Glycosylation Variants of a β-Glucosidase Secreted by a Taiwanese Fungus, Chaetomella raphigera, Exhibit Variant-Specific Catalytic and Biochemical Properties

Abstract

Cellulosic biomass is an abundant and promising energy source. To make cellulosic biofuels competitive against conventional fuels, conversion of rigid plant materials into sugars must become efficient and cost-effective. During cellulose degradation, cellulolytic enzymes generate cellobiose (β-(1→4)-glucose dimer) molecules, which in turn inhibit such enzymes by negative feedback. β-Glucosidases (BGLs) cleave cellobiose into glucose monomers, assisting overall cellulolytic activities. Therefore, BGLs are essential for efficient conversion of cellulosic biomass into biofuels, and it is important to characterize newly isolated BGLs for useful traits. Here, we report our discovery that the indigenous Taiwanese fungus Chaetomella raphigera strain D2 produces two molecular weight variants of a single BGL, D2-BGL (shortened to “D2”), which differ in O-glycosylation. The more extensively O-glycosylated form of native D2 (nD2L) has increased activity toward the natural substrate, cellobiose, compared to the less O-glycosylated form (nD2S). nD2L is more stable at 60°C, in acidic pH, and in the presence of the ionic detergent sodium dodecyl sulfate than nD2S. Furthermore, unlike nD2S, nD2L does not display substrate inhibition by an artificial substrate p-nitrophenyl glucopyranoside (pNPG), and the glucose feedback inhibition kinetics of nD2L is competitive (while it is non-competitive for nD2S), suggesting that these two glycovariants of D2 bind substrates differently. Interestingly, D2 produced in a heterologous system, Pichia pastoris, closely mimics properties of nD2S. Our studies suggest that O-glycosylation of D2 is important in determining its catalytic and biochemical properties.

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“…This observation is consistent with our previous findings that Pichia pastoris -produced recombinant D2-BGL activity was sensitive to SDS and could not be renatured even after SDS was removed [ 30 ]. This finding is likely to due to unusual glycosylation of this enzyme in Pichia [ 30 ].

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“…This phenomenon has been observed in many cases. Previous studies have shown that the C. raphigera expressed native D2-BGL has two glycosylation variants and the more extensively O -glycosylated form exhibited increased activity toward its natural substrate cellobiose [ 30 ]. The recombinant A. aculeatus Aa BGL1, a 91-kDa β-glucosidase with 16 potential N -glycosidation sites, showed a smeared band between 135 and 140 kDa during SDS-PAGE analysis [ 55 ].…”

Chaetomella raphigera β-glucosidase D2-BGL has intriguing structural features and a high substrate affinity that renders it an efficient cellulase supplement for lignocellulosic biomass hydrolysis

“…For example, N-glycosylation is essential for proper protein folding and the secretion of recombinant Aspergillus terreus β-glucosidase [43], whereas O-glycosylation decreases pH stability of T. leycettanus β-glucosidase [39]. In our previous study [27], we observed that C. raphigera -expressed native D2-BGL has two glycosylation variants, and the large-form native D2-BGL with greater O-glycosylation showed higher enzyme activity toward cellobiose. For P. pastoris -expressed D2-BGL, both N-glycosylation and O-glycosylation may function in enzyme stability and secretion.…”

“…Cloning of D2-BGL cDNA into pGAPZαC vector (Invitrogen, USA) was performed as described [27] to generate the expression vector Pp D2-BGL #1. To increase purification yield by affinity chromatography, a second vector ( Pp D2-BGL #5) was generated by inserting an additional 6-histidine tag at the N terminus of D2-BGL.…”

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“…raphigera was reported to produce pectinase, cellulase and xylanase activity, which could play a major virulence role for rot in pomegranates [41]. Yoneda et al [42] reported that β-glucosidase secreted by C . raphigera .…”