Esterases are widely distributed in nature and have important applications in medical, industrial and physiological. Recently, the increased demand for flavor esters has prompted the search of catalysts like lipases and esterases. Esterases from thermophiles also show thermal stability at elevated temperatures and have become enzymes of special interest in biotechnological applications. Although most of esterases catalyzed reactions are carried out in toxic and inflammable organic solvents, the solvent-free system owning many advantages such as low cost and easy downstream processing. The gene estGSU753 from Geobacillus subterraneus DSM13552 was cloned, sequenced and overexpressed into Escherichia coli BL21 (DE3). The novel gene has an open reading frame of 753 bp and encodes 250-amino-acid esterase (EstGSU753). The sequence analysis showed that the protein contains a catalytic triad formed by Ser97, Asp196 and His226, and the Ser of the active site is located in the conserved motif Gly95-X-Ser97-X-Gly99 included in most esterases and lipases. The protein catalyzed the hydrolysis of pNP-esters of different acyl chain lengths, and the enzyme specific activity was 70 U/mg with the optimum substrate pNP-caprylate. The optimum pH and temperature of the recombinant enzyme were 8.0 and 60 °C respectively. The resulting EstGSU753 showed remarkable stability against methanol. After the incubation at 50% methanol for 9 days, EstGSU753 retained 50% of its original activity. Even incubation at 90% methanol for 35 h, EstGSU753 retained 50% of its original activity. Also, the preliminary study of the transesterification shows the potential value in synthesis of short-chain flavor esters in a solvent-free system, and more than 99% conversion was obtained in 6 h (substrate: cinnamyl alcohol, 1.0 M). This is the first report of esterase gene cloning from Geobacillus subterraneus with detailed enzymatic properties. This methanol-stable esterase showed potential value in industrial applications especially in the perfume industry.

中文翻译：

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来自地下芽孢杆菌DSM13552的新型甲醇稳定酯酶的功能表达，用于在无溶剂系统中生物催化合成乙酸肉桂酯。

酯酶在自然界中广泛分布并且在医学，工业和生理学中具有重要应用。最近，对风味酯的需求增加促使人们寻找诸如脂肪酶和酯酶的催化剂。来自嗜热菌的酯酶在高温下也显示出热稳定性，并已成为生物技术应用中特别关注的酶。尽管大多数酯酶催化的反应都是在有毒和易燃的有机溶剂中进行的，但无溶剂系统具有许多优势，例如成本低廉且易于下游加工。来自地下芽孢杆菌DSM13552的基因estGSU753被克隆，测序并在大肠杆菌BL21（DE3）中过表达。该新基因的开放阅读框为753 bp，编码250个氨基酸的酯酶（EstGSU753）。序列分析表明该蛋白质含有由Ser97，Asp196和His226形成的催化三联体，并且活性位点的Ser位于大多数酯酶和脂肪酶中保守的基序Gly95-X-Ser97-X-Gly99中。该蛋白催化不同酰基链长度的pNP-酯的水解，在最佳的pNP-辛酸酯底物条件下，酶的比活为70 U / mg。重组酶的最适pH和温度分别为8.0和60℃。所得的EstGSU753对甲醇显示出显着的稳定性。在50％甲醇中孵育9天后，EstGSU753保留了其原始活性的50％。即使在90％甲醇中孵育35小时，EstGSU753仍保留其原始活性的50％。也，酯交换反应的初步研究显示了在无溶剂体系中合成短链风味酯的潜在价值，并在6小时内获得了99％以上的转化率（底物：肉桂醇，1.0 M）。这是第一个从地下芽孢杆菌克隆酯酶基因的报道，该酶具有详细的酶学性质。这种甲醇稳定的酯酶在工业应用中特别是在香料工业中显示出潜在的价值。