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Knockout of fadD Gene and Influence of ω-1-hydroxy Fatty Acid Yield of Bacillus Pumilus

Author: WangYan
Tutor: WuZuFang
School: Ningbo University
Course: Of Food Science
Keywords: ω-1- hydroxy fatty acid Bacillus pumilus fadD gene Clone Knockout
CLC: TQ921.7
Type: Master's thesis
Year: 2009
Downloads: 50
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Abstract


The the C18 long carbon chain hydroxy fatty acids , ω -1 ω -2 ω -3 - hydroxy fatty acids (ω -1 , ω -2 , ω -3 -HFA) can be applied to the machinery industry , the food industry ; as a monomer synthetic polymer polymer can provide monomer molecule longest carbon chain , so no single molecular side chains of the polymer material , the better the performance , the best in today's green polymer materials research and development of one of the monomer components . Bacillus pumilus (Bacillus pumilus) M-F641 is an after mutagenesis obtained after ω -1 - hydroxy fatty overproducing strain , the strain is easy to culture , and ω -1 - hydroxy fatty high conversion rate (16.8%) , ratio of the total ) , and the hydroxy fatty acid yield (hydroxy fatty acid with a fatty acid consumption ratio) in order to be able to further improve the conversion rate of the hydroxy fatty acid (hydroxy fatty acid with a fatty acid were added , and this paper fatty acid β-oxidation metabolic processes involved in activation of long-chain fatty acids , transmembrane transport and encoding acyl CoA synthase (Fatty acyl coenzyme A synthetases, FACS) , a key enzyme gene fadD study , the main contents are as follows : First , based on post Bacillus pumilus , and the genus Bacillus strain fadD gene design specific primers , PCR amplified the fadD gene sequence of the B. pumilus M-F641 , the length is 1720bp , and using MEGA 3.1, DNAStar software those the sequence of the gene were analyzed. Second, to construct the recombinant expression the plasmid pET32M - fadD. Base , was transformed into E. coli BL21 (DE3) prokaryotic expression after induction with IPTG obvious expression of the recombinant plasmid at approximately 65 kDa by SDS-PAGE electrophoresis analysis showed that the expression product , and the use of reagents the box measured the activity of the expression product of the fatty acyl- CoA synthetase activity reached 79.2U/mg . Finally, determine the knockout strategy successfully constructed a suicide vector pMD18-T - fadD. Base :: Tetr by insertional inactivation of B. pumilus M-F641 fadD gene knockout obtained its fadD genetic defect strains B. pumilus M-F641A, after 24h simple fermentation authentication deficient strain of B. pumilus M-F641A ω-1 hydroxy fatty acid with a yield of 16.95% , but does not prove to yield increased , the need verified by further fermentation .

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