A thermostable GH45 endoglucanase from yeast: impact of its atypical multimodularity on activity

Abstract

BackgroundThe gene encoding an atypical multi-modular glycoside hydrolase family 45 endoglucanase bearing five different family 1 carbohydrate binding modules (CBM1), designated PpCel45A, was identified in the Pichia pastoris GS115 genome.ResultsPpCel45A (full-length open reading frame), and three derived constructs comprising (i) the catalytic module with its proximal CBM1, (ii) the catalytic module only, and (iii) the five CBM1 modules without catalytic module, were successfully expressed to high yields (up to 2 grams per litre of culture) in P. pastoris X33. Although the constructs containing the catalytic module displayed similar activities towards a range of glucans, comparison of their biochemical characteristics revealed striking differences. We observed a high thermostability of PpCel45A (Half life time of 6 h at 80°C), which decreased with the removal of CBMs and glycosylated linkers. However, both binding to crystalline cellulose and hydrolysis of crystalline cellulose and cellohexaose were substantially boosted by the presence of one CBM rather than five.ConclusionsThe present study has revealed the specific features of the first characterized endo β-1,4 glucanase from yeast, whose thermostability is promising for biotechnological applications related to the saccharification of lignocellulosic biomass such as consolidated bioprocessing.

Lytic polysaccharide monooxygenases from Myceliophthora thermophila C1 differ in substrate preference and reducing agent specificity

“…Both Mt LPMO9B and Mt LPMO9C contain multiple glycosylations that were determined as hexoses based on LC/ESI-MS (Additional file 2). It is known that LPMOs can show glycosylation in the substrate-binding site, though the glycosylation could also appear at the serine/threonine containing linker between the carbohydrate-binding module (CBM), the C-terminal end of the LPMO or at the binding site of the CBM [29, 31, 32]. …”

The ectomycorrhizal basidiomycete Laccaria bicolor releases a secreted β‐1,4 endoglucanase that plays a key role in symbiosis development

“…The expression protocol is described by Couturier et al . (). Large scale (2.4 liter) production of each protein was performed in 500 ml nonbaffled flasks, each containing 100 ml of BMGY medium (1% yeast extract, 2% peptone, 1% glycerol, 400 μg l −1 biotin and 0.1 M potassium phosphate, pH 6.0).…”

Multiple horizontally acquired genes from fungal and prokaryotic donors encode cellulolytic enzymes in the bdelloid rotifer Adineta ricciae

“…The GH45 cellulases in the nematode Bursaphelenchus xylophilus were reported to be most closely related to sequences from ascomycete fungi (Palomares-Rius et al, 2014), but also show similarity to bdelloid and zygomycete sequences (although none of the studied groups seems monophyletic). The evolutionary origins of these sequences may be complicated by HGT of cellulolytic genes between different fungal groups, however (Garcia-Vallve et al, 2000;Couturier et al, 2011). It remains unknown whether the GH45-encoding genes were acquired from a common source by both bdelloids and nematodes.…”

“…It is uncommon for GH45 ENGs to contain more than two CBMs (Baba et al, 2005), although one GH45 ENG with five CBM1 domains has been reported in the yeast Pichia pastoris (Couturier et al, 2011), where the extra CBMs facilitate enzyme stability at higher temperatures.…”