Expression of a wheat β-1,3-glucanase in Pichia pastoris and its inhibitory effect on fungi commonly associated with wheat kernel

Abstract

β-1,3-glucanases, the plant PR-2 family of pathogenesis-related (PR) proteins, can be constitutively expressed and induced in wheat crop to enhance its anti-fungal pathogen defense. This study aimed to investigate the inhibitory effect of wheat β-1,3-glucanase on fungi most commonly associated with wheat kernel. A β-1,3-glucanase from wheat was successfully expressed in Pichia pastoris X-33 and its biochemical and antifungal properties were characterized herein. The molecular weight of recombinant β-1,3-glucanase is approximately 33 kDa. β-1,3-glucanase displays optimal activity at pH 6.5, remaining relatively high at pH 5.5-8.0. The optimal reaction temperature of β-1,3-glucanase is 50 °C, retaining approximately 84.0% residual activity after heat-treated at 50 °C for 1 h. The steady-state kinetic parameters of β-1,3-glucanase against laminarin was determined and the K_m and V_max were 1.32 ± 0.20 mg/ml and 96.4 ± 4.4 U mg^-1 protein, respectively. The inhibitory effect of purified β-1,3-glucanase against the seven fungi commonly associated with wheat kernel was assessed in vitro. β-1,3-glucanase exerted differential inhibitory effects on hyphal growth of Fusarium graminearum, Alternaria sp., A. glaucus, A. flavus, A. niger, and Penicillium sp. Spore formation and mycelial morphology of Alternaria sp., A. flavus, and A. niger were significantly affected by β-1,3-glucanase (1U). The present results would help elucidate the mechanism underlying the inhibition of wheat β-1,3-glucanases on pathogens.

Keywords: Fungal pathogen; Pathogenesis-related proteins; Pichia pastoris; Wheat; β-1,3-glucanase.