Preparation And Application Of Lipase Candida SP.99-125Cleas In Silica Materials

The technique of preparing CLEAs in the pores of suitable supports has beendeveloped as a new method for enzyme immobilization. It combined the CLEAs methodwith supports to realize high-efficient immobilization. In this study, Lipase Candida sp.99-125was chosen as modal enzyme, and three-dimensionally ordered macroporous silicaand mesoporous silica were chosen as supports. The preparing conditions, stabilities andcatalytic properties of immobilized enzyme were studied here and the immobilized enzymewas used for the transesterification of jatropha oil and dimethyl carbonate (DMC). Thedetails in this study were summarized as follows:Firstly, preparation of CLEAs in three-dimensionally ordered macroporous silica(CLEAs-LP@3DOM-SiO2) and the study of stabilities and catalytic properties. Whenthree-dimensionally ordered macroporous silica was chosen as support, saturatedammonium sulfate was used as precipitant and glutaraldehyde(GA) with a concentration of0.25%(w/w) was employed as cross-linker, the highest activity recovery ofCLEAs-LP@3DOM-SiO2was obtained. Compared with EAs-LP@3DOM-SiO2and nativelipase, CLEAs-LP@3DOM-SiO2exhibited excellent thermal and mechanical stability, andcould maintain more than85%of initial activity after16days’ shaking in organic andaqueous phase. The half-life of CLEAs-LP@3DOM-SiO2in70oC isooctane was60h.When CLEAs-LP@3DOM-SiO2was applied in the hydrolysis, esterification andtransesterification reactions, improved activity and reusability were achieved.Secondly, preparation of lipase Candida sp.99-125CLEAs in mesoporous silica(CLL@MPS): characterization and catalytic properties. Mesoporous silica materials (MPS)were synthesized with a diameter of300-1000nm and the pore size was5.43nm. Thepreparing conditions were that: enzyme concentration was12mg/mL, adsorption time was30min, GA concentration was0.5%and crosslinking time was60min. The stability ofADL@MPS and CLL@MPS was investigated. Compared with ADL@MPS and nativelipase, CLL@MPS showed outstanding stability under vigorous shaking condition and thethermal stability of in the presence of organic solvents was also improved. Additionally,CLL@MPS exhibited high catalytic performance in the hydrolysis, esterification, and transesterification reactions with increased stability and recyclability.Thirdly, CLL@MPS was used for the transesterification of jatropha oil and dimethylcarbonate. After optimizing the reaction conditions, when reaction temperature was50oCand the amount of DMC was16mL/g oil, the highest yield of biodiesel could reach81.6%.CLL@MPS showed good stability in reuse experiment. In the optimal condition,CLL@MPS was used to catalyze the transesterificstion of other oils with DMC andcorresponding yields would be obtained.Lastly, based on lipase immobilization, lipase Candida sp.99-125was coprecipitatedwith Cu3(PO4)2to form immobilized lipase with special structure-hybrid nanoflower andthe catalytic properties were studied. By changing enzyme concentration, hybridnanoflowers with different structure were obtained. When enzyme concentration was0.5mg/mL, the protein loading amount of hybrid nanoflower was57mg/g hybridnanoflower. Compared with native lipase, the thermal and pH stability of hybridnanoflower was improved and hybrid nanoflower exhibited excellent storage stability for itcould maintain72%of initial activity. Through the dynamics analysis, for hybridnanoflower, Kmis3.64mM, Eais7.21KJ/mol, Edis4.73KJ/mol; For native lipase, Kmis2.57mM, Eais7.45KJ/mol, Edis16.19KJ/mol. The yield of biodiesel could reach to67%after7times reuse, so this method has certain research value.