Optimization Of Solid-state Fermentation And Characteristics Of β-glucosidase From Aspergillus Oryzae FZ58

The solid-state fermentation medium of Aspergillus oryzae FZ58 producingβ-glucosidase was optimized.The single-factor test was carried out and the result indicated that the optimum carbon source was bagasse,nitrogen source was soybean cake powder.It also showed that the optimum initial fermentation pH was 6.5 and the ratio of solid to liquid was 1/3,all above the information made out the initial formulation of solidstate fermentation medium.Then the Plackett-Burman design was taken to investigate which factors contributed toβ-glucosidase solid- state fermentation of Aspergillus oryzae most and 3 most significant influence factors were found,those were wheat bran,water and FeSO₄.In the following experiments,Response surface methodology of Box-Behnken design was carried out to optimize the 3 significant factors and the optimum levels of those were found.The formulation of the optimum fermentation medium(g/L)was MgSO₄ 0.5,K₂HPO₄ 0.1,FeSO₄ 0.01,NaNO₃ 2,pH6.5 and solid shape object m(wheat bran):m(bagasse):m(soybean cake powder)= 4:1.5:0.2,the ratio of solid to liquid was 57:100.The optimum fermentation condition was obtained:the inoculum density was 1mL 5～7×10⁶ spore suspension/mL every 5g solid shape object, the fermentation temperature was 33℃and the fermentation time was 5 days.After the optimization,the enzyme activity was increased from 1002.6U/g to1401 U/g,39.7% more than before,the optimum result was more significant.Ammonium sulfate fractional precipitation,DEAE Sepharose Fast Flow ion-exchange chromatography and Sephacryl S-200 molecular column chromatography purification steps were carried out to do the initial purification of this enzyme.After the purification,the specific activity was 4125.72U/mg,the purification multiple was 7.68 and the coefficient of recovery was 1.81%.SDS—PAGE was done and showed that the protein molecular weight was about 120.23kDa.Enzyme characteristics research indicated that the best enzymatic reaction temperature was 60℃,the enzymatic reaction was relative stable under 60℃;the optimum pH of enzymatic reaction was 5.0,enzymatic reaction was stable between pH 3～9.Salicin and pNPG were used as the substrate and the enzymatic reaction fit the Michealis equation, the maximum reaction initial velocity V_m were 0.21mg/mL/min and 2500μmol/L/min. Michaelis constant(Km)were 0.81mg/mL and 1 mmol/L each.The activation energy(E_a) were 60.80 kJ/mol and 43.54kJ/mol each.Research on the metal ion's influence on the enzyme activity indicated that K⁺, Li²⁺,Mg²⁺,Mn²⁺,Zn²⁺ion at the concentration of 0-2.5mmol/L contributed to the enzymatic reaction insignificantly,Hg²⁺ion inhibited enzymatic reaction which indicated that the inhibitory action belongs to a reversible process.Research on the Hg²⁺ ion inhibited enzymatic reaction indicated that Hg²⁺not only had impact on K_m but also on V_m,its inhibition mechanism appeared to mixed pattern inhibition.Research on organic solvents' influence on the enzyme activity indicated that some organic solvent just like methanol,alcohol,1.2- propylene glycol,isopropyl alcohol and so on had impact on the enzyme activity too.When the concentration of those organic solvents went up,their impact on the enzymatic reaction went down.Those organic solvents' IC50(the inhibiting concentration when the emzyme activity descend 50%) were 11%,10%,11.3%,and 12.8%each.