Immobilization Of Lipases Onto The Halogen & Haloalkanes Modified SBA-15 And Their Performance In Glycerolysis

Lipase was a kind of enzyme that could hydrolyze triglyceride（TAG）to produce diglyceride（DAG）,glycerol monoester（MAG）and other products.In addition to catalytic hydrolysis,lipase could also catalyze synthetic reactions,such as esterification,acidolysis,alcoholysis and transesterification.Lipases are potential enzymatic catalysts due to their mild reaction conditions,high site selectivity and few by-products formation.Free lipase was however poor in stability,immobilization of lipase onto the solid supports,mesoporous material SBA-15,for example,could improve the stability and could be recycled.SBA-15 was hydrophilic,introduction of the hydrophobic group into SBA-15 could alter its properties.In this paper,the organic functional groups were introduced into SBA-15 by post-grafting,and then was used to immobilized lipases.The enzymatic properties of the obtained immobilized lipases and their application to catalyze glycerolysis of soybean oil for DAG production were studied.The main results are as follows:1.The SBA-15,SBA-15-R and CALB@SBA-15-R were characterized by X-Ray diffraction（XRD）analysis,X-ray photoelectron spectroscopy（XPS）,Fourier transform infrared spectroscopy（FT-IR）,scanning electron microscope（SEM）,frozen transmission electron microscope（TEM）and N₂physical adsorption-desorption analysis.At the same time,the tertiary structure of the immobilized enzyme was analyzed by fluorescence spectrum.The results showed that SBA-15-R and CALB@SBA-15-R did not destroy the original pore structure of SBA-15,and the organic functional groups were successfully introduced into the surface of SBA-15,and The CALB was also successfully immobilized into the pores of SBA-15 and organic functionalized SBA-15.The tertiary structure of immobilized lipase CALB was analyzed by fluorescence spectroscopy.The maximum emission wavelength of free CALB was 323.90±0.28 nm.The red shift of the maximum emission wavelength was observed in the immobilized enzyme,especially the maximum emission wavelength of A shifted from 323.90±0.28 nm to343.15±0.49 nm.The same phenomenon was found in SBA-15-CH₂CH₂（CF₂）₅CF₃and CALB@SBA-15-CH₂CH₂（CF₂）₇CF₃fixed CALB,which was red shifted from 323.90±0.28 nm to338.4±0.21nm and 329.85±1.34 nm,respectively.The occurrence of redshift showed that the polarity of the environment around TRP in the protein increased and the hydrophobicity decreased.The tertiary structure of lipase changed when it was adsorbed on the carrier.2.The SBA-15 was modified with 9 different functional groups and then immobilized with 4 lipases,including:CALB(Antarctic Candida lipase B,AOL（Aspergillus oryzae lipase）,RML（lipase from Rhizomucor miehei）and LU（Lecitase（?）Ultra）.The results showed that the enzyme CALB activity of SBA-15 modified by 1H,1H,2H,2H-heptafluorodecyl group was the highest,which was 5577.80±200.00 U/g;Chloromethyl modified SBA-15 immobilized AOL had the highest enzyme activity,which was 12000.00±581.19 U/g;The highest LU activity of SBA-15modified by 1H,1H,2H,2H-heptafluorodecyl group was 2822.20±200.00U/g;The RML enzyme activity of SBA-15 modified by1H,1H,2H,2H-heptafluorodecyl group was the highest,which was11577.78±234.13 U/g.The activity of the modified SBA-15 immobilized lipase was higher than that of the parent SBA-15 immobilized lipase,indicating that halogen and haloalkane modified SBA-15 was an effective way to improve the enzymatic properties of the immobilized enzyme.3.The lipase immobilized with halogen and haloalkane modified SBA-15 was applied to the producing of diglyceride（DAG）by catalytic glycerolysis.The reaction conditions:the reaction temperature was 60℃,the reaction time was 12h,and the amount of immobilized enzyme was0.2g.The results showed that the CALB@SBA-15-CH₂CH₂（CF₂）₇CF₃producing DAG by catalyze glycerolysis of soybean oil,the content of DAG was 54.19±1.91wt%and the ratio of DAG/MAG was 2.75±0.59;The AOL@SBA-15-（CH₂）₂CH₂Cl producing DAG by glycerolysis of soybean oil,the content of DAG was 52.20±0.10 wt%and the ratio of DAG/MAG was 2.83±0.11;LU@SBA-15-CH₂CH₂（CF₂）₅CF₃ producing DAG by glycerolysis of soybean oil,the content of DAG was 52.55±0.73and the ratio of DAG/MAG was 1.59±0.02;RML@SBA-15-CH₂Cl catalyzes the glycerolysis reaction,and the DAG content was 59.22±0.23wt%and the ratio of DAG/MAG was 3.96±0.13.At the same time,the thermodynamic equations of glycerolysis catalyzed by CALB@SBA-15-CH₂CH₂（CF₂）₅CF₃and CALB@SBA-15-CH₂CH₂（CF₂）₇CF₃ were studied.The Arrhenius equations between 40℃and 90℃were ln V₀=3.13-3.07/T和ln V₀=7.90-4.64/T,respectively.4.In order to further increase the yield of DAG,the effects of reaction temperature、reaction time、the amount of immobilized enzyme and the molar ratio of soybean oil/glycerol on the producing DAG by glycerolysis of CALB@CH₂CH₂（CF₂）₅CF₃ were studied.It was found that when the reaction temperature was 80℃,the reaction time was 12h,the amount of enzyme was 0.3g and the molar ratio of soybean oil/glycerol was 2:1,the content rate of DAG content was 60.13±1.34 wt%and TAG conversion was 81.14%.Under the optimum reaction conditions,the reusability of CALB@SBA-15-CH₂CH₂（CF₂）₅CF₃ and CALB@SBA-15-CH₂CH₂（CF₂）₇CF₃ was studied.After 10 times of reuse,99.63±0.86%and 93.06±5.80%of the initial TAG conversion rate were retained.It shows that SBA-15-CH₂CH₂（CF₂）₅CF₃ adsorption of lipase can improve the stability of enzyme operation.