pH调控对里氏木霉菌产β-葡萄糖苷酶的影响机制

Mechanisms of pH control on β-glucosidase produced in Trichoderma reesei

研究了里氏木霉(Trichoderma reesei)发酵生产β-葡萄糖苷酶(β-glucosidase,BG)过程中,pH调控对产酶的影响与作用机制。以控制产酶阶段pH 5.0为实验组、不控制pH值为对照组,通过酶学实验、免疫胶体金分析和Raman光谱检测来进行分析。结果表明:pH调控能使发酵液中BG表达量提高1.8倍、酶活力提高2.5倍。免疫胶体金分析证实,对照组酶合成时间在发酵72h左右终止,而实验组合成时间可延至96h。Raman光谱分析显示,葡萄糖苷酶在pH 5.0状态下,酶蛋白主链结构主要为α-螺旋和无规则卷曲;而不控制pH状态下,酶蛋白主链结构的无规则卷曲发生较大变化,螺旋也受到一定影响。综合分析来看pH调控改变了BG酶的合成特性、延长了胞内酶蛋白的合成时间从而提高了酶的产量;而酶活的提高则得益于酶蛋白分子构象的较好保持。

The effects and mechanisms of pH control on β-glucosidase（BG） production in Trichoderma reesei were studied.The pH in the fermentation process in the experimental group was controlled at 5.0 but not control in the control group.The results show that pH control increases the expression of BG 1.8 fold and the activity 2.5 fold.Immunogold analyses confirm that the BG enzymatic synthesis ceased at 72 h in the control,but was extended to 96 h in the pH-controlled tests.Raman spectroscopy shows that with pH 5.0,the BG protein structure was mainly α-helix and random coil,pH 2.4 condition greatly attend the main random coil chain structure.Thus,pH regulation changes the BG biosynthesis model,extends the intracellular enzyme protein synthesis time,and enhances the production of the BG enzyme.The main mechanism for the improved activity was the main tendency of the BG protein molecular conformation.