为了更加高效低成本地制备茶黄素,以儿茶素作为多酚氧化酶蛋白的交联试剂,对巨大芽孢杆菌（Bacillus megaterium）来源的多酚氧化酶进行了酶交联聚集体的制备并应用于茶黄素-3,3ʹ-双没食子酸酯的合成。通过对酶交联参数优化以及交联前后的酶催化性能研究,结果表明,在pH 4.0,EGCG为0.5 mg·mL-1,多酚氧化酶活性200 U·mL-1,交联时间50 min时,可以获得交联酶的最佳酶活回收率。与游离酶相比,交联酶表现出更优异的催化性能（热稳定性、有机溶剂耐受性、底物耐受性）。交联酶被用于合成茶黄素-3,3ʹ-双没食子酸酯时,产物质量浓度可达800 μg·mL-1,且交联酶能被重复循环使用至少3个批次,利用该方法制备茶黄素,可以显著降低酶的应用成本,具有潜在工业化应用价值。
In order to prepare theaflavin more efficiently and economically, catechins were used as the cross-linking reagent in this study, and a polyphenol oxidase (tyrosinase) crossed-linked aggregate derived from Bacillus megaterium was prepared and used for the synthesis of theaflavin-3,3ʹ-digalate. Through optimization of enzyme cross-linking parameters and the catalytic performance before and after cross-linking, the results show that the optimal enzyme activity recovery rate of cross-linked enzyme (200 U·mL-1) could be obtained at pH 4.0, EGCG 0.5 mg·mL-1 and cross-linking time 50 min. Compared with free enzymes, cross-linked enzymes showed better catalytic performance (thermal stability, organic solvent tolerance, substrate tolerance). When the cross-linked enzyme was used to synthesize theaflavin-3,3′-digallate, the concentration of the product could reach 800 μg·mL-1, and the cross-linked enzyme could be reused for at least three batches. This method could significantly reduce the application cost of theaflavin, which has potential industrial application value.
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