基于结构改造来源于大阪伊德氏杆菌201-F6的PET水解酶  被引量:4

Structure-based engineering of PET hydrolase from Ideonella sakaiensis

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作  者:陈纯琪 韩旭 刘卫东[2] 马立新[1] 刘珂[3] 郭瑞庭 Chunqi Chen;Xu Han;Weidong Liu;Lixin Ma;Ke Liu;Rey-Ting Guo(Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources,Hubei Key Laboratory of Industrial Biotechnology,State Key Laboratory of Biocatalysis and Enzyme Engineering,School of Life Sciences,Hubei University,Wuhan 430062,Hubei,China;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;Hubei Key Laboratory of Genetic Regulation and Integrative Biology,School of Life Sciences,Central China Normal University,Wuhan 430079,Hubei,China)

机构地区:[1]湖北大学生命科学学院省部共建生物催化与酶工程国家重点实验室,工业生物技术湖北省重点实验室,生物资源绿色转化湖北省协同创新中心,湖北武汉430062 [2]中国科学院天津工业生物技术研究所,天津300308 [3]华中师范大学生命科学学院,遗传调控与整合生物学湖北省重点实验室,湖北武汉430079

出  处:《生物工程学报》2021年第9期3268-3275,共8页Chinese Journal of Biotechnology

基  金:国家重点研发计划(No.2019YFA0706900);国家自然科学基金(No.31800662)资助。

摘  要:聚对苯二甲酸乙二酯(Polyethylene terephthalate,PET)塑料是由对苯二甲酸(Terephthalic acid,TPA)和乙二醇(Ethyleneglycol,EG)通过酯键聚合而成的高分子聚合物,具有性质稳定、不易分解等特点,目前已成为广泛使用的一种聚酯,然而大量产生的PET废弃物同时也造成严重的环境污染。2016年,一种有效降解PET的PET水解酶在大阪伊德氏杆菌201-F6中被鉴定发现(命名为IsPETase),且多个IsPETase结构已被解析。为了设计获得PET降解效率更高的IsPETase,针对底物结合位点Ⅱc区的天冬氨酸233(N233)残基进行了多种突变测试,酶活性检测实验表明,用丙氨酸替代N233可以提高IsPETase的性能,尤其是结合R280A突变后,IsPETase活性增加更为显著。此外,解析了IsPETase N233A突变体的X射线晶体结构。IsPETase N233A突变体与IsPETase野生型整体结构相似,但丙氨酸取代N233后增加了底物识别位点Ⅱ末端的空间结构,推测因此增加了IsPETase的活性。这些结果为进一步基于结构改造PET水解酶提供了新的线索。Polyethylene terephthalate(PET) is a synthetic polymer consisting of ester bond-linked terephthalate and ethylene glycol. Tremendous amounts of PET have been produced and majority of them enters terrestrial and marine environment as wastes, posing serious threats to the global ecosystems. In 2016, a PET hydrolase from a PET-assimilating bacterium Ideonalla sakaiensis was reported and termed as IsPETase. This enzyme outperforms other PET-hydrolyzing enzymes in terms of its PET hydrolytic activity at ambient temperature, thus holds a great promise for PET biodegradation. In order to improve IsPETase activity, we conducted structure-based engineering to modify the putative substrate-binding tunnel. Among the several variants to the N233 residue of IsPETase, we discovered that the substitution of N233 with alanine increases its PET hydrolytic activity, which can be further enhanced when combined with a R280A mutation. We also determined the X-ray crystal structure of the IsPETase N233A variant, which shares nearly identical fold to the WT protein, except for an open end of subsite Ⅱ. We hypothesized that the smaller side chain of N233A variant might lead to an extended subsite Ⅱ for PET binding, which subsequently increases the enzymatic activity. Thus, this study provides new clues for further structure-based engineering of PETase.

关 键 词:聚对苯二甲酸乙二酯 聚对苯二甲酸乙二酯水解酶 降解 X射线晶体结构 N233突变体 

分 类 号:X172[环境科学与工程—环境科学]

 

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