The Research On The Immobilization And Application Of β-Glucosidase

There were three carriers, porous ceramic balls, mesoporous TiO₂(M-TiO₂),and mercapto-modified mesoporous TiO₂(SH-M-TiO₂), which were studied on theimmobilization of the enzyme β-glucosidase (BG). In additional, the hydrolysisprocess and model of packed-bed reactor, which was loaded with β-glucosidaseimmobilized onto the porous ceramic balls, were investigated. The main conclusionsare as follows:1. The β-glucosidase was immobilized onto the porous ceramic ball of type I-a bythe crossing-linking and enveloping operation. The maximum apparent activity ofimmobilized β-glucosidase was about1.75U/g. It was known that two operationsplayed key roles to the activity of the immobilized enzyme. One was crossing-linkedwith the glutaraldehyde, the other was enveloped with a goil-based adhesive. Theimmobilized β-glucosidase showed better stabilities on pH, thermal and storage thanthe free β-glucosidase. After reacted for30cycles on β-glucosidase assay, theretained activity of the immobilized β-glucosidase was over80%. In the batchhydrolysis experiment, the conversion was over90%in the former three batches andthen declined gradually. The conversion was only50%for the ninth batch. Theretained activity of the immobilized β-glucosidase was68.5%after10batches ofreaction.2. A single packed-bed reactor was constructed for the hydrolysis of cellubiose ina continuous mode, while the reactor was loaded with the immobilized β-glucosidaseonto the porous ceramic ball of type I-a. The optimum operation pH and temperatureof the reactor were4.8and60℃, respectively. As the flow rate was low, theconversion was high but the production efficiency on volume was low. For example,as the flow rate was3.53ml/min, the conversion attained97.5%, but the productionefficiency on volume was only6.29g/(l·h). As the flow rate raised up to11.2ml/min,the conversion was only82.1%, but the production efficiency on volume attained15.62g/(l·h). Furthermore, as the flow rate was stable at5.47ml/min, the conversionmaintained over90%with an average value of90.7%. In addition, a model ofone-dimensional steady-state axial dispersion was set up to describe the performanceof the single packed-bed reactor. The fitting factor R of the model was0.95. Theaverage deviation was9.15%between the calculated results and the experimentalresults.3. When the two packed-bed reactors were linked in a serial mode and the flow rate was7.67ml/min, the conversion was91.4%, and the production efficiency onvolume was12.69g/(l·h). Compared with the data on single packed-bed reactor,which were the conversion of91.7%and the production efficiency on volume of8.98g/(l·h),the production efficiency on volume of the double serial reactors increased by41%.4. When β-glucosidase was immobilized onto M-TiO₂(BG-M-TiO₂) with theabsorption action, the maximum apparent activity recovery of BG-M-TiO₂was64.14%, meanwhile the apparent activity was4.68U/g. As the initial enzymeconcentration was0.907U/ml, the apparent activity of BG-M-TiO₂was closed to themaximum value of7.6U/g. After the immobilization of BG, M-TiO₂got less on theabsorption volume to N2, the specific surface area (SBET) of M-TiO₂reduced from62.04m²/g to47.72m²/g, and the pore volume (Vp) of M-TiO₂reduced from0.37cm³/g to0.29cm³/g. After reacted for6cycles on BG assay, the retained activity ofBG-M-TiO₂was below50%.5. NH2-M-TiO₂and SH-M-TiO₂were obtained after the M-TiO₂were respectivelymodified by3-aminopropyltriethoxysilane and3-mercaptopropyltrimethoxysilane.BG-SH-M-TiO₂showed better results than BG-NH2-M-TiO₂on the enzyme activity,activity recovery and operation stability. After M-TiO₂was functionalized with3-mercaptopropyltrimethoxysilane, SBETreduced from62.04m²/g to53.50m²/g,pore diameter (DBJH) changed from20.3nm to18.2nm, and Vpreduced from0.37cm³/g to0.31cm³/g. The elements Si and S were present on the energy dispersivespectrum of SH-M-TiO₂, whose mass fractions were1.78%and1.81%, respectively.Therefore the total mass fraction of mercapto group was about3.6%. Furthermore,there was a peak for the mercapto group loss at355℃on the thermogravimetryfigure of SH-M-TiO₂.6. The maximum apparent activity recovery of β-glucosidase immobilized ontoSH-M-TiO₂(BG-SH-M-TiO₂) was92.84%, while the apparent activity was10.99U/g.As the initial enzyme concentration was about18U/ml, the apparent activity of BG-SH-M-TiO₂was closed to the maximum value of21.3U/g. After reacted for30cycles of BG assay, the BG-SH-M-TiO₂remained the activity of about50%. In thebatch hydrolysis experiment, the conversions were stable over90%in the former8batches. The retained activity of BG-SH-M-TiO₂was80.5%after10batches ofreaction.