Purification, Characterization And Structure Of β-1, 3-Glucanase From Trichoderma Strain

β-1,3-glucan is a biological response modifiers with high efficiency and low side-effect. However, its application is constrained because the glucan obtained by alkali method is of low solubility. Hydrolysing by β-1,3-glucanase is a great method to increase the solubility of β-1,3-glucan, which will widen its application in medicine, food and cosmetic industry. With Trichoderma strain LE02 as objective, β-1,3-glucanase was purified through precipitation, gel filtration chromatography, anion-exchange chromatography and electrophoresis. The enzymological properties of β-1,3-glucanase were investigated by thin-layer chromatography, isoelectric focusing electrophoresis, ultraviolet difference spectrum and circular dichroism (CD). The main results summarized as follows:1. β-1,3-Glucanase from Trichoderma strain LE02 was precipitated by three methods. Two-step salting-out using ammonium sulfate was better than ethanol or acetone precipitation. Crude enzyme was salted-out by ammonium sulfate, followed by dialysed, concentrated with polyethylene 6000, and further purified by Sephdex G-200 gel filtration chromatography. The enzyme obtained through the above steps was not homogeneous protein. If further purified by DEAE-Sepharose CL-6B anion-exchange chromatography, the purified activity recovery of β-1,3-glucanase was 78.71%, and specific activity increased from 12.84 U/mg to 689.93 U/mg. The purified β-1,3-glucanase was a single band on both Native-PAGE and SDS-PAGE, with molecular weight of 80.137 KDa. With Native-PAGE, the β-1,3-glucanase from Trichoderma strain was stained by commassie brilliant blue R-250, or by 2,3,5-triphenyl tetrazolium chloride after reacting with Laminarin, which indicated that this enzyme hadn't isoenzyme. Isoelectric focusing electrophoresis study showed that pI of β-1,3-glucanase was 7.0.2. Enzymological properties of the purified β-1,3-glucanase were studied. The optimal temperature was 55℃ and pH 5.0 for the enzyme reaction, with high stability under 50℃, and pH from 4.5 to 6.0. In addition, Co²⁺, K⁺, Zn²⁺, Li⁺, Ba²⁺, Cu²⁺, 1% p-mercaptoethanol and 1.0 mmol/L Fe²⁺ had no effect on β-1,3-glucanase activity. Cd²⁺, 10.0 mmol/L Mg²⁺, 5.0 mmol/L and 10.0 mmol/L Fe²⁺ partly inhibited the enzyme activity, whereas 0.6 mol/L urea, 10.0mmol/L Fe³⁺, Mn²⁺ over 5.0mmol/L and low concentration Hg²⁺ significantly inhibited the enzyme activity. 1.0 mmol/L dithiothreitol,...