Faculty of Biology, Department of Biochemistry and Molecular Biology-A, University of Murcia, Campus Espinardo, E-30100 Murcia, Spain.
J Agric Food Chem. 2012 Oct 31;60(43):10789-95. doi: 10.1021/jf3024968. Epub 2012 Oct 18.
An arylesterase from the wine-making acetic acid bacterium, Gluconobacter oxidans, was cloned and expressed into Escherichia coli. The soluble 76.8 kDa dimeric enzyme obtained, Est0881, was purified in only two steps with a 3.1-fold purification, 43% recovery, and a specific activity of 214 U/mg for the hydrolysis of p-nitrophenyl acetate. The optimum pH and temperature were 7.0 and 40 °C, respectively. The substrate specificity of this arylesterase was higher toward short chain p-nitrophenyl esters (C(2) to C(4)) and also toward aromatic esters, such as phenyl acetate. The deduced amino acid sequence shares high identity with esterases of the HSL family. The inhibition results obtained showed that the enzyme was a serine esterase, belonging to the A-esterases (arylesterases) and contains a catalytic triad composed of Ser163, Asp263, and His293 in the active site. Est0881 retained significant activity under conditions simulating those of wine-making (75% activity at 20% ethanol), making it a promising biocatalyst for modulating the final aroma of wine.
从酿酒醋酸菌 Gluconobacter oxidans 中克隆并表达了一种芳基酯酶。获得的可溶性 76.8 kDa 二聚体酶 Est0881 仅通过两步纯化即可得到，纯化倍数为 3.1 倍，回收率为 43%，对 p-硝基苯乙酸酯的水解比活为 214 U/mg。最适 pH 和温度分别为 7.0 和 40°C。该芳基酯酶对短链 p-硝基苯酯（C(2) 到 C(4)）和芳香酯，如乙酸苯酯具有较高的底物特异性。推导的氨基酸序列与 HSL 家族的酯酶具有高度的同源性。抑制结果表明，该酶是一种丝氨酸酯酶，属于 A 酯酶（芳基酯酶），在活性位点包含由 Ser163、Asp263 和 His293 组成的催化三联体。Est0881 在模拟酿酒条件下（在 20%乙醇下仍保持 75%的活性）保持显著的活性，使其成为调节葡萄酒最终香气的有前途的生物催化剂。
