Study The Characters Of Hyperthermophilic Lipase And It's High Expression

Lipases are widely distributed in animals, plants andmicroorganisms. For their activities in both aqueous and nonaqueoussolvent systems, lipases have developed into the most widely usedclass of enzymes in various industrial processes, finding use instereospecific hydrolysis, trans- esterification, ester synthesis,modification of physicochemical properties of triglycerides for the fatsand other organic biosynthesis reactions. The discovery of specificlipase working under extreme conditions is necessary, which willwidely extend the range of reactions of lipase. Aeropyrum pernix K1has been isolated in 1993 from coastal solfotaric thermal vent inKodakara-jima Island in Kyusyu, Japan. It grows in the range of 90 to98℃ with an optimal temperature of 95℃. From the genomesequences of Aeropyrum pernix K1, we found lipase motif; APE 1547was selected as the target protein. After the gene was cloned andexpressed in E. coli strain, the recombinant protein turned out to showlipase activities. We used the engineering bacterium constructed by our lab toferment and get the enzyme. Through the thermal denaturation, HitrapQ Sepharose ion exchange chromatography and Sephacryl S-200 gelfiltration chromatography we got the superior thermophilic lipase ofeletrophoretic purity. Its MW was determined by SDS-PAGE to be63KDa. It is proved to be monomer by non-denatured electrophoresisand active dying electrophoresis. The study for the zymologic characters of the superior thermophilic 38吉林大学硕士学位论文 韩四平lipase shows that its optimum temperature is 90℃; its optimum PH is8.0. It has strong thermostability. Its half-life is 160 hours at90℃(2.14mg/ml), and it is stable at alkalinous environment. To investigate the express regularity of engineered E. coli BL21 andoptimize the culture conditions of engineered E. coli BL21, adopting amethod of feeding the glucose and the tryptone, including the controlof pH, DO, Temperature. The optical density(600nm)of fermentationliquid reached 54; In 1L fermentation liquid the somatic weight was96g and the activity of lipase reached 6.8×105U.