Structural and functional analyses of microcystinase A:optimized heterologous expression,stability,and degradability  

作　　者：Siyu YANG Jun ZUO Licheng HUANG Jin LIU Nanqin GAN 

机构地区：[1]Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan 430072,China [2]National and Local Joint Engineering Research Center of Ecological Treatment Technology for Urban Water Pollution,Zhejiang Provincial Key Lab for Water Environment and Marine Biological Resources Protection,Institute for Eco-Environmental Research of Sanyang Wetland,Wenzhou University,Wenzhou 325035,China [3]University of Chinese Academy of Sciences,Beijing 100190,China [4]Kunming Dianchi and Plateau Lakes Institute,Kunming 650228,China

出　　处：《Journal of Oceanology and Limnology》2024年第6期1805-1816,共12页海洋湖沼学报（英文）

基　　金：Supported by the National Key R&D Program of China(No.2018YFA0903100);the National Natural Science Foundation of China(No.32071601)。

摘　　要：Microcystinase(MlrA)is a key endopeptidase that catalyzes microcystin degradation without generating harmful byproduct.However,the application of MlrA in the field is primarily impeded by its limited productivity and short lifespan.Therefore,the MlrA's function was studied by modelling its structure,which subsequently increased its heterologous expression and high-temperature stability.Results demonstrate that after the irregular sequence at the C-terminus of MlrA was removed,enzyme solubility was significantly decreased.In addition,three fusion tags,namely maltose-binding protein,glutathione S-transferase(GST),and N-utilization substance A(NusA)were used to enhance the overexpression of soluble recombinant MlrA,among which NusA-MlrA exhibited the highest solubility.Moreover,NusA-MlrA was active in pH 4-10 at 20-80℃;even at 80℃,approximately 35.8%of fusion protein remained active.NusA-MlrA retained 89%of MlrA's activity even after 7 d of storage at 50℃;and on day 7,the protein retained>90%of its activity at pH 7.Finally,a stable,soluble,and long-lasting heterologous MlrA was successfully constructed that could eliminate microcystins in Escherichia coli C43(DE3).This study enriched the comprehension of MlrA's structure and enzymatic properties,by particularly addressing the endopeptidase's low expression and short lifespan,which improved its suitability for future applications.

关 键 词：microcystinase(MlrA) BIODEGRADATION MICROCYSTIN AlphaFold2 N-utilization substance A(NusA)-MlrA 

分 类 号：X52[环境科学与工程—环境工程]