单宁酶的固定化及其在酯型儿茶素水解反应中的应用

采用自制的壳聚糖为载体对单宁酶（ＴＡ）固定化，ＴＡ与壳聚糖配比１：２．５，３０℃固定２ｈ，活力回收达２３．６％～３３．１％；偶联效率为８４．９％～８８．０％。固定化单宁酶（ＩＴＡ）的表观Ｋｍ值（以没食子酸丙酯为底物）为２２．２×１０－６ｍｏｌ／Ｌ，ＴＡ的Ｋｍ值（以没食子酸丙酯为底物）为１０×１０－６ｍｏｌ／Ｌ，ＴＡ和ＩＴＡ的最适反应温度分别为４０℃和５０℃；６０℃处理１５ｍｉｎ，残存活性分别为１３．６％和６０．３％。ＴＡ和ＩＴＡ的最适ｐＨ值分别为５．８和６．４；ＴＡ在ｐＨ４．８～７．８活力稳定，而ＩＴＡ活力稳定范围在ｐＨ４．８～６．８．ＩＴＡ作用于ＥＧＣＧ的半衰期为７８．７ｈ，ＥＧＣＧ水解率达９０．３％。对茶多酚提取物进行水解，其所含的酯型儿茶素ＥＧＣＧ和ＥＣＧ水解率分别为９６．４％和９６．８％，非酯型儿茶素ＥＧＣ和ＥＣ的含量显著增加。

Immobilization of Tannase and Its Applicationin Hydrolysis of Estern-catechines

Tannase(TA) was immobilized with self-made chitosan.The optimum conditions for immobilization were as follows:250 mg chitosan was dissolved in 1% solution of acetate acid,then 100 mg crude TA was added and the mixture was incubated at 30℃ for 2 hours.Enzyme activity recovery was 23.6%-33.1% and the efficiency of enzyme protein linked on chitosan was 84.9%-88.0%.The apparant Km of immobilized tannase(ITA) was 22.2×10-6 mol/L,while Km of TA was 10×10-e mol/L.The optimum reaction temperatures for TA and ITA were 40℃ and 50℃,respectively.Treated at 60℃ for 15 min,TA only retained 13.6% of the original activity.while ITA was more stable and retained 60.3% of the original activity. The optimum pHs for TA and ITA were 5. 8 and 6.4,respectively.TA was stable in the PH range of 4.8-7.8,while ITA was in pH 4.86.8 range.The half-life time of ITA(EGCG was used as substrate) was 78.7 hours.In the mean time the percentage of hydrolysis of EGCG was 90.3%.When treated by ITA,EGCG and ECG contained in tea polyphenol extracts were hydrolyzed up to 96.4% and 96.8%,respectively.The contents of EGC and EC in hydrolysate were increased greatly.