Enzymatic Synthesis Of Biodiesel And Relative Studies

Object: Isolating and screening of the activity transesterify lipase-producing strains. On the one hand, optimizing of fermentation conditions to increase the activity of lipase and isolating lipase to catalyze biodiesel.On the other hand, the lipase gene was amplified by PCR.Methods: Enrichment using olive oil as sole carbon source, plate isolation using Rhodamine B as indicator and flask fermentation and detection using titration and plate diffusion, one lipase-producing strain with higher lipase activity was screened. Optimizing of fermentation conditions by orthogonal experiment. Using acetone and (NH4)2SO4 sedimentation method to isolate crude lipase. The lipase catalyzes cotton oil and methanol to be biodiesel.in tert-butyl alcohol as solvent. The lipase gene was amplified by PCR, and transformed into DH5α. Choosing the positive clone and sequencing it.Results:1. Many lipase-producing strains were screened. Through a series of steps including plate isolation using olive oil as sole carbon source and Rhodamine B as indicator, flask fermentation and detection of the activity of lipase, one lipase-producing strain B2 with higher lipase activity was screened. The B2 strain was identified as Bacillus sp. according to morphological observation and phylogenetic analysis using its 16S rDNA.2. Flask fermentation conditions of B2 lipase was as follows:1.0g sucrose, 1.0g soybean powder, 0.5g peptone, 0.1g K2HPO4, 0.05g MgSO4·7H2O, pH6.5, 100mL water. The inoculated medium was incubated at 30℃,225r/min of rotation speed, 30mL medium in 250mL flask and 48h of culture time.3. The activity of lipase increases from 10.12U/mL to 22.33U/mL, amplitude by 120.65%. Using acetone and (NH4)2SO4 sedimentation method to isolate crude lipase; B2 lipase showed transersterification activity using cotton oil and methanol as substrates in tert-butyl alcohol as solvent.4. The B2 lipase gene span 635bp amplified by PCR, the identity with EU482151.1 can reach 94% blast in Gene Bank.Conclusion:1. Screening one lipase-producing strain B2 with higher lipase activity. It was identified Bacillus sp.2. Optimizing of fermentation conditions the activity is increasing to 22.33 U/mL.B2 lipase showed transersterification activity using cotton oil and methanol as substrates in tert-butyl alcohol as solvent.3. The B2 lipase gene spans 635bp amplified by PCR.