Effect ofN-linked glycosylation on the activity and other properties of recombinant endoglucanase IIa (Cel5A) fromPenicillium verruculosum

Abstract

Endoglucanase IIa from Penicillium verruculosum (PvCel5A) has three potential N-glycosylation sites: Asn19, Asn42 and Asn194. In order to study the role of N-glycosylation, the wild type (wt) PvCel5A and its mutant forms, carrying Asn to Ala substitutions, were cloned into Penicillium canescens All forms of the rPvCel5A were successfully expressed and purified for characterization. The MALDI-TOF mass spectrometry peptide fingerprinting showed that N-glycans linked to Asn42 and Asn194 represent variable oligosaccharides, according to the formula (Man)(GlcNAc) No evidence for Asn19 glycosylation was found. Mutations had no notable effect on the enzyme thermostability; however, the N-linked glycans stabilized the enzyme against proteolytic attack. For N42A and N194A mutants, a slight shift of pH-optimum to pH 5.0 was observed (from pH-optimum of 4.5 for the native enzyme, rPvCel5A-wt and N19A mutant). The N19A mutation led to a notable decrease in the specific activity against carboxymethylcellulose and barley β-glucan (by 26% and 12% relative to the rPvCel5A-wt), while the N42A and N194A mutants displayed 12-13% and 32-35% increase in the activities. Similar effects of the mutations were observed in prolonged hydrolysis of β-glucan and milled aspen wood by rPvCel5A forms in the presence of purified β-glucosidase.

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

“…In particular, these properties were demonstrated for CBHs from P. funiculosum, P. pulvirrolum, P. verruculosum, and P. canescens [23,24]. It should be noted that one of the reasons for such a high specific activity in the case of CBH I and CBH II from P. verruculosum is the optimal distribution of N-linked glycans on the surface of the catalytic domain of these enzymes [25,26].…”

“…Although it has been shown that when the substrate is more recalcitrant, the synergism in reducing sugar production decreases [32], as the interaction between cellulase and cellulose is a complex process, the understanding of the molecular mechanisms of cellulose hydrolysis by cellulase mixtures is highly essential and remains to be elucidated. specific activity in the case of CBH I and CBH II from P. verruculosum is the optimal distribution of N-linked glycans on the surface of the catalytic domain of these enzymes [25,26].…”

Oxygen Activation by Cu LPMOs in Recalcitrant Carbohydrate Polysaccharide Conversion to Monomer Sugars

“…244 Similarly, glycosylation was correlated with improved activity of the cellobiohydrolases of GH families 5, 6, and 7. 245–248 …”

Aspergillus sydowii: Genome Analysis and Characterization of Two Heterologous Expressed, Non-redundant Xylanases

“…Frequently, more complex eukaryotic expression systems, like Komagataella phaffii (formerly known as Pichia pastoris) or Yarrowia lipolytica, have to be used to express eukaryotic xylanases from A. niger, Trichioderma harzianum, Hypocrea orientalis, or Trichioderma reesei (He et al, 2009;Wang et al, 2014;Li et al, 2018) as they have the advantage of correct N-and O-glycosylation. The glycosylation can have different effects like enhancement of solubility (Solá and Griebenow, 2009), increased activity, as shown for the endo-cellulase IIa from Penicillium verrucosum (Dotsenko et al, 2016), or enhanced stability (Sarkar and Wintrode, 2011). On the other hand, there are a number of reports showing the expression of xylanases in E. coli (Basaran et al, 2001;Le et al, 2011) but having the disadvantage of either being periplasm bound to the E. coli or having low activity (about 30 U/mg).…”