Mahardika Gita, Dewi Laksmi, Yohandini Heni, Widhiastuty Made Puspasari, Sakti Raden Aditya Wibawa, Wahyudi Setyanto Tri
Biochemistry Research Group, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung, Indonesia.
Department of Chemistry, Faculty of Science and Computer, Universitas Pertamina, Jl. Teuku Nyak Arief, Simprug Kebayoran, South Jakarta, Indonesia.
Heliyon. 2021 Mar 24;7(3):e06542. doi: 10.1016/j.heliyon.2021.e06542. eCollection 2021 Mar.
A novel thioesterse gene was successfully cloned and sequenced directly from natural sample of Domas Hot Spring, West Java, Indonesia. Homological analysis of the sequence showed that the gene appeared high homology to thioesterase genes with the highest to a putative thioesterase gene from uncultured at 66% identity. However, phylogenetic analysis showed that the protein was separated from the branch with other known thioesterases. The size of the gene is around 500 base pairs, lied into 2 kb DNA fragment from a random PCR amplicon. The gene was overexpressed in , a dominant band appeared at 17 kDa in SDS-PAGE with expression level at around 32% of total proteins. The activity of the purified protein using acetyl-CoA as substrate showed that the protein exhibited thioesterase activity. Furthermore, the enzyme also showed esterase activity on p-nitrophenyl ester as substrate. Detail characterization of esterolytic activity showed that the enzyme preferred p-nitrophenyl decanoate as substrate. The optimum activity of the enzyme was at 80 °C and pH 8. Activity of the enzyme was maintained after incubation at 80 °C up to 24 h. In addition, the enzyme was favorable on polar organic solvents. All the data obtained suggested that the enzyme is a novel alkaline thermostable thioesterase.
一个新的硫酯酶基因已成功地从印度尼西亚西爪哇多马斯温泉的天然样本中直接克隆并测序。序列的同源性分析表明，该基因与硫酯酶基因具有高度同源性，与来自未培养菌的一个假定硫酯酶基因的同源性最高，同一性为66%。然而，系统发育分析表明，该蛋白质与其他已知硫酯酶的分支分离。该基因大小约为500个碱基对，位于一个随机PCR扩增子的2 kb DNA片段中。该基因在[具体宿主未提及]中过表达，在SDS-PAGE中17 kDa处出现一条主带，表达水平约占总蛋白的32%。以乙酰辅酶A为底物对纯化蛋白的活性检测表明，该蛋白具有硫酯酶活性。此外，该酶对以对硝基苯酯为底物也表现出酯酶活性。酯解活性的详细表征表明，该酶优先选择癸酸对硝基苯酯作为底物。该酶的最适活性温度为80℃，最适pH为8。在80℃孵育24小时后，该酶的活性仍得以保持。此外，该酶对极性有机溶剂具有耐受性。所有获得的数据表明，该酶是一种新型的碱性耐热硫酯酶。
