蛋白质谷氨酰胺酶基因的合成表达及性质研究

Gene Synthesis,Expression and Property Research of Protein-glutaminase

蛋白质谷氨酰胺酶(Protein-glutaminase,PG)是引起蛋白质脱酰胺作用的一种新型水解酶,在食品工业中具有十分广泛的应用前景。通过重叠延伸PCR法合成了PG全长基因,将其成熟肽序列克隆至原核表达载体pET32a(+)上,转化大肠杆菌BL21(DE3),经IPTG诱导,重组蛋白主要以包涵体形式表达。通过降低诱导温度和共表达分子伴侣两种策略,以期实现重组蛋白的可溶性表达,结果发现低温对可溶性的提高有一定的作用,而分子伴侣影响甚微。收集包涵体经变性、复性后获得活性PG。将PG与底物Cbz-Gln-Gly进行孵育反应,结果表明,PG可有效水解Cbz-Gln-Gly谷氨酰胺残基上的氨酰基,释放出氨;酶学性质的研究表明,此酶的最适反应温度为40℃,最适作用pH为6.0。实验为PG酶的研究和开发提供了理论依据和重要参考。

Protein-glutaminase, a novel protein-deamidating enzyme, which has potential for industrial applications. The gene encoding the protein was synthesized using overlap extension PCR and the mature PG gene was cloned into expression vector pET32a（ ＋ ）. The recombinant protein was expressed in Escherichia coli BL21 （DE3） as inclusion bodies, and the active PG was obtained after denaturation and renaturation. In order to improve the solubility of PG, the culture was incubated in cold-shocked condition and a chaperone plasmid pTfl6- tig was also cloned into pET32a-matPG/BL21 （DE3）. The results showed that low temperature can improve the solubility of PG slightly, but pTfl6-tig was futile. For deamidating activity assay, Cbz-Gln-Gly was used as substrate. The reaction showed that PG can effectively hydrolyzed glutaminyl residues in the Cbz-Gln-Gly and resulting in release of ammonia. The research on enzymatic properties of PG showed that the optimum temperature is 40℃ and the optimal pH is 6. 0. The gene encoding protein-glutaminase was synthesized and expressed successfully. The research provided a new idea for heterologous expression of this particular food-enzyme using genetic engineering.