Study On β-1,3-Glucanase Production On Solid-State Fermentation From Trichoderma TP-24 And Enzymological Characteristics

Yeast β-1,3-Glucan is a biological response modifiers with high efficiency and low side-effect , but its low solubility makes it be utilized difficultly. With strain LE02 as object, the culture condition was optimized by solid state fermentation. β-Glucanase was purified through salting-out, dialyses, gel filtration chromatography. Enzymological properties were studied in this research. Results summarized as following:1. β-Glucanase producing colonies was screened according to the transparent zone around the colony on plates containing Congo-red. A high β-Glucanase producing strain Tβ-24 was selected by solid state fermentation, which produces β-Glucanase as high as 335.37U/g.ssc .Strain Tβ-24 was primarily identified to be Trichoderma by morphology.2. The culture condition for producing β-Glucanase on solid state fermentation medium(SFM) was optimized by response surface analysis. The optimum conditions in 20g wheat bran on SFM were 1.91% yeast powder as additional carbon source, 1.99% NH4NO3 as additional nitrogen source, 1:0.79 proportion of wheat bran to water, fermenting continuously 4 days at 30C. Under this condition, β-Glucanase activity was 594.37U/g.ssc, increased by 273.55% than that of contrast sample.3. Crude enzyme solution was precipitated with ammonium sulfate fraction, dialyses, and Sephadex G-100 chromatographic filtration.. The recovery of β-Glucanase was almost 45.15%, the specific activity was increased from 11.96U/mg to 342.95U/mg. SDS-PAGE of the purified β-Glucanase showed a single stained band at approximately molecular weight of 54.56KD.4. Enzymological properties of the purified β-Glucanase were studied. The optimal temperature and pH for the enzyme reaction was 55C, and 5.0, respectively; and with high stability from 30C to 40C, and pH 3.0 to 5.0. It was inhibited by Fe3+, Mg2+, Mn2+, Cu2+ , and stimulated by Zn2+, Ca2+, Fe2+. The β-Glucanase has high specificity on β-1,3 linkage and could hydrolyze β-1,4 linkage slightly, but no activity to a -1,4 anda -1,6 linkage, when CMC-Na as reaction substrate, Km and Vmax are 3.85mg/ml and 20.88 u g/min, respectively; when yeast β-1,3-Glucan as reaction substrate, they were 8.02 u g/ml and 5.59 u g/min, respectively.