Screening Of Tannase-Producing Strains And Optimization Of Culture Conditions

Tannase was produced by microorganisms,it was a kind of enzyme which can hydrolyses tannins. Tannase has aroused people's extensive concern because of its wide-ranging application in food,beverage,alcohol making,feeding,cosmetic,pharmacy and tanning industries.This research progressed on screening a strain from nature where the resources were rich in tannins,then selecting a strain which had higher enzyme activity by UV,DES and both of the two inducing mutagenesis in turn,finally optimizing solid-state fermentation conditions and culture medium and preliminary researching on enzymology character.Forty-six tannase-producing strains were selected from the specific environment where was rich in tannins through enrichment-culture method,the plate transparent zone method and solid state fermentation.Then the strain 1-39 with the higher tannase activity had been selected from the forty-six strains by the secondary screening by solid state fermentation,the tannase activity of each gram dried fermentation solid culture medium achieved 1.58 IU. Colonies and morphologic observation showed that this strain belonged to Aspergillus,then named it DNM1-39.The Spore suspension liquid of original strain DNM1-39 was mutated by ultraviolet,diethyl sulfate and both of them in turn,the mutant strain UD-6 was selected finally with the enzyme activity reaching 2.55IU/g dried fermentation solid medium which was enhanced by 62.8%.The fermentation conditions and the culture medium of tannase production by mutant strain UD-6 were optimized by single factor experiment and orthogonal experiment.After poor analysis,variance analysis and multiple comparisons,The optimal fermentation conditions for tannase production were determined as follows:30℃,the pH of salt solution 6.0, weight in flask 12g/250mL,growth cycle 60h;The optimal culture medium for enzyme production were determined as follows(g/100mL):wheat bran,Solid:liquid(g:mL)1:1,salt solution (g/100mL):tannins 0.75,NaCl 0.5,MgSO₄·7H₂O 0.5,(NH₄)₂SO₄ 1.0,MnSO₄ 0.1,ZnSO₄ 0.05,NH₄Cl 1.5,the enzyme activity of mutant strain UD-6 was 5.61IU/g dried fermentation solid medium in this circumstance,with enzyme activity enhanced by 120%.The tannase was stable between 23℃and 30℃,it would be devitalized quickly above 50℃;The optimal situation of the reaction was citric acid buffer with pH5.0.The enzyme activity was strongly inhibited by Cu²⁺,Al³⁺,Ag⁺ ;Mn²⁺,Zn²⁺ can increase it a little,other metal ions had no effection on it.