食品与发酵工业 >

2023 , Vol. 49 >Issue 3: 22 - 30

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.031920

研究报告

萎缩芽孢杆菌Rk1壳聚糖酶高效制备及特性表征

  • 王建荣 ,
  • 祝木金 ,
  • 王平 ,
  • 陈微 ,
  • 余思 ,
  • 钟斌
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  • 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)
博士,高级工程师(通信作者,E-mail:wangyiqing0728@163.com)

收稿日期: 2022-04-11

  修回日期: 2022-05-19

  网络出版日期: 2023-03-03

基金资助

深圳市科技攻关面上项目(20201024145915001)

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

  • WANG Jianrong ,
  • ZHU Mujin ,
  • WANG Ping ,
  • CHEN Wei ,
  • YU Si ,
  • ZHONG Bin
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  • 1(Shenzhen Raink Ecology & Environment Co.Ltd., Shenzhen 518102, China)
    2(School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China)

Received date: 2022-04-11

  Revised date: 2022-05-19

  Online published: 2023-03-03

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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。

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

本文引用格式

王建荣 , 祝木金 , 王平 , 陈微 , 余思 , 钟斌 . 萎缩芽孢杆菌Rk1壳聚糖酶高效制备及特性表征[J]. 食品与发酵工业, 2023 , 49(3) : 22 -30 . DOI: 10.13995/j.cnki.11-1802/ts.031920

Abstract

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.

Key words： Bacillus atrophaeus; chitosanase; codon optimization; Pichia pastoris; chitosan oligosaccharides

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