灰色链霉菌胰蛋白酶在变铅青链霉菌中的异源表达及酶学性质分析

Heterologous expression and enzymatic analysis of Streptomyces griseus trypsin in Streptomyces lividans

摘要:

胰蛋白酶作为一种重要的丝氨酸蛋白酶被广泛应用于食品、医药和皮革等工业领域。本文成功实现了灰色链霉菌来源的胰蛋白编码基因在变铅青链霉菌中的高效活性表达，并对其酶学性质进行分析比较。以灰色链霉菌ATCC10137基因组为模板，获得胰蛋白酶编码基因sprT并克隆至表达质粒pIJ86，成功构建了重组链霉菌工程菌TK24/pIJ86-sprT。以R2YE和SELF为发酵培养基，最高酶活分别达9.21 U/mL和8.61 U/mL。酶学性质分析表明，和牛胰蛋白酶 (BT) 相比，重组链霉菌胰蛋白酶 (rSGT) 的耐酸能力强，具有较广的pH；且rSGT对酰胺键具有更高的特异性；此外，Zn2+和有机溶剂分别对rSGT的酯酶活力和酰胺酶活力具有促进作用；本研究结果为rSGT的性质改造以及工业应用提供了依据。

Abstract:

Trypsin as an important serine protease has been widely used in food, pharmaceutical and tanning industries. In this study, we successfully expressed trypsin (cloning from Streptomyces griseus ATCC10137) in Streptomyces lividans TK24 and comparatively investigated its enzymatic properties. Specifically, applying S. griseus ATCC 10137 genome as template, we obtained the sprT gene and sub-cloned it into the expression plasmid pIJ86, generating the recombinant strain S. lividans TK24/pIJ86-sprT. When cultivated in R2YE and SELF, the activity of rSGT reached 9.21 U/mL and 8.61 U/mL, respectively. Meanwhile, the results of the enzymatic analysis showed that rSGT exhibited a higher acid tolerance and a higher specificity to hydrolyze amide bonds compared with bovine trypsin (BT). In addition, Zn2+ and organic solvents up-regulated esterase and amidase of rSGT. Taken together, the results obtained herein provide meaningful information for further modification of rSGT and its industrial application.