Sinorhizobium morelens S-5中N-氨甲酰基水解酶基因的克隆、表达和纯化  

Cloning,expression and purification of D-carbamoylase from Sinorhizobium morelens S-5

作　　者：吴胜[1] 王建军[1] 杨柳[1] 孙万儒[1] 

机构地区：[1]中国科学院微生物研究所微生物资源国家重点实验室,北京100080

出　　处：《微生物学报》2006年第4期565-570,共6页Acta Microbiologica Sinica

摘　　要：N-氨甲酰基水解酶是一种非常具有工业应用价值的水解酶,可用于制备光学纯氨基酸。通过LA PCR从Sinorhizobium morelensS-5菌中克隆到1.3kb的DNA片段,测序表明该片段上含有一个完整的N-氨甲酰基水解酶的基因(hyuC)序列。将hyuC基因克隆到表达载体pET30a上,重组质粒pET30a-HyuC在大肠杆菌中获得了高水平表达。重组的N-氨甲酰基水解酶经过热处理和三步柱色谱分离而纯化。纯化倍数为16.1倍,收率21.2%。该酶为同源四聚体,亚基分子量是38kDa。最适温度是60℃,最适pH为7.0。该酶有较高的热稳定性和氧化稳定性。Fe2+和Ca2+对酶的活性有一定的促进作用,而金属螯合剂和巯基试剂对酶活无明显影响。A N-carbamoyl-D-amino acid amidohydrolase gene （hyuC） from Sinorhizobium morelens S-5 was cloned by LA PCR, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the hyuC gene exhibited high homology to the amino acid sequences of D-carbamoylase from other sources. The gene could be highly expressed in Escherichia cog and the recombinant enzyme was purified 16.1-fold to homogeneity with a yield of 21.2% by heat treatment and three steps of column chromatography. The results of gel filtration on Superdex 200 HR and SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a tetramer protein of identical 38-kDa subunits. The recombinant enzyme catalyzed the hydrolysis of N-carbamoyl alpha- amino acid to the corresponding free amino acid, and it was strictly D-specific. The enzyme showed broad substrate specificity, and exhibited high activity in the hydrolysis of N-carbamoyI-D-p-hydroxyphenylglycine as substrate. The enzyme did not hydrolyze N- carbamoyl-β-alanine. The optimum pH and temperature of the enzyme were pH ？.0 and 60℃, respectively. Enzyme activity was slightly improved by Ca^2＋ and Fe^2＋ , and nearly not affected by metal chelators and sulfhydryl reagents. The enzyme showed high thermal and oxidative stability. These results show that the enzyme has great potential for industrial application.

关 键 词：SINORHIZOBIUM morelens N-氨甲酰基水解酶基因 蛋白纯化 表达 

分 类 号：Q933[生物学—微生物学] Q78