Recombinant thermo-alkali-stable endoglucanase of Myceliopthora thermophila BJA (rMt-egl): Biochemical characteristics and applicability in enzymatic saccharification of agro-residues.

Codon adaptation index (CAI) of a 1263bp long endoglucanase encoding gene from the thermophilic mould Myceliopthora thermophile BJA has been improved from 0.44 to 0.76 by in vitro gene synthesis. The codon optimized endoglucanase gene (Mt-egl) has been constitutively expressed in Pichia pastoris under the regulation of GAP promoter. Recombinant endoglucanase (rMt-egl), purified by size exclusion chromatography, has been confirmed to be a monomeric protein of ∼47kDa. rMt-egl is optimally active at pH 10 and 50°C, displaying stability in broad pH and temperature ranges, with a t1/2 of 60 and 15min at 90 and 100°C, respectively. This retained ∼70% of activity after 3h incubation at pH 5-12. The Km, Vmax, kcat and kcat/Km of rMt-egl were 5mgmL-1, 20μmolesmin-1mg-1, 1.02×103s-1 and 204s-1mg-1mL-1, respectively. Homology modeling and bioinformatics analysis confirmed catalytically important role of glutamate 234 and 344. rMt-egl released high amounts of reducing sugars from wheat bran and corn cobs (421 and 382mgg-1), thus making it a useful biocatalyst for producing bioethanol and fine chemicals from agro-residues.