分子研究来自溶杆菌属的重组耐冷、耐去污剂和耐碱酯酶（EstRag）：家族 VI 的一个成员。
Molecular study on recombinant cold-adapted, detergent- and alkali stable esterase (EstRag) from Lysinibacillus sp.: a member of family VI.
机构信息
Environmental Studies Department, Institute of Graduate Studies and Research, Alexandria University, 163 Horreya Avenue, P.O. Box 832, Chatby, 21526, Alexandria, Egypt.
Biotechnology Department, Institute of Graduate Studies and Research, Alexandria University, 163 Horreya Avenue, P.O. Box 832, Chatby, 21526, Alexandria, Egypt.
出版信息
World J Microbiol Biotechnol. 2022 Sep 7;38(12):217. doi: 10.1007/s11274-022-03402-5.
Cold-adapted esterases have potential industrial applications. To fulfil the global continuous demand for these enzymes, a cold-adapted esterase member of family VI from Lysinibacillus sp. YS11 was cloned on pET-28b (+) vector and expressed in E. coli BL21(DE3) Rosetta cells for the first time. The open reading frame (654 bp: GenBank MT120818.1) encodes a polypeptide (designated EstRag: 217 amino acid residues). EstRag amino acid sequence has conserved esterase signature motifs: pentapeptide (GFSQG) and catalytic triad Ser-Asp-His. EstRag 3D predicted model, built with LOMETS3 program, showed closest structural similarity to PDB 1AUO_A (esterase: Pseudomonas fluorescens); TM-align score program inferences. Purified EstRag to 9.28-fold, using Niaffinity agarose matrix, showed a single protein band (25 kDa) on SDS-PAGE, Km (0.031 mM) and Kcat/Km (657.7 s mM) on p-NP-C2. Temperature and pH optima of EstRag were 35 °C and 8.0, respectively. EstRag was fully stable at 5-30 °C for 120 min and at pH(s) 8.0-10.0 after 24 h. EstRag activity (391.46 ± 0.009%) was impressively enhanced after 30 min preincubation with 5 mM Cu. EstRag retained full stability after 30 min pre-incubation with 0.1%(v/v) SDS, Triton X-100, and Tween-80. EstRag promising characteristics motivate performing guided evolution and industrial applications prospective studies.
冷适应酯酶具有潜在的工业应用价值。为了满足全球对这些酶的持续需求，首次将来自Lysinibacillus sp. YS11 的家族 VI 的冷适应酯酶成员克隆到 pET-28b（+）载体上，并在大肠杆菌 BL21(DE3) Rosetta 细胞中表达。开放阅读框（654bp：GenBank MT120818.1）编码一个多肽（命名为EstRag：217 个氨基酸残基）。EstRag 氨基酸序列具有保守的酯酶特征基序：五肽（GFSQG）和催化三联体 Ser-Asp-His。使用 LOMETS3 程序构建的 EstRag 3D 预测模型与 PDB 1AUO_A（酯酶：荧光假单胞菌）具有最接近的结构相似性；TM-align 得分程序推断。使用 Ni-affinity 琼脂糖基质将 EstRag 纯化至 9.28 倍，在 SDS-PAGE 上显示出单一的蛋白质条带（25kDa），Km（0.031mM）和 Kcat/Km（657.7s mM）在 p-NP-C2 上。EstRag 的最适温度和 pH 值分别为 35°C 和 8.0。EstRag 在 5-30°C 下稳定 120 分钟，在 pH(s)8.0-10.0 下稳定 24 小时。经过 30 分钟 5mM Cu 预孵育后，EstRag 活性（391.46±0.009%）显著增强。经过 30 分钟 0.1%（v/v）SDS、Triton X-100 和 Tween-80 预孵育后，EstRag 仍保持完全稳定性。EstRag 具有令人印象深刻的特性，这促使我们进行定向进化和工业应用的前瞻性研究。
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