Study On Purification And Characterization Of Tannase From Aspergillus Niger And Application In Non-aqueous Media

Tannase (EC3.1.1.20), also known as tannic acid ester hydrolase, is an adaptive enzyme synthesized by certain microorganisms in the presence of antileptic such as tannic acid. Tannase has also been widely used in the food, beverage, fine chemicals, leather, feed, cosmetics and other fields. Tannase not only catalyze hydrolysis reaction, but also catalyze esterification. For example, it can transfer ester bone of tannic acid to alcohols to synthesis gallic acid esters directly. Gallic acid esters, one of important derivatives of gallic acid, can promote the dissolution of fibrin and thrombosis, dilate blood vessels, increasing blood flow and inhibit the crown platelet aggregation and are also widely used in the treatment of cardiovascular and cerebrovascular diseases. Gallic acid esters have broad and important application perspective in medicine field, in which propyl gallate(PG) is particularly conspicuous.First of all, the experiment tannase-producing strain was selected from the four strains of Aspergillus niger, Aspergillus flavus, Aspergillus oryzae and Rhizopus through primary hydrolysis circle method and UV spectrophotometry check.The tannase activity of broth is 2.414 U/ml in basic medium; Fermentation conditions of Aspergillus niger with high tannase yield were further optimized by orthogonal test,in which seven factors of tannic acid, KH PO content, (NH₄)₂SO₄ concentration, MgCl₂·6H₂O content, temperature, pH, shaking speed affect on the yield of tannase were investigated; The electrophoresis pure tannase was obtained and purified at a simple and efficient procedure, and high-performance liquid chromatography (HPLC) and SDS-PAGE were applied to the analysis of purity and Molecular Weight respectively.; Meanwhile, the characterization of three types enzymes of crude, purified and immobilized enzyme were studied, including the effects of temperature tolerance and stability, pH tolerance and stability, substrate specificity of the enzyme ,surface active agents, organic solvents, ionic liquids and so on. Eventually, tannase catalytic esterification reaction in non-aqueous medium with the substrate of gallic acid and propanol was studied. Characterization of Enzymatic esterification in four kinds of non-aqueous medium of chloroform, acetone, [bmim][BF₄] and [bupy][BF₄] were also explored, including effects of substrate molar ratio, water activity on enzyme activity.Results: The combination of primary and check method was used to choose the experimental strain of Aspergillus fungus; As orthogonal analysis of variance show, The effect of tannic acid on the yield was significant.The optimum conditions of tannase production by Aspergillus niger were as follows: 2% tannic acid; 0.1% KH2PO4 ;0.7%(NH₄)₂SO₄ ;0.1% MgCl₂·6H₂O ;temp 40℃;pH 5.5;rotation speed 140 rpm. In the optimal medium and culture conditions, the tannase activity reached 5.215 U/ml, which increased 2.16 times to that of basic medium. Electrophoretically pure tannase was obtained after filtration, centrifugalization, ultrafiltration and chromatography and the MW of electrophoretically pure tannase is 119 KD analyzed by SDS-PAGE, and 6.4 mg enzyme protein can be obtained from 1L culture medium eventually; The optimum temperature and pH value of crude, purified and immobilized enzyme were 45℃, 5.5, and the gradual loss of residue activity was happening over time. 12 hours later, the loss of activity was up to 9.9%, 10.2% and 8.3% separately. The Km of tannic acid and propyl gallate were 4.6 mM and 5 mM, so the affinity of tannase to tannic acid ester is greater than that of propyl gallate. Tannase activity was suppressed by sodium dodecylsulphate and Tween-80 completely, and enzyme activity remained stable in polyethylene glycol octyl phenyl ether. To place tannase in 20% of the organic solvents (acetone, chloroform and isopropyl alcohol) for 5 min, residual activity of tannase were 71%, 76% and 80% respectively. Hydrophilic [bmim] [BF₄] and [bupy] [BF₄] inhibited the enzyme activity. To place tannase in 20% [bmim] [BF₄] and [bupy] [BF₄] for 5 min, the remaining activity was 95% and 93% separately. To incubate tannase in 1 mMβ-mercaptoethanol, bromine and ammonia acrylic acid for 30 min, residual activity was 41.2%, 54.6% and 66% respectively. According to result of the enzymatic synthesis of propyl gallate, when gallic acid and propanol in the ratio of 1:2, the highest conversion rate was obtained and up to 92.6%; when initial water activity of 0.55 in the reaction, the max convertion rate and initial speed were up to 92.3% and 0.63 mM/h separately. By comparison of the esterification in different media, effects of esterification in ionic liquids were better than that of organic solvents, in which the max convertion rate and initial speed were obtained in [bmim][BF ] and up to 92.5% and 0.65 mM/h respectively. 4...