Screening Of Tannase-producing Strains

Tanin acyl hydrolase （E.C.3.11.20） commonly referred to as tannase, catalyzes thehydrolysis of ester and depside bonds in hydrolysable tannins and epicatechol gallate,releasing glucose and gallic acid. Many microorganisms in nature could excrete tanase.Currently, as the prime strains in the production of tannase, the Aspergillus andPenicillium were researched more deeply than others strains. Compared with the fungus,the yeast has certain advantages in producing tanase, such as the simple cell structure,the effortless genetic modification and the rapid breeding. The paper concentrated onthe the screening and identification of tanase-producing yeast, the optimization of theculture medium and cultivation conditions, the analysis of the enzymatic properties andthe application in the process of tea drinks. The main results were as follows:The strain FC6-1with higher tannase activity was successfully isolated from thesoil sample near tea plants by enrichment-culture technigue, assaying hydrolyzed circleon the selective medium, determing tannase activity under shake bottle fermentation.The initial enzyme activity of this strain FC6-1was2.86U/mL. The morphologica and26S rDNA P1/P2sequence analysis identified the strain FC6-1as Aureobasidium sp.This yeast strain was able to hydrolyze tannin into gallic acid and glucose.The conditions of fermentation were optimized by single factor and orthogonalexperiments. The optimum culture medium was as follows（g/L）: tannin30，corn starch10, soybean meal30, MgSO4·7H2O2.0, K2HPO4·3H2O1.0, NaCl0.5, MnSO4·H2O0.4,CaCl20.2; the optimal culture conditions in shake bottle were as follows: initial pH3.0,temperature28℃, inoculation amount10%（V/V）, rotation speed180rpm, broth’svolume in shake flask35mL/250mL and culture time72h. Under the optimumcondition, the enzyme activity of tannase was enhanced from2.86U/mL to4.83U/mL.The characterization of tannase from this yeast strain FC6-1was studied.Theenzyme was stable between20℃⁵0℃and pH4.0⁷.0. Its maximum enzyme activityreached5.48U/mL at50℃, pH5.5. The enzyme activity coule be advanced by Mg²⁺and Mn²⁺, and inhibited by Fe²⁺and Zn²⁺.The application of crude tannase for improving the clarification of tea extracts waspreliminary studied in this paper. Tea extracts was prepared by tea and water with theproportion of1:30（W/V）. Then, tea extracts was treated for20min at50℃with theproportion of crude enzyme and tea extracts8:100（W/V）. The clarity of tea extracts was improved by37.41%than before.