生物技术与生物材料

抽象的

Cloning and Characterization of Physarum Endo-Beta-1,3-1,4-Glucanase-1 Expression in E. coli and Trichoderma reesei

Jian-Hua Zhang, Xiao-qing Li, Yong-Xia Zhang, Miao Xing, Sheng-Li Tian and Shi-De Liu

The endo-β-1,3-1,4-glucanases have been found widely in bacteria, fungi, algae and plants, and have been applied to the hydrolysis of barley β-1,3-1,4-glucans in the beer brewing industry. Here, we report a novel endo-β-1,3- 1,4-glucanase gene (PEGase1) isolated from Physarum polycephalum and expressed in E. coli, and Trichoderma reesei. The molecular weights of the proteins are 34 and 66 kDa respectively by SDS-PAGE analysis. Enzymatic assays show that recombinant PEGase1 expressed by T. reesei was more efficient when hydrolyzing barley β-1,3- 1,4-glucans specifically, suggesting that modifications have a dramatic effect on the activity of PEGase1. We found that recombinant PEGase1 can be enriched by 40-70% ammonium sulfate precipitation and purified by HiTrapTM CaptoTM Q ion-exchange chromatography. Measurements of the enzymatic activity of purified PEGase1 under a range of conditions show that its optimal pH is around 5.5, and its optimal temperature is 55°C, with Km = 0.882 mg/ ml. PEGase1 activity is more stable in acidic solution and at low temperatures than it is in alkaline solution and at high temperatures. Metal ions and their concentration are able to affect the enzymatic activity, some metal ions, for example, Cu2+, Ni2+, Zn2+, Fe2+, Ba2+, Mg2+ and Ca2+ can enhance the enzymatic activity at 1 mmol/L concentration and inhabit the activity at 5 mmol/L concentration, but metal ion Mo2+and Co2+show inhabit the enzymatic activity at both concentration.