Advertisement

Highly efficient conversion of 1-cyanocycloalkaneacetonitrile using a “super nitrilase mutant”

  • Zhe Xu
  • Neng Xiong
  • Shu-Ping Zou
  • Yu-Xiao Liu
  • Zhi-Qiang liu
  • Ya-Ping XueEmail author
  • Yu-Guo Zheng
Research Paper
  • 57 Downloads

Abstract

Nitrilase is the member of carbon–nitrogen hydrogen hydrolase superfamily, which has been widely used for the hydrolysis of nitriles into corresponding carboxylic acids. But most nitrilases are plagued by product inhibition in the industrial application. In this study, a “super nitrilase mutant” of nitrilase with high activity, thermostability and improved product tolerance from Acidovorax facilis ZJB09122 was characterized. Then, an efficient process was developed by employing the whole cell of recombinant E. coli for the conversion of high concentration of 1-cyanocyclohexylacetonitrile-to-1-cyanocyclohexaneacetic acid, an important intermediate of gabapentin. Under the optimized conditions, the higher substrate concentrations such as 1.3 M, 1.5 M and 1.8 M could be hydrolyzed by 13.58 g DCW/L with outstanding productivity (> 740 g/L/day). This study developed a highly efficient bioprocess for the preparation of 1-cyanocyclohexaneacetic acid which has the great potential for industrial application.

Keywords

Nitrilase Product tolerance Biocatalysis 1-Cyanocyclohexaneacetic acid Gabapentin 

Notes

Acknowledgements

This work was funded by the National Natural Science Foundation of China (nos. 21476210 and 21706235).

References

  1. 1.
    Effenberger F, Oßwald S (2001) Selective hydrolysis of aliphatic dinitriles to monocarboxylic acids by a nitrilase from Arabidopsis thaliana. Synthesis 2001(12):1866–1872CrossRefGoogle Scholar
  2. 2.
    Chauhan S, Wu S, Blumerman S, Fallon RD, Gavagan JE, DiCosimo R, Payne MS (2003) Purification, cloning, sequencing and over-expression in Escherichia coli of a regioselective aliphatic nitrilase from Acidovorax facilis 72W. Appl Microbiol Biotechnol 61(2):118–122CrossRefGoogle Scholar
  3. 3.
    DeSantis G, Wong K, Farwell B, Chatman K, Zhu ZL, Tomlinson G, Huang HJ, Tan XQ, Bibbs L, Chen P, Kretz K, Burk MJ (2003) Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM). J Am Chem Soc 125(38):11476–11477CrossRefGoogle Scholar
  4. 4.
    Zhang XH, Liu ZQ, Xue YP, Zheng YG (2014) Activity improvement of a regioselective nitrilase from Acidovorax facilis and its application in the production of 1-(cyanocyclohexyl) acetic acid. Process Biochem 49(12):2141–2148CrossRefGoogle Scholar
  5. 5.
    Veselá AB, Rucká L, Kaplan O, Pelantová H, Nešvera J, Pátek M, Martínková L (2015) Bringing nitrilase sequences from databases to life: the search for novel substrate specificities with a focus on dinitriles. Appl Microbiol Biotechnol 100(5):2193–2202CrossRefGoogle Scholar
  6. 6.
    Brady D, Dube N, Petersen R (2006) Green chemistry: highly selective biocatalytic hydrolysis of nitrile compounds. S Afr J Sci 102(7–8):339–344Google Scholar
  7. 7.
    Singh R, Sharma R, Tewari N, Geetanjali, Rawat DS (2006) Nitrilase and its application as a ‘green’ catalyst. Chem Biodivers 3(12):1279–1287CrossRefGoogle Scholar
  8. 8.
    Martínková L, Kren V (2010) Biotransformations with nitrilases. Curr Opin Chem Biol 14(2):130–137CrossRefGoogle Scholar
  9. 9.
    Martínková L, Rucká L, Nešvera J, Pátek M (2016) Recent advances and challenges in the heterologous production of microbial nitrilases for biocatalytic applications. World J Microbiol Biotechnol 33(1):8CrossRefGoogle Scholar
  10. 10.
    Bhalla TC, Kumar V, Kumar V, Thakur N, Savitri (2018) Nitrile metabolizing enzymes in biocatalysis and biotransformation. Appl Biochem Biotechnol.  https://doi.org/10.1007/s12010-018-2705-7 CrossRefPubMedGoogle Scholar
  11. 11.
    Xue YP, Jiao B, Hua DE, Cheng F, Liu ZQ, Zheng YG (2017) Improving catalytic performance of an arylacetonitrilase by semirational engineering. Bioprocess Biosyst Eng 40(10):1565–1572CrossRefGoogle Scholar
  12. 12.
    Zhang ZJ, Pan J, Li CX, Yu HL, Zheng GW, Ju X, Xu JH (2014) Efficient production of (R)-(−)-mandelic acid using glutaraldehyde cross-linked Escherichia coli cells expressing Alcaligenes sp. nitrilase. Bioprocess Biosyst Eng 37(7):1241–1248CrossRefGoogle Scholar
  13. 13.
    Zhang CS, Zhang ZJ, Li CX, Yu HL, Zheng GW, Xu J-H (2012) Efficient production of (R)-O-chloromandelic acid by deracemization of O-chloromandelonitrile with a new nitrilase mined from Labrenzia aggregata. Appl Microbiol Biotechnol 95(1):91–99CrossRefGoogle Scholar
  14. 14.
    Cooling FB, Fager SK, Fallon RD, Folsom PW, Gallagher FG, Gavagan JE, Hann EC, Herkes FE, Phillips RL, Sigmund A, Wagner LW, Wu W, DiCosimo R (2001) Chemoenzymatic production of 1,5-dimethyl-2-piperidone. J Mol Catal B Enzym 11(4–6):295–306CrossRefGoogle Scholar
  15. 15.
    Burns MP, Wong JW (2004) Biocatalytic preparation of 1-cyanocyclohexaneacetic acid. WO2004111256 A1. https://patents.google.com/patent/WO2004111256A1
  16. 16.
    Zhu D, Mukherjee C, Biehl ER, Hua L (2007) Nitrilase-catalyzed selective hydrolysis of dinitriles and green access to the cyanocarboxylic acids of pharmaceutical importance. Adv Synth Catal 349(10):1667–1670CrossRefGoogle Scholar
  17. 17.
    Duan YT, Yao PY, Ren J, Han C, Li Q, Yuan J, Feng JH, Wu QQ, Zhu DM (2014) Biocatalytic desymmetrization of 3-substituted glutaronitriles by nitrilases. A convenient chemoenzymatic access to optically active (S)-Pregabalin and (R)-Baclofen. Sci China Chem 57(8):1164–1171CrossRefGoogle Scholar
  18. 18.
    Xie ZY, Feng JL, Garcia E, Bernett M, Yazbeck D, Tao JH (2006) Cloning and optimization of a nitrilase for the synthesis of (3S)-3-cyano-5-methyl hexanoic acid. J Mol Catal B Enzym 41(3–4):75–80CrossRefGoogle Scholar
  19. 19.
    Ishikawa T, Okazaki K, Kuroda H, Itoh K, Mitsui T, Hori H (2007) Molecular cloning of Brassica rapa nitrilases and their expression during clubroot development. Mol Plant Pathol 8(5):623–637CrossRefGoogle Scholar
  20. 20.
    Zheng RC, Zheng YG, Zhang Q, Huang YM, Li Y, Weng J, Liu T, Fan W (2015) Nitrilase from Arabis alpina, its encoding gene, vector, recombinant bacterial strain and uses thereof. US20170355976 A1. https://patents.google.com/patent/US20170355976A1/
  21. 21.
    Kobayashi M, Nagasawa T, Yamada H (1988) Regiospecific hydrolysis of dinitrile compounds by nitrilase from Rhodococcus rhodochrous J1. Appl Microbiol Biotechnol 29(2–3):231–233Google Scholar
  22. 22.
    Bengis-Garber C, Gutman AL (1989) Selective hydrolysis of dinitriles into cyano-carboxylic acids by Rhodococcus rhodochrous NCIB 11216. Appl Microbiol Biotechnol 32(1):11–16CrossRefGoogle Scholar
  23. 23.
    Dadd MR, Claridge TDW, Walton R, Pettman AJ, Knowles CJ (2001) Regioselective biotransformation of the dinitrile compounds 2-, 3- and 4-(cyanomethyl) benzonitrile by the soil bacterium Rhodococcus rhodochrous LL100-21. Enzyme Microb Technol 29(1):20–27CrossRefGoogle Scholar
  24. 24.
    Rey P, Rossi JC, Taillades J, Gros G, Nore O (2004) Hydrolysis of nitriles using an immobilized nitrilase: applications to the synthesis of methionine hydroxy analogue derivatives. J Agric Food Chem 52(26):8155–8162CrossRefGoogle Scholar
  25. 25.
    Wang HL, Li GN, Li MY, Wei DZ, Wang XD (2014) A novel nitrilase from Rhodobacter sphaeroides LHS-305: cloning, heterologous expression and biochemical characterization. World J Microbiol Biotechnol 30(1):245–252CrossRefGoogle Scholar
  26. 26.
    Xue YP, Wang YP, Xu Z, Liu ZQ, Shu XR, Jia DX, Zheng YG, Shen YC (2015) Chemoenzymatic synthesis of gabapentin by combining nitrilase-mediated hydrolysis with hydrogenation over Raney-nickel. Catal Commun 66:121–125CrossRefGoogle Scholar
  27. 27.
    Xue YP, Zhong HJ, Zou SP, Zheng YG (2017) Efficient chemoenzymatic synthesis of gabapentin by control of immobilized biocatalyst activity in a stirred bioreactor. Biochem Eng J 125:190–195CrossRefGoogle Scholar
  28. 28.
    Zou SP, Huang JW, Xue YP, Zheng YG (2018) Highly efficient production of 1-cyanocyclohexaneacetic acid by cross-linked cell aggregates (CLCAs) of recombinant E. coli harboring nitrilase gene. Process Biochem 65:93–99CrossRefGoogle Scholar
  29. 29.
    Xue YP, Shu XR, Zou SP, Wang YJ, Zheng YG (2015) Efficient recovery of 1-cyanocyclohexaneacetic acid by ion-exchange process. Sep Sci Technol 50(17):2717–2725Google Scholar
  30. 30.
    Xu Z, Cai T, Xiong N, Zou SP, Xue YP, Zheng YG (2018) Engineering the residues on “A” surface and C-terminal region to improve thermostability of nitrilase. Enzyme Microb Technol 113:52–58CrossRefGoogle Scholar
  31. 31.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  32. 32.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680CrossRefGoogle Scholar
  33. 33.
    Sewell BT, Berman MN, Meyers PR, Jandhyala D, Benedik MJ (2003) The cyanide degrading nitrilase from Pseudomonas stutzeri AK61 is a two-fold symmetric, 14-subunit spiral. Structure 11(11):1413–1422CrossRefGoogle Scholar
  34. 34.
    Wu S, Fogiel AJ, Petrillo KL, Jackson RE, Parker KN, Dicosimo R, Ben-Bassat A, O’Keefe DP, Payne MS (2008) Protein engineering of nitrilase for chemoenzymatic production of glycolic acid. Biotechnol Bioeng 99(3):717–720CrossRefGoogle Scholar
  35. 35.
    Xue YP, Liu ZQ, Xu M, Wang YJ, Zheng YG, Shen YC (2010) Enhanced biotransformation of (R,S)-mandelonitrile to (R)-(−)-mandelic acid with in situ production removal by addition of resin. Biochem Eng J 53(1):143–149CrossRefGoogle Scholar
  36. 36.
    Yang C, Wang X, Wei D (2011) A new nitrilase-producing strain named Rhodobacter sphaeroides LHS-305: biocatalytic characterization and substrate specificity. Appl Biochem Biotechnol 165(7):1556–1567CrossRefGoogle Scholar
  37. 37.
    Sharma NN, Sharma M, Bhalla TC (2011) An improved nitrilase-mediated bioprocess for synthesis of nicotinic acid from 3-cyanopyridine with hyperinduced Nocardia globerula NHB-2. J Ind Microbiol Biotechnol 38(9):1235–1243CrossRefGoogle Scholar
  38. 38.
    Xue YP, Xu M, Chen HS, Liu ZQ, Wang YJ, Zheng YG (2013) A novel integrated bioprocess for efficient production of (R)-(–)-mandelic acid with immobilized Alcaligenes faecalis ZJUTB10. Org Process Res Dev 17(2):213–220CrossRefGoogle Scholar
  39. 39.
    Zhu XY, Gong JS, Li H, Lu ZM, Shi JS, Xu ZH (2014) Bench-scale biosynthesis of isonicotinic acid from 4-cyanopyridine by Pseudomonas putida. Chem Pap 68(6):739–744CrossRefGoogle Scholar
  40. 40.
    Vaughan PA, Knowles CJ, Cheetham PSJ (1989) Conversion of 3-cyanopyridine to nicotinic acid by Nocardia rhodochrous LL100-21. Enzyme Microb Technol 11(12):815–823CrossRefGoogle Scholar
  41. 41.
    Almatawah QA, Cowan DA (1999) Thermostable nitrilase catalysed production of nicotinic acid from 3-cyanopyridine. Enzyme Microb Technol 25(8):718–724CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Zhe Xu
    • 1
    • 2
  • Neng Xiong
    • 1
    • 2
  • Shu-Ping Zou
    • 1
    • 2
  • Yu-Xiao Liu
    • 1
    • 2
  • Zhi-Qiang liu
    • 1
    • 2
  • Ya-Ping Xue
    • 1
    • 2
    Email author
  • Yu-Guo Zheng
    • 1
    • 2
  1. 1.Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and BioengineeringZhejiang University of TechnologyHangzhouChina
  2. 2.Engineering Research Center of Bioconversion and Biopurification of Ministry of EducationZhejiang University of TechnologyHangzhouChina

Personalised recommendations