| In the previous studies, we found that bacterium Variovorax boronicumulans CGMCC 4969 could degrade the neonicotinoid insecticide thiactoprid (THI) and the major pathway involved hydrolysis of the N-cyanoimino group in THI to an N-carbamoylinino group, forming the metabolite THI amide and this biotransformation was mediated by a nitrile hydratase (TNHase). The TNHase gene cluster includes an α-and β-subunit genes named TnhA and TnhB respectively, the downstream of TnhB followed by a completely open reading frame encoding TnhC. TnhA and TnhB genes were expressed in Escherichia coli BL21, getting expressed proteins as inactive inclusion bodies and low THI hydrolysis acitivity. In present study, the expression conditions of TnhA and TnhB genes were furhter optimized in Escherichia coli. TnhC was also co-expressed with TnhA and TnhB to evaluate the effect on the expressing level and activity of TNHase.The TnhA, TnhB and TnhC genes were respectively recombined into plasmid pET28a and over-expressed in E. coli BL21 and E. coli Rosetta. The result showed the functional expression of TNHase was promoted by Rosetta than BL21. The TnhA, TnhB, TnhC genes and a SD sequence, respectively, according to different order, were recombined into plasmid pET28a and over expressed in E. coli Rosetta. The result showed that the order of TnhAB and TnhC gene and the absence of SD sequence had important influence on heterologous recombinant expression of THNase. The expressed TnhAB was in the form of inclusion bodies when it was expressed atone or co-expressed in combination with the TnhC gene followed by TnAB. The TnhC was largely expressed and active while the TnhAB was barely expressed when the TnhC gene located in front of the TnhAB gene. When a SD sequences seted between TnhC and TnhAB gene, TnhA, TnhB and TnhC were all expressed larger, and existed in the form of soluble proteins. The resting cells of recombinants pNAB, pNABC, pNCAB and pNCDAB were prepared to transform THI and the half-lifes of THI were calculated as 4.0,99.0,59.2 and 13.4 min, respectively. Although it could’t improve the expression level of recombinant proteins, the TNHase activity of pABC was 14.8 times higher than that of pNAB, meanwhile the TNHase activity of pCAB was 0.4 times lower than that of pNAB. When a SD sequence lied between TnhC and TnhAB, the solubility of recombinant proteins were significantly improved, but the TNHase activity of pNCDAB increased only 4.4 times than that of pAB. We further optimized the conditions of expression and conversion of resting cells of recombinants. The result of the experiments showed that the optimum condition of the THI transformation of TNHase resting cells was that the pH value, temperature and the concentration of Co2+were 7.0 to 8.0,25 to 35℃,0.2to 0.3 mmmol/L respectively.Recombinants could transform 100 g/L of 3-cyanopyridine to nicotinamide. The result showed that the functional expression of TNHase was promoted by Rosetta than BL21. The co-expressed of TnhC with TnhAB had great effect on the activity of TNHase. Rosetta-pNABC was grown in LB supplemented with 0.2 mmol/L Co2+, to an absorbance (600 nm) of 0.7, then IPTG was added to the culture medium (0.2 mmol/L final concentration) for induction of TNHase synthesis. The conversion conditions by resting cells are pH 7.0 and temperature of 25℃. They are optimal conditions for expression and transformation of 3-cyanopyridine by the TNHase form the resting cells of recombinant E. coli, respectively.Our present studies revealed that the co-expression of TnhC could significantly improve the expression level and activity of TNHase on transformation of THI and 3-cyanopyridine. |
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