奶牛瘤胃微生物元基因组文库中脂肪酶的筛选与酶学性质

Screening and characterization of lipase from a metagenome library of dairy rumen microflora

摘要:

利用含有三油酸甘油酯的脂肪酶选择性筛选培养基, 从奶牛瘤胃微生物元基因组文库15 360个克隆中, 筛选得到了18个脂肪酶阳性克隆, 其插入片段大约为60 kb, 并且各个克隆的插入片段各不一样。利用p-NPP法对脂肪酶克隆的脂肪酶活性分析, 表明均具有大小不等的脂肪酶活性。底物特异性分析表明Lipase6、Lipase7和Lipase8分别对C16底物(对硝基苯棕榈酸酯)、C12底物(对硝基苯月桂酸酯)和C16底物(对硝基苯棕榈酸酯)水解能力最强。Lipase 6、Lipase 7、Lipase 8的脂肪酶最适pH为7.5; Lipase8的脂肪酶活性半衰期随反应温度的升高而缩短, 70oC时能达到30 min。本研究所筛选的脂肪酶具有不同的底物特异性和较好的热稳定性, 这对于工业化生产具有一定的应用潜力。

Abstract:

Using lipase segregation agar containing trioleoylglycerol, we obtained 18 lipase positive clones by screening from a metagenome library of dairy rumen microflora containing 15 360 clones. The average insert size of lipase positive clones was about 60 kb. Lipase enzyme activity analysis by p-NPP method indicated that Lipase6, Lipase7 and Lipase8 had higher lipolytic activities to substrates of p-nitrophenyl palmitate (C16), p-nitrophenyl alaurate (C12) and p-nitrophenyl palmitate (C16) respectively. The optimum pH of Lipase 6, Lipase 7 and Lipase 8 were 7.5. The halflife period of Lipase 8 with the value of 15 min in 70oC decreased with the increase of temperature. In conclusion, the lipases screened in this study had different substrates specificity and good thermo stability, which laid a basis for large-scale industrial application.