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Cloning, purification and evaluation of the enzymatic properties of a novel arylacetonitrilase from Luminiphilus syltensis NOR5-1B: a potential biocatalyst for the synthesis of mandelic acid and its derivatives

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Abstract

Objective

To examine nitrilase-mediated hydrolysis of nitriles to produce optically pure α-hydroxycarboxylic acids.

Results

A novel nitrilase, GPnor51, from Luminiphilus syltensis NOR5-1B was discovered by genomic data mining. It could hydrolyze racemic o-chloromandelonitrile to (R)-o-chloromandelic acid with high enantioselectivity (ee 98.2 %). GPnor51 was overexpressed in Escherichia coli BL21 (DE3), purified, and its catalytic properties studied. GPnor51 had a broad substrate acceptance toward various nitriles with structure diversity. It was an arylacetonitrilase that uses arylacetonitriles as optimal substrates. The V max and K m of GPnor51 towards o-chloromandelonitrile were 1.9 μmol min−1 mg−1 protein and 0.38 mM, respectively. GPnor51 also demonstrated high enantioselectivity toward mandelonitrile and other substituted mandelonitrile.

Conclusion

This enzyme has a great potential for commercial production of optically pure (R)-mandelic acid and its derivatives.

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References

  • Banerjee A, Dubey S, Kaul P, Barse B, Piotrowski M, Banerjee UC (2005) Enantioselective nitrilase from Pseudomonas putida: cloning, heterologous expression, and bioreactor studies. Mol Biotechnol 41:35–41

    Article  Google Scholar 

  • Brenner C (2002) Catalysis in the nitrilase superfamily. Curr Opin Struct Biol 12:775–782

    Article  CAS  PubMed  Google Scholar 

  • DeSantis G, Zhu ZL, Greenberg WA, Wong K, Chaplin J, Hanson SR, Farwell B, Nicholson LW, Rand CL, Weiner DP, Robertson DE, Burk MJ (2002) An enzyme library approach to biocatalysis: development of nitrilases for enantioselective production of carboxylic acid derivatives. J Am Chem Soc 124:9024–9025

    Article  CAS  PubMed  Google Scholar 

  • Dong HP, Liu ZQ, Zheng YG, Shen YC (2010) Novel biosynthesis of (R)-ethyl-3-hydroxyglutarate with (R)-enantioselective hydrolysis of racemic ethyl 4-cyano-3-hydroxybutyate by Rhodococcus erythropolis. Appl Microbiol Biotechnol 87:1335–1345

    Article  CAS  PubMed  Google Scholar 

  • Ema T, Okita N, Ide S, Sakai T (2007) Highly enantioselective and efficient synthesis of methyl (R)-o-chloromandelate with recombinant E. coli: toward practical and green access to clopidogrel. Org Biomol Chem 5:1175–1176

    Article  CAS  PubMed  Google Scholar 

  • Gong JS, Lu ZM, Li H, Zhou ZM, Shi JS, Xu ZH (2013) Metagenomic technology and genome mining: emerging areas for exploring novel nitrilases. Appl Microbiol Biotechnol 97:6603–6611

    Article  CAS  PubMed  Google Scholar 

  • He YC, Zhang ZJ, Xu JH, Liu YY (2010) Biocatalytic synthesis of (R)-(−)-mandelic acid from racemic mandelonitrile by cetyltrimethylammonium bromide-permeabilized cells of Alcaligenes faecalis ECU0401. J Ind Microbiol Biotechnol 37:741–750

    Article  CAS  PubMed  Google Scholar 

  • Ju X, Yu HL, Pan J, Wei DZ, Xu JH (2010) Bioproduction of chiral mandelate by enantioselective deacylation of α-acetoxyphenylacetic acid using whole cells of newly isolated Pseudomonas sp. ECU1011. Appl Microbiol Biotechnol 86:83–91

    Article  CAS  PubMed  Google Scholar 

  • Kiziak C, Conradt D, Stolz A, Mattes R, Klein J (2005) Nitrilase from Pseudomonas fluorescens EBC191: cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme. Microbiology 151:3639–3648

    Article  CAS  PubMed  Google Scholar 

  • Schreiner U, Hecher B, Obrowsky S, Waich K, Klempier N, Steinkellner G, Gruber K, Rozzell JD, Glieder A, Winkler M (2010) Directed evolution of Alcaligenes faecalis nitrilase. Enzym Microbiol Technol 47:140–146

    Article  CAS  Google Scholar 

  • Wang MX (2005) Enantioselective biotransformations of nitriles in organic synthesis. Top Catal 35:117–130

    Article  Google Scholar 

  • Wang HL, Sun HH, Wei DZ (2013a) Discovery and characterization of a highly efficient enantioselective mandelonitrile hydrolase from Burkholderia cenocepacia J2315 by phylogeny-based enzymatic substrate specificity prediction. BMC Biotechnol 13:14

    Article  PubMed Central  PubMed  Google Scholar 

  • Wang HL, Sun HH, Gao WY, Wei DZ (2013b) Efficient production of (R)-o-chloromandelic acid by recombinant Escherichia coli cells harboring nitrilase from Burkholderia cenocepacia J2315. Org Process Res Dev 18:767–773

    Article  CAS  Google Scholar 

  • Weatherburn MW (1967) Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 39:971–974

    Article  CAS  Google Scholar 

  • Yamamoto K, Oishi K, Fujimatsu I, Komatsu K (1991) Production of R-(−)-mandelic acid from mandelonitrile by Alcaligenes faecalis ATCC 8750. Appl Environ Microbiol 57:3028–3032

    CAS  PubMed Central  PubMed  Google Scholar 

  • Zhang ZJ, Xu JH, He YC, Ouyang LM, Liu YY, Imanaka T (2010) Efficient production of (R)-(-)-mandelic acid with highly substrate/product tolerant and enantioselective nitrilase of recombinant Alcaligenes sp. nitrilase. Process Biochem 45:887–891

    Article  CAS  Google Scholar 

  • Zhang ZJ, Pan J, Liu JF, Xu JH, He YC, Liu YY (2011a) Significant enhancement of (R)-mandelic acid production by relieving substrate inhibition of recombinant nitrilase in toluene-water biphasic system. J Biotechnol 152:24–29

    Article  CAS  PubMed  Google Scholar 

  • Zhang ZJ, Xu JH, He YC, Ouyang LM, Liu YY (2011b) Cloning and biochemical properties of a highly thermostable and enantioselective nitrilase from Alcaligenes sp. ECU0401 and its potential for (R)-(-)-mandelic acid production. Bioproc Biosyst Eng 34:315–322

    Article  CAS  Google Scholar 

  • Zhang CS, Zhang ZJ, Li CX, Yu HL, Zheng GW, Xu JH (2012) Efficient production of (R)-o-chloromandelic acid by deracemization of o-chloromandelonitrile with a new nitrilase mined from Labrenzia aggregate. Appl Microbiol Biotechnol 95:91–99

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 21406068/B060804) and China Postdoctoral Science Foundation funded Project (No. 2014M560308) and National major science and technology projects of China (2012ZX09304009).

Supporting information

Supplementary Table 1—Chiral analytical conditions for the products of mandelonitrile derivates.

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Correspondence to Hualei Wang.

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Sun, H., Gao, W., Fan, H. et al. Cloning, purification and evaluation of the enzymatic properties of a novel arylacetonitrilase from Luminiphilus syltensis NOR5-1B: a potential biocatalyst for the synthesis of mandelic acid and its derivatives. Biotechnol Lett 37, 1655–1661 (2015). https://doi.org/10.1007/s10529-015-1830-4

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