Department of Agricultural Chemistry, College of Bio-Resource and Agriculture, National Taiwan University, Taipei 10617, Taiwan.
Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan 701401, Taiwan.
Molecules. 2023 Jul 14;28(14):5410. doi: 10.3390/molecules28145410.
sp. D01, capable of growing in tributyrin medium, was isolated from the gut microbiota of yellow mealworm. By using in silico analyses, we discovered a hypothesized esterase encoding gene in the D01 bacterium, and its encoded protein, EstD04, was classified as a bacterial hormone-sensitive lipase (bHSL) of the type IV lipase family. The study revealed that the recombinant EstD04-His(6x) protein exhibited esterase activity and broad substrate specificity, as it was capable of hydrolyzing -nitrophenyl derivatives with different acyl chain lengths. By using the most favorable substrate -nitrophenyl butyrate (C), we defined the optimal temperature and pH value for EstD04 esterase activity as 40 °C and pH 8, respectively, with a catalytic efficiency (/) of 6.17 × 10 mM s at 40 °C. EstD04 demonstrated high stability between pH 8 and 10, and thus, it might be capably used as an alkaline esterase in industrial applications. The addition of Mg and NH, as well as DMSO, could stimulate EstD04 enzyme activity. Based on bioinformatic motif analyses and tertiary structural simulation, we determined EstD04 to be a typical bHSL protein with highly conserved motifs, including a triad catalytic center (Ser, Glu, and His), two cap regions, hinge sites, and an oxyanion hole, which are important for the type IV enzyme activity. Moreover, the sequence analysis suggested that the two unique discrete cap regions of EstD04 may contribute to its alkali mesophilic nature, allowing EstD04 to exhibit extremely distinct physiological properties from its evolutionarily closest esterase.
D01 株能够在丁酸甘油酯培养基中生长，从黄粉虫肠道微生物群中分离得到。通过计算机分析，我们在 D01 菌中发现了一个假定的酯酶编码基因，其编码的蛋白质 EstD04 被归类为 IV 型脂肪酶家族的细菌激素敏感脂肪酶 (bHSL)。研究表明，重组 EstD04-His(6x)蛋白具有酯酶活性和广泛的底物特异性，因为它能够水解不同酰基链长的 -硝基苯衍生物。使用最有利的底物 -硝基苯丁酸酯 (C)，我们确定了 EstD04 酯酶活性的最佳温度和 pH 值分别为 40°C 和 pH 8，在 40°C 时催化效率 (/)为 6.17×10 mM s。EstD04 在 pH 8 到 10 之间表现出很高的稳定性，因此，它可能能够在工业应用中用作碱性酯酶。添加 Mg 和 NH 以及 DMSO 可以刺激 EstD04 酶的活性。基于生物信息学基序分析和三级结构模拟，我们确定 EstD04 是一种典型的 bHSL 蛋白，具有高度保守的基序，包括三核催化中心 (Ser、Glu 和 His)、两个帽区、铰链位点和氧阴离子穴，这对于 IV 型酶活性非常重要。此外，序列分析表明 EstD04 的两个独特离散帽区可能有助于其碱性嗜温特性，使 EstD04 表现出与其进化上最接近的酯酶截然不同的生理特性。
