Abstract
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.
Highlights
The methylotrophic yeast, Pichia pastoris, is widely used as a useful experimental tool in protein engineering and production
Because a Kex2 cleavage site at K66R67 is present in the prosequence, the R. chinensis prolipase expressed in P. pastoris was truncated by Kex2 endoprotease
The resulting product was the mature lipase attached with 27 amino acids of the carboxy-terminal part of the prosequence, containing an his tag, named r27RCLC (Figure 1C), in which three potential N-glycosylation sites in the propeptide were removed, retained only one potential glycosyaltion site (N-263) in the mature region
Summary
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. Lipases are well known hydrolases capable of hydrolyzing the ester bonds of water-insoluble substrates at the interface between substrate and water, which show remarkable levels of activity and stability in non-aqueous environments, in contrast to many other enzymes [15] Due to these unique properties, lipases are the most attractive enzymes for use in various industrial applications, such as in the food processing industry [16,17] and in the energy industry for biodiesel production [18,19]. The N-glycosylated Thermomyces lanuginosus lipase exhibited better thermostability than their non-glycosylated variants [29]
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