萎缩芽孢杆菌Rk1壳聚糖酶高效制备及特性表征  

High-level expression and biochemical properties of a chitosanase from Bacillus atrophaeus Rk1

作　　者：王建荣 祝木金 王平 陈微 余思 钟斌 WANG Jianrong;ZHU Mujin;WANG Ping;CHEN Wei;YU Si;ZHONG Bin(Shenzhen Raink Ecology&Environment Co.Ltd.,Shenzhen 518102,China;School of Food Science and Engineering,South China University of Technology,Guangzhou 510641,China)

机构地区：[1]深圳润康生态环境股份有限公司,广东深圳518102 [2]华南理工大学食品科学与工程学院,广东广州510641

出　　处：《食品与发酵工业》2023年第3期22-30,共9页Food and Fermentation Industries

基　　金：深圳市科技攻关面上项目(20201024145915001)。

摘　　要：壳聚糖酶在壳寡糖酶法制备过程中发挥重要作用,高效制备壳聚糖酶为其产业化应用奠定基础。采用同源克隆获得萎缩芽孢杆菌(Bacillus atrophaeus)Rk1壳聚糖酶基因(bacsn46)。将密码子优化和分子伴侣蛋白共表达进行结合,实现萎缩芽孢杆菌壳聚糖酶(BaCsn46)高效表达。通过表征分析获得重组BaCsn46酶学特性。bacsn46基因全长825个碱基,编码274个氨基酸,其中前32个氨基酸为信号肽序列。在7 L发酵罐条件下,重组菌最高发酵酶活力和总蛋白质量浓度分别达到7 356 U/mL和5.95 g/L。重组BaCsn46最适反应温度和pH分别是60℃和6.0,金属离子Mg2+和Mn2+对重组BaCsn46具有激活作用,重组BaCsn46最小和最适水解底物分别是壳四糖和95%脱乙酰度胶体壳聚糖。此外重组BaCsn46能够高效水解不同浓度胶体壳聚糖,制备不同聚合度壳寡糖。Chitosanase plays an important role on enzymatic preparation of chitosan oligosaccharides(COSs) with different degree of polymerization. High-level expression of chitosanase will provide foundation for its industrial application. Herein, a gene encoding chitosanase(bacsn46) from Bacillus atrophaeus Rk1 was cloned and analyzed. The chitosanase from Bacillus atrophaeus Rk1(BaCsn46) was overexpressed by combining codon optimization and co-expression of helper gene. In addition, the biochemical properties of BaCsn46 were characterized. The open reading frame of bacsn46 is 825 bp in length, which encodes a protein of 274 amino acid residues, including a potential signal sequence of 32 amino acid residues. The maximum activity was 7 356 U/mL and total protein content 5.95 g/L, which were obtained in fed-batch cultivation in a 7 L bioreactor. The optimal condition for purified BaCsn46 were 60 ℃ and pH 6.0. The activity of purified BaCsn46 was stimulated by Mg2+and Mn2+, respectively. The smallest and most favorable substrates of purified BaCsn46 were chitotetraose and colloidal chitosan with degrees of deacetylation of 95%, respectively. Furthermore, the purified BaCsn46 exhibited high efficiency to hydrolyze colloidal chitosan to prepare COSs with different degree of polymerization.

关 键 词：萎缩芽孢杆菌 壳聚糖酶 密码子优化 毕赤酵母 壳寡糖 

分 类 号：TQ925[轻工技术与工程—发酵工程]