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.
Similar content being viewed by others
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
Brenner C (2002) Catalysis in the nitrilase superfamily. Curr Opin Struct Biol 12:775–782
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
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
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
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
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
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
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
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
Wang MX (2005) Enantioselective biotransformations of nitriles in organic synthesis. Top Catal 35:117–130
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
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
Weatherburn MW (1967) Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 39:971–974
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
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
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
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
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
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.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-015-1830-4