title: Optimized expression of (S)-carbonyl reductase in Pichia pastoris for efficient production of (S)-1-phenyl-1, 2-ethanediol.
abstract: The recombinant (S)-carbonyl reductase (SCR) in Escherichia coli catalyzed the reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol (PED) with low efficiency. In this work, its 6× histidine fusion gene his6 -scr was cloned in Pichia pastoris under the control of the AOX1 methanol inducible promoter. The heterologous protein SCR was expressed through a Mut(s) phenotype. Under the optimal conditions: pH 7.0, initial OD600 2.5, methanol daily addition concentration 1.0% and induction duration 4-5 days, the recombinant protein SCR was produced at the highest level. The enzyme activity in the cell-free exacts of P. pastoris was 0.38, which was over twofold than that of the recombinant E. coli-SCR. The enzyme was purified to homogeneity with a specific activity of 3.41 U mg(-1) , and it catalyzed the biotransformation of (S)-PED with a high optical purity of 96.9% in a high yield of 89.7% at optimum pH of 7.0. The developed effective system of P. pastoris-SCR will facilitate the preparation of pure chiral alcohol in industry.
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education & School of Biotechnology, Jiangnan University, Wuxi, P. R. China; National Key Laboratory for Food Science, Jiangnan University, Wuxi, P. R. China.
authors.forename: Rongzhen
authors.initials: R
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.forename: Rong
authors.initials: R
authors.lastname: Xiao
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Botao
authors.initials: B
authors.lastname: Zhang
mesh_terms: D042931:Aldehyde Oxidase / Q000235:genetics; D000449:Aldehyde Reductase / Q000096:biosynthesis / Q000235:genetics*; D000074408:Aldo-Keto Reductases; D002175:Candida / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D005026:Ethylene Glycols / Q000378:metabolism*; D015870:Gene Expression; D000432:Methanol / Q000737:chemistry; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D005026:Ethylene Glycols; D011994:Recombinant Proteins; C031474:styrene glycol; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; D042931:Aldehyde Oxidase; D000432:Methanol
keywords: (S)-carbonyl reductase; Candida parapsilosis; Chiral alcohol; Expression; Pichia pastoris