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Lipase Producing Strain of Screening, Identification and Related Research

Author: XuanGuoYing
Tutor: GaoJianFeng
School: Shihezi University
Course: Biochemistry and Molecular Biology
Keywords: Biodiesel Klebsiella Lipase Lipase gene
CLC: TQ925
Type: Master's thesis
Year: 2009
Downloads: 55
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Abstract


The purpose: to olive oil as the sole carbon source, screened strains transesterification activity olive oil, and its identification. 1, corresponding to the strain body olive gene was amplified by means of polymerase chain reaction; 2, by cold acetone to extract the crude enzyme preparation of biodiesel Experimental. Method: olive oil as the sole carbon source for enrichment, as the indicator color effect color change indicator to select suitable strains, shake flask containing specific medium rescreening method to obtain higher production fat a wild strains of the enzyme. By using two methods, one of the second of the plate diffusion method is a comprehensive technical experimental methods of acid-base titration method and the transesterification reaction, filters having higher transesterification activity lipase wild strain of a to ; corresponding lipase, using laboratory methods and cold acetone to give the corresponding wild strain lipase gene in vivo laboratory conditions include t-butanol as the corresponding solvent, and the catalytic laboratory methanol and purchase cottonseed from the reaction of biodiesel; design specific primers to amplify the strain lipase gene in vivo, and is helpful for further tests. Means to obtain two complete lipase gene using polymerase chain reaction amplification, preparing competent and transformed into the target gene engineering bacteria, polymerase chain reaction to detect via the colonies, and finally will be screened A2 strain i.e. Klebsiella (Klebsiella sp.) appropriate lipase gene in GenBank Blast, and then two pairs of primers were designed, was amplified by polymerase chain reaction, the total length of 582bp and 1623bp two lipase gene , these two genes belong to the the MGH 78578 Klebsiella segment the CDS. The sequencing results sequence registered in GenBank accession number FJ615553 and FJ615554. Results and conclusions: 1. Collected soil samples of different places, a lipase bacteria large number of screening work. Olive oil as the sole carbon source, RhodamineB determination of enzyme activity as an indicator Flat screening, and the rescreening and acid-base titration shake flask eventually filter out a lipase producing strain A2. The lipase produced by the strain A2 vivo can catalyze the transesterification reaction. A2 morphological observation, physiological and biochemical tests initially identified A2 as Klebsiella (Klebsiella sp.) Screened A2 strains Klebsiella (Klebsiella sp.) By PCR amplification of 16S rDNA characteristic fragment analysis, confirmed the A2 strain of Klebsiella (Klebsiella sp.) Similarity of 99%, the sequencing of the resulting sequence registered in GenBank accession number is FJ615552 A2 strain intracellular lipase acetone, and with (NH) 2SO4 grading sedimentation strains extracellular lipase enzymatic assays , extracellular enzyme, lipase activity than the lipase activity in the intracellular. Followed by t-butanol as the solvent, catalytic cottonseed reaction with methanol was prepared biodiesel. 4 of the A2 strain screened i.e. Klebsiella (Klebsiella sp.) In GenBank Blast appropriate lipase gene, and two pairs of primers were designed by PCR amplified full-length, respectively two of 582bp and 1623bp lipase gene, these two genes belong to the the MGH 78578 Klebsiella segment the CDS. Sequencing results sequence registered in GenBank accession number FJ615553 and FJ615554 5. A2 strain conditions for enzyme production optimization, speed 200r/min under cultivation 48h production enzyme activity was the highest enzyme activity by initial 9.02U/mL to improve 12.33U/mL.

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