Biochemical characterization of a glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii

The glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii (AkCel61) is a modular enzyme that consists of a catalytic domain and a carbohydrate-binding module belonging to family 1 (CBM1) that are connected by a Ser-Thr linker region longer than 100 amino acids. We expressed the recombinant AkCel61, wild-type enzyme (rAkCel61), and a truncated enzyme consisting of the catalytic domain (rAkCel61ΔCBM) in Pichia pastoris and analyzed their biochemical properties. Purified rAkCel61 and rAkCel61ΔCBM migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were demonstrated to have apparent molecular masses of 81,000 and 34,000 Da, respectively. After treatment with endoglycosidase H, both proteins showed an increase in mobility, thus, demonstrating estimated molecular masses of 78,000 and 28,000 Da, respectively. Mass spectrometry analysis revealed that rAkCel61 and rAkCel61ΔCBM expressed in P. pastoris are heterogeneous due to protein glycosylation. The rAkCel61 protein bound to crystalline cellulose but not to arabinoxylan. The rAkCel61 and rAkCel61ΔCBM proteins produced small amounts of oligosaccharides from soluble carboxymethylcellulose. They also exhibited a slight hydrolytic activity toward laminarin. However, they showed no detectable activity toward microcrystalline cellulose, arabinoxylan, and pectin. Both recombinant enzymes also showed no detectable activity toward p-nitrophenyl β-d-glucoside, p-nitrophenyl β-d-cellobioside, and p-nitrophenyl β-d-cellotrioside. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.