Heterologous production and characterization of mannan acting enzymes from the white rot fungus Dichomitus squalens

The enzymatic degradation of plant cell walls has being extensively explored during the past decade chiefly due to the current and potential uses of plant biomass found in several industries. In this process, the effective removal of the hemicellulosic portion is of paramount importance. For this purpose, hemicellulases have been intensively studied and produced from bacterial and fungal sources. Within this group of enzymes, mannanases, which are mostly used during the processing of softwood, have been found in several industrial applications. Currently, white rot basidiomycetes have gained increasing attention due to their ability to fully decompose wood. One of these fungi, Dichomitus squalens, has been regarded as an effective wood degrader; nonetheless, its enzymatic arsenal and catalytic potential have only recently been explored. In the present study, six mannan acting enzymes were selected from the genome of D. squalens and heterologously produced using the methylotrophic yeast Pichia pastoris. Four of these enzymes, consisting of two β-1,4-endomannanases, one β-1,4-mannosidase and one α-galactosidase, were successfully produced. The biochemical characterization of the concentrated enzymes using synthetic p-nitrophenyl substrates corresponding to their predicted activities showed optimal pH ranging from 3 to 5 and optimal temperatures ranging from 50 to 60 °C. In addition, three of the enzymes, namely MAN1, MND1 and GAL2, showed high thermostability retaining the majority of their enzyme activities after an incubation of 60 minutes at 60 °C. In contrast, MAN2 lost all of its activity after an incubation of 30 minutes at 60 °C. Furthermore, MAN1 and GAL2 showed optimal enzyme activities of 1922.2 (± 26.6) and 903.7 (± 7.4) U/mL respectively, with specific activities of 11.1 (± 1.0) and 27.9 (± 0.5) U/mg respectively. The four enzymes had a molecular mass ranging from 47 to 110 kDa, without showing evidence that any of them formed dimers or more complex struct