Novel chiral tool, ( R )-2-octanol dehydrogenase, from Pichia finlandica : purification, gene cloning, and application f
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BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Novel chiral tool, (R)-2-octanol dehydrogenase, from Pichia finlandica: purification, gene cloning, and application for optically active α-haloalcohols Hiroaki Yamamoto & Masatake Kudoh
Received: 21 August 2012 / Revised: 4 December 2012 / Accepted: 6 December 2012 # Springer-Verlag Berlin Heidelberg 2012
Abstract A novel enantioselective alcohol dehydrogenase, (R)-2-octanol dehydrogenase (PfODH), was discovered among methylotrophic microorganisms. The enzyme was purified from Pichia finlandica and characterized. The molecular mass of the enzyme was estimated to be 83,000 and 30,000 by gel filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, respectively. The enzyme was an NAD+-dependent secondary alcohol dehydrogenase and showed a strict enantioselectivity, very broad substrate specificity, and high tolerance to SH reagents. A geneencoding PfODH was cloned and sequenced. The gene consisted of 765 nucleotides, coding polypeptides of 254 amino acids. The gene was singly expressed and coexpressed together with a formate dehydrogenase as an NADH regenerator in an Escherichia coli. Ethyl (S)-4chloro-3-hydroxybutanoate and (S)-2-chloro-1-phenylethanol were synthesized using a whole-cell biocatalyst in more than 99 % optical purity. Keywords (R)-2-octanol dehydrogenase . Pichia finlandica . Chiral alcohol . (S)-haloalcohol . Ethyl (S)-4-chloro-3-hydroxybutanoate
Introduction In the pharmaceutical industry, the current trend is the development of single enantiomer drugs. The production of single enantiomers of chiral raw materials and chiral intermediates has become increasingly important (Patel H. Yamamoto (*) : M. Kudoh Green Product Development Center, R&D Management, Daicel Corporation, 1-1 Shinko-cho, Myoko, Niigata 944-8550, Japan e-mail: [email protected] URL: http://www.daicel.com/en
2002; Rouhi 2004). Optically active alcohols are molecules with a wide range of synthetic applications and are synthesized by chiral pool method, optical resolution method, and asymmetric synthesis (Patel 2004, Yamamoto and Matsuyama 2006). Asymmetric reduction of ketones with biocatalysts, in particular, is more advantageous than other methods at the points of chemo-, regio-, and stereoselectivity (Devaux-Basseguy et al. 1997; Hummel 1999; Kataoka et al. 2003). Among enzymes to reduce carbonyl compounds, alcohol dehydrogenases (ADHs) are more advantageous than carbonyl reductases which exclusively catalyze the reduction of carbonyl compounds and aldehydes because ADHs also catalyze the stereoselective oxidation of chiral alcohols and is used to synthesize optically pure alcohols by enantioselective oxidation and kinetic resolution. A variety of (S)-specific secondary ADHs have ever been discovered, purified, and characterized from Candida parapsilosis (Peters et al. 1993; Yamamoto et al. 1995), Corynebacterium sp. (Itoh et al. 1997), Nocardia fusca (Xie et al. 1999), Rhodococus erythropolis (Zelinski et al. 1994), Rhodococcus ruber (Stampfer et al. 2002
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