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

Abstract

Background: The 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.

Results: PpCel45A (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.

Conclusions: The 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.

Figure 1

Modular representation of PpCel45A and derived constructs used in the study.

Figure 2

Alignment of the 5 CBMs amino-acid sequences. The three conserved aromatic amino acids (by referring to T. reesei Cel7a CBM1 sequence) forming a hydrophobic platform for interaction with cellulose are shown in grey.

Figure 3

Phylogenetic representation of family GH45 from fungal origin. The cladogram highlights the relative position of the proteins labelled with the abbreviation of the species name, the reference public database accession numbers (Genbank) and the identified activity for characterized enzymes (EC numbers). Subfamilies are indicated based on [28].

Figure 4

SDS-PAGE analysis of PpCel45A and its derived constructs. 10 μg of each purified recombinant protein was loaded onto a 12% Tris glycine SDS-PAGE and proteins were stained with Coomassie blue. PpCel45A (lane 1), PpCBM1⁵:GH (lane 2), PpGH (lane 3) and PpCBM1^1-5 (lane 4). Molecular masses (kDa) of marker proteins are shown on the right.

Figure 5

Thermostability of PpCel45A and its derived constructs. Thermostability profiles at 65°C (A) and 80°C (B). Light gray: PpGH, dark gray: PpCBM1⁵:GH, black: PpCel45A. Half-lives of the three constructions are indicated on the right.

Figure 6

Hydrolysis of crystalline cellulose at different temperatures. Hydrolysis was performed on Avicel for 16 hours at 45°C or 65°C with: PpGH (white bars), PpCBM1⁵:GH (light gray bars), PpCel45A (dark gray bars), PpCBM1^1-5 (black bars) and PpGH+PpCBM1^1-5 in equimolar amounts (striped bars).