[Molecular cloning of an amidase gene from Nocardia sp. and its expressionin Escherichia coli].
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 38.05 kD 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 BL21(DE3) as the host. SDS-PAGE analyses of both the whole cells 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 BL21(DE3) not more than 0.5 u/mg, because most of the enzymes expressed were formed as inclusion bodies.
Xu
Mi
M
Life Science and Technology Department, Bejing University of Chemical Technology, Beijing 100029, China.
Yu
Hui-Min
HM
Tan
Tian-Wei
TW
Zhu
Yan-Qin
YQ
Shen
Zhong-Yao
ZY
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D008970:Molecular Weight; D009615:Nocardia / Q000201:enzymology*; D010802:Phylogeny
D000581:Amidohydrolases; C019665:amidase