摘要
外泌几丁质酶在溶珊瑚弧菌生活史中发挥重要作用。本研究从蛋白质序列角度分析了多种来源于溶珊瑚弧菌的几丁质酶,并选其中蛋白质结构相对简单,容易进行序列改造和大肠杆菌重组表达、纯化的几丁质酶开展研究:(1)在保留野生型弧菌C端几丁质结合域的同时,N段增加陆生微生物几丁质结合域;(2)获得高表达重组几丁质酶的大肠杆菌菌株,采用一步层析柱工艺,重组蛋白纯度达90%以上;(3)不经过复杂脱盐换液处理,几丁质酶活性仍维持较高水平。经过对以上条件的优化,本研究确立了几丁质酶重组表达的工艺路线,分析了此重组酶活性匹配的含盐缓冲液条件,为今后几丁质酶大规模生产积累理论研究基础。
Exo-chitinase plays an important role in the life history of Vibrio coralliilyticus.In this study,a variety of chitinases from Vibrio were analyzed from the perspective of protein sequence,and one of them,with simple protein sequence and modifiable,was selected for expression and purification by recombinant E.coli.The results showed that:(1)The C-terminal chitin binding domain of Vibrio wild-type was retained,while the N-segment chitin binding domain of terrestrial microorganisms was increased;(2)High expression of recombinant chitinase was obtained by one-step chromatography,and the purity of recombinant protein was over 90%.(3)The chitinase activity remained at a high level without complex desalting and liquid exchange treatment.Through the optimization of the above conditions,this study established the process route of recombinant expression of chitinase and analyzed the conditions of the salt-containing buffer matching the recombinant enzyme activity,which provided the theoretical basis for large-scale production and accumulation of chitinase in the future.
作者
孔德颖
蔡燕飞
KONG De-ying;CAI Yan-fei(Shanghai Vocational College of Agriculture and Forestry,Shanghai 201699;College of Resources and Environment,South China Agricultural University,Guangzhou 510642,China)
出处
《浙江海洋大学学报（自然科学版）》
CAS
2021年第5期424-429,共6页
Journal of Zhejiang Ocean University：Natural Science
基金
上海农林职业技术学院(上海市农业学校)科研课题计划(KY2-0000-19-04)
上海市“科技创新行动计划”农业领域项目(20392001300)
上海市自然科学基金(20ZR1439600)。
关键词
溶珊瑚弧菌
几丁质酶
蛋白质工程
Vibrio coralliilyticus
chitinase
protein engineering
