Heterologous Expression, Purification and Characterization of Alpha-Glucanase from Chaetomium globosum

Abstract:

In this study, α-glucanase derived from Chaetomium globosum was synthesized and optimized according to the codon preference of <>Pichia pastoris, and then successfully heterologous expressed in Pichia pastoris GS115. Through the high-copy screening and single-factor optimization of shaike flask fermentation, the enzyme activity of recombinant α-glucanase was increased from 2.692 U/mL to 47.915 U/mL. The crude enzyme was purified to electrophoretic purity by two-step chromatography through Hitrap Q HP and Hitrap SP HP. The purification factor reached 40.83, with the recovery of 24.15%. And the specific activity of purified enzyme reached 277.61 U/mg. The optimal reaction temperature and pH were 60 ℃ and 5.5, respectively. The enzymatic activity kept stable within 20~50 ℃ and pH 4.5~8.5. The presence of 10 mM Fe²⁺ activated the enzyme, whereas 0.1~10 mM Cu²⁺ inhibited the enzyme. The higher concentration of Cu²⁺, the stronger inhibition of enzyme. Affinity study indicated that the recombinant enzyme had the higher affinity towards substrates with higher molecular weight. Dextran T2000 was the optimal substrate of the enzyme.