嗜热玫瑰红球菌嗜热脂肪酶的重组表达与酶学性质研究

Expression and characterization of a novel thermostable lipase from Thermomicrobium roseum

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T m和焓变ΔH分别为97.53℃和1637 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Ca^(2+)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K m为0.01 mmol/L,催化常数k cat为3.0 s^(-1),k cat/K m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 T m andΔH were 97.53°C and 1637 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 Ca^( 2+)also had a promotion effect on the enzyme activity.Based on kinetic study,the lipase showed high affinity to p-nitrophenol palmitate.The K m,k cat and k cat/K m 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.