Alteration of Electron Acceptor Preferences in the Oxidative Half-Reaction of Flavin-Dependent Oxidases and Dehydrogenases
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Glucose Oxidase
Catalog Number: 100289, 100330, 195196 Glucose Oxidase Molecular Weight: ~160,0001 CAS #: 9001-34-0 Physical Description: Yellow lyophilized powder Source: Aspergillus niger Isoelectric Point: 4.2 Michaelis Constants: phosphate buffer, pH 5.6; 25°C, air: Glucose: 3.3 x 10-2 mol/l(2) Glucose: 1.1 x 10-1 mol/l(3) 2-Deoxyglucose: 2.5 x 10-2 mol/l O2: 2.0 x 10-4 mol/l Structure: The enzyme contains 2 moles FAD/mole GOD. Inhibitors: Ag+, Hg2+, Cu2+ (4), 4-chloromercuribenzoate, D-arabinose (50%). FAD binding is inhibited by several nucleotides.5 pH Optimum: 6.5 pH Stability: 8.0 pH Range: 4-7 Thermal Stability: Below 40°C Description: Glucose oxidase is an FAD-containing glycoprotein. The enzyme is specific for b-D-glucose. O2 can be replaced by hydrogen acceptors such as 2.6-dichlorophenol indophenol. Relative Rates: D-glucose, 100; D-mannose, 20; 2-Deoxy-D-glucose, 20; negligible on other hexoses. Solubility: Dissolves readily at 5 mg/ml in 0.1 M potassium phosphate pH 7.0, giving a clear, yellow solution, also soluble in water Assay Procedure 1: Unit Definition: One unit of glucose oxidase is the activity which causes the liberation of 1 micromole of H2O2 per minute at 25°C and pH 7.0 under the specified conditions. Reaction: Reagents: 1. 0.1 M Phosphate Buffer, pH 6.8: dissolve 6.8 g of potassium phosphate, monobasic, anhydrous and 7.1 g of sodium phosphate, dibasic, anhydrous in about 800 ml of deionized water. Adjust the pH to 6.8 ± 0.05 @ 25°C with 1 N HCl or 1N NaOH if necessary. -
Adaptive Laboratory Evolution Enhances Methanol Tolerance and Conversion in Engineered Corynebacterium Glutamicum
ARTICLE https://doi.org/10.1038/s42003-020-0954-9 OPEN Adaptive laboratory evolution enhances methanol tolerance and conversion in engineered Corynebacterium glutamicum Yu Wang 1, Liwen Fan1,2, Philibert Tuyishime1, Jiao Liu1, Kun Zhang1,3, Ning Gao1,3, Zhihui Zhang1,3, ✉ ✉ 1234567890():,; Xiaomeng Ni1, Jinhui Feng1, Qianqian Yuan1, Hongwu Ma1, Ping Zheng1,2,3 , Jibin Sun1,3 & Yanhe Ma1 Synthetic methylotrophy has recently been intensively studied to achieve methanol-based biomanufacturing of fuels and chemicals. However, attempts to engineer platform micro- organisms to utilize methanol mainly focus on enzyme and pathway engineering. Herein, we enhanced methanol bioconversion of synthetic methylotrophs by improving cellular tolerance to methanol. A previously engineered methanol-dependent Corynebacterium glutamicum is subjected to adaptive laboratory evolution with elevated methanol content. Unexpectedly, the evolved strain not only tolerates higher concentrations of methanol but also shows improved growth and methanol utilization. Transcriptome analysis suggests increased methanol con- centrations rebalance methylotrophic metabolism by down-regulating glycolysis and up- regulating amino acid biosynthesis, oxidative phosphorylation, ribosome biosynthesis, and parts of TCA cycle. Mutations in the O-acetyl-L-homoserine sulfhydrylase Cgl0653 catalyzing formation of L-methionine analog from methanol and methanol-induced membrane-bound transporter Cgl0833 are proven crucial for methanol tolerance. This study demonstrates the importance of -
Analysis of the Impact of Silver Ions on Creatine Amidinohydrolase
ActaBIOMATERIALIA Acta Biomaterialia 1 (2005) 183–191 www.actamat-journals.com A stable three enzyme creatinine biosensor. 2. Analysis of the impact of silver ions on creatine amidinohydrolase Jason A. Berberich b,1, Lee Wei Yang a, Ivet Bahar a, Alan J. Russell b,* a Center for Computational Biology & Bioinformatics and Department of Molecular Genetics & Biochemistry, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA b Department of Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA Received 11 October 2004; received in revised form 26 November 2004; accepted 28 November 2004 Abstract The enzyme creatine amidinohydrolase is a clinically important enzyme used in the determination of creatinine in blood and urine. Continuous use biosensors are becoming more important in the clinical setting; however, long-use creatinine biosensors have not been commercialized due to the complexity of the three-enzyme creatinine biosensor and the lack of stability of its components. This paper, the second in a series of three, describes the immobilization and stabilization of creatine amidinohydrolase. Creatine amidinohydrolase modified with poly(ethylene glycol) activated with isocyanate retains significant activity after modification. The enzyme was successfully immobilized into hydrophilic polyurethanes using a reactive prepolymer strategy. The immobilized enzyme retained significant activity over a 30 day period at 37 °C and was irreversibly immobilized into the polymer. Despite being stabilized in the polymer, the enzyme remained highly sensitive to silver ions which were released from the amperometric electrodes. Computational analysis of the structure of the protein using the Gaussian network model suggests that the silver ions bind tightly to a cysteine residue preventing normal enzyme dynamics and catalysis. -
Fructose As an Endogenous Toxin
HEPATOCYTE MOLECULAR CYTOTOXIC MECHANISM STUDY OF FRUCTOSE AND ITS METABOLITES INVOLVED IN NONALCOHOLIC STEATOHEPATITIS AND HYPEROXALURIA By Yan (Cynthia) Feng A thesis submitted in the conformity with the requirements for the degree of Master of Science Graduate Department of Pharmaceutical Sciences University of Toronto © Copyright by Yan (Cynthia) Feng 2010 ABSTRACT HEPATOCYTE MOLECULAR CYTOTOXIC MECHANISM STUDY OF FRUCTOSE AND ITS METABOLITES INVOLVED IN NONALCOHOLIC STEATOHEPATITIS AND HYPEROXALURIA Yan (Cynthia) Feng Master of Science, 2010 Department of Pharmaceutical Sciences University of Toronto High chronic fructose consumption is linked to a nonalcoholic steatohepatitis (NASH) type of hepatotoxicity. Oxalate is the major endpoint of fructose metabolism, which accumulates in the kidney causing renal stone disease. Both diseases are life-threatening if not treated. Our objective was to study the molecular cytotoxicity mechanisms of fructose and some of its metabolites in the liver. Fructose metabolites were incubated with primary rat hepatocytes, but cytotoxicity only occurred if the hepatocytes were exposed to non-toxic amounts of hydrogen peroxide such as those released by activated immune cells. Glyoxal was most likely the endogenous toxin responsible for fructose induced toxicity formed via autoxidation of the fructose metabolite glycolaldehyde catalyzed by superoxide radicals, or oxidation by Fenton’s hydroxyl radicals. As for hyperoxaluria, glyoxylate was more cytotoxic than oxalate presumably because of the formation of condensation product oxalomalate causing mitochondrial toxicity and oxidative stress. Oxalate toxicity likely involved pro-oxidant iron complex formation. ii ACKNOWLEDGEMENTS I would like to dedicate this thesis to my family. To my parents, thank you for the sacrifices you have made for me, thank you for always being there, loving me and supporting me throughout my life. -
Supplementary Materials
Supplementary Materials COMPARATIVE ANALYSIS OF THE TRANSCRIPTOME, PROTEOME AND miRNA PROFILE OF KUPFFER CELLS AND MONOCYTES Andrey Elchaninov1,3*, Anastasiya Lokhonina1,3, Maria Nikitina2, Polina Vishnyakova1,3, Andrey Makarov1, Irina Arutyunyan1, Anastasiya Poltavets1, Evgeniya Kananykhina2, Sergey Kovalchuk4, Evgeny Karpulevich5,6, Galina Bolshakova2, Gennady Sukhikh1, Timur Fatkhudinov2,3 1 Laboratory of Regenerative Medicine, National Medical Research Center for Obstetrics, Gynecology and Perinatology Named after Academician V.I. Kulakov of Ministry of Healthcare of Russian Federation, Moscow, Russia 2 Laboratory of Growth and Development, Scientific Research Institute of Human Morphology, Moscow, Russia 3 Histology Department, Medical Institute, Peoples' Friendship University of Russia, Moscow, Russia 4 Laboratory of Bioinformatic methods for Combinatorial Chemistry and Biology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia 5 Information Systems Department, Ivannikov Institute for System Programming of the Russian Academy of Sciences, Moscow, Russia 6 Genome Engineering Laboratory, Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia Figure S1. Flow cytometry analysis of unsorted blood sample. Representative forward, side scattering and histogram are shown. The proportions of negative cells were determined in relation to the isotype controls. The percentages of positive cells are indicated. The blue curve corresponds to the isotype control. Figure S2. Flow cytometry analysis of unsorted liver stromal cells. Representative forward, side scattering and histogram are shown. The proportions of negative cells were determined in relation to the isotype controls. The percentages of positive cells are indicated. The blue curve corresponds to the isotype control. Figure S3. MiRNAs expression analysis in monocytes and Kupffer cells. Full-length of heatmaps are presented. -
Optimization of Immobilized Aldose Reductase Isolated from Bovine Liver Sığır Karaciğerinden İzole Edilen İmmobilize Aldoz Redüktazın Optimizasyonu
Turk J Pharm Sci 2019;16(2):206-210 DOI: 10.4274/tjps.galenos.2018.81894 ORIGINAL ARTICLE Optimization of Immobilized Aldose Reductase Isolated from Bovine Liver Sığır Karaciğerinden İzole Edilen İmmobilize Aldoz Redüktazın Optimizasyonu Marya Vakıl NASLIYAN, Sidar BEREKETOĞLU, Özlem YILDIRIM* Ankara University, Faculty of Science, Department of Biology, Ankara, Turkey ABSTRACT Objectives: Isolation of enzymes and experiments on them require great effort and cost and are time-consuming. Therefore, it is important to extend the usability of the enzymes by immobilizing them. In this study our purpose was to immobilize the enzyme aldose reductase (AR) and to optimize the experimental conditions of the immobilized AR and compare them to those of free AR. Materials and Methods: AR was isolated from bovine liver and the enzyme immobilized in photographic gelatin by cross-linking with glutaraldehyde. Then the optimum conditions for free and immobilized AR in terms of pH, temperature, and storage were characterized by determining the enzyme activity. Results: Following immobilization, the optimum pH and temperature levels for free AR, which were pH 7.0 and 60°C, slightly altered to pH 7.5 and 50°C. The enzyme activity of the immobilized AR was maintained at about 65% after reusing 15 times. Moreover, immobilized AR maintained 95% of its original activity after 20 days of storage at 4°C, while the retained activity of the free AR was 85% of the original. Conclusion: Our experiments indicated that the conditions that affect enzyme activity might alter following immobilization. Once the optimum experimental conditions are fixed, the immobilized AR can be stored and reused with efficiency higher than that of free AR. -
Differential Gene Expression in Tomato Fruit and Colletotrichum
Barad et al. BMC Genomics (2017) 18:579 DOI 10.1186/s12864-017-3961-6 RESEARCH Open Access Differential gene expression in tomato fruit and Colletotrichum gloeosporioides during colonization of the RNAi–SlPH tomato line with reduced fruit acidity and higher pH Shiri Barad1,2, Noa Sela3, Amit K. Dubey1, Dilip Kumar1, Neta Luria1, Dana Ment1, Shahar Cohen4, Arthur A. Schaffer4 and Dov Prusky1* Abstract Background: The destructive phytopathogen Colletotrichum gloeosporioides causes anthracnose disease in fruit. During host colonization, it secretes ammonia, which modulates environmental pH and regulates gene expression, contributing to pathogenicity. However, the effect of host pH environment on pathogen colonization has never been evaluated. Development of an isogenic tomato line with reduced expression of the gene for acidity, SlPH (Solyc10g074790.1.1), enabled this analysis. Total RNA from C. gloeosporioides colonizing wild-type (WT) and RNAi– SlPH tomato lines was sequenced and gene-expression patterns were compared. Results: C. gloeosporioides inoculation of the RNAi–SlPH line with pH 5.96 compared to the WT line with pH 4.2 showed 30% higher colonization and reduced ammonia accumulation. Large-scale comparative transcriptome analysis of the colonized RNAi–SlPH and WT lines revealed their different mechanisms of colonization-pattern activation: whereas the WT tomato upregulated 13-LOX (lipoxygenase), jasmonic acid and glutamate biosynthesis pathways, it downregulated processes related to chlorogenic acid biosynthesis II, phenylpropanoid biosynthesis and hydroxycinnamic acid tyramine amide biosynthesis; the RNAi–SlPH line upregulated UDP-D-galacturonate biosynthesis I and free phenylpropanoid acid biosynthesis, but mainly downregulated pathways related to sugar metabolism, such as the glyoxylate cycle and L-arabinose degradation II. -
Engineering Alcohol Oxidases for Substrate Scope and Their Application in Flow and Cascade Biocatalysis
ENGINEERING ALCOHOL OXIDASES FOR SUBSTRATE SCOPE AND THEIR APPLICATION IN FLOW AND CASCADE BIOCATALYSIS Rachel Heath, University of Manchester [email protected] Matthew Thompson, EnginZyme Jeremy Ramsden, University of Manchester Sebastian Cosgrove, University of Manchester William Finnigan, University of Manchester Nicholas Turner, University of Manchester Key Words: oxidation, primary alcohol, secondary alcohol, high-throughput screening, thermostability Alcohol oxidases have significant advantages over alcohol dehydrogenases (ADHs) for biocatalytic oxidation of alcohols: they don’t require addition of (expensive) nicotinamide cofactors (or a recycling system for cofactor regeneration) and the catalytic reaction is irreversible. Although alcohol oxidases generate hydrogen peroxide when they turn over, this issue can be alleviated by addition of catalase, which, not only removes the peroxide, but also creates more oxygen for cofactor regeneration. Alcohol oxidases are perceived to have a limited substrate scope preventing their wider use in synthesis. Thus, we present the engineering of two alcohol oxidases for increased substrate scope, one for the selective oxidation of primary alcohols and one for secondary alcohol oxidation. Arthrobacter chlorophenolicus choline oxidase and Streptomyces hygroscopicus cholesterol oxidase were selected as the primary and secondary alcohol oxidase, respectively. Examination of crystal structures of homologous alcohol oxidases revealed positions in the active site and entrance channel to target for saturation mutagenesis. Libraries were screened using a high-throughput assay based on the detection of hydrogen peroxide using hexanol as the substrate for evolution of choline oxidase and cyclohexanol for evolution of cholesterol oxidase. Mutations from positive hits were combined and, in these cases, resulted in variants with further improvements in both kcat and conversion to the carbonyl in biotransformations. -
Solarbio Catalogue with PRICES
CAS Name Grade Purity Biochemical Reagent Biochemical Reagent 75621-03-3 C8390-1 3-((3-Cholamidopropyl)dimethylammonium)-1-propanesulfonateCHAPS Ultra Pure Grade 1g 75621-03-3 C8390-5 3-((3-Cholamidopropyl)dimethylammonium)-1-propanesulfonateCHAPS 5g 57-09-0 C8440-25 Cetyl-trimethyl Ammonium Bromide CTAB High Pure Grade ≥99.0% 25g 57-09-0 C8440-100 Cetyl-trimethyl Ammonium Bromide CTAB High Pure Grade ≥99.0% 100g 57-09-0 C8440-500 Cetyl-trimethyl Ammonium Bromide CTAB High Pure Grade ≥99.0% 500g E1170-100 0.5M EDTA (PH8.0) 100ml E1170-500 0.5M EDTA (PH8.0) 500ml 6381-92-6 E8030-100 EDTA disodium salt dihydrate EDTA Na2 Biotechnology Grade ≥99.0% 100g 6381-92-6 E8030-500 EDTA disodium salt dihydrate EDTA Na2 Biotechnology Grade ≥99.0% 500g 6381-92-6 E8030-1000 EDTA disodium salt dihydrate EDTA Na2 Biotechnology Grade ≥99.0% 1kg 6381-92-6 E8030-5000 EDTA disodium salt dihydrate EDTA Na2 Biotechnology Grade ≥99.0% 5kg 60-00-4 E8040-100 Ethylenediaminetetraacetic acid EDTA Ultra Pure Grade ≥99.5% 100g 60-00-4 E8040-500 Ethylenediaminetetraacetic acid EDTA Ultra Pure Grade ≥99.5% 500g 60-00-4 E8040-1000 Ethylenediaminetetraacetic acid EDTA Ultra Pure Grade ≥99.5% 1kg 67-42-5 E8050-5 Ethylene glycol-bis(2-aminoethylether)-N,N,NEGTA′,N′-tetraacetic acid Ultra Pure Grade ≥97.0% 5g 67-42-5 E8050-10 Ethylene glycol-bis(2-aminoethylether)-N,N,NEGTA′,N′-tetraacetic acid Ultra Pure Grade ≥97.0% 10g 50-01-1 G8070-100 Guanidine Hydrochloride Guanidine HCl ≥98.0%(AT) 100g 50-01-1 G8070-500 Guanidine Hydrochloride Guanidine HCl ≥98.0%(AT) 500g 56-81-5 -
A High-Throughput Screening System Based on Droplet Microfluidics For
molecules Article A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene Libraries Radivoje Prodanovi´c 1,2,* , W. Lloyd Ung 2, Karla Ili´c Đurđi´c 1 , Rainer Fischer 3, David A. Weitz 2 and Raluca Ostafe 4 1 Faculty of Chemistry, University of Belgrade, Studentski trg 12, 11000 Belgrade, Serbia; [email protected] 2 Department of Physics, School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA; [email protected] (W.L.U.); [email protected] (D.A.W.) 3 Departments of Biological Sciences and Chemistry, Purdue University, 207 S. Martin Jischke Dr., West Lafayette, IN 47907, USA; fi[email protected] 4 Purdue Institute of Inflammation, Immunology and Infectious Disease, Molecular Evolution, Protein Engineering and Production, Purdue University, 207 S. Martin Jischke Dr., West Lafayette, IN 47907, USA; [email protected] * Correspondence: [email protected]; Tel.: +38-111-333-6660 Academic Editors: Goran T. Vladisavljevi´cand Guido Bolognesi Received: 20 April 2020; Accepted: 15 May 2020; Published: 22 May 2020 Abstract: Glucose oxidase (GOx) is an important industrial enzyme that can be optimized for specific applications by mutagenesis and activity-based screening. To increase the efficiency of this approach, we have developed a new ultrahigh-throughput screening platform based on a microfluidic lab-on-chip device that allows the sorting of GOx mutants from a saturation mutagenesis library expressed on the surface of yeast cells. GOx activity was measured by monitoring the fluorescence of water microdroplets dispersed in perfluorinated oil. The signal was generated via a series of coupled enzyme reactions leading to the formation of fluorescein. -
The Genome of an Industrial Workhorse
NEWS AND VIEWS The genome of an industrial workhorse Dan Cullen Sequencing of the filamentous fungus Aspergillus niger offers new opportunities for the production of specialty chemicals and enzymes. Few microbes compare with the filamentous fungus Aspergillus niger in its ability to pro Environment CAT (2) H O H O + O duce prodigious amounts of useful chemicals 2 2 2 2 and enzymes. This fungus is the principal GOX (3) GLN (1) source of citric acid for food, beverages and D-glucono Glucose Gluconate Oxalate pharmaceuticals1 and of several important 1,5-lactone Citrate http://www.nature.com/naturebiotechnology http://www.nature.com/naturebiotechnology commercial enzymes, including glucoamy lase, which is widely used for the conversion of starch to food syrups and to fermentative Oxalate + acetate PEP feedstocks for ethanol production. Although OAH most of these fermentation processes are well cMDH (3) established, the underlying genetics are still cPYC (1) cACO (2) cIDH (1) poorly understood. In this issue, Pel et al.2 Pyruvate OAA MAL Citrate Isocitrate 2-ketoglutarate report the genome sequence of A. niger strain Cytosol CBS 513.88. The availability of this sequence OAT (1) CMC (2) mPYC (1) should provide invaluable aid toward improv Pyruvate OAA MAL Citrate Isocitrate 2-ketoglutarate ing the production of chemicals and enzymes PDH Nature Publishing Group Group Nature Publishing 7 in this organism. mMDH (1) mACO (2) mIDH (3) Pel et al. sequenced tiled bacterial artificial CS (3) 200 Acetyl-CoA TCA cycle © chromosomes representing the entire A. niger genome to produce a high-quality assembly of Mitochondrion 19 supercontigs with a combined length of 33.9 Mb. -
WO 2012/174282 A2 20 December 2012 (20.12.2012) P O P C T
(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2012/174282 A2 20 December 2012 (20.12.2012) P O P C T (51) International Patent Classification: David [US/US]; 13539 N . 95th Way, Scottsdale, AZ C12Q 1/68 (2006.01) 85260 (US). (21) International Application Number: (74) Agent: AKHAVAN, Ramin; Caris Science, Inc., 6655 N . PCT/US20 12/0425 19 Macarthur Blvd., Irving, TX 75039 (US). (22) International Filing Date: (81) Designated States (unless otherwise indicated, for every 14 June 2012 (14.06.2012) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, English (25) Filing Language: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, Publication Language: English DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, (30) Priority Data: KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, 61/497,895 16 June 201 1 (16.06.201 1) US MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 61/499,138 20 June 201 1 (20.06.201 1) US OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, SD, 61/501,680 27 June 201 1 (27.06.201 1) u s SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, 61/506,019 8 July 201 1(08.07.201 1) u s TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.