Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases

Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca2+-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter Plac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65°C and retained ≥ 97% activity after incubation at 50°C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.

热稳定且耐有机溶剂的酶因在高温下固有的稳定性与耐受严苛有机溶剂的能力，在工业领域众多合成反应中具备显著应用潜力。本研究通过将嗜热厌氧芽孢杆菌（Aneurinibacillus thermoaerophilus）菌株HZ的基因组DNA文库构建至大肠杆菌（Escherichia coli）质粒载体中，成功分离得到一段编码新型真实脂肪酶的基因。序列分析结果显示，HZ脂肪酶与假单胞样芽孢杆菌（Bacillus pseudomycoides）的推定脂肪酶具有62%的序列一致性。与HZ脂肪酶基因亲缘关系最近的已表征脂肪酶，均来自热稳定的芽孢杆菌属（Bacillus）和地芽孢杆菌属（Geobacillus），隶属于亚家族I.5，序列一致性≤57%。对HZ脂肪酶的氨基酸序列进行分析，发现其包含一段携带活性丝氨酸的保守五肽GHSMG，以及酶内的Ca²⁺结合基序GCYGSD。蛋白质结构建模结果表明，HZ脂肪酶由α/β水解酶折叠结构（α/β hydrolase fold）与盖子结构域（lid domain）组成。蛋白质序列比对、保守区域分析、Clustal距离矩阵（clustal distance matrix）以及氨基酸组成分析均证实，HZ脂肪酶与其他热稳定脂肪酶之间存在差异。系统发育分析显示，该脂肪酶隶属于细菌真实脂肪酶家族I的一个全新亚家族，归类为家族I.9。HZ脂肪酶在Plac启动子调控下经IPTG诱导表达，并完成了酶学表征。重组酶的最适反应温度为65℃，在50℃下孵育1小时后仍可保留≥97%的酶活性。HZ脂肪酶在多种极性与非极性有机溶剂中均能保持稳定。