Study On Lipase Production By Bacteria Fermentation, Its Purification And Immobilization Processing

In this paper,in order to provide the foundation material to expand lipase production source,the screening of lipase-producing bacieria,the fermentation conditions,the purification and immobilization of lipase were investigated.And the other characterization of lipase was also studed.All of the results works were listed as follow:Soil samples were collected from oil refinery,vegetable gardens, grain andoil market near Guiyang and canteen of Guizhou Normal University.24 kinds of lipase-producing bacterial strains were separated and identificated from 78 kinds of soil samples by primary screening, secondary screening,and the activity ranged of lipase productal from those strains was 1.0～13.2U/mL,and the activity of enzyme production of bacterial strain was named A7 which was collected from the soil samples of oil refinery was the highest.A7 baceterial strain were cultured by different carbon and nitrogen sources,and the results showed that the best carbon and nitrogen source for bacteria fermenting and producing lipase was sucrose and yeast powder.The fermentation medium for A7 strain was optimized by the orthogonal experiment designs,and the optimal medium for the lipase production was(%):yeast powder(3.5g),sucrose(1g),rapeseed oil (2mL),magnesium sulfate(0.25g),potassium hydrogen phosphate(2g), ammonium sulfate(0.3g),pH8.0.And the optimal culture time was 48h. Lipase that was excreted from the borth of strain was purified to homogeneity by(NH₄)₂SO₄ precipitation and gel filtration with Sephadex G-75 as medium,the activity recovery of lipase was 49.5%and 22.32%, respectively.The molecular weigh of the lipase was 29.5kd by SDS-PAGE.The study showed that the embedding method was better than the physical adsorption method.Kaolin combined with sodium alginate Kaolin-alginate was the best way,and the relative enzyme activity was 62.55%,and it was higher near 30 percentage points than the diatomite adsorption.We found that the optimal temperature for the catalysis processing of lipase had not changed by comparing the free lipase and immobilized enzyme characteristics,and both were 40℃.The optimal pH value for free lipase was 8.0 and that for immobilized lipase was 7.0.We also found that immobilized enzyme was more stable than the free lipase. After re-use 5 times,the activity of the immobilized enzyme decreated from 30U/g to 15.48U/g,and the time of the reaction was 15 minutes,so the half-life of immobilized enzyme was 75minutes(5×15).