目的:通过基因工程方法获得高效表达腈水合酶的基因工程菌CtNHase,探究其对腈类物质的全细胞催化作用,并考察pH、温度对全细胞催化反应的影响。方法:对来源Comamonas testosteroni 5-MGAM-4D的腈水合酶基因进行PCR扩增,以pET-24a为载体,构建基因重组表达质粒后,转化大肠杆菌感受态细胞进行SDS-PAGE蛋白表达验证,并通过色谱检测分析对底物丙烯腈和己二腈的转化情况。结果:在pH=7.4,30 ℃条件下,基因重组菌CtNHase催化活性最高,可通过全细胞催化在5 min内完全转化50 mmol/L己二腈生成己二酰二胺,在5 h内完全转化500 mmol/L丙烯腈生成丙烯酰胺。结论:基因重组菌CtNHase可高效催化丙烯腈和己二腈生成丙烯酰胺和己二酰二胺,具有潜在的工业应用价值。
Objective:Constructed the gene recombinant E. coli CtNHase which expressed nitrile hydratase efficiently by gene cloning technology,then investigated its biocatalytic activity of nitriles and the effects of pH and temperature with the whole cell catalytic reaction. Methods:The nitrile hydratase gene of Commamonas testosteroni 5-MGAM-4D was amplified by polymerase chain reaction(PCR),the recombinant expression plasmid was constructed with pET-24a vector. After transformed into competent E. coli cells,the expression of protein was verified by SDS-PAGE. The conversion of the substrates of acrylonitrile and adiponitrile were analyzed by chromatography. Results:According to the whole cell catalysis,we found the recombinant E. coli CtNHase transformed 50 mmol/L adipiconitrile to adipamide in 5 min completely,while transformed 500 mmol/L acrylonitrile to acrylamide in 5 h. The optimum pH and reaction tempreture were 7.4,30 ℃,the E. coli exhibited the highest catalytic activity. Conclusion:The recombinant E. coli CtNHase could catalyze acrylonitrile and adiponitrile to acrylamide and adipamide high efficiently,which would show the potential industrial application prospects.