Purification and characterization of a novel NADH-dependent carbonyl reductase from Pichia stipitis involved in biosynthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate

Cao, Hou; Mi, Lan; Ye, Qi; Zang, Guanglou; Yan, Ming; Wang, Yan; Zhang, Yueyuan; Li, Ximu; Lin, Xu; Xiong, Jing; Ouyang, Pingkai; Ying, Hanjie

doi:10.1016/j.biortech.2010.08.072

Cited by 28 publications

(13 citation statements)

References 34 publications

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“…In contrast, some reductases preferred NADH such as the carbonyl reductase from Pichia stipitis [29]. The cofactor preferences exhibited by carbonyl reductases might be attributed to the electrostatic environment surrounding the 2′-hydroxyl (or phosphate) group of the adenosine ribose moiety of NADH (or NADPH) [31].…”

Section: Resultsmentioning

confidence: 99%

A Novel Carbonyl Reductase with Anti-Prelog Stereospecificity from Acetobacter sp. CCTCC M209061: Purification and Characterization

et al. 2014

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A novel carbonyl reductase (AcCR) catalyzing the asymmetric reduction of ketones to enantiopure alcohols with anti-Prelog stereoselectivity was found in Acetobacter sp. CCTCC M209061 and enriched 27.5-fold with an overall yield of 0.4% by purification. The enzyme showed a homotetrameric structure with an apparent molecular mass of 104 kDa and each subunit of 27 kDa. The gene sequence of AcCR was cloned and sequenced, and a 762 bp gene fragment was obtained. Either NAD(H) or NADP(H) can be used as coenzyme. For the reduction of 4′-chloroacetophenone, the Km value for NADH was around 25-fold greater than that for NADPH (0.66 mM vs 0.026 mM), showing that AcCR preferred NADPH over NADH. However, when NADH was used as cofactor, the response of AcCR activity to increasing concentration of 4′-chloroacetophenone was clearly sigmoidal with a Hill coefficient of 3.1, suggesting that the enzyme might possess four substrate-binding sites cooperating with each other The Vmax value for NADH-linked reduction was higher than that for NADPH-linked reduction (0.21 mM/min vs 0.17 mM/min). For the oxidation of isopropanol, the similar enzymological properties of AcCR were found using NAD+ or NADP+ as cofactor. Furthermore, a broad range of ketones such as aryl ketones, α-ketoesters and aliphatic ketones could be enantioselectively reduced into the corresponding chiral alcohols by this enzyme with high activity.

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Section: Resultsmentioning

confidence: 99%

A Novel Carbonyl Reductase with Anti-Prelog Stereospecificity from Acetobacter sp. CCTCC M209061: Purification and Characterization

et al. 2014

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“…Several short-chain dehydrogenases including carbonyl reductase and alcohol dehydrogenase could catalyze conversion of COBE to (S)-CHBE. Exception of the carbonyl reductase from Pichia stipitis showed the highest activity at 30°C [23], most of the enzymes have an optimal temperature between 45 and 50°C, which is the same as BaADH. Figure 2b showed the effect of pH on the BaADH activity.…”

Section: Characterization Of Baadhmentioning

confidence: 95%

Cloning, Expression and Characterization of a Highly Active Alcohol Dehydrogenase for Production of Ethyl (S)-4-Chloro-3-Hydroxybutyrate

et al. 2019

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A novel alcohol dehydrogenase from Bartonella apis (BaADH) was heterologous expressed in Escherichia coli. Its biochemical properties were investigated and used to catalyze the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), which is a chiral intermediate of the cholesterol-lowering drug atorvastatin. The purified recombinant BaADH displayed 182.4 U/mg of the specific activity using ethyl 4-chloroacetoacetate as substrate under the conditions of 50°C in pH 7.0 Tris-HCl buffer. It was stable in storage buffers of pH 7 to 9 and retains up to 96.7% of the initial activity after 24 h. The K m and V max values of BaADH were 0.11 mM and 190.4 lmol min-1 mg-1 , respectively. Synthesis of (S)-CHBE catalyzed by BaADH was performed with a cofactor regeneration system using a glucose dehydrogenase, and a conversion of 94.9% can be achieved after 1 h reaction. Homology modeling and substrate docking revealed that a typical catalytic triad is in contact with local water molecules to form a catalytic system. The results indicated this ADH could contribute to the further enzymatic synthesis of (S)-CHBE.

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“…Residual activities were normalized as percentages of the maximum residual activity. condition was not commonly seen for SDRs of mesophilic origins, which typically display stability at pH lower than 8.0 [30][31][32][33][34][35][36][37][38].…”

Section: Effects Of Ph and Temperaturementioning

confidence: 99%

“…The results indicated excellent adaptability of ChKRED20 under varied reaction conditions. The high tolerance against thermal treatment and pH variation compared well with the majority of SDRs from mesophilic microorganisms [30][31][32][33][34][35][36][37][38][39][40], which would be beneficial for large-scale application of this enzyme.…”

Section: Effects Of Ph and Temperaturementioning

confidence: 99%

Characterization of a robust anti-Prelog short-chain dehydrogenase/reductase ChKRED20 from Chryseobacterium sp. CA49

Tang

Liu

2014

Journal of Molecular Catalysis B: Enzymatic

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Purification and characterization of a novel NADH-dependent carbonyl reductase from Pichia stipitis involved in biosynthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate

Cited by 28 publications

References 34 publications

A Novel Carbonyl Reductase with Anti-Prelog Stereospecificity from Acetobacter sp. CCTCC M209061: Purification and Characterization

A Novel Carbonyl Reductase with Anti-Prelog Stereospecificity from Acetobacter sp. CCTCC M209061: Purification and Characterization

Cloning, Expression and Characterization of a Highly Active Alcohol Dehydrogenase for Production of Ethyl (S)-4-Chloro-3-Hydroxybutyrate

Characterization of a robust anti-Prelog short-chain dehydrogenase/reductase ChKRED20 from Chryseobacterium sp. CA49

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