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 70 degrees C 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.
Animals ; Bacteria ; genetics ; Cattle ; Cloning, Molecular ; Female ; Gene Library ; Lipase ; genetics ; metabolism ; Metagenome ; genetics ; Rumen ; microbiology ; Substrate Specificity ; Temperature