Immobilization Of Yarrowia Lipolytica Lipase Lip2and Its Application In Enrichment Of Docosahexenoic Acid In Fish Oil

Yarrowia lipolytica Lip2is one of the ideal catalysts for the enrichment of DHA inFish Oil. However, there are some shortcomings of free Yl Lip2, such as poor operationalstability, expensive price and inability to reuse and so on. These have been enormouslyhampered the wide application Yl Lip2. Hence，these disadvanteges must be overcame andnow, enzyme immobilization technology is the mainstream in this field. This researchsystematically investigated the immobilization process of Yl Lip2, using macroporousresin as carrier. In addition, the application of immobilized lipase in the enrichment ofdocosahexenoic acid was still researched. The main work is as following:(1) Through primarily screening on eight kinds of ion exchange resins includingD311、D301R、D418、D152H、D380、D151H、D401and D113and four kinds macroporousresin including D3520、D4020、AB-8. D152H was choosed as the best supporter. Yl Lip2was immobilized on the cation-exchange resin D152H by strong-acid cation exchangeresin adsorption. Factors as loading amount of the lipase, immobilized time, immobilizedtemperature, pH of immobilized medium and drying metholds were optimized in detail.The optimum levels for the factors were: lipase loading0.02g per gram carrier, adsorptionfor1h, temperature20℃, pH6.6of citric acid-citric acid sodium buffer, Vaccuum drying.The immobilization efficiency of the optimized immobilized enzyme was88.17%. Thespecific activity of the immobilized protein was764722U/g with its activity recovery177.66%.(2) Based on single factorial experiments, the Box-Behnken method was applied tofurther optimize the preparation conditions. Three factors: lipase loading、buffer pH andreaction temperature were selected as the key factors affecting the specific activity of theimmobilized protein and the optimal preparation conditions were: lipase loading0.02g/gresin, pH6.5of citric acid-citric acid sodium buffer,20℃, adsorption for1h andvacuum-drying. The immobilization efficiency of the optimized immobilized enzyme was89.81%. The specific activity of the immobilized protein was807099U/g, which wasincreased1.91times compared with free lipase (specific activity was379506U/g).(3) The characteristics of immobilized Yl Lip2were investigated. The results revealedthat the optimal temperature and pH of the immobilized lipase were the same as the free lipase, but the immobilized lipase showed a much better thermal and pH stabilitycompared with the free lipase. Then make a detection of the both on their substratespecificity, and the results showed that both of the free lipase and the immobilized lipasehad nice catalytic activity in carbon chain of nitro phenol ester (C8、C10、C12).In additionthat, the immobilized lipase also had a good catalytic activity in long carbon chain of nitrophenol ester (C16).(4) The immobilized Yl Lip2was used to enrich DHA from fish oil. The conditionswere optimized: lipase supply0.64g, oil/buffer volume ratio1:1, N2as protection gas,temperature30℃,200rpm for16h, the content of DHA could reach32%under theoptimum conditions. After10batch reaction, the immobilized Yl Lip2can still keep93%of its initial catalytic efficiency, indicating t a good operational stability.