Skip to main content
SpringerLink
Log in

Characterization and Application of a Robust Glucose Dehydrogenase from Paenibacillus pini for Cofactor Regeneration in Biocatalysis

  • Original research article
  • Published:
Indian Journal of Microbiology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Prier CK, Kosjek B (2019) Recent preparative applications of redox enzymes. Curr Opin Chem Biol 49:105–112. https://doi.org/10.1016/j.cbpa.2018.11.011

    Article  CAS  PubMed  Google Scholar 

  2. Wu H, Tian C, Song X, Liu C, Yang D, Jiang Z (2013) Methods for the regeneration of nicotinamide coenzymes. Green Chem 15:1773–1789. https://doi.org/10.1039/C3GC37129H

    Article  CAS  Google Scholar 

  3. Sengupta A, Sunder AV, Sohoni SV, Wangikar PP (2018) The effect of CO2 in enhancing photosynthetic cofactor recycling for alcohol dehydrogenase mediated chiral synthesis in cyanobacteria. J Biotechnol 289:1–6. https://doi.org/10.1016/j.jbiotec.2018.11.002

    Article  CAS  PubMed  Google Scholar 

  4. Han L, Liang B (2018) New approaches to NAD(P)H regeneration in the biosynthesis systems. World J Microbiol Biotechnol 34:141. https://doi.org/10.1007/s11274-018-2530-8

    Article  CAS  PubMed  Google Scholar 

  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

    Article  CAS  PubMed  PubMed Central  Google 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

    Article  CAS  Google 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

    Article  CAS  Google 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

    Article  CAS  PubMed  Google 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

    Article  CAS  PubMed  Google Scholar 

  10. Smith DL, Budgen N, Bungard JS, Danson JM, Hough WD (1989) Purification and characterization of glucose dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum. Biochem J 261:973–977. https://doi.org/10.1042/bj2610973

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Naeem M, Rehman AU, Shen B, Ye L, Yu H (2018) Semi-rational engineering of carbonyl reductase YueD for efficient biosynthesis of halogenated alcohols with in situ cofactor regeneration. Biochem Eng J 137:62–70. https://doi.org/10.1016/j.bej.2018.05.008

    Article  CAS  Google 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

    Article  CAS  PubMed  Google 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

    Article  CAS  Google 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

    Article  Google Scholar 

  15. Chanique AM, Parra LP (2018) Protein engineering for nicotinamide coenzyme specificity in oxidoreductases: attempts and challenges. Front Microbiol 9:194. https://doi.org/10.3389/fmicb.2018.00194

    Article  PubMed  PubMed Central  Google Scholar 

  16. Kratzer R, Woodley JM, Nidetzky B (2015) Rules for biocatalyst and reaction engineering to implement effective NAD(P)H-dependent, whole cell bioreductions. Biotechnol Adv 33:1641–1652. https://doi.org/10.1016/j.biotechadv.2015.08.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sohoni SV, Nelapati D, Sathe S, Javadekar-Subhedar V, Gaikaiwari RP, Wangikar PP (2015) Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli. Bioresour Technol 188:202–208. https://doi.org/10.1016/j.biortech.2015.02.038

    Article  CAS  PubMed  Google Scholar 

  18. Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3

    Article  CAS  PubMed  Google Scholar 

  19. Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227:680–685. https://doi.org/10.1038/227680a0

    Article  CAS  PubMed  Google Scholar 

  20. Vidal LS, Kelly CL, Mordaka PM, Heap JT (2018) Review of NAD(P)H dependent oxidoreductases: properties, engineering and application. BBA Prot Prot 1866:327–347. https://doi.org/10.1016/j.bbapap.2017.11.005

    Article  CAS  Google Scholar 

  21. Oppermann U, Filling C, Hult M, Shafqat N, Wu XQ, Lindh M, Shafqat J, Nordling E, Kallberg Y, Persson B, Jornvall H (2003) Short-chain dehydrogenases/reductases (SDR): the 2002 update. Chem Biol Interact 143:247–253. https://doi.org/10.1016/S0009-2797(02)00164-3

    Article  CAS  PubMed  Google Scholar 

  22. Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172. https://doi.org/10.3389/fmicb.2014.00172

    Article  PubMed  PubMed Central  Google Scholar 

  23. Geertz-Hansen HM, Blom N, Feist AM, Brunak S, Petersen TN (2014) Cofactory: sequence-based prediction of cofactor specificity of Rossmann folds. Prot Struct Func Bioinform 82:1819–1828. https://doi.org/10.1002/prot.24536

    Article  CAS  Google Scholar 

  24. Nishioka T, Yasutake Y, Nishiya Y, Tamura T (2012) Structure-guided mutagenesis for the improvement of substrate specificity of Bacillus megaterium glucose-1-dehydrogenase IV. FEBS J 279:3264–3275. https://doi.org/10.1111/j.1742-4658.2012.08713.x

    Article  CAS  PubMed  Google Scholar 

  25. Ding H, Gao F, Liu D, Li Z, Xu X, Wu M, Xhao Y (2013) Significant improvement of thermostability of glucose-1-dehydrogenase by introducing disulfide bonds at the tetramer interface. Enzym Microb Technol 53:365–372. https://doi.org/10.1016/j.enzmictec.2013.08.001

    Article  CAS  Google Scholar 

  26. Ramaley RF, Vasantha N (1983) Glycerol protection and purification of Bacillus subtilis glucose dehydrogenase. J Biol Chem 258:12558–12565

    CAS  PubMed  Google Scholar 

  27. Kumar A, Dhar K, Kanwar SS, Arora PK (2016) Lipase catalysis in organic solvents: advantages and applications. Biol Proced Online 18:2. https://doi.org/10.1186/s12575-016-0033-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Li BJ, Li YX, Bai DM, Zhang X, Yang HY, Wang J, Liu G, Yue JJ, Ling Y, Zhou DS, Chen HP (2014) Whole-cell biotransformation systems for reduction of prochiral carbonyl compounds to chiral alcohol in Escherichia coli. Sci Rep 4:5. https://doi.org/10.1038/srep06750

    Article  Google 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

    Article  CAS  PubMed  Google Scholar 

  30. Jakoblinnert A, Mladenov R, Paul A, Sibilla F, Schwaneberg U, Ansorge-Schumacher MB, De María PD (2011) Asymmetric reduction of ketones with recombinant E. coli whole cells in neat substrates. Chem Commun 47:12230–12232. https://doi.org/10.1039/c1cc14097c

    Article  CAS  Google 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

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.

Author information

Author notes

  1. Shikha Shah and Avinash Vellore Sunder have equally contributed to this manuscript.

Authors and Affiliations

  1. Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai, 400076, India

    Shikha Shah, Avinash Vellore Sunder, Pooja Singh & Pramod P. Wangikar

  2. Department of Biochemistry, Savitribai Phule Pune University, Pune, 411007, India

    Pooja Singh

Authors
  1. Shikha Shah
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Avinash Vellore Sunder
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pooja Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pramod P. Wangikar
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Pramod P. Wangikar.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 508 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shah, S., Sunder, A.V., Singh, P. et al. Characterization and Application of a Robust Glucose Dehydrogenase from Paenibacillus pini for Cofactor Regeneration in Biocatalysis. Indian J Microbiol 60, 87–95 (2020). https://doi.org/10.1007/s12088-019-00834-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12088-019-00834-w

Keywords

Search

Navigation