对羟基苯甲酸酯水解酶和单宁酶催化对羟基苯甲酸酯(4-羟基苯甲酸酯)和没食子酸(3,4,5-三羟基苯甲酸)酯的水解,分别。来自米曲霉的对羟基苯甲酸酯水解酶(AoPrbA)和单宁酶(AoTanB)属于ESTHER数据库中的单宁酶家族。然而,AoPrbA和AoTanB的底物特异性狭窄。根据黑曲霉单宁酶(PDB代码7k4o)的结构信息,我们构建了五个AoPrbA的单一变体(Thr200Glu,Phe231Gln,Leu232Gln,Ile361Tyr,和Leu428Ser)和AoTanB的四个(Glu203Asp,Glu203Thr,His237Ala,和Ser440Leu)来研究AoPrbA和AoTanB之间的底物区分。每种变体在巴斯德毕赤酵母中表达并从培养上清液中纯化。与AoPrbA和AoTanB野生型相比,AoPrbA的五个纯化变体和AoTanB的四个变体显示出降低的对羟基苯甲酸酯水解酶和单宁酶活性,分别。有趣的是,AoPrb野生型不水解没食子酸甲酯,而Thr200Glu,Leu232Gln,和Leu428Ser的变体,表明这三种变体获得了单宁酶活性。特别是,Leu428Ser变体表现出明显更大的没食子酸和原儿茶酸甲酯水解。同时,AoTanB野生型,和Glu203Asp,His237Ala和Ser440Leu变体水解了原儿茶酸甲酯和4-羟基苯甲酸乙酯;然而,Glu203Thr变体不水解上述底物。此外,Ser440Leu中对羟基苯甲酸酯水解酶活性与单宁酶活性的比率显着升高。
Paraben hydrolase and tannase catalyze the hydrolysis of parabens (4-hydroxybenzoic acid esters) and gallic acid (3,4,5-trihydroxybenzoic acid) esters, respectively. Paraben hydrolase (AoPrbA) and tannase (AoTanB) from Aspergillus oryzae belong to the tannase family in the ESTHER database. However, the substrate specificities of AoPrbA and AoTanB are narrow. Based on structural information of Aspergillus niger tannase (PDB code 7k4o), we constructed five single variants of AoPrbA (Thr200Glu, Phe231Gln, Leu232Gln, Ile361Tyr, and Leu428Ser) and four of AoTanB (Glu203Asp, Glu203Thr, His237Ala, and Ser440Leu) to investigate substrate discrimination between AoPrbA and AoTanB. Each variant was expressed in Pichia pastoris and were purified from the culture supernatant. Five purified variants of AoPrbA and four variants of AoTanB showed reduced paraben hydrolase and tannase activities compared with AoPrbA and AoTanB wild types, respectively. Interestingly, the AoPrbA wild type did not hydrolyze gallic acid methyl ester, whereas the Thr200Glu, Leu232Gln, and Leu428Ser variants did, indicating that these three variants acquired tannase activity. In particular, the Leu428Ser variant exhibited considerably greater hydrolysis of gallic acid and protocatechuic acid methyl esters. Meanwhile, the AoTanB wild type, and Glu203Asp, His237Ala and Ser440Leu variants hydrolyzed the protocatechuate methyl and 4-hydroxybenzoate ethyl esters; however, the Glu203Thr variant did not hydrolyze above-mentioned substrates. Additionally, the ratio of paraben hydrolase activity to tannase activity in Ser440Leu was markedly elevated.