Journal
JOURNAL OF BIOSCIENCE AND BIOENGINEERING
Volume 128, Issue 5, Pages 518-524Publisher
SOC BIOSCIENCE BIOENGINEERING JAPAN
DOI: 10.1016/j.jbiosc.2019.05.004
Keywords
N-glycosylation; Laccase; Specific activity; Pichia pastoris; Coprinopsis cinerea
Funding
- National Natural Science Foundation of China [31300044, 31600078]
- Open Fund for Discipline Construction, Institute of Physical Science and Information Technology, Anhui University
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Laccase Lcc9 from Coprinopsis cinerea heterologously expressed in Pichia pastoris (rLcc9) displayed different molecular weight and specific activity from the native laccase (nLcc9). Glycosylation may play a role in regulating the Lcc9 specific activity. To elucidate this hypothesis, in this study, firstly we demonstrated that rLcc9 and nLcc9 were glycoproteins, and then enzymatically deglycosylated them. The obtained drLcc9 and dnLcc9 showed an apparent decrease in their specific activities. Three putative N-glycosylation sites (N293, N313, and N454) were then predicted in Lcc9 and substituted to evaluate their roles in its specific activity. Molecular weight analysis on those mutants suggested that glycosylation should have occurred on N313 and N454 whereas not on N293 in rLcc9. Comparison of catalytic properties of those mutants revealed that glycosylation at N313 and N454 in rLcc9 could affect the binding affinity to substrates and the catalytic rate, respectively. In addition, the glycosylation could also affect the thermal stability of rLcc9 and nLcc9 since deglycosylation of those Lcc9s resulted in decreases in their thermal stability to some extent. These results will help us to understand the effect of glycosylation on biochemical characteristics of fungal laccases, and provide us support for the improvement of fungal laccase activity based on N-linked glycosylation modification. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.
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