Lipase Producing Strain, Cloning And Applied Research

In this paper,screening of lipase-producing strains,optimization of fermentation conditions,esterification and transesterification by immobilized lipase were carried out.The gene of isolated G5 lipase was also cloned,preparation of biodiesel from olive oil or fried oil and short-chain alcohol and structural modification of sophorolipid were investigated.First,many lipase-producing strains were screened.Through a series of steps including enrichment,plate isolation,flask fermentation and detection of transesterification activity to catalyze the production of biodiesel,two lipase-producing strains C3 and G5 with higher lipase activity were screened.The two strains were both identified as Galactomyces geotrichum according to the 18S rDNA and morphological observation.The properties of crude enzyme of this C3 lipase was also investigated.The optimum temperature and pH for this lipase were 40℃and pH 7.5,respectively,and the lipase showed high stability below 50℃and in the pH range of pH6.0～8.0.Then,Flask fermentation conditions of C3 lipase were optimized as follows:1.5 g sucrose,1.5 g soybean powder,0.5g peptone, 0.5g(NH4)2SO4,0.1g KH2PO4,0.05g MgSO4·7H2O,1.0 ml olive oil,100ml water,pH 6.5.Afterwards,the mutation breeding by EMS was proved to be better method compared with ultraviolet mutation.G5 Galactomyces geotrichum lipase activity was promoted to 168.6%by EMS mutation and successive cultivation for three generations.According to the conservative domain of nucleotide sequence in Galactomyces geotrichum lipase,by using the Thermal asymmetric interlaced PCR(TAIL-PCR), G5 lipase gene sequence is 1692bp long without intron and encodes 563 amino acids, bioinformatics of G5 lipase was analyzed.Finally,the immobilized enzyme G5 lipase was applied in the transesterification in non-aqueous media to transform olive oil,cotton seed oil or fried oil and short-chain alcohol into biodiesel.Biosurfactant sophorolipid was also modified by G5 lipase in non-aqueous media,and the antibacterial activities for some pathogenic microorganisms of modified sophorolipids were investigated.The results showed that the antifungal activities of modified sophorolipids were enhanced to Microsporum gypseum and Trichophyton rubrum,while has weaker activity to Monilia albican and has no activity to Microsporum Canis.