Characterization and Application of a Robust Glucose Dehydrogenase from Paenibacillus pini for Cofactor Regeneration in Biocatalysis
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Glucose dehydrogenases are important auxiliary enzymes in biocatalysis, employed in the regeneration of reduced nicotinamide cofactors for oxidoreductase catalysed reactions. Here we report the identification and characterization of a novel glucose-1-dehydrogenase (GDH) from Paenibacillus pini that prefers NAD+ as cofactor over NADP+. The purified recombinant P. pini GDH displayed a specific activity of 247.5 U/mg. The enzyme was stable in the pH range 4–8.5 and exhibited excellent thermostability till 50 °C for 24 h, even in the absence of NaCl or glycerol. Paenibacillus pini GDH was also tolerant to organic solvents, demonstrating its potential for recycling cofactors for biotransformation. The potential application of the enzyme was evaluated by coupling with a NAD+-dependent alcohol dehydrogenase for the reduction of acetophenone and ethyl-4-chloro-3-oxo-butanoate. Conversions higher than 95% were achieved within 2 h with low enzyme loading using lyophilized cell lysate, suggesting that P. pini GDH could be highly effective for recycling NADH in redox biocatalysis.
KeywordsCofactor regeneration Oxidoreductase Dehydrogenase Biocatalysis Paenibacillus pini
AVS acknowledges the award of National Postdoctoral Fellowship by the Science and Engineering Research Board (SERB) (Grant No. PDF_2016_003685), Government of India. The authors acknowledge financial support from the Wadhwani Research Center for Bioengineering at Indian Institute of Technology Bombay.
AVS and PW designed the research. SS, PS and AVS performed the research and analysed the data. SS, AVS and PW wrote the paper. All authors read and approved the final manuscript.
- 5.Nagao T, Mitamura T, Wang XH, Negoro S, Yomo T, Urabe I, Okada H (1992) Cloning, nucleotide-sequences, and enzymatic-properties of glucose-dehydrogenase isozymes from Bacillus megaterium IAM1030. J Bacteriol 174:5013–5020. https://doi.org/10.1128/jb.174.15.5013-5020.1992 CrossRefPubMedPubMedCentralGoogle Scholar
- 6.Diederichs S, Linn K, Luckgen J, Klement T, Grosch JH, Honda K, Ohtake H, Buchs J (2015) High-level production of (5S)-hydroxyhexane-2-one by two thermostable oxidoreductases in a whole-cell catalytic approach. J Mol Catal B Enzym 121:37–44. https://doi.org/10.1016/j.molcatb.2015.08.001 CrossRefGoogle Scholar
- 7.Pongtharangkul T, Chuekitkumchorn P, Suwanampa N, Payongsri P, Honda K, Panbangred W (2015) Kinetic properties and stability of glucose dehydrogenase from Bacillus amyloliquefaciens SB5 and its potential for cofactor regeneration. AMB Expr 5:68. https://doi.org/10.1186/s13568-015-0157-9 CrossRefGoogle Scholar
- 8.Ding HT, Du YQ, Liu DF, Li ZL, Chen XJ, Zhao YH (2011) Cloning and expression in E. coli of an organic solvent-tolerant and alkali-resistant glucose 1-dehydrogenase from Lysinibacillus sphaericus G10. Bioresour Technol 102:1528–1536. https://doi.org/10.1016/j.biortech.2010.08.018 CrossRefPubMedGoogle Scholar
- 9.Hyun J, Abigail M, Choo JW, Ryu J, Kim HK (2016) Effects of N-/C-terminal extra tags on the optimal reaction conditions, activity, and quaternary structure of Bacillus thuringiensis glucose-1-dehydrogenase. J Microbiol Biotechnol 26:1708–1716. https://doi.org/10.4014/jmb.1603.03021 CrossRefPubMedGoogle Scholar
- 12.Cui ZM, Zhang JD, Fan XJ, Zheng GW, Chang HH, Wei WL (2017) Highly efficient bioreduction of 2-hydroxyacetophenone to (S)- and (R)-1-phenyl-1,2-ethanediol by two substrate tolerance carbonyl reductases with cofactor regeneration. J Biotechnol 243:1–9. https://doi.org/10.1016/j.jbiotec.2016.12.016 CrossRefPubMedGoogle Scholar
- 13.Shah S, Agera R, Sharma P, Sunder AV, Bajwa H, James HM, Gaikaiwari RP, Wangikar PP (2018) Development of biotransformation process for asymmetric reduction with novel anti-Prelog NADH-dependent alcohol dehydrogenases. Proc Biochem 70:71–78. https://doi.org/10.1016/j.procbio.2018.04.016 CrossRefGoogle Scholar
- 14.Zhu YH, Liu CY, Cai S, Guo LB, Kim IW, Kalia VC, Lee JK, Zhang YW (2019) Cloning, expression and characterization of a highly active alcohol dehydrogenase for production of ethyl-(S)-4-chloro-3-hydroxybutyrate. Ind J Microbiol 59:225–233. https://doi.org/10.1007/s12088-019-00795-0 CrossRefGoogle Scholar
- 29.He YC, Tao ZC, Zhang X, Yang ZX, Xu JH (2014) Highly efficient synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate and its derivatives by a robust NADH-dependent reductase from E. coli CCZU-K14. Bioresour Technol 161:461–464. https://doi.org/10.1016/j.biortech.2014.03.133 CrossRefPubMedGoogle Scholar
- 31.Dascier D, Kambourakis S, Hua L, Rozzell JD, Stewart JD (2014) Influence of cofactor regeneration strategies on preparative scale, asymmetric carbonyl reductions by engineered Escherichia coli. Org Process Res Dev 18:793–800. https://doi.org/10.1021/op400312n CrossRefPubMedPubMedCentralGoogle Scholar