Lipase Immobilization On Three-dimensionally Ordered Macro/mesoporous Materials And Research Of Their Catalytic Performance

Hierarchically ordered macroporous/mesoporous silica materials,which have advantages of good mass transfer,large specific surface area,are novel candidates as supports for enzyme immobilization.In this paper,hierarchically ordered macroporous/mesoporous silica and organosilica materials were used as supports,and physical adsorption and crosslinking methods were employed to fabricate immobilized lipases.The details were summarized as follows:?1?Preparation of hierarchically ordered macroporous/mesoporous silica?3DOM/m-S?material and its catalytic performance.3DOM/m-S was prepared through the dual templating method with polystyrene?PS?colloidal crystals as the hard template and amphiphilic triblock copolymers?P123?as the soft template.As shown in SEM and TEM images,the achieved 3DOM/m-S possessed ordered macropores of 450 nm and ordered mesopores.N₂ adsorption and desorption test results showed the pore size was 5.5 nm,and the specific surface area and pore volume were 295.17 m²/g and 0.405 cm³/g,respectively.The ordered arrangement of mesopores was examined by SAXRD pattern.Lipase B from C.antarctica?CALB?was employed to be immobilized on 3DOM/m-S?CALB@3DOM/m-S?.The successful immobilization of CALB was verified by FTIR and CLSM.The thermal stability and tolerance to organic solvents of CALB@3DOM/m-S were investigated.After immersed in isooctane and acetone at 80 ^oC for 3 h,CALB@3DOM/m-S retained 69%and60%of its initial activity,respectively.After immersed in isooctane and acetone at 40 ^oC for288 h,CALB@3DOM/m-S retained 144%and 74%of its initial activity,respectively.Also,CALB@3DOM/m-S could be used for esterification reactions between acids and alcohols with different chain lengths,and 90%of conversion rate could be reached.In examining the reusability in esterification of oleic acid and ethanol,the conversion rate of oleic acid can retain 80%with the initial conversion rate 93%after 12 reaction cycles,indicating a remarkable reusability of the immobilized lipase.?2?Cross-linked lipases in hierarchically ordered macroporous/mesoporous silica?CLL@3DOM/m-S?material and its catalytic performance.Lipases were immobilized in the mesopores of 3DOM/m-S via cross linking method.Glutaraldehydes?GA?were prior to lipases adsorbed in the mesopores and this strategy made CLL@3DOM/m-S more active.Thermal stabilities in organic solvents and PBS of CLL@3DOM/m-S were investigated,and obviously improved stabilities were performed compared with CALB@3DOM/m-S.When immersed in PBS at 70 ^oC for 0.5 h,CLL@3DOM/m-S still retained 70%of the initial activity.After immersed in isooctane and acetone at 80 ^oC for 3 h,CLL@3DOM/m-S could retain 100%and 70%of the initial activity,respectively.Tolerance to organic solvents of CLL@3DOM/m-S was investigated.After immersed in isooctane and acetone at 40 ^oC for 288 h,CLL@3DOM/m-S retained 140%and 110%of its initial activity,respectively.Reusability of CLL@3DOM/m-S uesd for esterification of oleic acid and ethonal was been examined.And after CLL@3DOM/m-S was reused for 14 times,the conversion rate of oleic acid was still 90%with the initial conversion rate 97%.Compared with CALB@3DOM/m-S,CLL@3DOM/m-S showed improved reusability.?3?Preparation of hierarchically ordered macroporous/mesoporous organosilica?3DOM/m-OS?material and its catalytic performance.3DOM/m-OS was firstly prepared through the dual templating method.As shown in SEM and TEM images,the achieved3DOM/m-OS possessed ordered macropores of 400 nm and ordered mesopores.N₂adsorption and desorption test results showed the mesopore size was 5.0 nm,and the specific surface area and pore volume were 344.41 m²/g and 0.475 cm³/g,respectively.The ordered arrangement of mesopores was examined by SAXRD pattern.The difference between 3DOM/m-S and 3DOM/m-OS was verified by FTIR.Lipases were immobilized in3DOM/m-OS via physicaladsorptionandcrosslinkingmethod.Thus,CALB@3DOM/m-OS and CLL@3DOM/m-OS were obtained.Thermal stabilities and tolerance to organic solvents of the two forms of immobilized enzyme were investigated.After immersed in isooctane and acetone at 80 ^oC for 3 h,CALB@3DOM/m-OS retained75%and 65%of its initial activity,respectively,while CLL@3DOM/m-OS retained 85%and 75%of its initial activity,respectively.After immersed in isooctane and acetone at 40^oC for 288 h,CALB@3DOM/m-OS retained 110%and 60%of its initial activity,respectively,while CLL@3DOM/m-OS retained 120%and 80%of its initial activity,respectively.Reusabilities of CALB@3DOM/m-OS and CLL@3DOM/m-OS uesd for esterification of oleic acid and ethanol were also investigated,and the initial conversion rate was 97%for both lipase forms.The conversion rates of oleic acid could retain 90%and70%for CALB@3DOM/m-OS and CLL@3DOM/m-OS,respectively.All the results indicated that 3DOM/m-OS had great potential to be applied in enzyme immobilization field.