诺卡氏菌酰胺酶基因的分子克隆及在大肠杆菌中表达的初步研究

Molecular Cloning of an Amidase Gene from Nocardia sp. and Its Expressionin Escherichia coli

酰胺酶是一种重要的工业酶。利用生物信息学手段,在和已知酰胺酶基因序列分析比对的基础上,首次从Ncordiasp.YS-2002中成功地克隆得到酰胺酶基因ami,并对其基因序列及氨基酸序列的性质进行了分析。结果表明,所得酰胺酶基因ami片段大小共为1446bp,由启动子区、阅读框和回文结构终止区三部分构成。序列分析和进化树分析表明,Ncordiasp.YS-2002酰胺酶是一种比较特殊的酰胺酶,不含大多数酰胺酶共同具有的保守区序列。进一步将酰胺酶基因连接到pET-28a(+)上,转入大肠杆菌BL21(DE3)中筛选获得重组菌株PEAB。酶活测定结果表明重组菌具有酰胺酶酶活,但较低,其原因可能是因为大量表达的产物主要以包涵体的形式存在。

The amidase of Nocardia sp. is one of important industrial enzymes. Based on DNA and protein sequence alignment from different strains, a new gene of amidase was successfully cloned from Nocardia YS-2002, which is widely used for industrial production of acrylamide in China. DNA sequence analyses showed that the 1466bp cloned-fragment contains promoter, open reading frame and terminating-palindrome. Protein sequence alignment and phylogenetic tree analyses showed that the amidase coming from Nocardia sp. YS-2002 is a kind of specialamidase, without the typical conserved sequence of the amidases. Enzymatic characteristics predictions indicated that the molecular weight and pI of the new amidase is approximately 38.05kD and 4.88, respectively, and it would be stable when heterogeneously expressed in E. coli. By inserting the ORF of the amidase into plasmid pET-28a（ ＋ ）, a recombinant strain, pEAB, was selected using E. coli BI21 （DE3） as the host. SDS-PAGE analyses of both the whole ceils and ultrasonic-treated cells confirmed the feasibility of the heterogeneous expression of amidase in the recombinant E. coli. But the activity of amidase in E. coli BI21（DE3） not more than 0.5u/mg, because most of the enzymes expressed were formed as inclusion bodies.