背景:酯酶(EC3.1.1。X)是催化水解酯键的酶。这些酶在精细工业中具有巨大的应用潜力,特别是在药物方面,化妆品,和生物乙醇生产。
结果:在这项研究中,成功克隆了一个编码FuscaYX(TfEst)酯酶的基因,其产物在大肠杆菌中过表达,并用亲和层析纯化。TfEst动力学测定显示,对对硝基苯乙酸的催化效率为0.58s-1mM-1,1.09s-1mM-1和0.062s-1mM-1,对硝基苯丁酸酯,和乙酸1-萘酯底物,分别。此外,TfEst在6.0至10.0的pH范围内也表现出活性,在pH8.0时具有最大活性。该酶在70℃下的半衰期为20分钟。值得注意的是,TfEst显示乙酰木聚糖酯酶活性,如乙酰化木聚糖测定所证明的。使用AlphaFold对TfEst的结构预测表明具有α/β-水解酶折叠,这与其他酯酶一致。
结论:在较宽的pH范围内的酶稳定性及其在高温下的活性使其成为工业过程中具有吸引力的候选物。总的来说,TfEst作为一种有前途的酶促工具出现,对生物技术和生物燃料行业的发展具有重要意义。
BACKGROUND: Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fine industries, particularly in pharmaceuticals, cosmetics, and bioethanol production.
RESULTS: In this study, a gene encoding an esterase from Thermobifida fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purified using affinity chromatography. The TfEst kinetic assay revealed catalytic efficiencies of 0.58 s-1 mM-1, 1.09 s-1 mM-1, and 0.062 s-1 mM-1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/β-hydrolase fold, which is consistent with other
esterases.
CONCLUSIONS: The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with significant implications for the advancement of biotechnology and biofuels industries.