An alkaline esterase, designated as EstXT1, was identified through functional screening from a metagenomic library. Sequence analysis revealed that EstXT1 belonged to the family VIII carboxylesterases and contained a characteristic conserved S-x-x-K motif and a deduced catalytic triad Ser56-Lys59-Tyr165. EstXT1 exhibited the strongest activity toward methyl ferulate at pH 8.0 and temperature 55°C and retained over 80% of its original activity after incubation in the pH range of 7.0–10.6 buffers. Biochemical characterization of the recombinant enzyme showed that it was activated by Zn²⁺ and Co²⁺ metal ion, while inhibited by Cu²⁺ and CTAB. EstXT1 exhibited significant promiscuous acyltransferase activity preferred to the acylation of benzyl alcohol acceptor using short-chain pNP-esters (C2-C8) as acyl-donors. A structure–function analysis indicated that a WAG motif is essential to acyltransferase activity. This is the first report example that WAG motif plays a pivotal role in acyltransferase activity in family VIII carboxylesterases beside WGG motif. Further experiment indicated that EstXT1 successfully acylated cyanidin-3-O-glucoside in aqueous solution. The results from the current investigation provided new insights for the family VIII carboxylesterase and lay a foundation for the potential applications of EstXT1 in food and biotechnology fields.

通过宏基因组文库的功能筛选鉴定了一种碱性酯酶，命名为 EstXT1。序列分析显示 EstXT1 属于 VIII 羧酸酯酶家族，包含一个特征性的保守 SxxK 基序和一个推导的催化三联体 Ser56-Lys59-Tyr165。EstXT1 在 pH 8.0 和温度 55°C 时表现出对阿魏酸甲酯的最强活性，并且在 7.0–10.6 的缓冲液 pH 范围内孵育后保留其原始活性的 80% 以上。重组酶的生化表征表明，它被Zn ²⁺和Co ²⁺金属离子激活，而被Cu ^2+抑制和 CTAB。EstXT1 表现出显着的混杂酰基转移酶活性，优于使用短链p NP 酯 (C2-C8) 作为酰基供体的苯甲醇受体的酰化。结构-功能分析表明 WAG 基序对酰基转移酶活性至关重要。这是除 WGG 基序外，WAG 基序在家族 VIII 羧酸酯酶的酰基转移酶活性中起关键作用的第一个报告实例。进一步的实验表明，EstXT1 在水溶液中成功地酰化了 cyanidin-3-O-glucoside。目前的研究结果为家族 VIII 羧酸酯酶提供了新的见解，并为 EstXT1 在食品和生物技术领域的潜在应用奠定了基础。