枯草芽孢杆菌来源的碱性果胶酶在毕赤酵母中的高效表达  

High-Efficiency Expression of Alkaline Pectinase from Bacillus Subtilis in Pichia Pastoris

作　　者：陈洲 王亚森 汪步青 许向阳 高晓冬[1] 李子杰[1] CHEN Zhou;WANG Yasen;WANG Buqing;XU Xiangyang;GAO Xiaodong;LI Zijie(School of Biotechnology,Jiangnan University,Wuxi 214122,China;Zaozhuang Jienuo Biological Enzyme Co.,Ltd.,Zaozhuang 277100,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]枣庄市杰诺生物酶有限公司,山东枣庄277100

出　　处：《食品科技》2022年第2期1-9,共9页Food Science and Technology

基　　金：山东省重大科技创新工程项目(2019JZZY011006)。

摘　　要：以Bacillus subtilis 168来源的碱性果胶酶(PELA)为研究对象,首先采用毕赤酵母密码子偏好性优化,结合G418高浓度抗性筛选策略,构建了PELA突变体工程菌株Pp03;其次,通过信号肽(MF4I、HFBI、W1、Albumin、Invertase 2)优化和分子伴侣(BMH2、HAC1、PDI1、KEX2)共表达,获得一株高效表达PELA的工程菌株Pp13;接着对该菌株摇瓶表达PELA的诱导温度、甲醇添加量进行优化;最后,采用5 L发酵罐进行高密度发酵。结果显示,PELA的基因经过密码子、信号肽以及伴侣因子优化后,实现PELA的酶活分别提高至2.10、7.35、1.49倍,其酶活分别达到39.11、353.87、527.27 U/mL;最佳诱导温度和甲醇添加量分别为26 ℃和1.0%;在最佳诱导条件下,PELA的摇瓶水平酶活达到732.60 U/mL;采用5 L发酵罐进行高密度发酵,PELA的酶活达到2301.05 U/mL。研究结果为碱性果胶酶的工业化应用提供理论依据,同时也为毕赤酵母中异源蛋白的高效表达提供方法参考。In this study,alkaline pectinase (PELA) from Bacillus subtilis 168 was used as the research object.Firstly,an engineered strain Pp03 was constructed by optimization of codon bias in Pichia pastoris and resistance screening strategies with high concentration of G418.Then,an engineered strain Pp13 with high-efficiency expression of PELA was obtained based on signal peptides (MF4I,HFBI,W1,Albumin and Invertase 2) optimization and co-expression with molecular chaperones (BMH2,HAC1,Pdi1 and KEX2).Next,the induction temperature and the additive amount of methanol were optimized for the expression of PELA.Finally,high density fermentation was carried out in a 5 L fermenter.The results showed that the PELA activity was increased by 2.10,7.35,1.49 times after the optimization of codon,signal peptide and molecular chaperones,and the PELA activity reached 39.11,353.87,527.27 U/mL,respectively.The optimum induction temperature and methanol addition were 26 ℃ and 1.0%,respectively.Under optimum induction condition,the PELA activity reached 732.60 U/mL in shaking flask,the highest activity of PELA reached 2301.05 U/mL in a 5 L fermenter.The results can provide a theoretical basis for the industrial application of alkaline pectinase and provides a method reference for the efficient expression of heterologous protein in Pichia pastoris.

关 键 词：碱性果胶酶 毕赤酵母 信号肽 分子伴侣 高密度发酵 

分 类 号：TS201.3[轻工技术与工程—食品科学]