title: Role of N-linked glycosylation in the secretion and enzymatic properties of Rhizopus chinensis lipase expressed in Pichia pastoris.
abstract: BACKGROUND
  The 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  RCL 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.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China. ymin827@163.com.
authors.forename: Min
authors.initials: M
authors.lastname: Yang
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China. bioyuxw@aliyun.com.|State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China. bioyuxw@aliyun.com.
authors.forename: Xiao-Wei
authors.initials: XW
authors.lastname: Yu
authors.affiliation: Biological Mass Spectrometry Facility at Robert wood Johnson medical school and Rutgers, the state university of new jersey, Piscataway, NJ 08854, USA. Haiyanz@cabm.rutgers.edu.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Zheng
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China. jnshachong@163.com.
authors.forename: Chong
authors.initials: C
authors.lastname: Sha
authors.affiliation: Biological Mass Spectrometry Facility at Robert wood Johnson medical school and Rutgers, the state university of new jersey, Piscataway, NJ 08854, USA. zhaocf@cabm.rutgers.edu.
authors.forename: Caifeng
authors.initials: C
authors.lastname: Zhao
authors.affiliation: Biological Mass Spectrometry Facility at Robert wood Johnson medical school and Rutgers, the state university of new jersey, Piscataway, NJ 08854, USA. meiqian@cabm.rutgers.edu.
authors.forename: Meiqian
authors.initials: M
authors.lastname: Qian
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China. yxu@jiangnan.edu.cn.|State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China. yxu@jiangnan.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
mesh_terms: D006031:Glycosylation; D008049:Lipase / Q000378:metabolism*; D010843:Pichia / Q000378:metabolism*; D015202:Protein Engineering; D012233:Rhizopus / Q000201:enzymology*
chemical_list: D008049:Lipase