Abstract
Nitrilases have attracted tremendous attention for the preparation of optically pure carboxylic acids. This article aims to address the production and utilization of a highly enantioselective nitrilase from Pseudomonas putida MTCC 5110 for the hydrolysis of racemic mandelonitrile to (R)-mandelic acid. The nitrilase gene from P. putida was cloned in pET 21b(+) and over-expressed as histidine-tagged protein in Escherichia coli. The histidine-tagged enzyme was purified from crude cell extracts of IPTG-induced cells of E. coli BL21 (DE3). Inducer replacement studies led to the identification of lactose as a suitable and cheap alternative to the costly IPTG. Effects of medium components, various physico-chemical, and process parameters (pH, temperature, aeration, and agitation) for the production of nitrilase by engineered E. coli were optimized and scaled up to a laboratory scale bioreactor (6.6 l). Finally, the recombinant E. coli whole-cells were utilized for the production of (R)-(−)-mandelic acid.
Similar content being viewed by others
References
Kobayashi, M., & Shimizu, S. (2000). Nitrile hydrolases. Current Opinion Chemical Biology, 4, 95–102.
Banerjee, A., Sharma, R., & Banerjee, U. C. (2002). The nitrile degrading enzymes: Current status and future prospects. Applied Microbiology and Biotechnology, 60, 33–44.
Banerjee, A., Kaul, P., Sharma, R., & Banerjee, U. C. (2003). A high-throughput amenable colorimetric assay for enantioselective screening of nitrilase producing microorganisms. Journal of Biomolecular Screening, 8, 559–565.
Kaul, P., Banerjee, A., & Banerjee, U. C. (2004). Screening for enantioselective nitrilases: Kinetic resolution of racemic mandelonitrile to (R)-(−)-mandelic acid by new bacterial isolates. Tetrahedron: Asymmetry, 15, 207–211.
Kaul, P., Banerjee, A., & Banerjee, U. C. (2006). Enantioselective nitrile hydrolysis by immobilized whole-cell biocatalyst. Biomacromolecules, 7, 1536–1541.
Banerjee, A., Kaul, P., & Banerjee, U. C. (2006). Enhancing the catalytic potential of nitrilase from Pseudomonas putida for enantioselective nitrile hydrolysis. Applied Microbiology and Biotechnology, 72, 77–87.
Osprian, I., Jarret, C., Straussm, U., Kroutil, W., Orru, R., Felfer, U., et al. (1999). Large-scale preparation of a nitrile-hydrolysing biocatalyst: Rhodococcus R 312 (CBS 717.73). Journal of Molecular Catalysis B Enzymatic, 6, 555–560.
Aav, R., Parve, O., Pehk, T., Claesson, A., & Martin, I. (1999). Tetrahedron: Asymmetry preparation of highly enantiopure stereoisomers of 1-(2, 6-dimethylphenoxy)-2-aminopropane (Mexiletine). Tetrahedron: Asymmetry, 10, 3033–3038.
Matsugi, M., Fakuda, N., Maguruma, Y., Yamaguchi, T., Minamikawa, J., & Otsuka, S. (2001). Catalytic assymetric oxidation of sulfide with titanium-mandelic acid complex: Practical synthesis of (S)-3-[1-(2-methylphenyl) imidazole-2-ylsulfinyl]-propan-1-ol, a key intermediate of OPC 29030. Tetrahedron, 57, 2739–2744.
Terreni, M., Pegani, G., Ubiali, D., Fernandez, R., Mateo, C., & Guisán, J. M. (2001). Modulation of penicillin acylase properties via immobilization techniques: One pot chemo-enzymatic synthesis of cephamandole from cephalosporin C. Bioorganic and Medicinal Chemistry Letters, 11, 2429–2432.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., et al. (1987). Current protocols in molecular biology (Vol. I). New York: Wiley.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403–410.
Miller, G. L. (1959). Use of dinitrosalicylic reagent for the determination of reducing sugar. Analytical Chemistry, 31, 426–428.
Nagasawa, T., Mauger, J., & Yamada, H. (1990). A novel nitrilase, arylacetonitrilase, of Alcaligenes faecalis JM3, purification and characterization. European Journal of Biochemistry, 194, 765–772.
Acknowledgments
Anirban Banerjee gratefully acknowledges financial assistance provided by CSIR Govt. of India, and DAAD Fellowship, Sachin Dubey and Praveen Kaul gratefully acknowledge financial assistance provided by DBT and CSIR, Govt. of India. This is NIPER communication number 413.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Banerjee, A., Dubey, S., Kaul, P. et al. Enantioselective Nitrilase from Pseudomonas putida: Cloning, Heterologous Expression, and Bioreactor Studies. Mol Biotechnol 41, 35–41 (2009). https://doi.org/10.1007/s12033-008-9094-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12033-008-9094-z