Key Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China.
Arch Microbiol. 2021 Sep;203(7):4113-4125. doi: 10.1007/s00203-021-02398-0. Epub 2021 May 31.
A novel lipolytic gene, estq7, was identified from a fosmid metagenomic library. The recombinant enzyme EstQ7 consists of 370 amino acids with an anticipated molecular mass of 42 kDa. Multiple sequence alignments showed that EstQ7 contained a pentapeptide motif GHSMG, and a putative catalytic triad Ser174-Asp306-His344. Interestingly, EstQ7 was found to have very little similarity to the characterized lipolytic enzymes. Phylogenetic analysis revealed that EstQ7 may be a member of a novel family of lipolytic enzymes. Biochemical characterization of the recombinant enzyme revealed that it constitutes a slightly alkalophilic, moderate thermophilic and highly active carboxylesterase against short-chain fatty acid esters with optimum temperature 50 ℃ and pH 8.2. The Km and kcat values toward p-nitrophenyl acetate were determined to be 0.17 mM and 1910s, respectively. Moreover, EstQ7 was demonstrated to have acyltransferase activity by GC-MS analysis. Structural modeling of the three-dimensional structure of this new enzyme showed that it exhibits a typical α/β hydrolase fold, and the catalytic triad residues are spatially close. Molecular docking revealed the interactions between the enzyme and the ligand. The high levels of lipolytic activity of EstQ7, combined with its moderate thermophilic property and acyltransferase activity, render this novel enzyme a promising candidate biocatalyst for food, pharmaceutical and biotechnological applications.
从宏基因组文库中鉴定出一种新型脂肪酶基因 estq7。重组酶 EstQ7 由 370 个氨基酸组成，预计分子量为 42 kDa。多重序列比对表明，EstQ7 含有五肽基序 GHSMG 和一个推定的催化三联体 Ser174-Asp306-His344。有趣的是，EstQ7 与已鉴定的脂肪酶几乎没有相似性。系统发育分析表明，EstQ7 可能是一种新型脂肪酶家族的成员。重组酶的生化特性研究表明，它是一种略带碱性、中温、高度活跃的羧酸酯酶，对短链脂肪酸酯具有最佳温度 50℃和 pH8.2。对 p-硝基苯乙酸酯的 Km 和 kcat 值分别为 0.17 mM 和 1910 s。此外，通过 GC-MS 分析证明 EstQ7 具有酰基转移酶活性。该新酶的三维结构的结构建模表明，它表现出典型的 α/β 水解酶折叠，并且催化三联体残基空间上接近。分子对接揭示了酶与配体之间的相互作用。EstQ7 的脂肪酶活性高，同时具有中温特性和酰基转移酶活性，使其成为食品、制药和生物技术应用中很有前途的候选生物催化剂。
