| Lipase(EC 3.1.1)has superior characteristics in high selectivity and specificity,high catalytic efficiency,mild reaction conditions,little environmental pollution and so on,which were widely used in industries.Normally,immobilization strategies are used to improve the stability and reuse of enzymes in non-natural reaction systems.It is generally believed that the properties of the carrier have an effect on the catalytic properties of the immobilized enzyme.In this work,bentonite was used as enzyme immobilized material,modified by surfactant and used to immobilize lipase of Lipase OF and Candida rugosa lipase(Type VII)(CRL),and study on immobilization Lipase on surfactant modified bentonites and their properties.First,three kinds of surfactants,such as cationic,anionic and nonionic,modified Na-bentonite was investigated,the results showed that the hydrophobicity of the support is the key factor that affect the lipase activity and protein loading,octadecyltrimethylammonium chloride(OTAB)is the most suitable while its specific activity was 415.8 U·mg-1 in hydrolysis olive oil emulsion,twice the activity of Na-Bent-OF,In addition,by comparing the protein loading and immobilized enzyme activity of bentonite modified by different types of surfactants,it was considered that the chargeability of the support surface has no significant effect on the support adsorption performance,but the functional groups on the surface may affect the characteristics of the immobilized enzyme because of the hydrophobicity of the support and the non-specific binding of the enzyme molecule to the support.When the Lipase OF and CRL were respectively immobilized on the surfactant modified bentonites,it was found that for the two lipases,the modified Na-bentonite all had better immobilization effect,and the modified bentonite immobilize Lipase OF showed a relatively higher protein loading and specific activity recovery.By comparing the immobilization effect of bentonite modified by different surfactants,found that the Lipase OF immobilized on cation-nonion surfactant modified bentonite CTAB/TX 100-Bent was the best,and its protein loading was 0.68 mg·g-1 support,the recovery of specific activity was 133.4%.The catalytic properties of the modified bentonite immobilized lipases and free lipase were compared,it was found that the immobilized Lipase OF and CRL obtained with the cation-nonionic surfactant CTAB/TX 100 modified bentonite exhibited higher pH and temperature tolerance,and their storage stability was also improved.Determination of kinetic parameters,the Km and Vmax values of free lipase are 5.57 mg·mL-1 and 42.63 ?g·min-1,while the Km and Vmax values of CTAB/TX100-Bent-OF changed to 3.43 mg·mL-1 and 57.57?g·min-1.It indicates that surfactant modified bentonite enhanced the affinity ability of the lipase with substrate,and the maximum reaction rate was increased 1.35 times,showed a better catalytic activity.In addition,by the technology of scanning electron microscopy(SEM),X-ray diffraction(XRD),specific surface(BET),thermogravimetric analysis(TGA),Fourier transform infrared spectroscopy(FT-IR),Raman spectroscopy(RRS),the properties of modified bentonite and immobilize lipase were studied.It was found that the lipase was successfully immobilized on the modified bentonite,and the adsorption sites of the Na-bentonite modified by surfactant CTAB/TX100 were increased,the interlayer spacing increased from 1.20 nm to 1.59 nm,the properties of bentonite were improved,which facilitated the immobilization of lipases,and part of lipases adsorption on the surface of modified bentonite,part of lipases embedded in the bentonite layer. |
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