The Research Of Immobilization Candida Cylindracea Lipase On Bentonite

Lipase has superior characteristics in mild reaction conditions,high catalytic efficiency,high reaction specificity,little environmental pollution and so on.Immobilization free lipase on a support can overcome some disadvantages,such as low stability,can not be recycled and difficult separation of product and by-product.In this work,we choose lipase from Candida yeast as the research object to immobilize,and apply the immobilized lipase in biodiesel preparation and ?-pinene epoxidation system.First,Investigating three kinds of carriers(carboxyl carbon nanotube,magnetic microspheres and bentonite)to immobilize Lipase OF.The lipase activity and storage stability of immobilized lipases on carbon nanotubes with different types are different,MCNTs-COOH lipase is the best,storage at 4?for 12 days can keep 79.8%of its initial activity.After modification with the chitosan or SiO2,the activity and stability of magnetic Fe3O4 lipases were better than that of the unmodified.Employing two different kinds of bentonites(Na-bentonite and Ca-bentonite)with different ion exchange capacity as the carrier,the Ca-bentonite lipase had higner protein loading and specific activity than Na-bentonite lipase.Considering that the bentonite is cheap and easy get,so we choosed it as immobilization support.Eight kinds of amino acid modified bentonite was investigated,the results show that the "flexible chain" between lipase and the support is the key factor that affect the lipase activity and reusability,glutamic acid is most suitable while its specific activity was 62.1 U·mg-1 protein,which can maintain 68%of its initial activity after reusing six times in hydrolysis olive oil emulsion.The immobilization of lipase onto the glutamic acid midified bentonite(Glu-Bent-lipase)is confirmed by Fourier transform infrared(FT-IR)spectra and X-ray diffractometer(XRD).Determination of kinetic parameters,the Km and Vmax values of free lipase are 1.88 mg·ml-1 and 17.31 ?g·min-1,while the Km and Vmax values of Glu-Bent-lipase increased to 6.74 mg·ml-1 and 48.78?g·min-1.It indicates that although amino acid modified bentonite reduced the affinity ability of the lipase with substrate,but upon immobilization the lipase can be activated and improve the catalytic efficiency of the lipase.Moreover,nonionic surfactant can increase the "interfacial activation" of the immobilized lipase,such as X-100 and X-114,the immobilized lipase activity increases about 2 times while its best incubation time is 1 h.Moreover,immobilized lipases(Glu-Bent-lipase OF and Glu-Bent-CALB)was applied to the biodiesel preparation and a-pinene epoxidation:Glu-Bent-lipase OF is an effective biocatalyst for the transesterification reaction,at 40? for 24 h,conversion rate can increased from 52.8%to 99.9%and could be used for ten times with conversion to methyl esters of 53.8%.Glu-Bent-CALB can be used in a-pinene epoxidation system,the ?-pinene conversion rate is 70.7%,and can be reused in the system.