地衣芽孢杆菌谷氨酰内切酶的克隆表达与性质研究

Cloning and Characterization of Bacillus Licheniformis Glutamyl Endopeptidase

谷氨酰内切酶在食品行业应用广,但来源受限。以地衣芽孢杆菌ATCCl4580基因组DNA为模板,PCR扩增出670bp大小的谷氨酰内切酶基因,克隆入表达载体pET28a(+),转化Escherichia coli BL21(DE3),经IPTG诱导,重组蛋白主要以包涵体形式存在。通过与硫氧还蛋白融合、降低诱导温度和共表达分子伴侣三种策略,以期增加重组蛋白可溶性,结果表明分子伴侣对可溶性有明显提高作用,而低温和硫氧还蛋白影响甚微。将重组蛋白样品与底物(Z-Phe-Leu-Glu-pNA)进行孵育反应,结果表明,该酶可有效水解谷氨酸残基的羧基,释放出4-硝基苯胺。酶学性质研究表明,此酶的最适反应温度为42℃,最适作用pH为8.0,金属离子Mn2+对酶活有较好的激活作用。

The glutamyl endopeptidase gene was amplified by PCR with genomic DNA of Bacillus1icheniformis ATCCl4580 as template, and expressed in Escherichia coli BL21 （DE3） by yielding hybrid plasmid pET28a-GE. Induced with IPTG, the E.coli BL21（DE3） harboring pET28a-GE successfully expressed the glutamyl endopeptidase as inclusion bodies. In order to improve the solubility, the gene was expressed with thioredoxin protein or coexpressed with a chaperone plasmid pTf16-tig,or the culture was incubated at low temperature.. The results showed that the chaperone could improve the solubility of recombinant glutamyl endopeptidase, while thioredoxin and low temperature was futile. The measurement of enzyme activity with Benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide（Z-Phe-Leu-Glu-pNA）as substrate demonstrated that the recombinant glutamyl endopeptidase can effectively hydrolyze the alpha- carboxyl of glutamic acid residue, releasing p-nitroanilide. The optimum temperature and pH for the glutamyl endopeptidase is 42℃, 8.0, respectively, and Mn ion can improve the enzyme activity.