Cloning of the nitrile hydratase gene from Nocardia sp in Escherichia coli and Pichia pastoris and its functional expression using site-directed mutagenesis

To obtain a recombinant Rhodococcus or Nocardia with not only higher enzymatic activity but also better operational stability and product-tolerance ability for bioconversion of acrylamide from acrylonitrile, an active and stable expression system of nitrile hydratase (NHase) was tried to construct as the technical platform of genetic manipulations. Two NHase genes, NHBA and NHBAX, from Nocardia YS-2002 were successfully cloned, based on bioinformatics design of PCR primers, and inserted into plasmid pUC18 and pET32a, respectively. Then, two recombinant Escherichia coli strains, JM105 (pUC18-NHBA) and BL21 (DE3) (pET32a-NHBAX) were constructed and their expressions of NHase were focused. The induction results showed that there was either no NHase activity in JM105 (pUC18-NHBA), or as low as 0.04 U (1 U=1 mumol acrylamide min(-1) mg(-1) dry cell) in BL21 (DE3) (pET32a-NHBAX). SDS-PAGE results showed that the alpha-subunit of NHBA and NHBAX could not be efficiently expressed in both recombinant E. coli strains. The novel Pichia pastoris system was also applied to express NHase, but the expression level remained quite low (0.5-0.6 U) and the protein was unstable. For solving this problem, a possible genetic strategy, site-directed mutagenesis of the u-subunit of the NHase was carried out. After the successful mutagenesis of the original rare start codon gtg into atg, a new recombinant strain, E. coli XL1-Blue (pUC18-NHBA(M)), was screened and the NHase activity stably reached as high as 51 U under the same induction conditions. (C) 2004 Elsevier Inc. All rights reserved.