Research On Fermentation Process, Enzymatic Property And Immobilization Of Tannase From Aspergillus Ficuum

In this work, tanase production by Aspergillus ficuum Gim3.6was conducted in the uncooked material fermentation with gallnut as inducer. Optimization of fermentation process, reverse micellar extraction of tannase, enzymatic properties and immobilization of tannase were successively studied.Single factor experiment followed by response surface methodology was applied to the optimization of tanase production under uncooked material fermentation. The optimum conditions for tannase production were ammonium chloride as nitrogen source, wheat bran as carbon source,5g of culture medium and addition of0.55g gallmut as inducer,1.22ml of inoculum size at30℃and initial pH5.0. The enzyme activity in this culture could be as high as28.37U/gds after incubated for72h.Reverse micellar system of CTAB/n-butyl alcohol/isooctane was used for the extraction and purification of tannase. The optimum conditions for the forward extraction were50mmol/L of CTAB, addition of1/5cosolvent, and shaking for15min at45℃. As for the back extraction, the optimum conditions were aqueous phase pH3.8, addition of3ml octanol,1:1of the two phase,0.25mol/L of sodium chloride and mixing for10min. Under this condition more than5.64-fold purified tannase was obtained with a yield of75.5%.Enzymatic properties of tannase from A.ficuum Gim3.6were investigated. This enzyme showed temperature and pH optima of40℃and6.0, respectively. The effects of different metal ions on the enzyme activities was studied and the results showed that:Fe3+and Ba2+had no obvious impact on the enzyme activity, whereas Fe2+mildly activated tannase activity, and Mg2+, A13+and EDTA inhibited tannase activity. The catalytical property of tannase was evaluated using propyl gallate as substrate, and the maximal reaction velocity (Km) and Michaelis constant (Vmax) were found to be0.6144mmol/L and65.359μmol/L·min, respectively.Immobilization of tannase from A.ficuum Gim3.6was performed. The enzyme activity recovery of82.57%was attained under the optimum conditions of immobilization, namely1.5%of chitosan,2%of sodium alginate,0.2%of glutaraldehyde, and0.25mol/L of Ca2+, crosslinking for5.5h. Activity of the immobilized tannase remained51.25%after repeatedly used for7times, which indicated that the stability of the immobilized tannase was improved.