通过体外分子进化技术提高淀粉液化芽胞杆菌BS5582 β-1,3-1,4-葡聚糖酶热稳定性

Improvement of thermostability of β-1,3-1,4-glucanase from Bacillus amyloliquefaciens BS5582 through in vitro evolution

摘要:

应用基于易错PCR随机突变的体外分子进化技术，来提高淀粉液化芽胞杆菌β-1,3-1,4-葡聚糖酶的热稳定性。利用建立的基于96微孔板高通量筛选模型，经过两轮定向进化与高通量筛选，共筛选得到3株热稳定性明显提高的突变体2-JF-01、2-JF-02和2-JF-03。将野生型β-葡聚糖酶基因和热稳定性提高的突变基因的高效表达产物经镍亲和层析柱纯化后，酶学性质测定表明突变酶2-JF-01、2-JF-02和2-JF-03的T50值分别比野生酶 (53℃) 提高2.2℃、5.5℃和3.5℃。突变酶2-JF-01、2

Abstract:

In vitro evolution methods are often used to modify protein with improved characteristics. We developed a directed evolution protocol to enhance the thermostability of the β-1,3-1,4-glucanase. The thermostability of the enzyme was significantly improved after two rounds of directed evolution. Three variants with higher thermostability were obtained. The mutant enzymes were further analyzed by their melting temperature, halftime and kinetic parameters. Comparing to intact enzyme, the T50 of mutant enzymes 2-JF-01, 2-JF-02 and 2-JF-03 were increased by 2.2°C, 5.5°C and 3.5°C, respectively, the halftime (t1/2, 60°C) of mutant enzymes 2-JF-01, 2-JF-02 and 2-JF-03 were shortened by 4,13 and 17 min, respectively, the Vmax of mutant enzymes were decreased by 8.3%, 2.6% and 10.6%, respectively, while Km of mutant enzymes were nearly unchanged. Sequence analysis revealed seven single amino acid mutant happened among three mutant enzymes, such as 2-JF-01 (N36S, G213R), 2-JF-02 (C86R, S115I, N150G) and 2-JF-03 (E156V, K105R). Homology-modeling showed that five of seven substituted amino acids were located on the surface of or in hole of protein. 42.8% of substituted amino acids were arginine, which indicated that arginine may play a role in the improvement of the thermostability of the β-1,3-1,4-glucanase.This study provide some intresting results of the structural basis of the thermostability of β-1,3-1,4-glucanase,and provide some new point of view in modifying enzyme for future industrial use.