耐熱脂肪分解酵素分離與特性測定

• Main Author: 謝汎擎
• Other Authors: 趙維良
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/52932605604767018090

碩士 === 東吳大學 === 微生物學系 === 89 === Lipases（Triacylglycerol acyl hydrolases; EC 3.1.1.3）which hydrolyze triglyceride into glycerol and fatty acid, and are used widely in fat, food, oil and detergent industries. Most of the lipases are mesophilic enzyme which can not hydrolyze substrate that exists in solid form at room temperature and are unstable when chemical denaturants are present. This has severely restricted the application of lipase in industries. The discovery of thermostable lipase has circumvented this obstacle. Thermostable lipase can catalyze hydrolysis of fat at high temperature and is highly stable against chemical denaturants. Hence, it has a wide application potential in industry. Generally speaking, it is common strategy to isolate thermostable lipase from thermophile. However, mass cultivation of thermophile is usually energy intensive and the yield is poor. In this study, we have isolated lipase-producing organisms from natural environment. We characterized lipases produced from five bacterium which isolated in prevent. Lipase in the growth medium was concentrated by ultra-filtration using a pre-packed cartridge, and the influences of temperature and pH on lipase activity were determined. Plate puncture assay was used to detect lipase activity which consisted of DF minimal medium supplemented with 0.1﹪olive oil. Lipase activity was determined by measuring the size of clear zone due to hydrolysis of olive oil in the medium. The best temperature tolerance was detected in lipase produced by isolate G10 which maintained 15% activity at 55℃. In general, the optimum temperatures for all the tested lipase are between 30~40℃ which made them mesophilic enzymes. As for pH tolerance, lipase produced by isolate G10 and G11 maintain 73﹪and 93﹪of relative activity at pH11. Molecular weight of lipase produced by isolate G11 was determined through native PAGE using active stain technique, and two clear zones were observed which were estimated to be around 36 kDa and 77kDa. The ability to utilize different nitrogen and carbon sources for isolates G7, G10 and G11 is also investigated. All three bacteria tested can not grow in DF minimal medium without the addition of casamino acid. As for carbon sources, isolate G7 can utilized glucose and glycerol but not sucrose and citric acid while isolates G10 and G11 can not utilized all four of the test carbons descried above. When using BIOLOG identification systems, isolate G7 is identified as Bacillus cereus and isolates G10 and G11 are identified as Pseudomonas alcaligenes. The results indicated that lipase produced by Pseudomonas alcaligenes is an alkaline lipase and it can be used in detergent industry.