Abstract
Recombinant Escherichia coli cells harboring nitrilase from Alcaligenes faecalis were immobilized using tris(hydroxymethyl)phosphine (THP) as the coupling agent. The optimal pH and temperature of the THP-immobilized cells were determined at pH 8.0 and 55 °C. The half-lives of THP-immobilized cells measured at 35, 40, and 50 °C were 1800, 965, and 163 h, respectively. The concentration of R-mandelic acid (R-MA) reached 358 mM after merely 1-h conversion by the immobilized cells with 500 mM R,S-mandelonitrile (R,S-MN), affording the highest productivity of 1307 g L−1 day−1 and the space-time productivity of 143.2 mmol L−1 h−1 g−1. The immobilized cells with granular shape were successfully recycled for 60 batches using 100 mM R,S-MN as substrate at 40 °C with 64% of relative activity, suggesting that the immobilized E. coli cells obtained in this study are promising for the production of R-MA.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Chen, J., Zheng, R. C., Zheng, Y. G., & Shen, Y. C. (2009). Microbial transformation of nitriles to high-value acids or amides. Advances in Biochemical Engineering/Biotechnology, 113, 33–77.
Xue, Y. P., Yang, Y. K., Lv, S. Z., Liu, Z. Q., & Zheng, Y. G. (2016). High-throughput screening methods for nitrilases. Applied Microbiology and Biotechnology, 100, 3421–3432.
Zhang, Z. J., Pan, J., Li, C. X., Yu, H. L., Zheng, G. W., Ju, X., & Xu, J. H. (2014). Efficient production of (R)-(−)-mandelic acid using glutaraldehyde cross-linked Escherichia coli cells expressing Alcaligenes sp nitrilase. Bioprocess and Biosystems Engineering, 37, 1241–1248.
Liu, Z. Q., Dong, L. Z., Cheng, F., Xue, Y. P., Wang, Y. S., Ding, J. N., Zheng, Y. G., & Shen, Y. C. (2011). Gene cloning, expression, and characterization of a nitrilase from Alcaligenes faecalis ZJUTB10. Journal of Agricultural and Food Chemistry, 59, 11560–11570.
Zhang, X. H., Liu, Z. Q., Xue, Y. P., Yang, B., Xu, M., & Zheng, Y. G. (2016). R-mandelic acid production with immobilized recombinant Escherichia coli cells in a recirculating packed bed reactor. Biocatalysis and Biotransformation, 34, 205–211.
Zhang, Z. J., Yu, H. L., Imanaka, T., & Xu, J. H. (2015). Efficient production of (R)-(−)-mandelic acid by isopropanol-permeabilized recombinant E. coli cells expressing Alcaligenes sp nitrilase. Biochemical Engineering Journal, 95, 71–77.
Zhang, X. H., Liu, Z. Q., Xue, Y. P., Xu, M., & Zheng, Y. G. (2016). Nitrilase-catalyzed conversion of (R,S)-mandelonitrile by immobilized recombinant E. coli cells harboring nitrilase. Biotechnology and Applied Biochemistry, 63, 479–489.
Walt, D. R., & Agayn, V. I. (1994). The chemistry of enzyme and protein immobilization with glutaraldehyde. Trac-Tread Anal Chem, 13, 425–430.
Kirkeby, S., Jakobsen, P., & Moe, D. (1987). Glutaraldehyde—“pure and impure.” A spectroscopic investigation of two commercial glutaraldehyde solutions and their reaction products with amino acids. Analytical Letters, 20, 303–315.
Chen, S. C., & Duan, K. J. (2015). Production of galactooligosaccharides using beta-galactosidase immobilized on chitosan-coated magnetic nanoparticles with tris(hydroxymethyl)phosphine as an optional coupling agent. International Journal of Molecular Sciences, 16, 12499–12512.
Cheng, T. C., Duan, K. J., & Sheu, D. C. (2005). Immobilization of beta-fructofuranosidase from Aspergillus japonicus on chitosan using tris(hydroxymethyl)phosphine or glutaraldehyde as a coupling agent. Biotechnology Letters, 27, 335–338.
Cheng, T. C., Duan, K. J., & Sheu, D. C. (2006). Application of tris(hydroxymethyl)phosphine as a coupling agent for β-galactosidase immobilized on chitosan to produce galactooligosaccharides. Journal of Chemical Technology and Biotechnology, 81, 233–236.
Oswald, P. R., Evans, R. A., Henderson, W., Daniel, R. M., & Fee, C. J. (1998). Properties of a thermostable beta-glucosidase immobilized using tris(hydroxymethyl)phosphine as a highly effective coupling agent. Enzyme and Microbial Technology, 23, 14–19.
Liu, Z. Q., Zhang, X. H., Xue, Y. P., Xu, M., & Zheng, Y. G. (2014). Improvement of Alcaligenes faecalis nitrilase by gene site saturation mutagenesis and its application in stereospecific biosynthesis of (R)-(−)-mandelic acid. J Arg Food Chem, 62, 4685–4694.
Xue, Y. P., Xu, M., Chen, H. S., Liu, Z. Q., Wang, Y. J., & Zheng, Y. G. (2013). A novel integrated bioprocess for efficient production of (R)-(−)-mandelic acid with immobilized Alcaligenes faecalis ZJUTB10. Organic Process Research and Development, 17, 213–220.
Wang, H., Fan, H., Sun, H., Zhao, L., & Wei, D. (2015). Process development for the production of (R)-(−)-mandelic acid by recombinant Escherichia coli cells harboring nitrilase from Burkholderia cenocepacia J2315. Organic Process Research and Development, 19, 2012–2016.
Gong, J. S., Li, H., Lu, Z. M., Shi, J. S., & Xu, Z. H. (2015). Recent progress in the application of nitrilase in the biocatalytic synthesis of pharmaceutical intermediates. Progress in Chemistry, 27, 448–458.
Vesela, A. B., Krenkova, A., & Martinkova, L. (2015). Exploring the potential of fungal arylacetonitrilases in mandelic acid synthesis. Molecular Biotechnology, 57, 466–474.
DiCosimo, R., McAuliffe, J., Poulose, A. J., & Bohlmann, G. (2013). Industrial use of immobilized enzymes. Chemical Society Reviews, 42, 6437–6474.
Antrim, R. L., & Auterinen, A. L. (1986). A new regenerable immobilized glucose isomerase. Starch-Starke, 38, 132–137.
Zhang, Z. J., Xu, J. H., He, Y. C., Ouyang, L. M., Liu, Y. Y., & Imanaka, T. (2010). Efficient production of (R)-(−)-mandelic acid with highly substrate/product tolerant and enantioselective nitrilase of recombinant Alcaligenes sp. Process Biochemistry, 45, 887–891.
Funding
This work was financially supported by the National Natural Science Foundation of China (No. 21476210) and the Public Welfare Project of Zhejiang Province (No. 2014C33223).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of Interest
The authors declare that they have no conflicts of interest.
Rights and permissions
About this article
Cite this article
Zhang, XH., Liu, ZQ., Xue, YP. et al. Production of R-Mandelic Acid Using Nitrilase from Recombinant E. coli Cells Immobilized with Tris(Hydroxymethyl)Phosphine. Appl Biochem Biotechnol 184, 1024–1035 (2018). https://doi.org/10.1007/s12010-017-2604-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12010-017-2604-3