N-氨甲酰-D-氨基酸酰胺水解酶的融合表达、纯化和酶活性

Fusion expression,purification and enzymatic activity of D-carbamoylase

将菌株SS-ori的N-氨甲酰-D-氨基酸酰胺水解酶基因(DCase)插入到pMAL-c2X载体中,构建表达产物为麦芽糖结合蛋白(MBP)与DCase融合的重组子。该重组子在E.coliBL21中的表达产物为融合蛋白MBP-DCase(76.4ka),光密度扫描表明,其表达量约占菌体超声后离心上清总蛋白的19%。经Amy-lose亲和纯化和Superose12凝胶排阻层析后达到了电泳纯。根据pMAL-c2X的背景,用FactorXa剪切后,可得约41.7ka的MBP和34.7ka的DCase。酶活性检测表明当用N-氨甲酰-DL-缬氨酸做为底物,A600=10时,每升菌体每小时可产生D-缬氨酸0.78g。

A D-carbamoylase(DCase) gene was successfully amplified by PCR with the genomic DNA as its template from Sinorhizobium morelense SS-ori.The gene was inserted into a vector,pMAL-c2X and transformed into E.coli BL21.After screening its possible colony,the engineered strain(E.coli BL21/pMD) can produce an active DCase in the soluble form, reached 19%of total soluble protein in cells by GDS analysis.The cells from 1L culture at the concentration of 10A600 can converse N-carbamoyl-DL-valine to about 0.78g D-valine per an hour in this experimental condition.In addition,the fusion protein MBP-DCase was also purified by affinity chromatography and gel filtration.A 76.4ka band occurried on SDS-PAGEE after passing through amylose resin and Superose 12 columns.Meanwhile,the pure DCase(34.7ka) could be obtained by Factor Xa cleavage on fusion product,as described on SDS-PAGE analysis.Results showed that the recombinant DCase could be used to produce D-amino acids in future.