Transgenic Potato with Candida rugosa Lipase Gene and Characterization of the Recombinant Lipase

• Main Authors: Jia-Hung Wang, 王嘉宏
• Other Authors: Jei-Fu Shaw
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/32869070442455502748

碩士 === 國立海洋大學 === 水產生物技術研究所 === 90 === There is a tendency towards using genetically modified crops to replace microorganism for the production of high-value proteins and enzymes. The tuber of potato is a desirable plant organ as a bioreactor. Patatin is the most abundant soluble potato tuber protein, an estimated 30 ~ 40 % of total soluble protein. Patatin has been shown to exhibit lipid acyl hydrolase activity which can hydrolyze mono-acylglycerol but not di-acylglycerol or tri-acylglycerol. We predicted a potential synergistic effect on the hydrolysis of fat or oils by the combination of lipase and patatin. In this study, the Candida rugosa LIP4 gene encoding high lipase activity have been isolated and fused with the sporamin or Sus4 promoter, with or without targeting peptide such as transit peptide (TP) from rbcS gene and sequence-specific vacuolar sorting domain (spVSD) from sporamin gene, and inserted into binary vectors. These plasmids were transferred to Agrobaterium tumefaciens LBA4404, for transformation of potato. Molecular analyses showed that the transgenic plants expressed active recombinant LIP4. Results from the PCR, Southern blotting, RT-PCR analyses, and GUS activity staining demonstrated that we have obtained several independent transgenic potato lines expressing lipase genes. The RT-PCR assay showed that the LIP4 mRNA was accumulated in microtuber, root, stem, and leaf. The pH-stat assay also detected lipase activity in microtuber, root, stem, and leaf. The microtuber was the best tissue for the expression of active lipase. Biochemical analyses showed that the optimal pH value of the recombinant lipase from transgenic potato is 7.0 and the optimal temperature is 40oC. The enzyme retained 100, 98, 72, 66, 57, 34, 11 and 4 % relative activity after 10 minutes of incubation at 37, 40, 50, 60, 70, 80, 90 and 100 oC respectively. For the lipase substrate specificity, the recombinant lipase showed highest activity towards medium-chain triglycerides.