黑曲霉T3-5-1高活力单宁酶的制备及其性质研究

Preparation and Properties of High Activity Tannase with Aspergillus niger T3-5-1

摘要:

用黑曲霉T3-5-1生产单宁酶，将粗酶液分别采用硫酸铵沉淀法和透析法初步纯化，结果表明，使用截留分子量12 kDa的透析袋对粗酶液透析后的比活力(3108.58 U/mg)，高于硫酸铵沉淀法处理后的比活力(2939.38 U/mg)。然后对透析样品用Sephadex G-100葡聚糖凝胶层析法进一步纯化，得到纯度较高的单宁酶，通过与单宁酶A及单宁酶B进行GPC色谱图比较发现，其分子量基本一致，但黑曲霉T3-5-1单宁酶的比活力(5304.78 U/mg)，远高于单宁酶A(1788.15 U/mg)和单宁酶B(935.07 U/mg)的比活力。该单宁酶性质研究表明：其最适pH值为5.0，最适反应温度为40 ℃，且其酸碱稳定性和热稳定性良好。当底物为没食子酸丙酯时，最大酶促反应速率Vmax为81.96 μmol/(L?min)，米氏常数Km为0.85 mmol/L。最后考察了该单宁酶的催化合成能力，结果表明，在AOT异辛烷反胶束体系中，该单宁酶可催化没食子酸和丙醇反应合成没食子酸丙酯。

Abstract:

Tannase was produced by Aspergillus niger T3-5-1, then the crude enzyme was preliminary purified by ammonium sulfate precipitation and dialysis respectively. The results showed that, specific activity of the crude enzyme dealed with dialysis sack of 12 kDa MWCO was higher than that of ammonium sulfate precipitation. Further purification was performed by Sephadex G-100 dextran gel chromatography, and then high purity tannase was obtained. Compared the tannase with Tannase A and Tannase B by GPC chromatogram, it was found that their molecular weight were generally consistent. However, specific activity of the tannase(5304.78 U/mg) was far higher than those of Tannase A (1788.15 U/mg) and Tannase B (935.07 U/mg). The optimum pH and reaction temperature of the tannase were 5.0 and 45 ℃, respectively,, with favorable pH and thermal stability. When the substrate, the Vmax and Km were were propyl gallate (PG), 81.96 μmol/(L?min) and 0.85 mmol/L, respectively. At last, the catalytic synthesis ability of the tannase was investigated, which suggested that the enzyme could catalyze the reaction between gallic acid and propyl alcohol to synthesize PG in the reverse micelle of AOT- isooctane.