枯草杆菌NX-2聚谷氨酸解聚酶的克隆表达及其降解性质研究

Cloning and Expression of ywtD Gene from B. subtilis NX-2 and the Enzymatic Degradation of γ-Polyglutamic Acid

利用PCR技术从γ-聚谷氨酸产生菌Bacillus subtilisNX-2的基因组DNA上扩增出聚谷氨酸内切解聚酶基因(ywtD),克隆后测序,对该基因编码区进行了序列分析,比对结果表明扩增的ywtD基因编码与文献报道的序列相似性为99.0%,碱基的差异均表现为碱基取代,仅有一个碱基取代导致编码氨基酸的变化,即第349位密码子由GCC变为GTC。将ywtD基因克隆入表达载体pET15b中,该载体转化大肠杆菌Rosetta(DE3),经0.5mmol/LIPTG诱导,外源解聚酶蛋白获得高效表达。利用酶的初提液对聚谷氨酸进行降解,结果显示经72小时解聚酶作用后,γ-PGA分子量从700kDa降到20kDa,此后随作用时间的延长,聚谷氨酸分子量趋于定值。

The B. subtilis ywtD gene, encoding a γ-polyglutamic acid （γ-PGA） depolymerase, was amplified from the genome of B. subtilis NX-2 by PCR. The comparability between the cloned ywtD gene sequence to the reported sequence is high to 99.0%. Only one of the substituted nucleotide base caused the change to the amino acid sequence. The recombinant plasmid pET-15b-ywtD was then transformed into E. coli Rosetta （DE3） and the ywtD gene product could be expressed with the induction of 0. 5mmol/L IPTG. The YwtD protein exhibited a remarkable activity in γ-polyglutamic acid degradation. The molecular weight of γ-PGA could be reduced from 700kDa to 20kDa after 72h through the enzymatic hydrolysis and consequently trended to be constant.