N‐glycosylation affects the proper folding, enzymatic characteristics and production of a fungal β‐glucosidase

Abstract

Heterologous expression of ß-glucosidase is one of the approaches to enhance the efficiency of fungal cellulase preparations. It has been reported that N-glycosylation affects the structure framework, function and stability of proteins. In this study, a ß-glucosidase from Aspergillus terreus (GenBank: XP_001216552, BglS) was heterologously expressed in Pichia pastoris and Trichoderma reesei. The four asparagine residues were all linked with high-mannose-type oligosaccharides in P. pastoris, whereas only N224 carried high-mannosetype glycan in T. reesei (the other three sites carried one N-acetylglucosamine). The long N-glycan chains on PpBglS weakened its substrate affinity, activity and thermostability. The moderate post-translational and post-secretory glycan modification in T. reesei makes it a suitable expression system for BglS. The N224 glycan played a critical role in BglS folding. The elucidation of the correlation between the different N-glycosylation patterns of BglS and their corresponding enzymatic characteristics is an important step towards improving the activity, thermostability and even production of heterologous ß-glucosidase by glycan engineering.

RNA Sequencing Reveals Xyr1 as a Transcription Factor Regulating Gene Expression beyond Carbohydrate Metabolism

“…In T. reesei RutC-30, the expression of endo T was potentially activated by Xyr1, as well as lignocellulose degradation-related genes, when cultured on lignocellulose. Thus, the resulted single GlcNAc on the heterologous expressed TrBglS benefitted from its enzymatic activity and thermostability, compared with PpBglS [69]. After deletion of xyr1 , the upregulation of endo T might be due to starvation of carbon sources, which might be an effort to deglycosylate the glycan coat of the glycoprotein composed of the cell wall, and contribute to further protease degradation [67].…”

Role of glycosylation in secretion and stability of micromycetes α -galactosidase

“…The influence of glycosylation inhibitors on secretion and properties of proteins were studied before on insects, mushrooms and уeasts [2,4,[13][14][15]. Tunicamicyn was most often used as an inhibitor of glycosylation.…”

“…It is known [1] that carbohydrate component plays a substantial role in formation of quaternary structure of protein; it is responsible for protection from degrading influence of proteolytic enzymes during protein synthesis. The presence of N-glycosylated sites in polypeptide may determine the stability of enzyme and create a possibility for creation of supramolecular structures [2,3]. It is shown [2] that N-bound carbohydrates play an important role in secretion, because they are mainly revealed in extracellular enzymes as a part of linker site of a molecule.…”

“…The presence of N-glycosylated sites in polypeptide may determine the stability of enzyme and create a possibility for creation of supramolecular structures [2,3]. It is shown [2] that N-bound carbohydrates play an important role in secretion, because they are mainly revealed in extracellular enzymes as a part of linker site of a molecule. О-glycosylation prevents accumulation of molecules of substrate in binding centers of enzymes and provides their stechiometric binding.…”

N-Glycosylation Sited at Residues of Catalytic Center Could Repress The Activity of Endoglucanase from Rhizopus Stolonifer

“…It has been confirmed that N-glycosylation maintains the stability of proteins via the steric hindrance caused by N-linked glycan avoiding the hydrolysis of protease [10][11][12]. The importance of N-glycosylation in regulating the structure and function of cellulases has been reported, which indicated the unique and diversified role that N-glycosylation played [13][14][15][16].…”