Immobilization Of Lipase On Ceramic Membrane Surface And Its Stability Study

Lipase is widely used in biocatalytic industry because of its green and environmental protection,mild reaction conditions,etc.However,free lipase has problems such as difficult recovery,enzyme activity is easily affected by external environment and poor stability,etc.Immobilizing the enzyme on a carrier can solve these problems,in which choosing a suitable immobilization method and carrier is the key.Inorganic ceramic membranes have good chemical stability and strong mechanical properties and can be used as lipase immobilization carriers.In addition,the membrane can separate the products and substrates produced by enzyme catalysis in time and promote the positive reaction.Therefore,in this paper,inorganic ceramic ultrafiltration membranes were selected as carriers for lipase immobilization,and immobilized lipase with high heat resistance,acid and alkali resistance,and stability were constructed by physical adsorption and chemical cross-linking.The main research of this topic is as follows:A hydrophobically modified ceramic membrane with methyltrimethoxysilane(MTMS)was used as the carrier base membrane for the immobilization of lipase by physical adsorption,and the lipase was immobilized on the membrane by reverse filtration using a homemade staggered flow filtration device.Finally,the enzyme aggregates were immobilized by cross-linking with glutaraldehyde(GA).Fourier transform infrared spectroscopy(FTIR),X-ray photoelectron spectroscopy(XPS),thermogravimetric analysis(TGA)and X-ray diffraction analysis(XRD)were used to characterize the surface chemical structure,thermal properties and structure of the hydrophobically modified enzyme membrane reactor(GA-EMR)and various composite ceramic membranes,and the results showed the successful modification of silane,the successful immobilization of lipase and the successful cross-linking of GA.And the immobilized lipase has good thermal properties and stable physicochemical structure.The optimum immobilization conditions were determined as follows: MTMS concentration of 0.1 M,modification time of 120 min,immobilization time of 80 min,enzyme solution concentration of 3 mg/m L,cross-linking agent concentration of 2%,and the immobilized lipase membrane had a stable pure water flux.The catalytic conditions of the immobilized lipase were optimized to obtain the optimal catalytic conditions: 45 ℃,p H 7,0.2 MPa of transmembrane pressure and 0.05 M of triglyceride(TA)concentration,Finally,comparing the blank membrane(CM),the enzyme membrane reactor(L-CM,EMR-1)prepared by hydrophobic membrane(MTMS-CM)and the GA cross-linked enzyme membrane reactor(GA-EMR),it was found that the stability of GA-EMR was better than the rest two,and after 7 cycles,GA-EMR still retained more than 60% of the initial enzyme activity,this result proved that The hydrophobic treatment and GA cross-linking were beneficial to improve the reusability of the prepared enzyme membrane reactors.Although the immobilization of lipase by physical adsorption method is easy to operate,because the connection between the enzyme and the carrier only depends on the various interaction forces between them,this method of immobilizing lipase is prone to enzyme loss.To obtain a more stable immobilized lipase,we considered the immobilization of lipase by chemical cross-linking method,using MTMS and(3-aminopropyl)-triethoxysilane(APTES)to The membrane was modified to increase the hydrophobicity of the membrane,while the membrane was aminated to make it with amino groups to provide binding sites for subsequent GA cross-linking,and finally the lipase was immobilized on the membrane surface by reverse filtration through a staggered flow filtration device to obtain the enzyme membrane reactor(EMR-2).The surface chemical structure,thermal properties and physicochemical structure of the prepared enzyme membrane reactor(EMR-2)and various composite ceramic membranes were characterized by FTIR,XPS,TGA and XRD.The optimal conditions for lipase immobilization and catalytic conditions were optimized as follows: APTES/MTMS concentration ratio of 1:1,modification time of 120 min,GA concentration of 0.5%,immobilization time of 100 min,enzyme solution concentration of 5 mg/m L,catalytic temperature of 65 ℃,p H of 7,transmembrane pressure of 0.2MPa,and TA concentration of 0.15 M.Under the Under the optimum conditions,the immobilized lipase activity could reach 18 U/mg;compared with the free lipase,the immobilized lipase had higher thermal stability,and under alkaline environment,the immobilized lipase had higher stability than the free lipase.The reusability of three enzyme membrane reactors,namely,blank membrane(CM),enzyme membrane reactor constructed with APTES/MTMS hybrid modified membrane(APTES/MTMS-CM)(L-CM,L-APTES/MTMS-CM)and enzyme membrane reactor prepared with cross-linker GA(EMR-2),was compared,and the results showed that EMR-2,after 7 cycles,retained More than 65% of the initial enzyme activity remained even after 9 cycles,and about 45% of the initial enzyme activity remained even after 9cycles.Finally,the catalytic kinetics of the immobilized lipase obtained from the two methods were investigated,and the results showed that the immobilized lipase using the physisorption method had higher substrate affinity and greater maximum reaction rate than the immobilized lipase using the chemical cross-linking method,however,chemical cross-linking of the immobilized lipase was finally chosen for stability reasons.