A novel carboxylesterase gene, named dlfae4, was discovered and sequenced from a soil metagenomic library. The dlfae4 gene was composed of 1017 base pairs encoding 338 amino acid residues with a predicted molecular mass of 37.2 kDa. DLFae4 exhibited strong hydrolytic activity towards methyl ferulate under optimum pH and temperature conditions (pH 8.6, 50 °C) and displayed remarkable thermostability, with residual activity as high as 50% after incubation for 3 h at 60 °C. A family VIII esterase DLFae4 was found to contain a typical serine residue within the S-X-X-K motif, which serves as a catalytic nucleophile in class C β-lactamases and family VIII esterases. As a consequence of its high sequence similarity with β-lactamases, DLFae4 exhibited significant hydrolytic activity towards ampicillin. In addition, DLFae4 was found to be the first known member of family VIII carboxylesterases with phthalate-degrading ability. Site-directed mutagenesis studies revealed that Ser11, Lys14, and Tyr121 residues play an essential catalytic role in DLFae4. These new findings, which are of great importance for further in-depth research and engineering development of carboxylesterases, should advance the implementation of biotechnological applications.

中文翻译：

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一种新型的VIII羧基酯酶，对土壤氨苄青霉素具有很高的水解活性，来自土壤基因组学文库。

从土壤宏基因组文库中发现了一个名为dlfae4的新型羧酸酯酶基因并对其进行了测序。dlfae4基因由1017个碱基对组成，编码338个氨基酸残基，预测分子量为37.2 kDa。DLFae4在最佳的pH和温度条件（pH 8.6，50°C）下对阿魏酸甲酯具有很强的水解活性，并显示出显着的热稳定性，在60°C孵育3 h后残留活性高达50％。发现VIII族酯酶DLFae4在SXXK基序内含有典型的丝氨酸残基，其充当C类β-内酰胺酶和VIII族酯酶的催化亲核试剂。由于其与β-内酰胺酶的高度序列相似性，DLFAe4对氨苄青霉素显示出显着的水解活性。此外，发现DLFae4是具有邻苯二甲酸酯降解能力的VIII族羧酸酯酶的第一个已知成员。定点诱变研究表明，Ser11，Lys14和Tyr121残基在DLFae4中起着重要的催化作用。这些新发现对于进一步深入研究羧酸酯酶的研究和工程开发具有重要意义，应促进生物技术应用的实施。