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Upscale production of (R)-mandelic acid with a stereospecific nitrilase in an aqueous system

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Abstract

(R)-Mandelic acid (R-MA) is a key precursor for the synthesis of semi-synthetic penicillin, cephalosporin, anti-obesity drugs, antitumor agents, and chiral resolving agents for the resolution of racemic alcohols and amines. In this study, an enzymatic method for the large-scale production of R-MA by a stereospecific nitrilase in an aqueous system was developed. The nitrilase activity of the Escherichia coli BL21(DE3)/pET-Nit whole cells reached 138.6 U/g in a 20,000-L fermentor. Using recombinant E. coli cells as catalyst, 500 mM R,S-mandelonitrile (R,S-MN) was resolved into 426 mM (64.85 g/L) R-MA within 8 h, and the enantiomeric excess (ee) value of R-MA reached 99%. During the purification process, pure R-MA with a recovery rate of 78.8% was obtained after concentration and crystallization. This study paved the foundation for the upscale production of R-MA using E. coli whole cells as biocatalyst.

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References

  1. Bornscheuer UT, Huisman GW, Kazlauskas RJ, Lutz S, Moore JC, Robins K (2012) Engineering the third wave of biocatalysis. Nature 485:185–194

    Article  CAS  Google Scholar 

  2. Liu ZQ, Ye JJ, Shen ZY, Hong HB, Yan JB, Lin Y, Chen ZX, Zheng YG, Shen YC (2015) Upscale production of ethyl (S)-4-chloro-3-hydroxybutanoate by using carbonyl reductase coupled with glucose dehydrogenase in aqueous-organic solvent system. App Microbiol Biotechnol 5:2119–2129

    Article  Google Scholar 

  3. Zhang ZJ, Yu HL, Imanaka T, Xu JH (2015) Efficient production of (R)-(-)-mandelic acid by isopropanol-permeabilized recombinant E. coli cells expressing Alcaligenes sp nitrilase. Biochem Eng J 95:71–77

    Article  CAS  Google Scholar 

  4. Yang ZY, Ni Y, Lu ZY, Liao XR, Zheng YG, Sun ZH (2011) Industrial production of S-2,2-dimethylcyclopropanecarboxamide with a novel recombinant R-amidase from Delftia tsuruhatensis. Process Biochem 46:182–187

    Article  CAS  Google Scholar 

  5. Gong JS, Li H, Lu ZM, Shi JS, Xu ZH (2015) Recent progress in the application of nitrilase in the biocatalytic synthesis of pharmaceutical intermediates. Prog Chem 27:448–458

    CAS  Google Scholar 

  6. Gong JS, Li H, Zhu XY, Lu ZM, Wu Y, Shi JS, Xu ZH (2012) Fungal his-tagged nitrilase from Gibberella intermedia: gene cloning, heterologous expression and biochemical properties. PLoS ONE 7:11

    CAS  Google Scholar 

  7. Gong JS, Zhang Q, Gu BC, Dong TT, Li H, Li H, Lu ZM (2018) Efficient biocatalytic synthesis of nibotinic acid by recombinant nitrilase via high density culture. Bioresour Technol 260:427–431

    Article  CAS  Google Scholar 

  8. Fan H, Chen L, Sun H, Wang H, Liu Q, Ren Y, Wei D (2017) Characterization of a novel nitrilase, BGC4, from Paraburkholderia graminis showing wide-spectrum substrate specificity, a potential versatile biocatalyst for the degradation of nitriles. Biotechnol Lett 39:1725–1731

    Article  CAS  Google Scholar 

  9. Mathew S, Nadarajan SP, Sundaramoorthy U, Jeon H, Chung T, Yun H (2017) Biotransformation of beta-keto nitriles to chiral (S)-beta-amino acids using nitrilase and omega-transaminase. Biotechnol Lett 39:535–543

    Article  CAS  Google Scholar 

  10. Xu Z, Xiong N, Zou SP, Liu YX, Liu ZQ, Xue YP, Zheng YG (2019) Highly efficient conversion of 1-cyanocycloalkaneacetonitrile using a "super nitrilase mutant". Bioprocess Biosyst Eng 42:455–463

    Article  CAS  Google Scholar 

  11. Piotrowski M (2008) Primary or secondary? Versatile nitrilases in plant metabolism. Phytochemistry 69:2655–2667

    Article  CAS  Google Scholar 

  12. Liu ZQ, Zhang XH, Xue YP, Xu M, Zheng YG (2014) Improvement of Alcaligenes faecalis nitrilase by gene site saturation mutagenesis and its application in stereospecific biosynthesis of (R)-(−)-mandelic acid. J Agric Food Chem 62:4685–4694

    Article  CAS  Google Scholar 

  13. Liu ZQ, Dong LZ, Cheng F, Xue YP, Wang YS, Ding JN, Zheng YG, Shen YC (2011) Gene cloning, expression, and characterization of a nitrilase from Alcaligenes faecalis ZJUTB10. J Agric Food Chem 59:11560–11570

    Article  CAS  Google Scholar 

  14. Liu ZQ, Baker PJ, Cheng F, Xue YP, Zheng YG, Shen YC (2013) Screening and improving the recombinant nitrilases and application in biotransformation of iminodiacetonitrile to iminodiacetic acid. PLoS ONE 8:6

    Google Scholar 

  15. Pace HC, Hodawadekar SC, Draganescu A, Huang J, Bieganowski P, Pekarsky Y, Croce CM, Brenner C (2000) Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers. Curr Biol 10:907–917

    Article  CAS  Google Scholar 

  16. O'Reilly C, Turner PD (2003) The nitrilase family of CN hydrolysing enzymes—a comparative study. J Appl Microbiol 95:1161–1174

    Article  CAS  Google Scholar 

  17. Ni KF, Wang HL, Zhao L, Zhang MJ, Zhang SY, Ren YH, Wei DZ (2013) Efficient production of (R)-(−)-mandelic acid in biphasic system by immobilized recombinant E. coli. J Biotechnol 167:433–440

    Article  CAS  Google Scholar 

  18. Chen HB, Lin YB, Liu ZP (2002) Study of synthesis of mandelic acid. Chin J Syn Chem 10:185

    CAS  Google Scholar 

  19. Yadav GD, Sivakumar P (2004) Enzyme-catalysed optical resolution of mandelic acid via R, S-methyl mandelate in non-aqueous media. Biochem Eng J 19:101–107

    Article  CAS  Google Scholar 

  20. Zhang GY, Tao J, Zheng LY, Cao SG (2011) Molecular basis for stereospecific hydrolysis of ethyl mandelate by thermophilic esterase. Chem Res Chin U 27:841–844

    Google Scholar 

  21. Mark AKP, Szajewski RP, Whitesides GM (1981) Enzymic conversion of α-keto aldehydes to optically active α-hydroxy acids using glyoxalase I and II. J Org Chem 46:4682–4685

    Article  Google Scholar 

  22. Martinkova L, Kren V (2018) Biocatalytic production of mandelic acid and analogues: a review and comparison with chemical processes. Appl Microbiol Biotechnol 102:3893–3900

    Article  CAS  Google Scholar 

  23. Zhang XH, Liu ZQ, Xue YP, Xu M, Zheng YG (2015) Nitrilase-catalyzed conversion of (R, S)-mandelonitrile by immobilized recombinant Escherichia coli cells harboring nitrilase. Biotechnol Appl Biochem 63:479–489

    Article  Google Scholar 

  24. 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

    Article  CAS  Google Scholar 

  25. Xue YP, Liu ZQ, Xu M, Wang YJ, Zheng YG (2011) Efficient separation of (R)-(−)-mandelic acid biosynthesized from (R, S)-mandelonitrile by nitrilase using ion-exchange process. J Chem Technol Biot 86:391–397

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was financially supported by grants from the Natural Science from Education Department of Anhui Province (No. KJ2018A0563), Talent Research Foundation of Hefei University (No. 18-19RC10), Key Research and Development Plan of Anhui Province (No. 1804a07020120), Major Science and Technology Projects in Anhui Province (No. 18030701145).

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Authors and Affiliations

  1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China

    Xin-Hong Zhang, Xue Cai, Ya-Ping Xue, Zhi-Qiang Liu & Yu-Guo Zheng

  2. School of Biology, Food and Environment, Hefei University, Hefei, 230630, China

    Xin-Hong Zhang & Chu-Yan Wang

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  1. Xin-Hong Zhang
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  2. Chu-Yan Wang
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  3. Xue Cai
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  4. Ya-Ping Xue
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  5. Zhi-Qiang Liu
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Correspondence to Zhi-Qiang Liu.

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Zhang, XH., Wang, CY., Cai, X. et al. Upscale production of (R)-mandelic acid with a stereospecific nitrilase in an aqueous system. Bioprocess Biosyst Eng 43, 1299–1307 (2020). https://doi.org/10.1007/s00449-020-02326-4

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  • DOI: https://doi.org/10.1007/s00449-020-02326-4

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