Role of N-linked glycosylation in the secretion and enzymatic properties of Rhizopus chinensis lipase expressed in Pichia pastoris

Microbial Cell Factories

Research

Haiyan Zheng¹  Caifeng Zhao¹  Meiqian Qian¹  Min Yang²  Chong Sha²  Xiao-Wei Yu³  Yan Xu³ 

[1] Biological Mass Spectrometry Facility at Robert wood Johnson medical school and Rutgers, the state university of new jersey, NJ 08854, Piscataway, USA;The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, 214122, Wuxi, Jiangsu, China;The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, 214122, Wuxi, Jiangsu, China;State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, 214122, Wuxi, Jiangsu, China;

关键词: N-glycosylation;    Pichia pastoris;    Rhizopus chinensis;    Enzyme activity;    Secretion;    Thermostability;    LC-MS/MS;   

DOI  :  10.1186/s12934-015-0225-5

 received in 2014-11-18, accepted in 2015-03-11,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundThe methylotrophic yeast, Pichia pastoris, is widely used as a useful experimental tool in protein engineering and production. It is common for proteins expressed in P. pastoris to exhibit N-glycosylation. In recent years, glycosylation studies in P. pastoris have attracted increasing attention from scholars. Rhizopus chinensis lipase (RCL) is one of the most important industrial lipases, and it has four potential N-linked glycosylation sites. The aim of the present study was to determine whether RCL undergoes asparagine-linked (N-linked) glycosylation and to examine the role of this modification in RCL expression and function.ResultsIn this study, we demonstrated that RCL expressed in Pichia pastoris was N-glycosylated at the sites N-14, N-48 and N-60. The majority of the sites N-14 and N-60 were glycosylated, but the glycosylation degree of the site N-48 was only a very small portion. The glycan on N-60 played a key role in the expression and secretion of RCL. RT-PCR results showed that the mRNA level of proRCLCN60Q remained unchanged even though the protein secretion was hampered. Although the N-glycan on N-14 had no effect on the secretion of RCL, this glycan was beneficial for the lipase catalytic activity. On the other hand, the little amount of N-glycan on N-48 had no effect both on the secretion and activity of RCL in P. pastoris. Moreover, the thermostability analysis of RCL revealed that the lipase with more N-glycan was more thermostable.ConclusionsRCL was N-glycosylated when expressed in P. pastoris. The N-glycans of RCL on the different sites had different functions for the secretion and enzymatic properties of the lipase. Our report may also provide theoretical support for the improvement of enzyme expression and stability based on the N-linked glycosylation modification to meet the future needs of the biotechnological industry.

【 授权许可】

Unknown   
© Yang et al.; licensee BioMed Central. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.