogenase, is reported. This acyl enzyme undergoes all of the catalyzed reactions characteristic of the intermediate of the physiological acyl enzyme, 3-phospho-D-glyceroyl-glyceraldehyde-3-phosphate dehydrogenease. The rates of reactions of both these acyl enzymes depend strongly on the extent of interaction of the acyl enzyme with the oxidized coenzyme, NAD+, even where the “redox” properties of the coenzyme are not required. Likewise, the spectral properties of chromophoric acyl enzyme are affected by the extent of bound NAD. Under the pseudophysiological conditions reported herein, there is a stoichiometric limitation of two furylacryloyl-acyl groups per enzyme molecule containing four covalently-equivalent subunits. The binding of NAD both to the apoenzyme and to the diacyl enzyme is heterogeneous: at low extents of NAD occupancy, NAD binding is stronger. The binding to acyl enzyme can be quantitatively described by an enzyme model involving a tetramer with 2-fold symmetry, and consequently containing equal numbers of two classes of sites. NAD binding to difurylacryloyl-enzyme occurs virtually discretely, first to the two unmodified (tight-binding) sites, followed by looser binding to the two acyl-sites. NAD occupancy at these latter sites transforms the chromophoric acyl spectrum from that characteristic of a model furylacryloyl-thiol ester in H2O to a highly perturbed furylacryloyl spectrum characteristic of monomeric native “active-thiol” furylacryloyl-enzymes. Likewise the acyl reactivity towards arsenolysis depends on the extent of NAD bound to the loose sites. Elimination of the tight binding of NAD to the difurylacryloyl enzyme tetramer by alkylation of the remaining two free SH groups with iodoacetate has no apparent influence on the NAD-dependent furylacryloyl-spectral perturbation at the “two equivalent

  13. A basic motif in the N-terminal region of RAG1 enhances V(D)J recombination activity.

    PubMed Central

    McMahan, C J; Difilippantonio, M J; Rao, N; Spanopoulou, E; Schatz, D G

    1997-01-01

    The variable portions of antigen receptor genes are assembled from component gene segments by a site-specific recombination reaction known as V(D)J recombination. The RAG1 and RAG2 proteins are the critical lymphoid cell-specific components of the recombination enzymatic machinery and are responsible for site-specific DNA recognition and cleavage. Previous studies had defined a minimal, recombinationally active core region of murine RAG1 consisting of amino acids 384 to 1008 of the 1,040-residue RAG1 protein. No recombination function has heretofore been ascribed to any portion of the 383-amino-acid N-terminal region that is missing from the core, but it seems likely to be of functional significance, based on its evolutionary conservation. Using extrachromosomal recombination substrates, we demonstrate here that the N-terminal region enhances the recombination activity of RAG1 by up to an order of magnitude in a variety of cell lines. Deletion analysis localized a region of the N terminus critical for this effect to amino acids 216 to 238, and further mutagenesis demonstrated that a small basic amino acid motif (BIIa) in this region is essential for enhancing the activity of RAG1. Despite the fact that BIIa is important for the interaction of RAG1 with the nuclear localization factor Srp-1, it does not appear to enhance recombination by facilitating nuclear transport of RAG1. A variety of models for how this region stimulates the recombination activity of RAG1 are considered. PMID:9234712

  14. A new methodology for the determination of enzyme activity based on carbon nanotubes and glucose oxidase.

    PubMed

    Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

    2015-11-10

    In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device.

  15. Physics-based enzyme design: predicting binding affinity and catalytic activity.

    PubMed

    Sirin, Sarah; Pearlman, David A; Sherman, Woody

    2014-12-01

    Computational enzyme design is an emerging field that has yielded promising success stories, but where numerous challenges remain. Accurate methods to rapidly evaluate possible enzyme design variants could provide significant value when combined with experimental efforts by reducing the number of variants needed to be synthesized and speeding the time to reach the desired endpoint of the design. To that end, extending our computational methods to model the fundamental physical-chemical principles that regulate activity in a protocol that is automated and accessible to a broad population of enzyme design researchers is essential. Here, we apply a physics-based implicit solvent MM-GBSA scoring approach to enzyme design and benchmark the computational predictions against experimentally determined activities. Specifically, we evaluate the ability of MM-GBSA to predict changes in affinity for a steroid binder protein, catalytic turnover for a Kemp eliminase, and catalytic activity for α-Gliadin peptidase variants. Using the enzyme design framework developed here, we accurately rank the most experimentally active enzyme variants, suggesting that this approach could provide enrichment of active variants in real-world enzyme design applications.

  16. Role of enzyme-substrate flexibility in catalytic activity: an evolutionary perspective.

    PubMed

    Demetrius, L

    1998-09-21

    Site-directed mutagenesis has proved an effective experimental technique to investigate catalytic mechanisms and to determine relations between enzyme structure and function. This article invokes an analytical model based on evolution by mutation and natural selection-Nature's analogue of site-directed mutagenesis-to derive a set of general rules relating enzyme structure and activity. The catalysts are described in terms of the structural parameters, rigidity and flexibility, and the functional variables, reaction rate and substrate specificity. The evolutionary model predicts the following structure-activity relations: (a) rigid enzyme-flexible substrate: large variation in reaction rates, broad substrate specificity; (b) rigid enzyme-rigid substrate: diffusion controlled rates, absolute specificity; (c) flexible enzyme-rigid substrate: intermediate reaction rates, group specificity; (d) flexible enzyme-flexible substrate: slow rates, absolute specificity. Spectroscopic methods and X-ray crystallography now yield important characteristics of enzyme-substrate complexes such as molecular flexibility. The evolutionary analysis we have exploited provides general principles for inferring catalytic activity from structural studies of enzyme-substrate complexes.

  17. Dosage Compensation in Drosophila: Nadp-Enzyme Activities and Cross-Reacting Material

    PubMed Central

    Williamson, John H.; Bentley, Michael M.

    1983-01-01

    The relationships between gene dosage, enzyme activities and CRM levels have been determined for G6PD and 6PGD. Enzyme activities and CRM levels were directly proportional and increased in genotypes carrying duplications of the respective structural genes. When a duplication consisting of the distal 45% of the X chromosome was used to duplicate Pgd+, 6PGD activity and CRM increased and G6PD activity decreased. When the proximal 55% of the X chromosome was duplicated, G6PD activity and CRM increased whereas 6PGD activity and CRM levels decreased. These observations support the model of dosage compensation of X-linked genes that invokes an autosomal activator in limited concentrations for which X-linked loci compete. The distal 45% of the X chromosome, when duplicated, caused a significant increase in NADP-malic enzyme activity and CRM levels, as if a structural gene for NADP-ME is sex-linked. PMID:6406296

  18. Production of an active recombinant thrombomodulin derivative in transgenic tobacco plants and suspension cells.

    PubMed

    Schinkel, Helga; Schiermeyer, Andreas; Soeur, Raphael; Fischer, Rainer; Schillberg, Stefan

    2005-06-01

    Thrombomodulin is a membrane-bound protein that plays an active role in the blood coagulation system by binding thrombin and initiating the protein C anticoagulant pathway. Solulin is a recombinant soluble derivative of human thrombomodulin. It is used for the treatment of thrombotic disorders. To evaluate the production of this pharmaceutical protein in plants, expression vectors were generated using four different N-terminal signal peptides. Immunoblot analysis of transiently transformed tobacco leaves showed that intact Solulin could be detected using three of these signal peptides. Furthermore transgenic tobacco plants and BY2 cells producing Solulin were generated. Immunoblot experiments showed that Solulin accumulated to maximum levels of 115 and 27 microg g(-1) plant material in tobacco plants and BY2 cells, respectively. Activity tests performed on the culture supernatant of transformed BY2 cells showed that the secreted Solulin was functional. In contrast, thrombomodulin activity was not detected in total soluble protein extracts from BY2 cells, probably due to inhibitory effects of substances in the cell extract. N-terminal sequencing was carried out on partially purified Solulin from the BY2 culture supernatant. The sequence was identical to that of Solulin produced in Chinese hamster ovary cells, confirming correct processing of the N-terminal signal peptide. We have demonstrated that plants and plant cell cultures can be used as alternative systems for the production of an active recombinant thrombomodulin derivative.

  19. Investigations on the activity of poly(2-oxazoline) enzyme conjugates dissolved in organic solvents.

    PubMed

    Konieczny, Stefan; Krumm, Christian; Doert, Dominik; Neufeld, Katharina; Tiller, Joerg C

    2014-07-10

    The use of enzymes in organic solvents offers a great opportunity for the highly selective synthesis of complex organic compounds. In this study we investigate the POXylation of several enzymes with different polyoxazolines ranging from the hydrophilic poly(2-methyl-oxazoline) (PMOx) to the hydrophobic poly(2-heptyl-oxazoline) (PHeptOx). As reported previously on the examples of model enzymes POXylation mediated by pyromellitic acid dianhydride results in highly modified, organosoluble protein conjugates. This procedure is here extended to a larger number of proteins and optimized for the different polyoxazolines. The resulting polymer-enzyme conjugates (PEC) became soluble in different organic solvents ranging from hydrophilic DMF to even toluene. These conjugates were characterized regarding their solubility and especially their activity in organic solvents and in some cases the PECs showed significantly (up to 153,000 fold) higher activities than the respective native enzymes.

  20. Mesoporous silica-encapsulated gold nanoparticles as artificial enzymes for self-activated cascade catalysis.

    PubMed

    Lin, Youhui; Li, Zhenhua; Chen, Zhaowei; Ren, Jinsong; Qu, Xiaogang

    2013-04-01

    A significant challenge in chemistry is to create synthetic structures that mimic the complexity and function of natural systems. Here, a self-activated, enzyme-mimetic catalytic cascade has been realized by utilizing expanded mesoporous silica-encapsulated gold nanoparticles (EMSN-AuNPs) as both glucose oxidase- and peroxidase-like artificial enzymes. Specifically, EMSN helps the formation of a high degree of very small and well-dispersed AuNPs, which exhibit an extraordinarily stability and dual enzyme-like activities. Inspired by these unique and attractive properties, we further piece them together into a self-organized artificial cascade reaction, which is usually completed by the oxidase-peroxidase coupled enzyme system. Our finding may pave the way to use matrix as the structural component for the design and development of biomimetic catalysts and to apply enzyme mimics for realizing higher functions.

  1. Hepatic biotransformation and antioxidant enzyme activities in Mediterranean fish from different habitat depths.

    PubMed

    Ribalta, C; Sanchez-Hernandez, J C; Sole, M

    2015-11-01

    Marine fish are threatened by anthropogenic chemical discharges. However, knowledge on adverse effects on deep-sea fish or their detoxification capabilities is limited. Herein, we compared the basal activities of selected hepatic detoxification enzymes in several species (Solea solea, Dicentrarchus labrax, Trachyrhynchus scabrus, Mora moro, Cataetix laticeps and Alepocehalus rostratus) collected from the coast, middle and lower slopes of the Blanes Canyon region (Catalan continental margin, NW Mediterranean Sea). The xenobiotic-detoxifying enzymes analysed were the phase-I carboxylesterases (CbEs), and the phase-II conjugation activities uridine diphosphate glucuronyltransferase (UDPGT) and glutathione S-transferase (GST). Moreover, some antioxidant enzyme activities, i.e., catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR), were also included in this comparative study. Because CbE activity is represented by multiple isoforms, the substrates α-naphthyl acetate (αNA) and ρ-nitrophenyl acetate (ρNPA) were used in the enzyme assays, and in vitro inhibition kinetics with dichlorvos were performed to compare interspecific CbE sensitivity. Activity of xenobiotic detoxification enzymes varied among the species, following a trend with habitat depth and body size. Thus, UDPGT and some antioxidant enzyme activities decreased in fish inhabiting lower slopes of deep-sea, whereas UDPGT and αNA-CbE activities were negatively related to fish size. A trend between CbE activities and the IC50 values for dichlorvos suggested S. solea and M. moro as potentially more sensitive to anticholinesterasic pesticides, and T. scabrus as the most resistant one. A principal component analysis considering all enzyme activities clearly identified the species but this grouping was not related to habitat depth or phylogeny. Although these results can be taken as baseline levels of the main xenobiotic detoxification enzymes in Mediterranean fish, further research is

  2. Seasonal variation in the temperature sensitivity of proteolytic enzyme activity in temperate forest soils

    NASA Astrophysics Data System (ADS)

    Brzostek, Edward R.; Finzi, Adrien C.

    2012-03-01

    Increasing soil temperature has the potential to alter the activity of the extracellular enzymes that mobilize nitrogen (N) from soil organic matter (SOM) and ultimately the availability of N for primary production. Proteolytic enzymes depolymerize N from proteinaceous components of SOM into amino acids, and their activity is a principal driver of the within-system cycle of soil N. The objectives of this study were to investigate whether the soils of temperate forest tree species differ in the temperature sensitivity of proteolytic enzyme activity over the growing season and the role of substrate limitation in regulating temperature sensitivity. Across species and sampling dates, proteolytic enzyme activity had relatively low sensitivity to temperature with a mean activation energy (Ea) of 33.5 kJ mol-1. Ea declined in white ash, American beech, and eastern hemlock soils across the growing season as soils warmed. By contrast, Eain sugar maple soil increased across the growing season. We used these data to develop a species-specific empirical model of proteolytic enzyme activity for the 2009 calendar year and studied the interactive effects of soil temperature (ambient or +5°C) and substrate limitation (ambient or elevated protein) on enzyme activity. Declines in substrate limitation had a larger single-factor effect on proteolytic enzyme activity than temperature, particularly in the spring. There was, however, a large synergistic effect of increasing temperature and substrate supply on proteolytic enzyme activity. Our results suggest limited increases in N availability with climate warming unless there is a parallel increase in the availability of protein substrates.

  3. Studies on antioxidant activity of teasaponins after hydrolyzed by enzyme

    NASA Astrophysics Data System (ADS)

    Tian, Jing; Zhao, Sen; Xu, Longquan; Fei, Xu; Wang, Xiuying; Wang, Yi

    The biological activity of teasaponins and their molecular structure are closely related, and the activity of saponins may be increased with the change of their molecular structure. In this report, teasaponins were hydrolyzed by Aspergillus niger for increasing the antioxidant activity. The antioxidant activity of teasaponins before and after hydrolyzed was tested by DPPH, and the result showed four new teasaponins were produced after hydrolysis, and their antioxidant activity was increased significantly than the original teasaponins before hydrolysis, the radical scavenging capacity (RSC) was partly up to 95 %.

  4. Detection of Extracellular Enzyme Activity in Penicillium using Chromogenic Media.

    PubMed

    Yoon, Ji Hwan; Hong, Seung Beom; Ko, Seung Ju; Kim, Seong Hwan

    2007-09-01

    A total of 106 Penicillium species were tested to examine their ability of degrading cellobiose, pectin and xylan. The activity of β-glucosidase was generally strong in all the Penicillium species tested. P. citrinum, P. charlesii, P. manginii and P. aurantiacum showed the higher ability of producing β-glucosidase than other tested species. Pectinase activity was detected in 24 Penicillium species. P. paracanescens, P. sizovae, P. sartoryi, P. chrysogenum, and P. claviforme showed strong pectinase activity. In xylanase assay, 84 Penicillium species showed activity. Strong xylanase activity was detected from P. megasporum, P. sartoryi, P. chrysogenum, P. glandicola, P. discolor, and P. coprophilum. Overall, most of the Penicillium species tested showed strong β-glucosidase activity. The degree of pectinase and xylanase activity varied depending on Penicillium species.

  5. Novel ferulate esterase from Gram-positive lactic acid bacteria and analyses of the recombinant enzyme produced in E. coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using a plate containing ethyl ferulate as sole carbon source, various bacteria cultures were screened for ferulate esterase (FAE). Among a dozen of species showing positive FAE, one Lactobacillus fermentum strain NRRL 1932 demonstrated the strongest activity. Using a published sequence of ferulate ...

  6. Novel feruloyl esterase from Lactobacillus fermentum NRRL B-1932 and analysis of the recombinant enzyme produced in Escherichia coli.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using agar plates containing ethyl ferulate as the sole carbon source, 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity. Among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate, Lactobacillus fermentum NRRL B-1932 demonstrated the stronge...

  7. Modeling in situ soil enzyme activity using continuous field soil moisture and temperature data

    NASA Astrophysics Data System (ADS)

    Steinweg, J. M.; Wallenstein, M. D.

    2010-12-01

    Moisture and temperature are key drivers of soil organic matter decomposition, but there is little consensus on how climate change will affect the degradation of specific soil compounds under field conditions. Soil enzyme activities are a useful metric of soil community microbial function because they are they are the direct agents of decomposition for specific substrates in soil. However, current standard enzyme assays are conducted under optimized conditions in the laboratory and do not accurately reflect in situ enzyme activity, where diffusion and substrate availability may limit reaction rates. The Arrhenius equation, k= A*e(-Ea/RT), can be used to predict enzyme activity (k), collision frequency (A) or activation energy (Ea), but is difficult to parameterize when activities are measured under artificial conditions without diffusion or substrate limitation. We developed a modifed equation to estimate collision frequency and activation energy based on soil moisture to model in-situ enzyme activites. Our model was parameterized using data we collected from the Boston Area Climate Experiment (BACE) in Massachusetts; a multi-factor climate change experiment that provides an opportunity to assess how changes in moisture availability and temperature may impact enzyme activity. Soils were collected from three precipitation treatments and four temperature treatments arranged in a full-factorial design at the BACE site in June 2008, August 2008, January 2009 and June 2009. Enzyme assays were performed at four temperatures (4, 15, 25 and 35°C) to calculate temperature sensitivity and activation energy over the different treatments and seasons. Enzymes activities were measured for six common enzymes involved in carbon (β-glucosidase, cellobiohydrolase, xylosidase), phosphorus (phosphatase) and nitrogen cycling (N-acetyl glucosaminidase, and leucine amino peptidase). Potential enzyme activity was not significantly affected by precipitation, warming or the interaction of

  8. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    SciTech Connect

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  9. Defying the activity-stability trade-off in enzymes: taking advantage of entropy to enhance activity and thermostability.

    PubMed

    Siddiqui, Khawar Sohail

    2017-05-01

    The biotechnological applications of enzymes are limited due to the activity-stability trade-off, which implies that an increase in activity is accompanied by a concomitant decrease in protein stability. This premise is based on thermally adapted homologous enzymes where cold-adapted enzymes show high intrinsic activity linked to enhanced thermolability. In contrast, thermophilic enzymes show low activity around ambient temperatures. Nevertheless, genetically and chemically modified enzymes are beginning to show that the activity-stability trade-off can be overcome. In this review, the origin of the activity-stability trade-off, the thermodynamic basis for enhanced activity and stability, and various approaches for escaping the activity-stability trade-off are discussed. The role of entropy in enhancing both the activity and the stability of enzymes is highlighted with a special emphasis placed on the involvement of solvent water molecules. This review is concluded with suggestions for further research, which underscores the implications of these findings in the context of productivity curves, the Daniel-Danson equilibrium model, catalytic antibodies, and life on cold planets.

  10. Activity of recombinant and natural defensins from Vigna unguiculata seeds against Leishmania amazonensis.

    PubMed

    Souza, Géssika Silva; do Nascimento, Viviane Veiga; de Carvalho, Laís Pessanha; de Melo, Edésio José Tenório; Fernandes, Keysson Vieira; Machado, Olga Lima Tavares; Retamal, Claudio Andres; Gomes, Valdirene Moreira; Carvalho, André de Oliveira

    2013-09-01

    Antimicrobial peptides (AMPs), which are differentiated from other antibiotic peptides, such as gramicidins and polymyxins, because they are synthesized by large enzymatic complex and bear modified amino acids including d-amino acids, are short polymers of l-amino acids synthesized by ribosomes upon which all living organisms rely to defend themselves from invaders or competitor microorganisms. AMPs have received a great deal of attention from the scientific community as potential new drugs for neglected diseases such as Leishmaniasis. In plants, they include several families of compounds, including the plant defensins. The aim of the present study was to improve the expression of recombinant defensin from Vigna unguiculata seeds (Vu-Defr) and to test its activity against Leishmania amazonensis promatigotes. Recombinant expression was performed in LB and TB media and under different conditions. The purification of Vu-Defr was achieved by immobilized metal ion affinity and reversed-phase chromatography. The purified Vu-Defr was analyzed by circular dichroism (CD), and its biological activity was tested against L. amazonenis promastigotes. To demonstrate that the recombinant production of Vu-Defr did not interfere with its fold and biological activity, the results of all experiments were compared with the results from the natural defensin (Vu-Def). The CD spectra of both peptides presented good superimposition indicating that both peptides present very similar secondary structure and that the Vu-Defr was correctly folded. L. amazonensis treated with Vu-Defr led to the elimination of 54.3% and 46.9% of the parasites at 24 and 48h of incubation time, respectively. Vu-Def eliminated 50% and 54.8% of the parasites at 24 and 48 h, respectively. Both were used at a concentration of 100 μg/mL. These results suggested the potential for plant defensins to be used as new antiparasitic substances.

  11. The importance of myeloperoxidase enzyme activity in the pathogenesis of Crimean-Congo haemorrhagic fever.

    PubMed

    Guven, F M K; Aydin, H; Yildiz, G; Engin, A; Celik, V K; Bakir, D; Deveci, K

    2013-03-01

    Crimean-Congo haemorrhagic fever (CCHF) is a disease with a severe course including acute viral haemorrhagic fever, ecchymosis, thrombocytopenia, hepatic function disorder and high mortality. Myeloperoxidase (MPO) is an enzyme located in neutrophil granulocytes and plays an important role in the destruction of phagocytosed micro-organisms. The aim of this study was to analyse MPO enzyme activity in CCHF cases compared with a control group. A total of 47 randomly selected CCHF patients admitted to the Department of Infectious Diseases of Cumhuriyet University Hospital in Sivas, Turkey, were studied, and as a control group, 41 age- and sex-matched individuals without any systemic disease were included in this study. MPO enzyme activity was measured in plasma and leukocytes for both groups by the ELISA method. MPO plasma and MPO leukocyte values were calculated as 57.62 ± 8.85 and 44.84 ± 9.71 in CCHF patients, and 0.79 ± 0.29 and 0.49 ± 0.11 in the controls, respectively. MPO enzyme activity was statistically significantly higher in patients with CCHF when compared to the control group. In conclusion, MPO enzyme activity is directly related to the activation of phagocytic leukocytes, and increases in both the plasma and leukocytes in CCHF patients. The increase of the MPO enzyme activity in leukocytes due to viral load leads to the destruction of the leukocyte. It is thought that MPO enzyme activity in plasma was higher in CCHF patients due to the destruction of leukocytes. MPO enzyme activity may be important in terms of the prognosis in patients with CCHF; however, more extensive studies are required on this subject.

  12. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  13. Estrogenic and mutagenic activities of Crotalaria pallida measured by recombinant yeast assay and Ames test

    PubMed Central

    2013-01-01

    Background Crotalaria pallida Ailton is a plant belonging to the Fabaceae family, popularly known as “rattle or rattlesnake” and used in traditional medicine to treat swelling of the joints and as a vermifuge. Previous pharmacological studies have also reported anti-inflammatory, antimicrobial and antifungal activities. Nevertheless, scientific information regarding this species is scarce, and there are no reports related to its possible estrogenic and mutagenic effects. Thus, the purpose of the present study was to investigate the estrogenic potential of C. pallida leaves by means of the Recombinant Yeast Assay (RYA), seeking an alternative for estrogen replacement therapy during menopause; and to reflect on the safe use of natural products to assess the mutagenic activity of the crude extract from C. pallida leaves, the dichloromethane fraction and stigmasterol by means of the Ames test. Methods The recombinant yeast assay with the strain BY4741 of Saccharomyces cerevisiae, was performed with the ethanolic extract, dichloromethane fraction and stigmasterol isolated from the leaves of C. pallida. Mutagenic activity was evaluated by the Salmonella/microsome assay (Ames test), using the Salmonella typhimurium tester strains TA100, TA98, TA97 and TA102, with (+S9) and without (-S9) metabolization, by the preincubation method. Results All samples showed estrogenic activity, mainly stigmasterol. The ethanolic extract from C. pallida leaves showed mutagenic activity in the TA98 strain (-S9), whereas dichloromethane fraction and stigmasterol were found devoid of activity. Conclusion Considering the excellent estrogenic activity performed by stigmasterol in the RYA associated with the absence of mutagenic activity when evaluated by the Ames test, stigmasterol becomes a strong candidate to be used in hormone replacement therapy during menopause. PMID:24134316

  14. Quantum dot based enzyme activity sensors present deviations from Michaelis-Menten kinetic model

    NASA Astrophysics Data System (ADS)

    Díaz, Sebastián. A.; Brown, Carl W.; Malanoski, Anthony P.; Oh, Eunkeu; Susumu, Kimihiro; Medintz, Igor L.

    2016-03-01

    Nanosensors employing quantum dots (QDs) and enzyme substrates with fluorescent moieties offer tremendous promise for disease surveillance/diagnostics and as high-throughput co-factor assays. Advantages of QDs over other nanoscaffolds include their small size and inherent photochemical properties such as size tunable fluorescence, ease in attaching functional moieties, and resistance to photobleaching. These properties make QDs excellent Förster Resonance Energy Transfer (FRET) donors; well-suited for rapid, optical measurement applications. We report enzyme sensors designed with a single FRET donor, the QD donor acting as a scaffold to multiple substrates or acceptors. The QD-sensor follows the concrete activity of the enzyme, as compared to the most common methodologies that quantify the enzyme amount or its mRNA precursor. As the sensor reports on the enzyme activity in real-time we can actively follow the kinetics of the enzyme. Though classic Michaelis-Menten (MM) parameters can be obtained to describe the activity. In the course of these experiments deviations, both decreasing and increasing the kinetics, from the common MM model were observed upon close examinations. From these observations additional experiments were undertaken to understand the varying mechanisms. Different enzymes can present different deviations depending on the chosen target, e.g. trypsin appears to present a positive hopping mechanism while collagenase demonstrates a QD caused reversible inhibition.

  15. Development of Activity-based Cost Functions for Cellulase, Invertase, and Other Enzymes

    NASA Astrophysics Data System (ADS)

    Stowers, Chris C.; Ferguson, Elizabeth M.; Tanner, Robert D.

    As enzyme chemistry plays an increasingly important role in the chemical industry, cost analysis of these enzymes becomes a necessity. In this paper, we examine the aspects that affect the cost of enzymes based upon enzyme activity. The basis for this study stems from a previously developed objective function that quantifies the tradeoffs in enzyme purification via the foam fractionation process (Cherry et al., Braz J Chem Eng 17:233-238, 2000). A generalized cost function is developed from our results that could be used to aid in both industrial and lab scale chemical processing. The generalized cost function shows several nonobvious results that could lead to significant savings. Additionally, the parameters involved in the operation and scaling up of enzyme processing could be optimized to minimize costs. We show that there are typically three regimes in the enzyme cost analysis function: the low activity prelinear region, the moderate activity linear region, and high activity power-law region. The overall form of the cost analysis function appears to robustly fit the power law form.

  16. Improved complementary polymer pair system: switching for enzyme activity by PEGylated polymers.

    PubMed

    Kurinomaru, Takaaki; Tomita, Shunsuke; Kudo, Shinpei; Ganguli, Sumon; Nagasaki, Yukio; Shiraki, Kentaro

    2012-03-06

    The development of technology for on/off switching of enzyme activity is expected to expand the applications of enzyme in a wide range of research fields. We have previously developed a complementary polymer pair system (CPPS) that enables the activity of several enzymes to be controlled by a pair of oppositely charged polymers. However, it failed to control the activity of large and unstable α-amylase because the aggregation of the complex between anionic α-amylase and cationic poly(allylamine) (PAA) induced irreversible denaturation of the enzyme. To address this issue, we herein designed and synthesized a cationic copolymer with a poly(ethylene glycol) backbone, poly(N,N-diethylaminoethyl methacrylate)-block-poly(ethylene glycol) (PEAMA-b-PEG). In contrast to PAA, α-amylase and β-galactosidase were inactivated by PEAMA-b-PEG with the formation of soluble complexes. The enzyme/PEAMA-b-PEG complexes were then successfully recovered from the complex by the addition of anionic poly(acrylic acid) (PAAc). Thus, dispersion of the complex by PEG segment in PEAMA-b-PEG clearly plays a crucial role for regulating the activities of these enzymes, suggesting that PEGylated charged polymer is a new candidate for CPPS for large and unstable enzymes.

  17. Recombinant activated factor VII in the management of acute fatty liver of pregnancy: A case report.

    PubMed

    K, Supriya; Thunga, Suchitra; Narayanan, Athira; Singh, Prakhar

    2015-07-01

    A 20-year-old woman, primigravida at 36(+4) weeks' gestation presented with malaise, vomiting for 1 week, yellowish discoloration of the eyes for 3 days and loss of fetal movements. A clinical diagnosis of acute fatty liver with intrauterine fetal demise was made. Labor was induced with prostaglandin E2 gel and delivered vaginally. The post-partum period was complicated by atonic post-partum hemorrhage, an episode of seizure, recurrent hypoglycemic attack, hypokalemia and continuing coagulopathy. Supportive management in the intensive care unit using blood and blood products and injection recombinant activated factor VIIa to arrest the bleeding resulted in a successful outcome.

  18. Variation of dislocation etch-pit geometry: An indicator of bulk microstructure and recombination activity in multicrystalline silicon

    NASA Astrophysics Data System (ADS)

    Castellanos, S.; Kivambe, M.; Hofstetter, J.; Rinio, M.; Lai, B.; Buonassisi, T.

    2014-05-01

    Dislocation clusters in multicrystalline silicon limit solar cell performance by decreasing minority carrier diffusion length. Studies have shown that the recombination strength of dislocation clusters can vary by up to two orders of magnitude, even within the same wafer. In this contribution, we combine a surface-analysis approach with bulk characterization techniques to explore the underlying root cause of variations in recombination strength among different clusters. We observe that dislocation clusters with higher recombination strength consist of dislocations with a larger variation of line vector, correlated with a higher degree of variation in dislocation etch-pit shapes (ellipticities). Conversely, dislocation clusters exhibiting the lowest recombination strength contain mostly dislocations with identical line vectors, resulting in very similar etch-pit shapes. The disorder of dislocation line vector in high-recombination clusters appears to be correlated with impurity decoration, possibly the cause of the enhanced recombination activity. Based on our observations, we conclude that the relative recombination activity of different dislocation clusters in the device may be predicted via an optical inspection of the distribution and shape variation of dislocation etch pits in the as-grown wafer.

  19. Effects of Curcuma xanthorrhiza Extracts and Their Constituents on Phase II Drug-metabolizing Enzymes Activity

    PubMed Central

    Salleh, Nurul Afifah Mohd; Ismail, Sabariah; Ab Halim, Mohd Rohaimi

    2016-01-01

    Background: Curcuma xanthorrhiza is a native Indonesian plant and traditionally utilized for a range of illness including liver damage, hypertension, diabetes, and cancer. Objective: The study determined the effects of C. xanthorrhiza extracts (ethanol and aqueous) and their constituents (curcumene and xanthorrhizol) on UDP-glucuronosyltransferase (UGT) and glutathione transferase (GST) activities. Materials and Methods: The inhibition studies were evaluated both in rat liver microsomes and in human recombinant UGT1A1 and UGT2B7 enzymes. p-nitrophenol and beetle luciferin were used as the probe substrates for UGT assay while 1-chloro-2,4-dinitrobenzene as the probe for GST assay. The concentrations of extracts studied ranged from 0.1 to 1000 μg/mL while for constituents ranged from 0.01 to 500 μM. Results: In rat liver microsomes, UGT activity was inhibited by the ethanol extract (IC50 =279.74 ± 16.33 μg/mL). Both UGT1A1 and UGT2B7 were inhibited by the ethanol and aqueous extracts with IC50 values ranging between 9.59–22.76 μg/mL and 110.71–526.65 μg/Ml, respectively. Rat liver GST and human GST Pi-1 were inhibited by ethanol and aqueous extracts, respectively (IC50 =255.00 ± 13.06 μg/mL and 580.80 ± 18.56 μg/mL). Xanthorrhizol was the better inhibitor of UGT1A1 (IC50 11.30 ± 0.27 μM) as compared to UGT2B7 while curcumene did not show any inhibition. For GST, both constituents did not show any inhibition. Conclusion: These findings suggest that C. xanthorrhiza have the potential to cause herb-drug interaction with drugs that are primarily metabolized by UGT and GST enzymes. SUMMARY Findings from this study would suggest which of Curcuma xanthorrhiza extracts and constituents that would have potential interactions with drugs which are highly metabolized by UGT and GST enzymes. Further clinical studies can then be designed if needed to evaluate the in vivo pharmacokinetic relevance of these interactions Abbreviations Used: BSA: Bovine serum albumin

  20. Associations between PON1 enzyme activities in human ovarian follicular fluid and serum specimens

    PubMed Central

    Kim, Keewan; Fujimoto, Victor Y.; Browne, Richard W.

    2017-01-01

    The importance of high-density lipoprotein (HDL) particle components to reproduction is increasingly recognized, including the constituent paraoxonase 1 (PON1). However, the reliability characteristics of PON1 enzymes in ovarian follicular fluid (FF) as biomarkers for clinical and epidemiologic studies have not been described. Therefore, we characterized PON1 enzymes in FF and serum and assessed the impact of the PON1 Q192R polymorphism on associations between enzyme activities in two compartments. We also evaluated associations between HDL particle size and enzyme activities. We collected FF and serum from 171 women undergoing in vitro fertilization. PON1 activities were measured as paraoxonase and arylesterase activities, and HDL particle size was determined by 1H NMR spectrometry. Reliability indices for PON1 activities were characterized and we evaluated HDL particle sizes as predictors of PON1 enzyme activities. We found that PON1 enzyme activities were correlated between compartments, but higher in serum than in FF. For FF, the index of individuality (II) was low and the coefficient of variation (CV%) was high for paraoxonase activity overall (0.12 and 11.51%, respectively). However, IIs increased (0.33–1.30) and CV%s decreased (5.58%-8.52%) when stratified by PON1 Q192R phenotype. The intraclass correlation coefficient (ICC) for FF paraoxonase activity was high overall (0.89) but decreased when stratified by PON1 Q192R phenotype (0.43–0.75). We found similar, although more modest, patterns for FF arylesterase activity. For enzyme activities in serum, ICCs were close to 1.00 across all phenotypes. Additionally, different HDL particle sizes predicted PON1 enzyme activities according to PON1 Q192R phenotype. Overall, stratification by PON1 Q192R phenotype improved the reliability characteristics of FF PON1 enzymes as biomarkers for use in clinical investigations but diminished usefulness for epidemiologic studies. Thus, we recommend stratification by PON1 Q

  1. [Soil enzyme activities under two forest types as affected by different levels of nitrogen deposition].

    PubMed

    Zhao, Yu-tao; Li, Xue-feng; Han, Shi-jie; Hu, Yan-ling

    2008-12-01

    A simulation test was conducted to study the change trends of soil cellulase, polyphenol oxidase, and sucrase activities under natural broadleaf-Korean pine (Pinus koraiensis) and secondary poplar (Populus davidiana) -birch (Betula platyphylla) mixed forests as affected by 0, 25, and 50 kg x hm(-2) x a(-1) of N deposition. The results showed that the effects of elevated N deposition on test enzyme activities varied with forest type, and short-term nitrogen addition could significantly affect the test enzyme activities. High N deposition decreased soil polyphyneol oxidase activity, and correspondingly, soil cellulase and sucrase activities also had a trend of decrease.

  2. Seroprevalence of Cryptosporidium parvum infection of dairy c