Applied Microbiology and Biotechnology

, Volume 98, Issue 10, pp 4437–4443 | Cite as

Development of an improved phenylacetaldehyde reductase mutant by an efficient selection procedure

  • Yoshihide MakinoEmail author
  • Nobuya Itoh
Biotechnologically relevant enzymes and proteins


Chiral alcohols are valuable as diverse chemicals and synthetic intermediate materials. Phenylacetaldehyde reductase (PAR) is an enzyme that converts a wide variety of ketones into chiral alcohols with high optical purity. When an alcohol such as 2-propanol is used as a hydrogen donor, PAR itself will also mediate the regeneration of the coenzyme NADH in situ. Perceiving a capacity for improvement, we sought to develop a PAR that is able to convert higher concentrations of substrates in the presence of high concentrations of 2-propanol. The selection procedure for mutants was re-examined and a procedure able to select an effective amino acid substitution was established. Two advantageous amino acid substitutions were successfully selected using the procedure. When high-concentration substrate conversion reaction was subjected with a mutant that integrated both the two amino acid substitutions, near-complete conversions of m-chlorophenacyl chloride (m-CPC) (2.1 mmol/ml) and ethyl 4-chloro-3-oxobutanoate (ECOB) (1.9 mmol/ml) were achieved.


Phenylacetaldehyde reductase (PAR) Rhodococcus sp Optically pure alcohols Asymmetric reduction Coupled NADH regeneration Engineered enzymes 2-Propanol 



We thank Sumitomo Chemical for the supply of m-CPC.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. De Wildeman SM, Sonke T, Schoemaker HE, May O (2007) Biocatalytic reductions: from lab curiosity to “first choice”. Acc Chem Res 40:1260–1266PubMedCrossRefGoogle Scholar
  2. Goldberg K, Schroer K, Lütz S, Liese A (2007a) Biocatalytic ketone reduction—a powerful tool for the production of chiral alcohols—part I: processes with isolated enzymes. Appl Microbiol Biotechnol 76:237–248PubMedCrossRefGoogle Scholar
  3. Goldberg K, Schroer K, Lütz S, Liese A (2007b) Biocatalytic ketone reduction—a powerful tool for the production of chiral alcohols—part II: whole-cell reductions. Appl Microbiol Biotechnol 76:249–255PubMedCrossRefGoogle Scholar
  4. Itoh N, Isotani K, Nakamura M, Inoue K, Isogai Y, Makino Y (2012) Efficient synthesis of optically pure alcohols by asymmetric hydrogen-transfer biocatalysis: application of engineered enzymes in a 2-propanol-water medium. Appl Microbiol Biotechnol 93:1075–1085. doi: 10.1007/s00253-011-3447-4 PubMedCrossRefGoogle Scholar
  5. Itoh N, Matsuda M, Mabuchi M, Dairi T, Wang J (2002) Chiral alcohol production by NADH-dependent phenylacetaldehyde reductase coupled with in situ regeneration of NADH. Eur J Biochem 269:2394–2402PubMedCrossRefGoogle Scholar
  6. Itoh N, Morihama R, Wang J, Okada K, Mizuguchi N (1997) Purification and characterization of phenylacetaldehyde reductase from a styrene-assimilating Corynebacterium strain, ST-10. Appl Environ Microbiol 63:3783–3788Google Scholar
  7. Itoh N, Nakamura M, Inoue K, Makino Y (2007) Continuous production of chiral 1,3-butanediol using immobilized biocatalysts in a packed bed reactor: promising biocatalysis method with an asymmetric hydrogen-transfer bioreduction. Appl Microbiol Biotechnol 75:1249–1256PubMedCrossRefGoogle Scholar
  8. Kataoka M, Yamamoto K, Kawabata H, Wada M, Kita K, Yanase H, Shimizu S (1999) Stereoselective reduction of ethyl 4-chloro-3-oxobutanoate by Escherichia coli transformant cells coexpressing the aldehyde reductase and glucose dehydrogenase genes. Appl Microbiol Biotechnol 51:486–490Google Scholar
  9. Kroutil W, Mang H, Edegger K, Faber K (2004) Recent advances in the biocatalytic reduction of ketones and oxidation of sec-alcohols. Curr Opin Chem Biol 8:120–126PubMedCrossRefGoogle Scholar
  10. Makino Y, Dairi T, Itoh N (2007) Engineering the phenylacetaldehyde reductase mutant for improved substrate conversion in the presence of concentrated 2-propanol. Appl Microbiol Biotechnol 77:833–843PubMedCrossRefGoogle Scholar
  11. Makino Y, Inoue K, Dairi T, Itoh N (2005) Engineering of phenylacetaldehyde reductase for efficient substrate conversion in concentrated 2-propanol. Appl Environ Microbiol 71:4713–4720PubMedCentralPubMedCrossRefGoogle Scholar
  12. Matsuda T, Yamanaka T, Nakamura K (2009) Recent progress in biocatalysis for asymmetric oxidation and reduction. Tetrahedron Asymmetry 20:513–557CrossRefGoogle Scholar
  13. Moore JC, Pollard DJ, Kosjek B, Devine PN (2007) Advances in the enzymatic reduction of ketones. Acc Chem Res 40:1412–1419PubMedCrossRefGoogle Scholar
  14. Ni Y, Xu JH (2012) Biocatalytic ketone reduction: a green and efficient access to enantiopure alcohols. Biotechnol Adv 30:1279–1288. doi: 10.1016/j.biotechadv.2011.10.007 PubMedCrossRefGoogle Scholar
  15. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning—a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  16. Tseng WC, Lin JW, Wei TY, Fang TY (2008) A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method. Anal Biochem 375:376–378. doi: 10.1016/j.ab.2007.12.013 PubMedCrossRefGoogle Scholar
  17. Wang JC, Sakakibara M, Liu JQ, Dairi T, Itoh N (1999) Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding phenylacetaldehyde reductase from styrene-assimilating Corynebacterium sp. strain ST-10. Appl Microbiol Biotechnol 52:386–392Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.Biotechnology Research Center and Department of BiotechnologyToyama Prefectural UniversityImizuJapan

Personalised recommendations