Knockout And Function Of Lipase Genes From Burkholderia Sp. ZYB002

Whole cell biocatalyst displayed many advantages, including easy recycle, good stability and convenient produce, etc. In our previous research, we found that Burkholderia sp. ZYB002produced large amount of cell-bound lipase. In this dissertation, the cell-bound lipase of Burkholderia sp. ZYB002was qualitatively assayed.The results of this research are as follows:1. The tmp gene from plasmid pBBR1TP and gfp gene from plasmid pEGFP was cloned, respectively, which would be used as select marker in the following work. Three different lipase gene, lip A,lipC21and lipC24was cloned from Burkholderia sp. ZYB002, respectively. Based on the suicide plasmid pJQ200SK, three plasmid, pBCMB-S3, pBCMB-S5and pBCMB-S7, was constructed, respectively, which would be used to inactivate the corresponding lipase gene.2. Three suicide plasmids, pBCMB-S3, pBCMB-S5and pBCMB-S7, was introduced into the wild-type Burkholderia sp. ZYB002strain with three triparental mating technology, respectively, which resulted in the corresponding lipase gene inactivation, including lip A,lipC21and lipC24.3. The cell-bound lipase activity and the extracellular lipase activity was compared between the mutant strain and the wild-type strain, respectively. The extracellular lipase activity and the cell-bound lipase activity of Burkholderia sp. ZYB002-△lipA decreased to50.6%and42.0%, respectively. Burkholderia sp ZYB002-△lipC24strain decreased the extracellular lipase activity and cell-bound lipase activity by14.3%and20%, respectively.4. The swarming ability of Burkholderia sp ZYB002-△lipC21and Burkholderia sp ZYB002-△lipC24decreased slightly when the strains were cultured on the solid swarming media.