| Lipase, which can catalyzes triglyceride to diacylglycerol and single glycerol esters of fatty acids. It’s may be comes from animals, plants and microorganisms, and has important application in food, pharmaceutical, textile and so on. The lipase studied in this article is a low temperature alkaline lipase, it’s gene comes from an ocean microorganism named Bohai sea-9145. With the intention of prepare the lipase effectively and improve the enzyme activity, we optimized the fermentation, refolding the inclusion body protein and fixed the lipase.1.To effectively improve the production of the Bohai sea-9145 lipase, single factor experiment, PB experiment and BB surface response experiment were used to optimize the fermentation conditions of the recombinant strain Bohai sea-9145. The optimal conditions were as follows: the initial pH value of the medium is 8.0, the inoculation amount is 1.5%, and after 3.5 hours of incubation, the expression of the lipase was induced by adding IPTG to a final concentration of 0.5 mmol/L at 16 ℃ for 15 hours with the shaking speed of 200 r/min. After optimization, the final activity of the recombinant lipase was increased by 9.72 times.2.To improve the recombined lipase more effectively,we analyzed the recombined bacterial cell’s crushing conditions and inclusion body protein’s refolding methods.The results show that the optimal concentration of cell crushing solution is 20 mM,the optimal pH value is 8.0. It can be broken more completely at the power of 300 W after three times freezing and thawing repeatedly, break 1 minutes every 1 minutes, a total of 10 times. The inclusion body protein after washing and dissolving were refolding by two different methods, one was refolding by dilution methods, another was refolding by urea gradient dialysis methods. We found that when using the urea gradient dialysis method,the protein content and enzyme activity will higher than dilution method.3.On the basis of above test results,the recombined lipase was fixed by two methods: solid loading cross linked enzyme aggregates(CLEAs)、Chitosan cross-linking and adsorption method. The former determined immobilized carrier is resin MA-WP8, the settling agent is DMSO(VDMSO:Venzyme is 1:2), settling time is 2h, the concentration of glutaraldehyde is 0.15%, the fixed temperature is 25℃ and fixed time is 6h, the maximum amount of enzyme is 5m L, the maximum lipase recovery activity is 73%, and the maxmum enzyme activity is 705U/g. For the consideration of improving the activity of fixed lipase and saving free enzyme, the experiment determined the enzyme adding amount is 4mL, at this addding amount, the activity recovery is 62%,the enzyme activity is 650U/g. Chitosan cross-linking and adsorption method determined the concentration of chitosan, acetic acid and NaOH are 0.15%, 0.15%, 15%, the glutaraldehyde’s concentration is 0.2%, and it need cross-linking with chitosan for 2h before adding enzyme. The optimal temperature and time of immobilized is 20℃, 10 h, the maximum adding amount of free enzyme is 3m L, under these conditions the maximum activity recovery is 50%.This results indicated that the solid loading cross linked enzyme aggregates can fix the recombined lipase enzyme effectively.4.Compare the stability of CLEAs,chitosan immobilized enzyme and free enzyme. The results showed that the optimal temperature of immobilized enzyme are both 40℃, 5℃ higher than free enzyme; the immobilized optimal pH value is 8.0,which is same as free enzyme; the metal ions can influence the enzyme stability, Ca2+、Mg2+、Zn2+ can improve the enzyme activity, and Cu2+、Mn2+、Ag+ can inhibit the enzyme’s activity, but the inhibiting influence on immobilized enzyme is lower than that on free enzyme; The two method for enzyme immobilized both can improve the storage stability, and the stability of CLEAs is better than chitosan immobilized Enzyme... |
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