Lipase Producing Strain Of Screening, Identification And Related Research

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, plate diffusion and transesterification activity to catalyze the production of biodiesel, one lipase-producing strain with higher lipase activity was screened. Optimizing of fermentation conditions by orthogonal experiment. Using acetone and (NH)2SO4 sedimentation method to isolate crude lipase. The lipase catalyzes cotton oil and methanol to be biodiesel.in tert-butyl alcohol as solvent.Results and Conclusion: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 A2 with higher lipase activity was screened. The A2 strain was identified as Klebsiella sp. according to morphological observation.2. On the screening of the A2 strain carried out through PCR amplification of 16S rDNA fragments characteristic of a comparative analysis confirmed that strains A2 and Klebsiella 99% similarity to sequences at registration, register number is FJ615552.3. The inner lipase from strain isolated by acetone method, and the outer lipase isolated by (NH)2SO4 sedimentation method, the activity of latter is higher than the former. A2 lipase showed transersterification activity using cotton oil and methanol as substrates in tert-butyl alcohol as solvent.4. On the screening of the A2 strain Blast on GenBank at the appropriate lipase gene, then the design of merger of two pairs of primers, by PCR amplification of full-length 582bp and 1623bp, respectively of the two lipase genes, two genes that belong to g Lei MGH 78578 bacteria Borrelia section on the CDS. The results will be sequenced , registration number are FJ615553 and FJ615554 separately.5. A2 strains on the optimization of fermentation conditions, speed 200r/min cultured 48h in the middle the highest enzyme activity of the enzyme activity from the initial 9.02U/mL to 12.33U/mL.