Study On Extracellular Lipase From Yarrowia Lipolitica W₂₉

Yarrowia lipolitica is an unconventional and strictly aerobic kind of fungi. It can grow at a temperature interval from 25℃ to 30℃ and a pH interval from 3.0 to 8.0. It has the capability of widely using the inexpensive substrates like fats, oils and hydrocarbons as carbon sources. the As lipase produced by Y.lipolitica can involve in the catalytic hydrolysis, esterification, transesterification and other chemical reactions of fat, especially degrading the salad oil with high efficiency, it has been widely used in treatment of fats wastewater. Therefore, it is of practical and applied importance to obtain and extract the low-cost lipase from Y.lipolitica.In this study, we used the Y.lipolitica W29 which was kept by the laboratory as starting strain to produce the lipase, and lipase activity was used as the indicator. We used the orthogonal and single factor experiment to optimize the lipase production conditions by utilizing the salad-oil water as basic fermentation medium In view of the result from the single factor test, we optimized the fermentation process and usd ammonium sulfate salting out, dialysis, DEAE-Sepharose FF ion chromatography, PEG20000 concentration and other steps to pure and concentrate the lipase from the fermentation supernatant, and had a preliminary study about its enzymatic properties.The test results from preliminary laboratory showed that the Y.lipolitica W29 could efficiently degrade the salad-oil, so under the conditions of culture temperature at 28℃, fermentation shaking speed at 150 r/min, initial pH of the medium naturally, inoculum of 2.5%, in salad-oil+water fermentation medium, fermentation for 48 h, we found that the lipase activity was only 0.52 U/mL. The results suggested that the conditions was suitable to degrade the salad oil, but not for the production of lipase. In order to using the strain for further improvement of the lipase production, we respectively test the YPD+salad-oil and YPD liquid strain and the maximum lipase activity were respectively 1.38 U/mL and 1.75 U/mL. The results suggested that early induction of beneficial bacteria was in favor of lipase production, but the lipase production was still very low. Then we respectively test incubation temperature, rotation speed, initial pH of the medium. The result showed that under the condition of incubation temperature at 30℃, fermentation shaking speed at 100 r/min, medium with initial pH of 8.0, the lipase activity was up to 1.70 U/mL, increasing three-fold when compared with the lipase under the non-optimized condition. In order to investigate the interaction between culture conditions, we test the results of accordance based on the single factor orthogonal experiment, and the results showed that under the condition of the culture temperature at 25℃, fermentation shaking speed at 50 r/min, medium with initial pH of 8.0, the highest lipase activity could get to 2.44 U/mL, increasing nearly five-fold when compared to the activity of the before optimization. Overall, though the lipase activity was increased, the lipase activity from the strain to using the salad oil +water remained to be low. The results suggested that it was necessary to optimize the fermentation conditions.YPD, YPD+ salad oil and the reference medium was respectively used as the fermentation medium for Y.lipolitica W29 to optimize the condition of lipase production, and we found that the reference medium is suitable for lipase production. The optimized medium was composed of 0.1% sucrose,2.5% peptone,0.1% urea, 0.1% MgSO4,1.5% NaCl and 0.1% Tween-80, and the optimal culture conditions were culture temperature at 30℃, shaking speed at 150 r/min, the initial medium pH of 7.0 and inoculum of 2.5%. In this condition, by using 7g/L olive oil as inducers and fermenting for 60 h, the yield of lipase was improved. Its enzymatic activity was 7.70 U/mL; when using 7 g/L salad oil as inducing agent and fermenting for 36 h, enzyme production reached its highest and the activity is 4.04 U/mL.After purification, there are two kind of purified proteins and both of them have lipase activity. We respectively marked them as the purified enzyme 1 and 2. The purified enzymes were detected by SDS-PAGE and the result show that the molecular weight of purified enzyme 1 was 37-38 kD while the other is 41-42 kD. Further study showed that:the optimum temperature of both two enzymes were 40℃ and the optimum temperature was at 30-50℃, the optimum pH is 80,and in pH 6.5-8.0, the activity of both two enzymes were remain relatively stable. We also found that Mg2+ Ca2+, Li+ have a strong activating effect on both of the purified enzymes while Fe3+ Na+ and Cu2+ presented a certain inhibition for both enzymes. Moreover, it is worthy that Zn2+ played a significant role on improving the activity of the purified enzyme 1, but for pure enzyme 2, it had a strong inhibition. SDS, CTAB, Tween-80 are inhibited for both activity while PEG1750,0.5% PEG20000 were activated for both of the enzymes.