海洋弧菌HN897β-琼脂糖酶基因vas1-1339异源表达及活性分析  

Heterologous expression and enzymatic characterization of marine Vibrio astriarenae-derivedβ-Agarase gene vas1-1339

作　　者：喻飞 金兴坤 雷天影 曹海航 陈锵辉 阳耀帆 李佳航 赵哲 YU Fei;JIN Xingkun;LEI Tianying;CAO Haihang;CHEN Qianghui;YANG Yaofan;LI Jiahang;ZHAO Zhe(Department of Marine Biology,College of Oceanography,Hohai University,Nanjing 210098,China)

机构地区：[1]河海大学海洋学院,海洋生物技术与资源利用研究所,江苏南京210098

出　　处：《热带海洋学报》2022年第2期170-176,共7页Journal of Tropical Oceanography

基　　金：国家自然科学基金(31872597);江苏农业自主创新基金[CX(19)2033]。

摘　　要：海洋弧菌HN897株是一株高产琼脂糖酶的Vibrio astriarenae菌,前期功能缺失实验证明其β-琼脂糖酶基因vas1-1339的缺失可显著降低弧菌HN897株的琼脂水解活性。文章在大肠杆菌中异源表达Vas1-1339基因编码蛋白,分析该基因的表达及蛋白功能活性。首先,构建含vas1-1339基因开放阅读框全长的表达载体pET28a-1339,利用原核表达技术在大肠杆菌中成功地异源表达了Vas1-1339琼脂糖酶;然后,利用卢戈氏碘液染色分析发现异源表达Vas1-1339琼脂糖酶的大肠杆菌能够高效水解琼脂,且异丙基硫代半乳糖苷(IPTG)最优诱导浓度为10μmol·L^(-1);免疫印迹分析发现胞内基因表达产物羧基端可能存在切割现象,推测Vas1-1339琼脂糖酶存在复杂的成熟过程。对纯化的琼脂糖酶活性分析发现海洋弧菌HN897株β-琼脂糖酶Vas1-1339能够独立发挥琼脂糖降解功能。The marine Vibrio HN897 strain is an agarose-producing Vibrio astriarenae.Studies indicated that the absence of the gene Vas1-1339,encoding aβ-agarase,significantly reduced the hydrolysis effect of Vibrio HN897 strain on agarose.Herein,we further analyzed the expression and enzymatic characterization of theβ-agarase gene in heterologous bacteria,Escherichia coli(E.coli).Vas1-1339 agarase was successfully expressed in E.coli,and the optimal concentration of inducer,IPTG,was at 10μmol·L^(-1).Immunoblotting analysis showed that the C-terminal of the gene expression product might be cleavage in E.coli,suggesting complex maturation process of this protein.Lugol’s iodine staining analysis revealed that E.coli,expressing Vas1-1339 agarase,was highly effective in degrading agarose,and purified protein also had this similar function,which suggests that theβ-agarase Vas1-1339 of the HN897 strain can independently play the role on agarolytic degradation.These results lay a preliminary foundation for the functional study and related technology application ofβ-agarase derived from marine vibrion.

关 键 词：β-琼脂糖酶 海洋弧菌 琼脂糖酶解 异源蛋白表达 

分 类 号：P735.51[天文地球—海洋生物学]