Purification And Characterization Of β-1,3-1,4-Glucanase By Mucor Petrinsularis

Malting microbiology is a new technology for wheat, is malting microbiologysoul starter strains. Previous research has shown, Mucor petrinsularis by M-28was aneffective starter strains. In the thesis, using single factor experiment, purificationconditions of β-1,3-1,4-glucanase produced from Mucor petrinsularis M-28based onthe solid-state fermentation were optimized, and the enzymatic properties werestudied in partly, in order to provide scientific basis for its application in maltingprocess. The main results are as follows:(1) The optimal extraction conditions of Mucor petrinsularisβ-1,3-1,4–glucanase.Specific for pH5.5acetic acid sodium acetate buffer solution, shaking for30min at200r/min, leaching time30min, finally the crude enzyme activity of9.041U/ml,protein content is0.140mg/ml, than the specific activity for64.583U/mg.(2) The optimal purification methods by Mucor petrinsularisβ-1,3-1,4-glucanase,the crude enzyme prepared by the saturation of80%ammonium sulfate salting out,dialysis for24h, and column chromatography separation with Sephadex G-100. Thepurified sample by SDS-PAGE electrophoresis purity determination, the specificactivity reached225.02U/mg, the enzyme purity increased3.48times compared withthe crude enzyme.(3) The basic characteristics of β-1,3-1,4-glucanase，The enzyme and substrate isthe best reaction time is10min, this enzyme can not endure high temperature,relatively stable at40-50℃, the optimum temperature is55℃; enzyme activityvalues under4.5-5.5condition is relatively stable in the pH, the optimal pH value is5.5; Preliminary identified the kinetic parameters of the enzyme, Km=263.16ug/mL,Vm=28.54ug/min.mL, Different concentrations of Fe3+on the β-1,3-1,4-glucanasereaction process, just change the reaction rate of Vm, does not change the Km,belonging to the non of competitive inhibition; different concentrations of urea on theenzyme reaction process, not only change the reaction speed, but also change the Km,belongs to the typical mixed type inhibition.(4) The effect of different materials on the thermal stability of the enzyme,different materials have different thermal stability. The results show that: gelatin,albumin, xanthan gum has better protective effect on the enzyme, glycerol, trehalose,ascorbic acid, mannitol and potassium sorbateon has no protective effect on the enzyme, According to the orthogonal experiment the optimum enzyme protectiveagent: albumin1.8%, gelatin2.0%, xanthan gum0.25%; Fe3+and Al3+had inhibitoryeffect on the Fe3+, the inhibition strong in Al3+, Fe2+has the activation function to its,other metal ions had little effect on the; urea, mercaptoethanol has inhibitory effect onthe inhibition of urea, was stronger than the mercaptoethanol, the effect of SDS on theenzyme is not significant.Shows that the substrate specificity of the enzymeexperiment, with β-glycosidic bond type.