耐盐芽孢杆菌R1高效壳聚糖酶异源表达及特性分析

Heterologous Expression and Characterization Analysis of a High-Efficiency Chitosanase from Bacillus halotolerans R1

摘要:

采用同源克隆获得耐盐芽孢杆菌壳聚糖酶基因，并异源表达制备重组耐盐芽孢杆菌壳聚糖酶（CsnBh46），通过表征分析获得重组CsnBh46酶学特性。重组CsnBh46全长278个氨基酸，三维结构显示其分为上下两个半球，包含9个α螺旋和2个β折叠。在7 L发酵罐中，重组工程菌bh-5的最高酶活力和总蛋白质质量浓度分别为6 375 U/mL和4.3 g/L。纯化后的重组CsnBh46最适反应pH和温度分别为6.0和60 ℃，金属离子Mn²⁺、Ca²⁺和Mg²⁺对重组CsnBh46具有激活作用。重组CsnBh46最适底物为脱乙酰度95%胶体壳聚糖。重组CsnBh46能够高效水解胶体壳聚糖并且根据反应时间可以定向制备不同聚合度壳寡糖。本研究为CsnBh46产业化应用奠定基础。

Abstract:

The present study employed homologous cloning to obtain the chitosanase gene from Bacillus subtilis, and prepared recombinant Bacillus subtilis chitosanase(CsnBh46) through heterologous expression. The recombinant CsnBh46 enzyme was characterized to determine its enzymatic properties. The full-length recombinant CsnBh46 consists of 278 amino acids, and its three-dimensional structure reveals it is divided into upper and lower domains, each containing nine α-helices and two β-strands. The maximum enzymatic activity of recombinant CsnBh46 expressed in Pichia pastoris X33 was 6 375 U/mL, with a total protein mass concentration of 4.3 g/L. The optimal reaction pH and temperature for purified recombinant CsnBh46 were 6.0 and 60 ℃, respectively. Meanwhile, CsnBh46 was activated by Mn²⁺, Ca2+ ^{and Mg2+. The favorite substrate for recombinant CsnBh46 was colloidal chitosan with 95% degree of deacetylation. Furthermore, recombinant CsnBh46 efficiently hydrolyzed colloidal chitosan and allowed for the targeted preparation of chitosan oligosaccharides with different degrees of polymerization based on reaction time. This study provides a foundation for the industrial application of CsnBh46.}