Cholesterol Oxidase: Source, Properties and Applications
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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 -
Deletion of the Gene Encoding the Reductase Component of 3
Yeh et al. Microbial Cell Factories 2014, 13:130 http://www.microbialcellfactories.com/content/13/1/130 RESEARCH Open Access Deletion of the gene encoding the reductase component of 3-ketosteroid 9α-hydroxylase in Rhodococcus equi USA-18 disrupts sterol catabolism, leading to the accumulation of 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid and 1,4-androstadiene-3,17-dione Chin-Hsing Yeh1, Yung-Shun Kuo1, Che-Ming Chang1, Wen-Hsiung Liu2, Meei-Ling Sheu3 and Menghsiao Meng1* Abstract The gene encoding the putative reductase component (KshB) of 3-ketosteroid 9α-hydroxylase was cloned from Rhodococcus equi USA-18, a cholesterol oxidase-producing strain formerly named Arthrobacter simplex USA-18, by PCR according to consensus amino acid motifs of several bacterial KshB subunits. Deletion of the gene in R. equi USA-18 by a PCR-targeted gene disruption method resulted in a mutant strain that could accumulate up to 0.58 mg/ml 1,4-androstadiene-3,17-dione (ADD) in the culture medium when 0.2% cholesterol was used as the carbon source, indicating the involvement of the deleted enzyme in 9α-hydroxylation of steroids. In addition, this mutant also accumulated 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (Δ1,4-BNC). Because both ADD and Δ1,4-BNC are important intermediates for the synthesis of steroid drugs, this mutant derived from R. equi USA-18 may deserve further investigation for its application potential. Background generally believed to be carried out by cholesterol oxidase, Steroid drugs, including androgens, anabolic steroids, es- which catalyzes the oxidation of the 3β-hydroxyl moiety of trogens and corticosteroids, have been widely used for a sterols with the simultaneous isomerization of Δ5 to Δ4, variety of health purposes. -
Production of 4-Ene-3-Ketosteroids in Corynebacterium Glutamicum
catalysts Article Production of 4-Ene-3-ketosteroids in Corynebacterium glutamicum Julia García-Fernández 1, Beatriz Galán 1, Carmen Felpeto-Santero 1, José L. Barredo 2 and José L. García 1,* 1 Department of Environmental Biotechnology, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain; [email protected] (J.G.-F.); [email protected] (B.G.); [email protected] (C.F.-S.) 2 Department of Biotechnology, Crystal Pharma, A Division of Albany Molecular Research Inc. (AMRI), 47151 Boecillo Valladolid, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-918-373-112 Received: 29 September 2017; Accepted: 20 October 2017; Published: 27 October 2017 Abstract: Corynebacterium glutamicum has been widely used for the industrial production of amino acids and many value-added chemicals; however, it has not been exploited for the production of steroids. Using C. glutamicum as a cellular biocatalyst we have expressed the 3β-hydroxysteroid dehydrogenase/isomerase MSMEG_5228 from Mycobacterium smegmatis to demonstrate that the resulting recombinant strain is able to oxidize in vivo C19 and C21 3-OH-steroids to their corresponding keto-derivatives. This new approach constitutes a proof of concept of a biotechnological process for producing value-added intermediates such as 4-pregnen-16α,17α-epoxy-16β-methyl-3,20-dione. Keywords: 3-OH-steroids; biotransformation; dehydrogenase; Rhodococcus and Corynebacterium expression systems 1. Introduction Corynebacterium glutamicum has been widely used for the industrial production of amino acids, but the publication of its complete genome [1,2] has provided the basis for an enormous progress in the use of this microorganism for other biotechnological applications placing it as an ideal chassis within the top ranking of cell factories [3]. -
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. -
Insight V-CHEM General Health Profile
【Product Name】 General Health Profile 【Packing Specification】 1 Disc / Sample 【Instrument】 See the InSight V-CHEM chemistry analyser Operator’s Manual for complete information on use of the analyser. 【Intended Use】 The General Health Profile used with the InSight V-CHEM chemistry analyser is intended to be used for the in vitro quantitative determination of total Protein (TP), albumin (ALB), total bilirubin (TBIL), alanine aminotransferase (ALT), blood urea (BUN), creatinine (CRE), amylase (AMY), creatinekinase (CK), calcium (Ca2+), phosphorus (P), alkaline Phosphatase (ALP), glucose (GLU), and total cholesterol (CHOL)in heparinised whole blood, heparinised plasma, or serum in a clinical laboratory setting or point of care location. The General Health Profile and the InSight V-CHEM chemistry analyser comprise an in vitro diagnostic system that aids the physician in the following disorders: liver and gall bladder diseases, urinary system diseases, carbohydrate metabolism disorders, lipid metabolism disorders, cardiovascular disease and pancreatic diseases. 【Test Principles】 This product, which is based on spectrophotometry, is used to quantitatively determine the concentration or activity of the 13 biochemical indicators in the sample. The test principles are as follows: (1) Total Protein (TP) The total protein method is a Biuret reaction, the protein solution is treated with cupric [Cu(II)] ions in a strong alkaline medium. The Cu(II) ions react with peptide bonds between the carbonyl oxygen and amide nitrogen atoms to form a coloured Cu-protein complex. The amount of total protein present in the sample is directly proportional to the absorbance of the Cu-protein complex. The total protein test is an endpoint reaction and the absorbance is measured as the difference in absorbance between 550 nm and 800 nm. -
Western Blot Sandwich ELISA Immunohistochemistry
$$ 250 - 150 - 100 - 75 - 50 - 37 - Western Blot 25 - 20 - 15 - 10 - 1.4 1.2 1 0.8 0.6 OD 450 0.4 Sandwich ELISA 0.2 0 0.01 0.1 1 10 100 1000 Recombinant Protein Concentration(mg/ml) Immunohistochemistry Immunofluorescence 1 2 3 250 - 150 - 100 - 75 - 50 - Immunoprecipitation 37 - 25 - 20 - 15 - 100 80 60 % of Max 40 Flow Cytometry 20 0 3 4 5 0 102 10 10 10 www.abnova.com June 2013 (Fourth Edition) 37 38 53 Cat. Num. Product Name Cat. Num. Product Name MAB5411 A1/A2 monoclonal antibody, clone Z2A MAB3882 Adenovirus type 6 monoclonal antibody, clone 143 MAB0794 A1BG monoclonal antibody, clone 54B12 H00000126-D01 ADH1C MaxPab rabbit polyclonal antibody (D01) H00000002-D01 A2M MaxPab rabbit polyclonal antibody (D01) H00000127-D01 ADH4 MaxPab rabbit polyclonal antibody (D01) MAB0759 A2M monoclonal antibody, clone 3D1 H00000131-D01 ADH7 MaxPab rabbit polyclonal antibody (D01) MAB0758 A2M monoclonal antibody, clone 9A3 PAB0005 ADIPOQ polyclonal antibody H00051166-D01 AADAT MaxPab rabbit polyclonal antibody (D01) PAB0006 Adipoq polyclonal antibody H00000016-D01 AARS MaxPab rabbit polyclonal antibody (D01) PAB5030 ADIPOQ polyclonal antibody MAB8772 ABCA1 monoclonal antibody, clone AB.H10 PAB5031 ADIPOQ polyclonal antibody MAB8291 ABCA1 monoclonal antibody, clone AB1.G6 PAB5069 Adipoq polyclonal antibody MAB3345 ABCB1 monoclonal antibody, clone MRK16 PAB5070 Adipoq polyclonal antibody MAB3389 ABCC1 monoclonal antibody, clone QCRL-2 PAB5124 Adipoq polyclonal antibody MAB5157 ABCC1 monoclonal antibody, clone QCRL-3 PAB9125 ADIPOQ polyclonal antibody -
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 -
Cholesterol Oxidase Modified Microelectrodes for Detection of Cholesterol in The
CHOLESTEROL OXIDASE MODIFIED MICROELECTRODES FOR DETECTION OF CHOLESTEROL IN THE PLASMA MEMBRANE OF SINGLE CELLS by ANANDO DEVADOSS Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Dr. James David Burgess Department of Chemistry CASE WESTERN RESERVE UNIVERSITY January, 2006 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Dedicated to my parents and brother TABLE OF CONTENTS TABLE OF CONTENTS .................................................................................................. i LIST OF FIGURES .......................................................................................................... v LIST OF SCHEMES .....................................................................................................xiii LIST OF ABBREVIATIONS ....................................................................................... xiv ACKNOWLEDGEMENTS .......................................................................................... -
Purification and Some Properties of Alcohol Oxidase from Alkane-Grown Candida Tropicalis
Biochem. J. (1992) 282, 325-331 (Printed in Great Britain) 325 Purification and some properties of alcohol oxidase from alkane-grown Candida tropicalis Francis M. DICKINSON and Catherine WADFORTH Department of Applied Biology, University of Hull, Cottingham Road, Hull HU6 7RX, U.K. Alcohol oxidase was purified to homogeneity from membrane fractions obtained from alkane-grown Candida tropicalis. The enzyme appears to be a dimer ofequal-sized subunits of Mr 70000. The purified enzyme is photosensitive and contains flavin-type material which is released by a combination of boiling and proteolytic digestion. The identity of the flavin material is not yet known, but it is not FMN, FAD or riboflavin. The enzyme is most active with dodecan-l-ol, but other long-chain alcohols are also attacked. The enzyme shows a weak, but significant activity towards long-chain aldehydes. Detailed kinetic studies with decan-1-ol as substrate suggest a group-transfer (Ping-Pong)-type mechanism of catalysis. INTRODUCTION portions of this culture were inoculated into each of six 3-litre conical flasks containing 500 ml of minimal salts Work from this laboratory has shown that growth of the yeast medium, which contained (g/l) KH2PO4, 7.0; Na2HPO4, 2.0; MgSO4, 1.5; yeast Candida tropicalis on long-chain alkane substrates induces the extract, 1.5; CaCI2, 0.1; FeCl2, 0.008; ZnSO4, 0.0001; synthesis of a long-chain-specific alcohol oxidase (Kemp et al., diammonium tartrate, 15; in distilled water at pH 6.5. The 1988). The enzyme presumably catalyses one of the steps in the pathway in which alkanes undergo monoterminal oxidation carbon source was hexadecane (1 %). -
Cholesterol Oxidase (C8649)
Cholesterol Oxidase from Streptomyces sp. Catalog Number C8649 Storage Temperature –20 °C CAS RN 9028-76-6 Cholesterol oxidase is a monomeric flavoprotein EC 1.1.3.6 containing FAD.1 Synonyms: Cholesterol:oxygen oxidoreductase; Molecular mass:10 50 kDa (SDS-PAGE) 3b-hydroxy steroid oxidoreductase; CHOD; Cofactor:10 FAD 3b-hydroxysteroid:oxygen oxidoreductase; 10 cholesterol-O2 oxidoreductase pH Optimum: 6.0 10 Product Description pH Range: 6.0–8.0 Cholesterol oxidase (CHOD) catalyzes the first step in cholesterol catabolism. Some non-pathogenic bacteria, Temperature optimum:10 60 °C such as Streptomyces are able to utilize cholesterol as a carbon source. Pathogenic bacteria, such as Substrates:7 Rhodococcus equi, require CHOD to infect a host's cholesterol estrone macrophage.1 cholest-5-en-3b-ol-7-one dihydrocholesterol dehydroisoandrosterone pregnenolone CHOD is bifunctional. Cholesterol is initially oxidized to cholest-5-en-3-one in an FAD-requiring step. The KM (mM): cholest-5-en-3-one is isomerized to cholest-4-en- Cholesterol10 13.0 3-one.1 The isomerization reaction may be partially Pregnenolone7 0.023 reversible.2 The activity of CHOD depends on the Dehydroepiandrosterone11 0.0275 physical properties of membrane to which the substrate is bound.3 The net reaction is: Inhibitors: Fenpropimorph:6 50 mg/l, 50% inhibition CHOD Sarkosyl:12 1%, 56% inhibition Cholesterol + O2 cholest-4-en-3-one + H2O2 This product is purified from Streptomyces sp. and is supplied as a lyophilized powder containing ~60% Typically cholesterol oxidase is isolated from protein (biuret), BSA, sodium cholate, and borate. Gram-positive bacteria. CHOD from Streptomyces, Cellulomonas, and Brevibacterium have been found to Specific activity: ³20 units/mg protein be essentially equivalent analytically.4 4,5 Unit definition: one unit will convert 1.0 mmole of CHOD is used to determine serum cholesterol. -
Biomarkers of Arachidonic Acid Metabolism As Predictors for Presence of Cardiovascular Disease
Aus dem Institut für Laboratoriumsmedizin Klinik der Ludwig-Maximilians-Universität München Direktor: Prof. Dr. med. Daniel Teupser Biomarkers of Arachidonic Acid Metabolism as Predictors for Presence of Cardiovascular Disease Dissertation zum Erwerb des Doktorgrades der Medizin an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München vorgelegt von Alisa Kleinhempel aus Erfurt 2019 Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatterin: Prof. Dr. Dr. Lesca M. Holdt Mitberichterstatter: PD Dr. Christoph Bidlingmaier PD Dr. Martin Orban Mitbetreuung durch die promovierten Mitarbeiter: Prof. Dr. Daniel Teupser Dr. Mathias Brügel Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 09.05.2019 Eidesstattliche Versicherung Kleinhempel, Alisa Name, Vorname Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema Biomarkers of Arachidonic Acid Metabolism as Predictors for Presence of Cardiovascular Disease selbständig verfasst, mich außer der angegebenen keiner weiteren Hilfsmittel bedient und alle Erkenntnisse, die aus dem Schrifttum ganz oder annähernd übernommen sind, als solche kenntlich gemacht und nach ihrer Herkunft unter Bezeichnung der Fundstelle einzeln nachgewiesen habe. Ich erkläre des Weiteren, dass die hier vorgelegte Dissertation nicht in gleicher oder in ähnlicher Form bei einer anderen Stelle zur Erlangung eines akademischen Grades eingereicht wurde. München, 17.05.2019 Alisa Kleinhempel Ort, Datum Unterschrift Doktorandin/Doktorand -
Generate Metabolic Map Poster
Authors: Zheng Zhao, Delft University of Technology Marcel A. van den Broek, Delft University of Technology S. Aljoscha Wahl, Delft University of Technology Wilbert H. Heijne, DSM Biotechnology Center Roel A. Bovenberg, DSM Biotechnology Center Joseph J. Heijnen, Delft University of Technology An online version of this diagram is available at BioCyc.org. Biosynthetic pathways are positioned in the left of the cytoplasm, degradative pathways on the right, and reactions not assigned to any pathway are in the far right of the cytoplasm. Transporters and membrane proteins are shown on the membrane. Marco A. van den Berg, DSM Biotechnology Center Peter J.T. Verheijen, Delft University of Technology Periplasmic (where appropriate) and extracellular reactions and proteins may also be shown. Pathways are colored according to their cellular function. PchrCyc: Penicillium rubens Wisconsin 54-1255 Cellular Overview Connections between pathways are omitted for legibility. Liang Wu, DSM Biotechnology Center Walter M. van Gulik, Delft University of Technology L-quinate phosphate a sugar a sugar a sugar a sugar multidrug multidrug a dicarboxylate phosphate a proteinogenic 2+ 2+ + met met nicotinate Mg Mg a cation a cation K + L-fucose L-fucose L-quinate L-quinate L-quinate ammonium UDP ammonium ammonium H O pro met amino acid a sugar a sugar a sugar a sugar a sugar a sugar a sugar a sugar a sugar a sugar a sugar K oxaloacetate L-carnitine L-carnitine L-carnitine 2 phosphate quinic acid brain-specific hypothetical hypothetical hypothetical hypothetical