The Experts below are selected from a list of 3039 Experts worldwide ranked by ideXlab platform

Edmund Maser - One of the best experts on this subject based on the ideXlab platform.

  • the drosophila Carbonyl Reductase sniffer is an efficient 4 oxonon 2 enal 4one Reductase
    Chemico-Biological Interactions, 2011
    Co-Authors: Hans Jorg Martin, Marta Ziemba, Michael Kisiela, Jose A Botella, Stephan Schneuwly, Edmund Maser
    Abstract:

    Studies with the fruit-fly Drosophila melanogaster demonstrated that the enzyme sniffer prevented oxidative stress-induced neurodegeneration. Mutant flies overexpressing sniffer had significantly extended life spans in a 99.5 oxygen atmosphere compared to wild-type flies. However, the molecular mechanism of this protection remained unclear. Sequence analysis and database searches identified sniffer as a member of the short-chain dehydrogenase/Reductase superfamily with a 27.4 identity to the human enzyme Carbonyl Reductase type I (CBR1). As CBR1 catalyzes the reduction of the lipid peroxidation products 4HNE and 4ONE, we tested whether sniffer is able to metabolize these lipid derived aldehydes by Carbonyl reduction. To produce recombinant enzyme, the coding sequence of sniffer was amplified from a cDNA-library, cloned into a bacterial expression vector and the His-tagged protein was purified by Ni-chelate chromatography. We found that sniffer catalyzed the NADPH-dependent Carbonyl reduction of 4ONE (K(m)=24+/-2muM, k(cat)=500+/-10min(-1), k(cat)/K(m)=350s(-1)mM(-1)) but not that of 4HNE. The reaction product of 4ONE reduction by sniffer was mainly 4HNE as shown by HPLC- and GC/MS analysis. Since 4HNE, though still a potent electrophile, is less neurotoxic and protein reactive than 4ONE, one mechanism by which sniffer exerts its neuroprotective effects in Drosophila after oxidative stress may be enzymatic reduction of 4ONE.

  • regulation of human Carbonyl Reductase 3 cbr3 sdr21c2 expression by nrf2 in cultured cancer cells
    Biochemistry, 2010
    Co-Authors: Bettina Ebert, Michael Kisiela, Petra Malatkova, Yasser Elhawari, Edmund Maser
    Abstract:

    Carbonyl reduction is a central metabolic process that controls the level of key regulatory molecules as well as xenobiotics. Carbonyl Reductase 3 (CBR3; SDR21C2), a member of the short-chain dehydrogenase/Reductase (SDR) superfamily, has been poorly characterized so far, and the regulation of its expression is a complete mystery. Here, we show that CBR3 expression is regulated via Nrf2, a key regulator in response to oxidative stress. In human cancer cell lines, CBR3 mRNA was expressed differentially, ranging from very high (A549, lung) to very low (HT-29, colon; HepG2, liver) levels. CBR3 protein was highly expressed in SW-480 (colon) cells but was absent in HCT116 (colon) and HepG2 cells. CBR3 mRNA could be induced in HT-29 cells by Nrf2 agonists [sulforaphane (SUL, 7-fold) and diethyl maleate (DEM, 4-fold)] or hormone receptor ligand Z-guggulsterone (5-fold). Aryl hydrocarbon receptor agonist B[k]F failed to induce CBR3 mRNA after incubation for 8 h but elevated CBR3 levels after 24 h, most likely mediated by B[k]F metabolites that can activate Nrf2 signaling. Inhibition of Nrf2-activating upstream kinase MEK/ERK by PD98059 weakened DEM-mediated induction of CBR3 mRNA. Proteasome inhibitors MG-132 (5 μM) and bortezomib (50 nM) dramatically increased the level of CBR3 mRNA, obviously because of the increase in the level of Nrf2 protein. While siRNA-mediated knockdown of Nrf2 led to a decrease in the level of CBR3 mRNA in A549 cells (30% of control), Keap1 knockdown increased the level of CBR3 mRNA expression in HepG2 (9.3-fold) and HT-29 (2.7-fold) cells. Here, we provide for the first time evidence that human CBR3 is a new member of the Nrf2 gene battery.

  • cis and trans regulatory elements of 3α hydroxysteroid dehydrogenase Carbonyl Reductase as biosensor system for steroid determination in the environment
    Chemico-Biological Interactions, 2009
    Co-Authors: Guangming Xiong, Edmund Maser
    Abstract:

    3 alpha-Hydroxysteroid dehydrogenase/Carbonyl Reductase from Comamonas testosteroni is a key enzyme in the degradation of steroids in the environment. The encoding gene, hsdA, is expressed only at very low levels in the absence of steroids, but undergoes a several fold induction in the presence of steroid substrates. In previous investigations, we have elucidated the mechanism of hsdA regulation that involves several activators and repressors. In the present study, the hsdA gene was replaced by the green fluorescent protein (GFP) gene which was inserted downstream from the hsdA regulatory region. By homologous integration into the chromosomal DNA, the C testosteroni mutant strain CT-GFP5-1 was generated and used as fluorescence based biosensor system for steroid determination. With this cell-based system we could determine testosterone in a range between 57 and 450 ng/ml, estradiol between 1.6 and 12.8 ng/ml, and cholesterol between 19.3 and 154.4 ng/nl. Interestingly, the sensitivity of this bioassay could be further increased by using only the cytosol of the mutant. With the resulting cell-free system we could determine testosterone in a range between 28 and 219 pg/ml, estradiol between 0.029 and 0.430 fg/ml, and cholesterol between 9.7 and 77.2 fg/ml. The recovery ratio of the extraction was around 95% and the maximum fluorescence signals were obtained as early as after 30 min. Limitations of the established steroid biosensor system were quenching at higher steroid concentrations and the relatively high background of fluorescence, which are currently being improved in our lab. Combined, by exploiting the regulatory region of the gene hsdA that codes for the enzyme 3 alpha-hydroxysteroid dehydrogenase/Carbonyl Reductase we have constructed a mutant C testosteroni strain that can be used as a sensitive biosensor system for steroid determination in the environment. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

  • Carbonyl Reductase 1 is a predominant doxorubicin Reductase in the human liver
    Drug Metabolism and Disposition, 2008
    Co-Authors: Nina Kassner, Hans Jorg Martin, Klaus Huse, Ute Godtelarmbrust, Annegret Metzger, Ingolf Meineke, Jurgen Brockmoller, Kathrin Klein, Ulrich M Zanger, Edmund Maser
    Abstract:

    A first step in the enzymatic disposition of the antineoplastic drug doxorubicin (DOX) is the reduction to doxorubicinol (DOX-OL). Because DOX-OL is less antineoplastic but more cardiotoxic than the parent compound, the individual rate of this reaction may affect the antitumor effect and the risk of DOX-induced heart failure. Using purified enzymes and human tissues we determined enzymes generating DOX-OL and interindividual differences in their activities. Human tissues express at least two DOX-reducing enzymes. High-clearance organs (kidney, liver, and the gastrointestinal tract) express an enzyme with an apparent Km of approximately 140 microM. Of six enzymes found to reduce DOX, Km values in this range are exhibited by Carbonyl Reductase 1 (CBR1) and aldo-keto Reductase (AKR) 1C3. CBR1 is expressed in these three organs at higher levels than AKR1C3, whereas AKR1C3 has higher catalytic efficiency. However, inhibition constants for DOX reduction with 4-amino-1-tert-butyl-3-(2-hydroxyphenyl)pyrazolo[3,4-d]pyrimidine (an inhibitor that can discriminate between CBR1 and AKR1C3) were identical for CBR1 and human liver cytosol, but not for AKR1C3. These results suggest that CBR1 is a predominant hepatic DOX Reductase. In cytosols from 80 human livers, the expression level of CBR1 and the activity of DOX reduction varied >70- and 22-fold, respectively, but showed no association with CBR1 gene variants found in these samples. Instead, the interindividual differences in CBR1 expression and activity may be mediated by environmental factors acting via recently identified xenobiotic response elements in the CBR1 promoter. The variability in the CBR1 expression may affect outcomes of therapies with DOX, as well as with other CBR1 substrates.

  • human Carbonyl Reductase catalyzes reduction of 4 oxonon 2 enal
    Biochemistry, 2004
    Co-Authors: Jonathan A Doorn, Edmund Maser, Andreas Blum, David J Claffey, Dennis R Petersen
    Abstract:

    4-Oxonon-2-enal (4ONE) was demonstrated to be a product of lipid peroxidation, and previous studies found that it was highly reactive toward DNA and protein. The present study sought to determine whether Carbonyl Reductase (CR) catalyzes reduction of 4ONE, representing a potential pathway for metabolism of the lipid peroxidation product. Recombinant CR was cloned from a human liver cDNA library, expressed in Escherichia coli, and purified by metal chelate chromatography. Both 4ONE and its glutathione conjugate were found to be substrates for CR, and kinetic parameters were calculated. TLC analysis of reaction products revealed the presence of three compounds, two of which were identified as 4-hydroxynon-2-enal (4HNE) and 1-hydroxynon-2-en-4-one (1HNO). GC/MS analysis confirmed 4HNE and 1HNO and identified the unknown reaction product as 4-oxononanal (4ONA). Analysis of oxime derivatives of the reaction products via LC/MS confirmed the unknown as 4ONA. The time course for CR-mediated, NADPH-dependent 4ONE ...

Yu-guo Zheng - One of the best experts on this subject based on the ideXlab platform.

  • biosynthesis of tert butyl 3r 5s 6 chloro 3 5 dihydroxyhexanoate by Carbonyl Reductase from rhodosporidium toruloides in mono and biphasic media
    Bioresource Technology, 2018
    Co-Authors: Zhi-qiang Liu, Xiaojian Zhang, Ling Zheng, Wenzhong Wang, Liqun Jin, Yu-guo Zheng
    Abstract:

    Abstract tert -Butyl (3 R ,5 S )-6-chloro-3,5-dihydroxyhexanoate ((3 R ,5 S )-CDHH) is the key intermediate for synthesis of atorvastatin and rosuvastatin. Carbonyl Reductase exhibits excellent activity toward tert -butyl ( S )-6-chloro-5-hydroxy-3-oxohexanoate (( S )-CHOH) to synthesize (3 R ,5 S )-CDHH. In this study, a whole cell biosynthesis reaction system to produce (3 R ,5 S )-CDHH was constructed in organic solvents. A solution of 10% ( v/v ) Tween-80 was introduced to the reaction system as a co-solvent, which greatly enhanced biotransformation process, giving 98.9% yield, >99% ee and 1.8-fold higher space time yield in 5 h bioconversion of 1 M ( S )-CHOH, compared with 98.7% yield and >99% ee in 9 h bioconversion of a purely aqueous reaction system. Moreover, a water-octanol biphasic reaction system was built and 20% of octanol was added as reservoir of substrate resulting in 98% yield, >99% ee and 4.08 mmol L −1  h −1  g −1 (wet cell weight) space time yield. This study paved a way for the whole cell biosynthesis of (3 R ,5 S )-CDHH in mono and biphasic media.

  • large scale synthesis of tert butyl 3r 5s 6 chloro 3 5 dihydroxyhexanoate by a stereoselective Carbonyl Reductase with high substrate concentration and product yield
    Biotechnology Progress, 2017
    Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xiaojian Zhang, Xiaoling Tang, Feng Cheng, Yajun Wang, Dankai Yao, Yiteng Zhou, Yu-guo Zheng
    Abstract:

    To biosynthesize the (3R,5S)-CDHH in an industrial scale, a newly synthesized stereoselective short chain Carbonyl Reductase (SCR) was successfully cloned and expressed in Escherichia coli. The fermentation of recombinant E. coli harboring SCR was carried out in 500 L and 5000 L fermenters, with biomass and specific activity of 9.7 g DCW/L, 15749.95 U/g DCW, and 10.97 g DCW/L, 19210.12 U/g DCW, respectively. The recombinant SCR was successfully applied for efficient production of (3R,5S)-CDHH. The scale-up synthesis of (3R,5S)-CDHH was performed in 5000 L bioreactor with 400 g/L of (S)-CHOH at 30°C, resulting in a space-time yield of 13.7 mM/h/g DCW, which was the highest ever reported. After isolation and purification, the yield and d.e. of (3R,5S)-CDHH reached 97.5% and 99.5%, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:612-620, 2017.

  • directed evolution of Carbonyl Reductase from rhodosporidium toruloides and its application in stereoselective synthesis of tert butyl 3r 5s 6 chloro 3 5 dihydroxyhexanoate
    Journal of Agricultural and Food Chemistry, 2017
    Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xiaojian Zhang, Yu-guo Zheng
    Abstract:

    tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key intermediate of atorvastatin and rosuvastatin synthesis. Carbonyl Reductase RtSCR9 from Rhodosporidium toruloides exhibited excellent activity toward tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH). For the activity of RtSCR9 to be improved, random mutagenesis and site-saturation mutagenesis were performed. Three positive mutants were obtained (mut-Gln95Asp, mut-Ile144Lys, and mut-Phe156Gln). These mutants exhibited 1.94-, 3.03-, and 1.61-fold and 1.93-, 3.15-, and 1.97-fold improvement in the specific activity and kcat/Km, respectively. Asymmetric reduction of (S)-CHOH by mut-Ile144Lys coupled with glucose dehydrogenase was conducted. The yield and enantiomeric excess of (3R,5S)-CDHH reached 98 and 99%, respectively, after 8 h bioconversion in a single batch reaction with 1 M (S)-CHOH, and the space-time yield reached 542.83 mmol L–1 h–1 g–1 wet cell weight. This study presents a new Carbonyl Reductase for efficient sy...

  • enzymatic synthesis of an ezetimibe intermediate using Carbonyl Reductase coupled with glucose dehydrogenase in an aqueous organic solvent system
    Bioresource Technology, 2017
    Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xiaojian Zhang, Xiaoling Tang, Sichuan Dong, Huanhuan Yin, Yu-guo Zheng
    Abstract:

    Abstract (4 S )-3-[(5 S )-5-(4-Fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one (( S )-ET-5) is an important chiral intermediate in the synthesis of chiral side chain of ezetimibe. Recombinant Escherichia coli expressing Carbonyl Reductase (CBR) was successfully constructed in this study. The total E. coli biomass and the specific activity of recombinant CBR in 5 L fermenter culture were 10.9 g DCW L −1 and 14900.3 U g −1  DCW, respectively. The dual-enzyme coupled biocatalytic process in an aqueous-organic biphasic solvent system was first constructed using p -xylene as the optimal organic phase under optimized reaction conditions, and 150 g L −1 (4 S )-3-[5-(4-fluorophenyl)-1,5-dioxophentyl]-4-phenyl-1,3-oxazolidin-2-one (ET-4) was successfully converted to ( S )-ET-5 with a conversion of 99.1% and diastereomeric excess of 99% after 24-h, which are the highest values reported to date for the production of ( S )-ET-5.

Zhi-qiang Liu - One of the best experts on this subject based on the ideXlab platform.

  • biosynthesis of tert butyl 3r 5s 6 chloro 3 5 dihydroxyhexanoate by Carbonyl Reductase from rhodosporidium toruloides in mono and biphasic media
    Bioresource Technology, 2018
    Co-Authors: Zhi-qiang Liu, Xiaojian Zhang, Ling Zheng, Wenzhong Wang, Liqun Jin, Yu-guo Zheng
    Abstract:

    Abstract tert -Butyl (3 R ,5 S )-6-chloro-3,5-dihydroxyhexanoate ((3 R ,5 S )-CDHH) is the key intermediate for synthesis of atorvastatin and rosuvastatin. Carbonyl Reductase exhibits excellent activity toward tert -butyl ( S )-6-chloro-5-hydroxy-3-oxohexanoate (( S )-CHOH) to synthesize (3 R ,5 S )-CDHH. In this study, a whole cell biosynthesis reaction system to produce (3 R ,5 S )-CDHH was constructed in organic solvents. A solution of 10% ( v/v ) Tween-80 was introduced to the reaction system as a co-solvent, which greatly enhanced biotransformation process, giving 98.9% yield, >99% ee and 1.8-fold higher space time yield in 5 h bioconversion of 1 M ( S )-CHOH, compared with 98.7% yield and >99% ee in 9 h bioconversion of a purely aqueous reaction system. Moreover, a water-octanol biphasic reaction system was built and 20% of octanol was added as reservoir of substrate resulting in 98% yield, >99% ee and 4.08 mmol L −1  h −1  g −1 (wet cell weight) space time yield. This study paved a way for the whole cell biosynthesis of (3 R ,5 S )-CDHH in mono and biphasic media.

  • large scale synthesis of tert butyl 3r 5s 6 chloro 3 5 dihydroxyhexanoate by a stereoselective Carbonyl Reductase with high substrate concentration and product yield
    Biotechnology Progress, 2017
    Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xiaojian Zhang, Xiaoling Tang, Feng Cheng, Yajun Wang, Dankai Yao, Yiteng Zhou, Yu-guo Zheng
    Abstract:

    To biosynthesize the (3R,5S)-CDHH in an industrial scale, a newly synthesized stereoselective short chain Carbonyl Reductase (SCR) was successfully cloned and expressed in Escherichia coli. The fermentation of recombinant E. coli harboring SCR was carried out in 500 L and 5000 L fermenters, with biomass and specific activity of 9.7 g DCW/L, 15749.95 U/g DCW, and 10.97 g DCW/L, 19210.12 U/g DCW, respectively. The recombinant SCR was successfully applied for efficient production of (3R,5S)-CDHH. The scale-up synthesis of (3R,5S)-CDHH was performed in 5000 L bioreactor with 400 g/L of (S)-CHOH at 30°C, resulting in a space-time yield of 13.7 mM/h/g DCW, which was the highest ever reported. After isolation and purification, the yield and d.e. of (3R,5S)-CDHH reached 97.5% and 99.5%, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:612-620, 2017.

  • directed evolution of Carbonyl Reductase from rhodosporidium toruloides and its application in stereoselective synthesis of tert butyl 3r 5s 6 chloro 3 5 dihydroxyhexanoate
    Journal of Agricultural and Food Chemistry, 2017
    Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xiaojian Zhang, Yu-guo Zheng
    Abstract:

    tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key intermediate of atorvastatin and rosuvastatin synthesis. Carbonyl Reductase RtSCR9 from Rhodosporidium toruloides exhibited excellent activity toward tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH). For the activity of RtSCR9 to be improved, random mutagenesis and site-saturation mutagenesis were performed. Three positive mutants were obtained (mut-Gln95Asp, mut-Ile144Lys, and mut-Phe156Gln). These mutants exhibited 1.94-, 3.03-, and 1.61-fold and 1.93-, 3.15-, and 1.97-fold improvement in the specific activity and kcat/Km, respectively. Asymmetric reduction of (S)-CHOH by mut-Ile144Lys coupled with glucose dehydrogenase was conducted. The yield and enantiomeric excess of (3R,5S)-CDHH reached 98 and 99%, respectively, after 8 h bioconversion in a single batch reaction with 1 M (S)-CHOH, and the space-time yield reached 542.83 mmol L–1 h–1 g–1 wet cell weight. This study presents a new Carbonyl Reductase for efficient sy...

  • enzymatic synthesis of an ezetimibe intermediate using Carbonyl Reductase coupled with glucose dehydrogenase in an aqueous organic solvent system
    Bioresource Technology, 2017
    Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xiaojian Zhang, Xiaoling Tang, Sichuan Dong, Huanhuan Yin, Yu-guo Zheng
    Abstract:

    Abstract (4 S )-3-[(5 S )-5-(4-Fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one (( S )-ET-5) is an important chiral intermediate in the synthesis of chiral side chain of ezetimibe. Recombinant Escherichia coli expressing Carbonyl Reductase (CBR) was successfully constructed in this study. The total E. coli biomass and the specific activity of recombinant CBR in 5 L fermenter culture were 10.9 g DCW L −1 and 14900.3 U g −1  DCW, respectively. The dual-enzyme coupled biocatalytic process in an aqueous-organic biphasic solvent system was first constructed using p -xylene as the optimal organic phase under optimized reaction conditions, and 150 g L −1 (4 S )-3-[5-(4-fluorophenyl)-1,5-dioxophentyl]-4-phenyl-1,3-oxazolidin-2-one (ET-4) was successfully converted to ( S )-ET-5 with a conversion of 99.1% and diastereomeric excess of 99% after 24-h, which are the highest values reported to date for the production of ( S )-ET-5.

Xiaojian Zhang - One of the best experts on this subject based on the ideXlab platform.

  • biosynthesis of tert butyl 3r 5s 6 chloro 3 5 dihydroxyhexanoate by Carbonyl Reductase from rhodosporidium toruloides in mono and biphasic media
    Bioresource Technology, 2018
    Co-Authors: Zhi-qiang Liu, Xiaojian Zhang, Ling Zheng, Wenzhong Wang, Liqun Jin, Yu-guo Zheng
    Abstract:

    Abstract tert -Butyl (3 R ,5 S )-6-chloro-3,5-dihydroxyhexanoate ((3 R ,5 S )-CDHH) is the key intermediate for synthesis of atorvastatin and rosuvastatin. Carbonyl Reductase exhibits excellent activity toward tert -butyl ( S )-6-chloro-5-hydroxy-3-oxohexanoate (( S )-CHOH) to synthesize (3 R ,5 S )-CDHH. In this study, a whole cell biosynthesis reaction system to produce (3 R ,5 S )-CDHH was constructed in organic solvents. A solution of 10% ( v/v ) Tween-80 was introduced to the reaction system as a co-solvent, which greatly enhanced biotransformation process, giving 98.9% yield, >99% ee and 1.8-fold higher space time yield in 5 h bioconversion of 1 M ( S )-CHOH, compared with 98.7% yield and >99% ee in 9 h bioconversion of a purely aqueous reaction system. Moreover, a water-octanol biphasic reaction system was built and 20% of octanol was added as reservoir of substrate resulting in 98% yield, >99% ee and 4.08 mmol L −1  h −1  g −1 (wet cell weight) space time yield. This study paved a way for the whole cell biosynthesis of (3 R ,5 S )-CDHH in mono and biphasic media.

  • large scale synthesis of tert butyl 3r 5s 6 chloro 3 5 dihydroxyhexanoate by a stereoselective Carbonyl Reductase with high substrate concentration and product yield
    Biotechnology Progress, 2017
    Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xiaojian Zhang, Xiaoling Tang, Feng Cheng, Yajun Wang, Dankai Yao, Yiteng Zhou, Yu-guo Zheng
    Abstract:

    To biosynthesize the (3R,5S)-CDHH in an industrial scale, a newly synthesized stereoselective short chain Carbonyl Reductase (SCR) was successfully cloned and expressed in Escherichia coli. The fermentation of recombinant E. coli harboring SCR was carried out in 500 L and 5000 L fermenters, with biomass and specific activity of 9.7 g DCW/L, 15749.95 U/g DCW, and 10.97 g DCW/L, 19210.12 U/g DCW, respectively. The recombinant SCR was successfully applied for efficient production of (3R,5S)-CDHH. The scale-up synthesis of (3R,5S)-CDHH was performed in 5000 L bioreactor with 400 g/L of (S)-CHOH at 30°C, resulting in a space-time yield of 13.7 mM/h/g DCW, which was the highest ever reported. After isolation and purification, the yield and d.e. of (3R,5S)-CDHH reached 97.5% and 99.5%, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:612-620, 2017.

  • directed evolution of Carbonyl Reductase from rhodosporidium toruloides and its application in stereoselective synthesis of tert butyl 3r 5s 6 chloro 3 5 dihydroxyhexanoate
    Journal of Agricultural and Food Chemistry, 2017
    Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xiaojian Zhang, Yu-guo Zheng
    Abstract:

    tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key intermediate of atorvastatin and rosuvastatin synthesis. Carbonyl Reductase RtSCR9 from Rhodosporidium toruloides exhibited excellent activity toward tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH). For the activity of RtSCR9 to be improved, random mutagenesis and site-saturation mutagenesis were performed. Three positive mutants were obtained (mut-Gln95Asp, mut-Ile144Lys, and mut-Phe156Gln). These mutants exhibited 1.94-, 3.03-, and 1.61-fold and 1.93-, 3.15-, and 1.97-fold improvement in the specific activity and kcat/Km, respectively. Asymmetric reduction of (S)-CHOH by mut-Ile144Lys coupled with glucose dehydrogenase was conducted. The yield and enantiomeric excess of (3R,5S)-CDHH reached 98 and 99%, respectively, after 8 h bioconversion in a single batch reaction with 1 M (S)-CHOH, and the space-time yield reached 542.83 mmol L–1 h–1 g–1 wet cell weight. This study presents a new Carbonyl Reductase for efficient sy...

  • enzymatic synthesis of an ezetimibe intermediate using Carbonyl Reductase coupled with glucose dehydrogenase in an aqueous organic solvent system
    Bioresource Technology, 2017
    Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xiaojian Zhang, Xiaoling Tang, Sichuan Dong, Huanhuan Yin, Yu-guo Zheng
    Abstract:

    Abstract (4 S )-3-[(5 S )-5-(4-Fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one (( S )-ET-5) is an important chiral intermediate in the synthesis of chiral side chain of ezetimibe. Recombinant Escherichia coli expressing Carbonyl Reductase (CBR) was successfully constructed in this study. The total E. coli biomass and the specific activity of recombinant CBR in 5 L fermenter culture were 10.9 g DCW L −1 and 14900.3 U g −1  DCW, respectively. The dual-enzyme coupled biocatalytic process in an aqueous-organic biphasic solvent system was first constructed using p -xylene as the optimal organic phase under optimized reaction conditions, and 150 g L −1 (4 S )-3-[5-(4-fluorophenyl)-1,5-dioxophentyl]-4-phenyl-1,3-oxazolidin-2-one (ET-4) was successfully converted to ( S )-ET-5 with a conversion of 99.1% and diastereomeric excess of 99% after 24-h, which are the highest values reported to date for the production of ( S )-ET-5.

Guangming Xiong - One of the best experts on this subject based on the ideXlab platform.

  • cis and trans regulatory elements of 3α hydroxysteroid dehydrogenase Carbonyl Reductase as biosensor system for steroid determination in the environment
    Chemico-Biological Interactions, 2009
    Co-Authors: Guangming Xiong, Edmund Maser
    Abstract:

    3 alpha-Hydroxysteroid dehydrogenase/Carbonyl Reductase from Comamonas testosteroni is a key enzyme in the degradation of steroids in the environment. The encoding gene, hsdA, is expressed only at very low levels in the absence of steroids, but undergoes a several fold induction in the presence of steroid substrates. In previous investigations, we have elucidated the mechanism of hsdA regulation that involves several activators and repressors. In the present study, the hsdA gene was replaced by the green fluorescent protein (GFP) gene which was inserted downstream from the hsdA regulatory region. By homologous integration into the chromosomal DNA, the C testosteroni mutant strain CT-GFP5-1 was generated and used as fluorescence based biosensor system for steroid determination. With this cell-based system we could determine testosterone in a range between 57 and 450 ng/ml, estradiol between 1.6 and 12.8 ng/ml, and cholesterol between 19.3 and 154.4 ng/nl. Interestingly, the sensitivity of this bioassay could be further increased by using only the cytosol of the mutant. With the resulting cell-free system we could determine testosterone in a range between 28 and 219 pg/ml, estradiol between 0.029 and 0.430 fg/ml, and cholesterol between 9.7 and 77.2 fg/ml. The recovery ratio of the extraction was around 95% and the maximum fluorescence signals were obtained as early as after 30 min. Limitations of the established steroid biosensor system were quenching at higher steroid concentrations and the relatively high background of fluorescence, which are currently being improved in our lab. Combined, by exploiting the regulatory region of the gene hsdA that codes for the enzyme 3 alpha-hydroxysteroid dehydrogenase/Carbonyl Reductase we have constructed a mutant C testosteroni strain that can be used as a sensitive biosensor system for steroid determination in the environment. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

  • characterization and recombinant expression of the translational repressor repb of 3α hydroxysteroid dehydrogenase Carbonyl Reductase in comamonas testosteroni
    Chemico-Biological Interactions, 2003
    Co-Authors: Guangming Xiong, Edmund Maser, Hans Jorg Martin
    Abstract:

    Abstract 3α-Hydroxysteroid dehydrogenase/Carbonyl Reductase (3α-HSD/CR) from Comamonas testosteroni is a key enzyme involved in the degradation of steroids and xenobiotic Carbonyl compounds. The gene of 3α-HSD/CR ( hsdA ) was cloned and characterized by our group. We have also reported that two repressor proteins (RepA and RepB) have been identified which regulate hsdA expression. To further characterize RepB, the protein was expressed in Escherichia coli and purified in an active state. Gel shift experiments showed that RepB binds to a 16 nucleotide sequence downstream of AUG of the hsdA mRNA, providing evidence that RepB acts on the translational level. The addition of testosterone to the culture medium led to a derepression. Furthermore, a plasmid was prepared containing a point mutation that inactivates only repA , but has no effect on hsdA , with which it happens to partly overlap. The result of coexpression experiments with this construct and a plasmid containing the genetic information for RepB showed that RepB is still active and is therefore not dependent on a functional RepA. In conclusion, RepB is a novel regulatory protein that inhibits the translation of hsdA mRNA, thereby leading to a decreased expression of 3α-HSD/CR.

  • regulation of the steroid inducible 3α hydroxysteroid dehydrogenase Carbonyl Reductase gene incomamonas testosteroni
    Journal of Biological Chemistry, 2001
    Co-Authors: Guangming Xiong, Edmund Maser
    Abstract:

    Abstract The Comamonas testosteroni3α-hydroxysteroid dehydrogenase/Carbonyl Reductase gene (hsdA) codes for an adaptive enzyme in the degradation of steroid compounds. However, no information was available on the molecular regulation of steroid-inducible genes nor on the mechanism of steroid signaling in procaryotes. We, therefore, investigated thecis- and trans-acting elements ofhsdA expression to infer the mechanism of its molecular regulation by steroids. The gene was localized on a 5.257-kilobase EcoRI fragment of C. testosteroni chromosomal DNA. The promoter was characterized, and the transcriptional start site was identified. Two palindromic operator domains were found upstream of hsdA. A new gene coding for a trans-acting negative regulator (repressor A, RepA) ofhsdA expression was characterized. The specific interaction between RepA, testosterone, and the operator domain is demonstrated. From our results we conclude that hsdA is under negative transcriptional control by an adjacent gene product (RepA). Accordingly, induction of hsdA by steroids in fact is a derepression, where steroidal inducers bind to the repressor, thereby preventing its binding to the hsdA operator.

  • regulation of the steroid inducible 3α hydroxysteroid dehydrogenase Carbonyl Reductase gene in comamonas testosteroni
    Journal of Biological Chemistry, 2001
    Co-Authors: Guangming Xiong, Edmund Maser
    Abstract:

    Abstract The Comamonas testosteroni3α-hydroxysteroid dehydrogenase/Carbonyl Reductase gene (hsdA) codes for an adaptive enzyme in the degradation of steroid compounds. However, no information was available on the molecular regulation of steroid-inducible genes nor on the mechanism of steroid signaling in procaryotes. We, therefore, investigated thecis- and trans-acting elements ofhsdA expression to infer the mechanism of its molecular regulation by steroids. The gene was localized on a 5.257-kilobase EcoRI fragment of C. testosteroni chromosomal DNA. The promoter was characterized, and the transcriptional start site was identified. Two palindromic operator domains were found upstream of hsdA. A new gene coding for a trans-acting negative regulator (repressor A, RepA) ofhsdA expression was characterized. The specific interaction between RepA, testosterone, and the operator domain is demonstrated. From our results we conclude that hsdA is under negative transcriptional control by an adjacent gene product (RepA). Accordingly, induction of hsdA by steroids in fact is a derepression, where steroidal inducers bind to the repressor, thereby preventing its binding to the hsdA operator.

  • a model on the regulation of 3α hydroxysteroid dehydrogenase Carbonyl Reductase expression in comamonas testosteroni
    Chemico-Biological Interactions, 2001
    Co-Authors: Guangming Xiong, Hans Jorg Martin, Andreas Blum, Christina Schafers, Edmund Maser
    Abstract:

    Abstract 3α-Hydroxysteroid dehydrogenase/Carbonyl Reductase (3α-HSD/CR) from Comamonastestosteroni is a key enzyme involved in the degradation of steroids and xenobiotic Carbonyl compounds. The enzyme has recently been cloned and characterized by our group. A strong induction of enzyme activity is observed in the presence of steroids like testosterone. In the present investigation, two repressor proteins (Rep1 and Rep2) containing 78 and 420 amino acids, respectively, were found to regulate 3α-HSD/CR gene (hsdA) expression. Gel shift experiments showed that Rep2 binds to a 10 nucleotide sequence 9 bp upstream of the hsdA promoter. The deletion of this cis-regulating sequence significantly increases hsdA expression. About 1633 bp further upstream, a second ten nucleotide sequence, complementary to the first one, was found, which is also recognized by Rep2 and increases hsdA expression, if deleted. To purify the repressor proteins, the genes encoding each were cloned into His-tag expression vectors and overexpressed in Escherichiacoli. Rep1 does not bind to DNA but may bind to 3α-HSD/CR mRNA as predicted by its secondary structure. Concluding from our data, induction of 3α-HSD/CR in C.testosteroni by steroids in fact appears to be a de-repression, where the steroidal ‘inducer’ prevents the binding of the two repressor proteins to the hsdA promoter and mRNA, respectively.