黄色短杆菌GDK-9谷氨酸脱氢酶基因的克隆、序列分析及表达

Cloning, Sequence Analysis and Expression of Glutamate Dehydrogenase in Brevibacterium flavum GDK-9

利用PCR技术从黄色短杆菌GDK-9的基因组DNA中扩增出谷氨酸脱氢酶基因(gdh)片段(EC.1.4.1.4),连到pUCm-T载体上测序。核酸序列分析结果表明,该片段全长1927bp,包含一个ORF,推测此ORF区编码一条448个氨基酸的多肽,分子量约为48kD。与已报道的gdh序列相似性为99.55%,其中1190位碱基(C→A)突变导致了编码氨基酸的变化(Thr→Asn),其它的碱基变化不影响编码的氨基酸。将gdh基因克隆入穿梭质粒pXMJ19中,并转化E.coli XL-Blue和Brevibacterium flavum GDK-9,经IPTG诱导后,SDS-PAGE电泳结果显示,在预计位置出现明显的诱导蛋白条带,分子量约为48.7kD。谷氨酸发酵实验表明,尽管谷氨酸脱氢酶GDH能明显提高胞内的谷氨酸含量,但其不影响谷氨酸的分泌。

The glutamate dehydrogenase （EC.1.4.1.4） gene which amplified from the genome of Brevibacterium flavum GDK-9 by polymerase chain reaction was linked with pUCm-T for sequence alignment. Analysis of gdh sequences revealed that the whole sequence is 1927 bp, only one ORF existed, which used ATG as the initiation codon and coded a peptide of 448 amino acids with a calculated molecular weight of 48 kD. The comparability between the cloned gdh sequence to the reported sequence is high to 99.55%. Only the 1190^th base mutation （C→A） lead to the change of amino acid sequence （Thr→Asn）, the others are not. The recombinant plasmid pXG was then transformed into E. coli XL-Blue and Brevibacterium flavum GDK-9 which was induced by IPTG. SDS-PAGE analysis revealed that there was a clear induced protein band with molecular mass of 48.7 kD on expected position. Standard glutamate fermentations indicated that although the level of GDH increases the intracellular glutamate pool, the level of GDH has no influence on glutamate secretion.