嗜热脂肪酶能克服常温酶生物学性质不稳定的缺陷,在工业应用中有较大的优势,因而成为最近研究的热点。通过合成嗜热玫瑰红球菌Thermomicrobium roseum DSM 5159中的预测脂肪酶基因,在大肠杆菌BL21(DE3)中异源表达,并对该酶进行分离纯化和酶学性质表征。重组脂肪酶的熔融温度Tm和焓变ΔH分别为97.53 ℃和1 637 kJ/mol,二级结构由56.3% α-螺旋、4.3% β-折叠、19.9% β-转角和20.3%的无规则卷曲组成。以对硝基苯酚棕榈酸酯为底物时,脂肪酶最适反应温度为85 ℃,最适反应pH为8.5,在80 ℃孵育12 h后酶活力保留率在80%以上,具有良好的热稳定性;脂肪酶对实验中大部分有机溶剂具有较强的耐受力;K+和Ca2+对酶活力有促进作用;动力学研究证实以对硝基苯酚棕榈酸酯为底物时,亲和力最高,米氏常数Km为0.01 mmol/L,催化常数kcat为3.0 s-1,kcat/Km为299.0 L/(mmol·s)。研究结果有助于更好地了解嗜热菌脂肪酶的基因和酶学性质,为其在生物柴油合成、洗涤剂及高温环境工业等多领域的应用奠定基础。
Thermophilic lipases have great industrial application advantages on the stability of biological property over mesophilic enzyme and attracting a lot of attention. A predicted lipase from Thermomicrobium roseum DSM 5159 was expressed in E. coil BL21(DE3). The lipase enzymatic property was characterized after separation and purification. The Tm and ΔH were 97.53°C and 1 637 kJ/mol, respectively. The secondary structure of the lipase was 56.3% α-helix, 4.3% β-sheet, 19.9% β-turn and 20.3% random coil. The optimal temperature and pH of the lipase on p-nitrophenol palmitate were 85°C and 8.5, respectively. The enzyme activity could be maintained at 80% after an incubation at 80°C for 12 hours, suggesting a good thermal stability. Moreover, the lipase was verified with effective resistance ability to most of organic solvents. K+ and Ca2+ also had a promotion effect on the enzyme activity. Based on kinetic study, the lipase showed high affinity to p-nitrophenol palmitate. The Km, kcat and kcat/Km for this substrate were 0.01 mmol/L, 3.0 s-1 and 299.0 L/(mmol·s), respectively. The results could contribute to understanding the thermophilic lipases and its enzyme property, which providing the basis for its further application in biodiesel synthesis, detergents and industrial environment operated under high temperature.
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