Identification, sequencing and mutagenesis of the gene for a D-carbamoylase from Agrobacterium radiobacter

被引:20
作者
Buson, A [1 ]
Negro, A [1 ]
Grassato, L [1 ]
Tagliaro, M [1 ]
Basaglia, M [1 ]
Grandi, C [1 ]
Fontana, A [1 ]
Nuti, MP [1 ]
机构
[1] UNIV PADUA,CTR BIOTECHNOL,CRIBI BIOTECHNOL,I-35121 PADUA,ITALY
关键词
D-carbamoylase; D-amino acid; Agrobacterium radiobacter; Escherichia coli expression; cauA;
D O I
10.1111/j.1574-6968.1996.tb08556.x
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
A clone positive for D-carbamoylase activity (2.7 kb HindIII-BamHI DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli. This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The D-carbamoylase gene, named cauA, was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21(DE3). After induction with isopropyl-thio-beta-o-galactopyranoside, the synthesis of D-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced stability with respect to that of the wild-type enzyme derived from A. radiobacter. Site-directed mutagenesis experiments allowed us to establish that a pro(14)-->Leu(14) exchange leads to an inactive enzyme species, while a Cys(279)-->Ser(279) exchange did not impair the functional properties of the enzyme.
引用
收藏
页码:55 / 62
页数:8
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