| Conjugated linoleic acid ethyl ester(CLAEE)is lipophilic,stable and easily to be absorbed,which has been of wide interest for potential applications in the fields of drug,cosmetic,food and feed.To some extent,it will become good replacement of conjugated linoleic acid(CLA).Lipase-catalyzed synthesis of CLAEE under mild reaction conditions takes place of traditional production process,which can improve the quality of product and be friendly to the environment.In addition,it gives efficient method to separate two main isomers of 9c,11t-CLA and 10t,12c-CLA.This paper focuses on immobilization of Candida rugosa lipase(CRL)on mesoporous molecular sieves,in order to solve the instability of the free CRL,and to achieve the reusability of enzyme.The main purpose is preparation of immobilized CRL with high activity and good stability to obtain higher catalytic activity in esterification between CLA and ethanol.The main contents are as follows:In the first part including the third and fourth chapters,the supports for immobilization of CRL and the catalytic properties of immobilized CRL were investigated.Firstly,different structural mesoporous silicas of HMS,SBA-15 and MSU-H(7.2)were used as supports for immobilized CRL prepared by physical adsorption.The results showed not only the pore size,but also the morphology and particle size of mesoporous silicas influenced the activity of immobilized CRL.CRL immobilized on MSU-H(7.2)afforded higher activity of 1978.1U/g,protein loading of 46.8mg/g,activity recovery of 28.1%,and esterification of the isomer 9c,11t-CLA of 67.2%,and that of another isomer 10t,12c-CLA of 17.3%at the total esterification of 32.4%under the reaction condition.Moreover,the effect of different pore sizes of MSU-H silicas on CRL immobilization via physical adsorption and cross-linking method were studied.The pore sizes of MSU-H materials had an influence on activity of immobilized-CRL.The activity and amount of protein loading of the immobilized CRL were increased with an increase in the pore size of MSU-H silicas.The CRL immobilized on MSU-H with 13.3nm of pore size showed the highest activity of 2104.3U/g,protein loading of 47.7mg/g and activity recovery of 29.9%among the MSU-H silicas with pore sizes of 6,7.2 and 13.3nm.The cross-linking method led to lower activity and higher stability of immobilized-CRL than the physical adsorption method.Based on the activity and stabilty of the immobilized-CRL,MSU-H silica with BJH pore size of 7.2 nm was suitable for support to immobilize CRL with cross-linking method.In the second part containing the fifth chapter,a series of amino-functionalized MSU-H silicas with amino-containing of 7.0～18.0 mol%were prepared by co-condensation method and used as supports for immobilizing CRL.The CRL immobilized on 15.0%NH2-MSU-H prepared by physical adsorption exhibited higher activity of 2496.6U/g,protein loading of 34.4mg/g and activity recovery of 35.5%,and the total esterification of 27.8%.The amount of amino-loading of MSU-H silicas was responsible for catalytic property of immobilized CRL.However,the amino functionalization of MSU-H could not improve the total esterification of CLA in the reaction condition.In the third part including the sixth chapter,the amount of the surfactant contained in MSU-H silicas had great influence on the properties of immobilized CRL via cross-linking method.The CRL immobilized on MSU-H silica containing 38 wt.%of surfactant provided the highest total esterification of 56.7%and good recyclability,meanwhile,the 9c,11t-CLA and 10t,12c-CLA esterifications were 96.5%and 40%,respectively.After recycled 4 times in the reaction,the total CLA esterification decreased to 46.7%,and the residual activity retained 82.4%correspondingly.The structure and properties of mesoporous molecular sieves used as supports and immobilized CRL mentioned above were characterized by powder X-ray diffraction(XRD),nitrogen adsorption-desorption isotherms,Fourier transformation infrared spectra(FT-IR),scanning electron micrograph(SEM)and thermogravimetric and differential thermal analysis(TG-DTA). |
