Heterologous expression and biochemical characterization of a GHF9 endoglucanase from the termite Reticulitermes speratus in Pichia pastoris.

BACKGROUND: Cellulases are of great significance for full utilization of lignocellulosic biomass. Termites have an efficient ability to degrade cellulose. Heterologous production of the termite-origin cellulases is the first step to realize their industrial applications. The use of P. pastoris for the expression of recombinant proteins has become popular. The endoglucanase from Reticulitermes speratus (RsEG), belonging to glycoside hydrolase family 9 (GHF9), has not been produced in P. pastoris yet. RESULTS: A mutant RsEGm (G91A/Y97W/K429A) was successfully overexpressed in P. pastoris. RsEGm, with optimum pH 5.0, was active over the pH range of 4.0 to 9.0, and exhibited superior pH stability over between pH 4.0 and pH 11.0. It displayed the highest activity and good stability at 40 °C, but lost activity quickly at 50 °C. The apparent kinetic parameters of RsEGm against Carboxymethyl Cellulose (CMC) were determined, with K m and V max of 7.6 mg/ml and 5.4 µmol/min•mg respectively. Co2+, Mn2+ and Fe2+ enhanced the activity of RsEGm by 32.0, 19.5 and 11.2% respectively, while Pb2+ and Cu2+ decreased its activity by 19.6 and 12.7% separately. CONCLUSIONS: RsEGm could be overexpressed in P. pastoris. It was stable between pH 4.0 and pH 11.0, and exhibited higher stability at temperatures ≤ 40 °C. This endoglucanase may have potential to be used in the field of laundry, textile and lignocellulose-based biofuels and chemicals.