Instituto de Biotecnología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, UANL, Av. Universidad S/N, Col. Ciudad Universitaria, 66455, San Nicolás de los Garza, Nuevo León, Mexico.
Mol Biotechnol. 2022 Apr;64(4):388-400. doi: 10.1007/s12033-021-00416-6. Epub 2021 Oct 15.
Tannin acyl hydrolases or tannases (E.C.3.1.1.20) are enzymes that hydrolyze the ester bond of tannins to produce gallic acid and glucose. We engineered the Aspergillus niger GH1 tannase sequence and Pichia pastoris strains to produce and secrete the enzyme as a single-chain protein. The recombinant tannase was N-glycosylated, had a molecular mass after N-deglycosylation of 65.4 kDa, and showed activity over broad pH and temperature ranges, with optimum pH and temperature of 5.0 and 20 °C. Furthermore, the single-chain tannase had an 11-fold increased specific activity in comparison to the double-chain A. niger GH1 tannase, which was also produced in P. pastoris. Structural analysis suggested that the high specific activity may be due to the presence of a flexible loop in the lid domain, which can control and drive the substrate to the active site. In contrast, the low specific activity of the double-chain tannase may be due to the presence of a disordered and flexible loop that could hinder the substrate's access to the binding site. Based on its biochemical properties, high specific activity, and the possibility of its production in P. pastoris, the tannase described could be used in food and beverage processing at low and medium temperatures.
单宁酰基水解酶或单宁酶(E.C.3.1.1.20)是一类能够水解单宁酯键生成没食子酸和葡萄糖的酶。我们对黑曲霉 GH1 单宁酶序列和毕赤酵母菌株进行了工程改造,使其作为单链蛋白进行生产和分泌。重组单宁酶发生了 N-糖基化,经 N-糖基化酶切后分子质量为 65.4 kDa,在较宽的 pH 和温度范围内具有活性,最适 pH 和温度分别为 5.0 和 20°C。此外,与同样在毕赤酵母中生产的黑曲霉 GH1 双链单宁酶相比,单链单宁酶的比活性提高了 11 倍。结构分析表明,高比活性可能是由于在盖子结构域中存在一个灵活的环,该环可以控制并将底物导向活性位点。相比之下,双链单宁酶的低比活性可能是由于存在一个无序且灵活的环,这可能会阻碍底物与结合位点的结合。根据其生化特性、高比活性以及在毕赤酵母中生产的可能性,所描述的单宁酶可用于食品和饮料加工,在低温和中温下使用。