Construction And Optimization Of Immobilized Lipase For The Preparation Of 1,3-Dioleoyl-2-Palmitoylglycerol

Healthy human milk is the best way to provide infants under 6 months of age with the nutrients they need for growth and development.The high content of palmitic acid at sn-2 position in human milk fat（HMF）is of great significance for infants to absorb and utilize nutrients.In recent years,the rate of breast-feeding continues to decline,and the research on human milk fat substitudes（HMFs）has attracted more and more attention.1,3-Dioleoyl-2-palmitoylglycerol（OPO）is the main triglyceride component in HMFs.Adding OPO to infant formula may improve the content of palmitic acid at sn-2 position,which is conducive to the healthy development of infants.At present,the enzymatic synthesis of HMFS is still dominated by commercial lipase.In this study,five lipases were immobilized on different carrier materials to catalyze the acidolysis of glycerol tripalmitate（PPP）and oleic acid（OA）.The immobilized enzymes with high selectivity and reaction efficiency were selected to further optimize the reaction conditions and evaluate the feasibility of preparing HMFs rich in OPO.The main research contents are as follows:1.According to the existing methods of our research group,support materials such as silane coupling agent modified SBA-15,mesoporous metal organic frameworks（MOFs）and activated macroporous resin were prepared for the immobilization of lipases such as Thermomyces lanuginosus（TLL）,Rhizomucor miehei（RML）,Candida rugosa（CRL）,Porcine Pancreas Lipase（PPL）and AOL Aspergillus oryzae（AOL）.The obtained immobilized lipases were then used to catalyze the acidolysis of PPP and OA.The conditions of acidolysis reaction were as follows:substrates molar ratio PPP:OA=1:12,enzyme addition was 10%of the reaction system,reaction temperature was 70°C,the reaction time was 12 h.The results showed that:1)among the immobilized lipases supported by organic functionalized SBA-15,the catalytic efficiency of,,andwas higher,and the yields of OPO were64.73%,59.70%,64.07%and 59.52%respectively;2)In the immobilized lipases with mesoporous MOFs as carrier,TLL@GMP-Zn and AOL@GMP-Zn showed good catalytic activity,and the yields of OPO were 47.08%and 53.14%respectively;3)In the immobilized lipases with macroporous resin as carrier,TLL@ADS-17,TLL@DA-201,TLL@D3520 exhibited good catalytic activity,and the OPO yields were 56.23%,44.70%and 53.88%,respectively.Withand AOL@GMP-Zn as catalysts,OPO yields at 94.94%and 77.94%were respectively obtained,which were higher than that of with Lipozyme RM IM as catalyst.2.The enzyme protein loading of TLL@SBA-15-C₁₈H₃₇ and AOL@GMP-Zn were determined,which were 0.049±0.006 mg/mg support and 0.012±0.006 mg/mg support respectively.Then,TLL@SBA-15-C₁₈H₃₇ was characterized by XRD,FT-IR,TGA,and EDS.The XRD results showed that functional group modification and lipase immobilization did not destroy the pore structure of SBA-15.The EDS,FT-IR and TGA characterization confirmed that TLL was successfully immobilized onto the SBA-15-C₁₈H₃₇.3.The reaction conditions of the above TLL@SBA-15-C₁₈H₃₇ and AOL@GMP-Zn catalyzed acidolysis were optimized.The effects of substrate molar ratio（PPP:OA）,enzyme addition,reaction temperature and reaction time on the yield of OPO and the palmitic acid content at sn-2 position in the acidolysis products were studied.At the same time,the reusability of immobilized enzymes for acidolysis under the optimal reaction conditions were determined.The results were as follows:1)The optimal conditions for TLL@SBA-15-C₁₈H₃₇ catalyzed acidolysis reaction were:substrate PPP:OA=1:12,reaction temperature 50°C,enzyme addition 10%and reaction time 8 h.Under these conditions,the conversion of PPP was 99.07%,and the yield of OPO was 73.15%,which was higher than that of Lipozyme RM IM（68.18%）,while the content of palmitic acid at sn-2 position was 90.09%.After 5 times of reuse,the yield of OPO still retained 81.04%of the initial yield,indicating that the immobilized enzyme TLL@SBA-15-C₁₈H₃₇ was stable.2)The optimal conditions for AOL@GMP-Zn catalyzed acidolysis reaction were:substrate PPP:OA=1:10,reaction temperature 60°C,enzyme addition 14%and reaction time 12 h.Under these conditions,the conversion of PPP was 98.08%,and the yield of OPO was 63.15%,which was 92.62%of that with Lipozyme RM IM as catalyst,while the content of palmitic acid at sn-2 position was 86.13%.Howevewr,after only four times of reuse,the yield of OPO decreased rapidly,which was only 45.49%of the initial yield.It indicated that the reaction stability of AOL@GMP-Zn was relatively poor.