Laboratorio de Biotecnología Bacteriana, Instituto de Ciencia y Tecnología de Alimentos y Nutrición, ICTAN-CSIC, Juan de la Cierva 3, 28006, Madrid, Spain.
Appl Microbiol Biotechnol. 2014;98(14):6329-37. doi: 10.1007/s00253-014-5603-0. Epub 2014 Mar 1.
The gene in the locus GALLO_1609 from Streptococcus gallolyticus UCN34 was cloned and expressed as an active protein in Escherichia coli BL21 (DE3). The protein was named TanSg1 since it shows similarity to bacterial tannases previously described. The recombinant strain produced His-tagged TanSg1 which was purified by affinity chromatography. Purified TanSg1 protein showed tannase activity, having a specific activity of 577 U/mg which is 41 % higher than the activity of Lactobacillus plantarum tannase. Remarkably, TanSg1 displayed optimum catalytic activity at pH 6-8 and 50-70 °C and showed high stability over a broad range of temperatures. It retained 25 % of its relative activity after prolonged incubation at 45 °C. The specific activity of TanSg1 is enhanced by the divalent cation Ca(2+) and is dramatically reduced by Zn(2+) and Hg(2+). The enzyme was highly specific for gallate and protocatechuate esters and showed no catalytic activity against other phenolic esters. The protein TanSg1 hydrolyzes efficiently tannic acid, a complex and polymeric gallotanin, allowing its complete conversion to gallic acid, a potent antioxidant. From its biochemical properties, TanSg1 is a tannase with potential industrial interest regarding the biodegradation of tannin waste or its bioconversion into biologically active products.
从牛链球菌 UCN34 的 GALLO_1609 基因中克隆并在大肠杆菌 BL21 (DE3) 中表达为活性蛋白。由于它与先前描述的细菌单宁酶具有相似性，因此将该蛋白质命名为 TanSg1。重组菌株产生了带有 His 标签的 TanSg1，该蛋白通过亲和层析进行纯化。纯化的 TanSg1 蛋白显示出单宁酶活性，比植物乳杆菌单宁酶的活性高 41%，具有 577 U/mg 的比活性。值得注意的是，TanSg1 在 pH 6-8 和 50-70°C 下表现出最佳的催化活性，并且在较宽的温度范围内具有很高的稳定性。在 45°C 下长时间孵育后，它保留了其相对活性的 25%。TanSg1 的比活性通过二价阳离子 Ca(2+) 增强，而被 Zn(2+) 和 Hg(2+) 显著降低。该酶对没食子酸酯和原儿茶酸酯具有高度特异性，对其他酚酯没有催化活性。TanSg1 蛋白有效地水解单宁酸，一种复杂的聚合没食子单宁，允许其完全转化为具有强抗氧化作用的没食子酸。根据其生化特性，TanSg1 是一种具有潜在工业价值的单宁酶，可用于降解单宁废物或将其生物转化为具有生物活性的产品。
