荧光假单胞菌TM5-2中D-型氨基酸脱氢酶的鉴定和表达

从荧光假单胞菌TM5-2中得到一个含丙氨酸消旋酶基因的DNA片段(8.8kb),相邻的一个开读框(ORF)与甘氨酸/D-型氨基酸氧化酶基因相似。该ORF经过克隆、表达,并没有检测到甘氨酸/D-型氨基酸氧化酶的活性,推导而得的氨基酸序列与D-型氨基酸脱氢酶序列比较发现,ORF含有D-型氨基酸脱氢酶的所有重要的保守序列。经TTC培养基鉴定,其具有D-型氨基酸脱氢酶的活性,并对一系列D-型氨基酸有作用,最佳作用底物是D-组氨酸。

Identification and expression a D-amino acid dehydrogenase gene from Pseudomonas fluorescens TM5-2

In the previous study we have isolated DNA fragment containing an alanine racemase gene (dadX) from Pseudomonas fluorescens TM5-2. Adjacent to dadX one ORF similar to a putative glycine/D-amino acid oxidase gene have been found. The same gene organization is found in several Pseudomonas species. Here, author would characterize this ORF to determine what kind of enzyme this gene encodes.DNA fragment containing gene encoding putative glycine/D-amino acid oxidase was cloned into the expression vector. Firstly oxidase activity in cell lysates prepared from the recombinant cells was measured, however, neither glycine nor D-alanine were oxidized judging from hydrogen peroxide formation. Secondly when the amino acid sequence deduced from the oxidase gene was compared to dye-linked D-amino acid dehydrogenases, all the important residues including FAD-binding motif were conserved. This gene was transformed and checked on TTC plate, it showed some activities of D-amino acid dehydrogenase. D-amino acid dehydrogenase activity was also detected when D-alanine and DCIP were used. The best substrate of this enzyme is D-histdine, which is different from some reports. Author will be in progress to purify the dehydrogenase and determine enzyme characteristics.