Abstract
N-Glycosylation is a posttranslational modification commonly occurred in fungi and plays roles in a variety of enzyme functions. In this study, a xylanase (Af-XYNA) of glycoside hydrolase (GH) family 10 from Aspergillus fumigatus harboring three potential N-glycosylation sites (N87, N124 and N335) was heterologously produced in Pichia pastoris. The N-glycosylated Af-XYNA (WT) exhibited favorable temperature and pH optima (75°C and pH 5.0) and good thermostability (maintaining stable at 60°C). To reveal the role of N-glycosylation on Af-XYNA, the enzyme was deglycosylated by endo-β-N-acetylglucosaminidase H (DE) or modified by site-directed mutagenesis at N124 (N124T). The deglycosylated DE and mutant N124T showed narrower pH adaptation range, lower specific activity, and worse pH and thermal stability. Further thermodynamic analysis revealed that the enzyme with higher N-glycosylation degree was more thermostable. This study demonstrated that the effects of glycosylation at different degrees and sites were diverse, in which the glycan linked to N124 played a key role in pH and thermal stability of Af-XYNA.
Highlights
Hemicellulose, is the second most abundant polysaccharide after cellulose in nature
The results showed that N-glycosylated xylanase has higher activity over a broader pH range and better stability
The amino acid sequence of Af-XYNA exhibited the highest identity of 100% with a putative glycoside hydrolase (GH) 10 xylanase from A. fumigatus Af293
Summary
Hemicellulose, is the second most abundant polysaccharide after cellulose in nature. The efficient conversion of hemicellulosic biomass in various agro-industrial processes has been received much attention [1, 2]. The most abundant hemicellulose polymer, is composed of homopolymeric backbone substituted with side chains of different substituents. Due to the complex of xylan, which require a variety of hydrolytic enzymes to act synergistically. The branch degrading enzymes includes α-L-arabinofuranosidase, α-D-glucuronidase, acetylxylan esterase, and feruloyl or coumaroyl esterase [3, 4]. Β-1,4-endoxylanase (EC 3.2.1.8) catalyze the hydrolysis of β-1,4-linked D-xylopyranose units from the homopolymeric backbone of xylan. According to the carbohydrate-active enzyme (CAZy) database
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