需钠弧菌WPAGA4菌株3条琼胶酶基因克隆、表达与酶学性质分析  

Cloning,expression,and characterization of three agarases in Vibrio natriegens WPAGA4

作　　者：童秀芳 王宁 张孟源 王雯欣 张牟莹珂 邓帅 王健鑫 曾润颖 曲武 TONG Xiufang;WANG Ning;ZHANG Mengyuan;WANG Wenxin;ZHANG Mouyingke;DENG Shuai;WANG Jianxin;ZENG Runying;QU Wu(Marine Science and Technology College,Zhejiang Ocean University,Zhoushan 316000,Zhejiang,China;Technology Innovation Center for Exploitation of Marine Biological Resources,Ministry of Natural Resources,Xiamen 361000,Fujian,China)

机构地区：[1]浙江海洋大学海洋科学与技术学院,浙江舟山316000 [2]自然资源部海洋生物资源开发利用工程技术创新中心,福建厦门361000

出　　处：《微生物学报》2023年第9期3667-3678,共12页Acta Microbiologica Sinica

基　　金：浙江省“尖兵”“领雁”研发攻关计划(2022C02040);浙江省教育厅一般科研项目(Y202147737);舟山市科技局项目(2021C21009);大学生创新创业训练项目(202110340051)。

摘　　要：【目的】本文拟从西太平洋深海沉积物中分离得到的需钠弧菌(Vibrio natriegens) WPAGA4菌株中克隆并表达3条β-琼胶酶基因agaW3418、agaW3419和agaW3472,并对其酶学性质进行研究。【方法】通过克隆表达技术将得到的3条琼胶酶基因在大肠杆菌BL21(DE3)细胞中进行表达,通过二硝基水杨酸(DNS)法测定重组琼胶酶的酶活,并对其中活性最优的一种琼胶酶进行热稳定性和产酶条件优化。【结果】三种琼脂酶均为属于GH50家族。AgaW3418、AgaW3419和AgaW3472在温度分别为50、60和30℃以及pH值分别为6.0、7.0和7.0条件下,发挥作用的能力最强。其中琼胶酶AgaW3472表现出最优的酶活性质,在20℃下保持良好的稳定性,且在SOB (super optimal broth)培养基条件下以1%(质量体积分数)的乳糖作为碳源,同时添加20 mmol/L MgCl_(2),并将诱导温度和异丙基-1硫代-β-D-半乳糖苷(isopropyl-1 thio-β-D-galactoside, IPTG)浓度分别设置为37℃和0.1 mmol/L时能够获得最高的表达量。【结论】三条重组酶具有琼胶酶活性,为WPAGA4菌株代谢琼胶多糖并参与海洋碳循环过程提供物质基础。琼胶酶AgaW3472具有较高的酶活性、低温适应性与稳定性,为琼胶降解相关产业的发展提供了潜在的生物工具酶。[Objective]To clone and express threeβ-agarase genes agaW3418,agaW3419,and agaW3472 in Vibrio natriegens WPAGA4 isolated from the deep-sea sediment and study the enzymatic properties of the proteins.[Methods]The three agarase genes were expressed in Escherichia coli BL21(DE3)cells,and the 3,5-dinitrosalicylic acid(DNS)method was employed to measure the activities of the recombinant agarases.[Results]The three agarases,AgaW3418,AgaW3419,and AgaW3472,belonged to the GH50 family,with the highest activities at 50,60,and 30°C and pH 6.0,7.0,and 7.0,respectively.AgaW3472 showed the highest activity and maintained good stability at 20°C.The highest production of AgaW3472 was detected in the super optimal broth(SOB)medium with 1%(W/V)lactose as the carbon source,20 mmol/L MgCl_(2),and 0.1 mmol/L isopropyl-β-D-thiogalactoside(IPTG)at the induction temperature of 37°C.[Conclusion]The agarase AgaW3472 has high enzyme activity,low temperature adaptability,and good stability,serving as a potential bio-tool for the development of agar degradation-related industries.The three recombinant agarases provided a foundation for understanding the agarose metabolism in V.natriegens WPAGA4 and the role of this strain in the marine carbon cycling.

关 键 词：琼胶酶 琼胶寡糖 深海弧菌 冷适应性 

分 类 号：Q936[生物学—微生物学]