Screening Of Tannase-Producing Strains, Fermentation Optimization And Enzyme Purification

The present research involves the isolation and screening of tannase-producing strains, medium composition and process optimization for tannase production, purification, and characterization of the enzyme tannase, and the main results obtained are presented below.Through primary plate screening and secondary screening using solid state fermentation method, a fungal strain CY-3 with high tannase productivity was isolated from soil samples and identified as Aspergillus sp., on the basis of colonial and mciromorphologic analysis. Optimization of medium composition and fermentation conditions for tannase production by one-factor-at-a-time and response surface methodology resulted in the production of 84.90 U/gds of tannase in the medium containing 5 g of wheat bran as solid substrate moistured with salt solution (consisted of 59.4 g/L tannic acid,30 g/L glucose,6 g/L NaNO3, 1.5 g/L NaH2PO4,0.5 g/L MgSO4·7H2O,0.5 g/L KC1 and 0.01 g/L FeSO4·7H2O, pH 5.26) at a solid-liquid ratio of 1:1.5, inoculated with 20%(v/w) inoculum and incubated at 31.6℃ for 60 h. This level of tannase was 9.38 fold higher as against the initial yield under unoptimized conditions.Purification of tannase was carried out via aqueous two-phase extraction technique. Optimization of the process parameters resulted in a purification fold of 5.31 with an activity recovery of 86.70% in the system containing 18%(w/w) of PEG 600,24%(w/w) of sodium citrate and 20%(w/w) of enzyme extract, at pH 7.0. Ten times scale up of this extraction system did not lead to significant changes in the purification fold and activity recovery.Enzymatic characterization of tannase from strain CY-3 revealed that this enzyme showed optimal activity at 70℃. More than 80% of tannase activity was retained after 28 h of incubation at 50℃. The maximum activity was recorded at pH 7.0 and good stability was observed at the pH range of 5.0～7.0. The tannase was activated in the presence of 1 mM of Na+, Ba2+, Ca2+, Mg2+ and K+, whereas it was inhibited by Zn2+, Fe3+, Ag+ and Fe2+. All the organic solvents tested incruding methanol, ethanol, acetone, acetonitrile, glycerol, isooctane, propyl alcohol and islpropanol restrainted tannase activity, among which isooctane showed an inhibitory rate of 27.64%. SDS and EDTA at a concentration of 1mM exhibited inhibitory effects on tannase activity, whereas Tween 80 and Triton X-100 at 1%(v/v) had slight impact on the enzyme.