| Nitrile hydratase(nitrile hydratase, NHase, EC 4.2.1.84) is a metalloenzyme that hydrates nitriles to more valuable amides, mainly used in industry to produce acrylamide and nicotinamide. According to the metal ion contained in the catalytic center, NHases are generally divided into iron type nitrile hydratase(Fe-NHase) and conbalt type nitrile hydratase(Co-NHase). Currently, the R. rhodochrous J1, as the third generation industrial bacteria, was applied to produce acrylamide. Two kinds of NHases with different molecular mass, the high molecular mass nitrile hydratase(H-NHase) and the low-molecular mass nitrile hydratase(L-NHase) were classified in R. rhodochrous J1, and they were all Co-NHases. Compared with L-NHase, H-NHase was more stable and inclined to catalyze aliphatic nitriles which was mainly used in industrial to produce acrylamide. However, the Rhodococcus was hindered by its longer time for cultivation, low expression and cumbersome purification procedure. Contrary to the complex expression system of Rhodococcus, Escherichia coli was prominent for fast cell growth, high level protein expression and simple purification procedure. Moreover, H-NHase has not been over-expressed in E. coli.In this study, the gene nhhBAG, coding for H-NHase from R. rhodochrous J1, was greatly modified to be expressed in E. coli. The whole-cell catalysis under fed-batch substrate was initially tested. Meanwhile, the thermostability of H-NHase was improved by fusing the subunits of H-NHase. The main contents of this study were summarized as followed:(1) The native Shine-Dalgarno sequence of the α-subunit gene nhhA and the activator gene nhhG was substituted with a stronger SD sequence, and the intergenic spacer sequence of the two genes was optimized. In addition, the rare codons within the gene were optimized. The H-NHase was over-expressed in E. coli BL21(DE3) pET-24a(+)-nhh BrbsArbsG with the enzyme activity of 85.5 ± 4.3 U·mg-1 in cell-free extracts. By ion-exchange chromatography, the specific activity of the purified enzyme were 319.5 ± 11.7 U·mg-1. The molecular mass of the recombinant enzyme was determined as 504.5 ± 9.8 kDa by the size-exclusion chromatography, speculated as 24-multimers α12β12.(2) The catalytic properties of the recombinant strains were preliminarily studied. When the cell growth(OD600) reached to 1.0, 0.4 mM isopropyl-β-D-thiogalactoside(IPTG) and 0.1 g·L CoCl2·6H2O were added into the medium, and then the cells were cultivated at 30 oC for 18 h for expression. The optimum catalysis conditions were pH 7.5, 25 oC and 400 mM substrate. In addition, the product tolerance of the recombinant E. coli was stronger than that of the wild cells. In the fed-batch catalytic process, the final conversion rate of the productin the recombinant strains was about 99.9% which was much higher than that of the wild strain. These results were suggested that the recombinant E. coli was more likely to be applied in industry to improve the amide productivity owing to its rapid growth, short fermentation period and high conversion rate.(3) The subunits of H-NHase were fused to improve the thermostability of H-NHase. The genes of α and β subunit were fused and the Strep-tag was inserted just upstream of the β subunit gene to obtain pET-24a-nhh(BA)rbsG. Then, the recombinant plasmid was transformed into E. coli BL21(DE3). The fusion protein Nhh(BA)G was induced according to the same procedure as for E. coli pET-24a(+)-nhhBrbsArbsG. Afterwards, Nhh(BA)G was purified by Strep-tag affinity chromatography and the specific activity was detected as 82.8 ± 4.7 U·mg-1. The relative activity of the purified NhhBAG and Nhh(BA)G were reduced to 80% and 40%, respectively, with 50 oC for 40 min. These results were indicated that the subunits fusion could improve the thermostability of H-NHase. |
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