A Study On Immobilization Of Lipase MAS1 And Its Application In Synthesis Of N-3 PUFA-Rich Functional Lipids

n-3 PUFA-rich functional lipids have presented important health benefits in the human.Compared with ethyl esters(EE),the major n-3 PUFA-rich products in the market,n-3PUFA-rich triacylglycerols(TAG)and n-3 PUFA-rich phospholipids(PL)have higher bioavailability.However,n-3 PUFA-rich TAG and n-3 PUFA-rich PL are difficult to be synthesized efficiently by most of lipases.Lipase MAS1 is a novel enzyme from marine Streptomyces sp.strain W007 and its regiospecificity is different from other lipases when it was used as biocatalysts.In this paper,immobilized MAS1 lipase was prepared in the first time and the process was optimized.Then,immobilized MAS1 lipase was used to catalyze the synthesis of n-3 PUFA-rich TAG and n-3 PUFA-rich PL in different reaction system.This is the first report on the application of lipase MAS1 in synthesis of n-3 PUFA-rich TAG and n-3PUFA-rich PL.The parameters of the reaction and process route were also optimized systematically.The yield of the final products under optimal condition can meet the needs of industrialized production.This paper provided plenty of information for enzymatic preparation of n-3 PUFA-rich functional lipids.The main research contents and results are as follows:(1)Immobilization of lipase MAS1 and its characterization with enzymatic propertiesThe method of immobilization of lipase MAS1 was investigated in this study.It was found that the resin XAD1180 was the best support material for the immobilization of lipase MAS1 and the esterification activity of immobilized MAS1 lipase was 2700 U/g under the optimal immobilization conditions.The results of FT-IR analysis showed that the absoption of lipase MAS1 onto XAD1180 resin was successful with the optimal method.It was also found that the optimal temperature,pH value and regiospecificity of immobilized MAS1 lipase are as same as the free form.However,the thermal stability of immobilized MAS1 lipase was improved significantly compared with that of the free lipase.Immobilized MAS1 lipase showed a lower selectivity for long-chain polyunsaturated fatty acids(EPA and DHA).(2)Synthesis of n-3 PUFA-rich TAG by immobilized MAS1 lipase-catalyzed esterification reactionIn this study,immobilized MAS1 lipase was utilized to catalyze esterification of glycerolwith n-3 PUFA to produce n-3 PUFA-rich TAG and the reaction conditions were optimized.The highest esterification degree(99.31%)and TAG content(92.26%)were achieved under the optimal conditions,which were significantly higher than those(82.16% and 47.26%,respectively)of Novozym 435-catalyzed reaction.In addition,immobilized MAS1 lipase showed better catalytic efficiency and reusability than its free form.TAG rich in more than ninety percent of n-3 PUFA could be produced by immobilized MAS1 lipase-catalyzed esterification reaction.(3)The synthesis of n-3 PUFA-rich TAG by immobilized MAS1 lipase-catalyzed glycerolysis reactionEE are the main available form of high grade purity PUFA found in the market.Therefore,immobilized MAS1 lipase-catalyzed glycerolysis of n-3 PUFA-rich EE was investigated to prepare TAG.Higher TAG content(73.9%)and EE conversion(82%)were obtained by immobilized MAS1 lipase than those by Novozym 435(29.6% and 54.8%,respectively)and Lipozyme RM IM(10% and 49%,respectively)under the same conditions.The maximum TAG content(76.5%)was achieved under the optimal conditions.The final product consisted of 96.2% TAG with 3.8% DAG after purification of the glycerolysis reaction mixtures by molecular distillation.In addition,the final products had low acid value and peroxide value.n-3 PUFA-rich TAG in the final product showed similar fatty acids composition to the substrate.(4)Synthesis of n-3 PUFA-rich PC by immobilized MAS1 lipase-catalyzed transesterification reactionThe bioactivities of n-3 PUFA-rich PL in vivo were very different from those of n-3PUFA-rich TAG.Therefore,immobilized MAS1 lipase was also used to catalyze transesterification of n-3 PUFA-rich EE and phosphatidylcholine(PC)for production of n-3PUFA-rich PC and the reaction conditions were optimized.After 72 h,the incorporation of n-3 PUFA in PC and LPC reached 43.65% and 42.95% under the optimized conditions,respectively.The PL composition of the final reaction product consisted of 32.68% PC,28.76% sn-1 LPC,4.9% sn-2 LPC,and 33.66% GPC.The total yield of PC and LPC was66.34%.(5)Synthesis of n-3 PUFA-rich LPC by immobilized MAS1 lipase-catalyzedesterification reactionLarge amounts of sn-glycero-3-phosphatidylcholine(GPC)were produced by hydrolysis of PC and LPC during enzymatic transesterification reaction processes.Therefore,immobilized MAS1 lipase-catalyzed esterification of GPC and n-3 PUFA was also investigated.Higher GPC conversion(50.42%)and LPC content(47.6%)were obtained by immobilized MAS1 lipase than those obtained by Novozym 435(GPC conversion of 20%and LPC content of 18.4%).After 24 h,the maximal LPC content(90.77%)and GPC conversion(93.12%)by immobilized MAS1 lipase-catalyzed esterification of GPC and n-3PUFA were obtained under the optimized conditions.More than 89% n-3 PUFA was incorporated into LPC.