Heterologous expression and biochemical characterization of a novel thermostable Sclerotinia sclerotiorum GH45 endoglucanase in Pichia pastoris

Abstract Enzymatic saccharification of lignocellulosic biomass has been widely studied. Mainly endoglucanases were found to be a prerequisite for the quick initial biomass liquefaction. In the present study, Pichia pastoris was used as a host for the heterologous expression of a Sclerotinia sclerotiorum GH45 endoglucanase, Endo 2. The recombinant plasmid p PICZαA was used to transform Pichia pastoris . Pichia culture supernatants expressing the recombinant Endo 2 (r Endo 2) were used for the purification and biochemical characterization of this enzyme. Therefore, r Endo 2 was purified 6.7 fold to homogeneity with 34% yield and gave 19 U/mg specific activity. It also showed maximum activity at pH 7.0 and 60 °C (against pH 5.0 and 50 °C for the native enzyme) and was thermostable at relatively high temperatures. Furthermore, r Endo 2 retained its activity in a wide pH range (from 5 to 8). Besides, the recombinant endoglucanase was produced as an active 47 kDa enzyme. This molecular weight differs from the one of the native enzyme (34 kDa), which suggested a potential glycosylation of the recombinant enzyme. Moreover, r Endo2 was able to produce fermentable sugars after enzymatic assay on various cellulosic substrates with an interesting yield. Therefore, all these features offer prospects for large-scale production and industrial application of the recombinant endoglucanase.