海洋弧菌HN897 β-琼脂糖酶基因vas1-1339异源表达及活性分析

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。

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

The marine Vibrio HN897 strain is an agarose-producing Vibrio astriarenae. Studies indicated that the absence of the gene Vas1-1339, encoding a β-agarase, significantly reduced the hydrolysis effect of Vibrio HN897 strain on agarose. Herein, we further analyzed the expression and enzymatic characterization of the β-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 10 μmol·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 the β-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 of β-agarase derived from marine vibrion.