Dissecting the essential role of N-glycosylation in catalytic performance of xanthan lyase

Bioresources and Bioprocessing

Research

Shaonian Shen¹  Xianzhen Li¹  Fan Yang¹  Xiaoyi Chen¹  Xin Ni¹  Chenchen Nan¹  Jingjing Zhao¹  Qian Wang² 

[1] School of Biological Engineering, Dalian Polytechnic University, 116034, Ganjingziqu, Dalian, People’s Republic of China;School of Biological Engineering, Dalian Polytechnic University, 116034, Ganjingziqu, Dalian, People’s Republic of China;Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 116023, Dalian, People’s Republic of China;

关键词: Xanthan lyase;    N-Glycosylation;    Site-directed mutagenesis;    Structure regulation;    Catalytic properties;   

DOI  :  10.1186/s40643-022-00620-5

 received in 2022-08-11, accepted in 2022-12-08,  发布年份 2022

来源: Springer

【 摘 要 】

Modified xanthan produced by xanthan lyase has broad application prospects in the food industry. However, the catalytic performance of xanthan lyase still needs to be improved through rational design. To address this problem, in this work, the glycosylation and its influences on the catalytic performance of a xanthan lyase (EcXly), which was heterologously expressed in Escherichia coli, were reported. Liquid chromatography coupled to tandem mass spectrometry analysis revealed that the N599 site of EcXly was modified by a single N-glycan chain. Based on sequence alignment and three-dimensional structure prediction, it could be deduced that the N599 site was located in the catalytic domain of EcXly and in close proximity to the catalytic residues. After site-directed mutagenesis of N599 with alanine, aspartic acid and glycine, respectively, the EcXly and its mutants were characterized and compared. The results demonstrated that elimination of the N-glycosylation had diminished the specific activity, pH stability, and substrate affinity of EcXly. Fluorescence spectra further revealed that the glycosylation could significantly affect the overall tertiary structure of EcXly. Therefore, in prokaryotic hosts, the N-glycosylation could influence the catalytic performance of the enzyme by changing its structure. To the best of our knowledge, this is the first report about the post-translational modification of xanthan lyase in prokaryotes. Overall, our work enriched research on the role of glycan chains in the functional performance of proteins expressed in prokaryotes and should be valuable for the rational design of xanthan lyase to produce modified xanthan for industrial application.Graphical Abstract

【 授权许可】

CC BY   
© The Author(s) 2022

【 图 表 】

12982_2022_119_Article_IEq62.gif

12982_2022_119_Article_IEq58.gif

12982_2022_119_Article_IEq57.gif

Fig. 1

Fig. 1

12982_2022_119_Article_IEq32.gif

12982_2022_119_Article_IEq27.gif

12982_2022_119_Article_IEq24.gif

Fig. 6