N-糖基化改造碱性果胶酶的热稳定性

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  • 1(安徽工程大学 生物与化学工程院,安徽 芜湖,241000) 2(江南大学,工业生物技术教育部重点实验室,江苏 无锡,214122)
硕士研究生

网络出版日期: 2018-07-04

基金资助

国家自然科学基金面上项目(31671797);安徽省高等学校省级自然科学研究重点项目(KJ2016A801、KJ2017A 123);国家级大学生创新创业训练计划项目(2016103630062);安徽工程大学中青年拔尖人才项目(2016BJRRC006)

Improving the thermostability of alkaline polygalacturonate lyase byN-glycosylation modification

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  • 1(School of Biochemical Engineering, Anhui Polytechnic University, Wuhu 24100, China) 2(Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China)

Online published: 2018-07-04

Supported by


摘要

在软件模拟分析的基础上,运用半理性设计转换传统去除N-糖基化的方式,同时在反向转角区引入具有增强芳香族序列(enhanced aromatic sequence, EAS)特征的新N-糖基化,提高了碱性果胶酶(alkaline polygalacturonate lyase, PGL)的热稳定性,获得了高品质的PGL工程菌。将355位点的苏氨酸突变成突变能低的色氨酸来去除353位点的N-糖基化,酶的半衰期提高了176%,但与目标值仍相距甚远;再将127和137位点同时引入新N-糖基化,使酶的半衰期达到1.35 h,与目标值接近。在酶的反向转角区引入具有EAS特征的新N-糖基化,对提高酶的热稳定性具有显著促进作用。

本文引用格式

昂安娜, 堵国成, 葛飞, 等 . N-糖基化改造碱性果胶酶的热稳定性[J]. 食品与发酵工业, 2018 , 44(5) : 22 -27 . DOI: 10.13995/j.cnki.11-1802/ts.016290

Abstract

On the basis of modeling analysis by software, the traditional deglycosylation way was changed while new N-glycosylation with enhanced aromatic sequences (EAS) was introduced into the reverse turn of PGL through semi-rational design.In consequence, the thermostability of alkaline polygalacturonate lyase (PGL) was improved and a PGL engineering bacteria with higher quality was obtained.Removing the N-glycosylation in N353 by replacing the threonine at site 355 with tryptophan that has low mutation energy raised the half-life of PGL by 177%, which was still far from the target yet.Then, introducing new N-glycosylation in site 127 and 137 brought the half-life of recombinase PGL up to 1.35 h that was close to the target value.Introducing N-glycosylation with EAS into the reverse turn of enzyme could play a significant role in improving its thermostability.
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