| β-Glucosidase, existed widely in various types of living organisms, can hydrolyze various glycosides, and multiple potential applications have been developed in cellulose degradation, isoflavone glycoside hydrolysis, and enhancement of aroma in tea and wine. However, the free P-glucosidase has some limitations for industrial applications, such as unreusability, and poor stability. Enzyme immobilization technology can overcome these drawbacks and therefore is a good choice for wider applications. Enzyme immobilization is considered to be the promising method to overcome these drawback. In precent study, the immobilized P-glucosidase was investigated, and the effects of the immobilization carrier and methods on the activity of β-glucosidase was studied. The response surface method was used to optimize the preparation conditions of immobilized enzyme, and the enzymatic properties of immobilized P-glucosidase prepared under optimum process was investigated. In addition, we focused on the analyses of immobilization mechanism, which based on various characterization results reveal the relationship between the carrier structure and enzyme immobilization efficiency. Further more, we designed the boiling-bed reactor for the continuous bioconversion of isoflavones and evaluated its bioconversion efficiency. These researches would lay the theoretical foundation for the industrial application of immobilized β-glucosidase.The major results are as follow:1. β-Glucosidase was immobilized into chitosan beads. A factorial design was employed for screening significant factors, and then response surface methodology was applied to obtain the maximum activity recovery by central composite design. In precent study, crosslinking-adsorption-crosslinking method was chosen to immobilize β-glucosidase and the activity recovery reached 32.55%. To further improve the activity recovery of immobilized β-glucosidase, response surface methodology was used to optimize the conditions for the maximum activity and understand the significance and interaction of the factors affecting the specific activity of immobilized P-glucosidase. Enzyme concentration and adsorption time were found to be significant influence factors, and the maximum activity recovery (50.75%) was obtained at the optimum levels of enzyme concentration (67.15 μg/mL) and adsorption time (5.54 h), which is in excellent agreement with experimental value (50.81%). Furthermore, Various characteristics of immobilized P-glucosidase such as the pH-activity curve, the temperature-activity curve, thermal stability, storage stability and reusability were evaluated. Compared to the free β-glucosidase, the immobilized enzyme exhibited broader pH and temperature ranges, enhanced thermal stability, better storage stability and reusability. The decrease in Km after immobilization revealed higher accessibility of the substrate to the immobilized P-glucosidase. In summary, the activity recovery and enzymatic properties of immobilized β-glucosidase were improved in compared with free P-glucosidase.2. This study presented a novel and efficient P-glucosidase immobilization method using magnetic Fe3O4 nanoparticles as a carrier. Based on response surface methodology, the optimal immobilization conditions obtained were:glutaraldehyde concentration, 0.20%; enzyme concentration,50.25 μg/mL; cross-linking time,2.21 h; and the maximum activity recovery reached 89.35%. The properties of the immobilized P-glucosidase were improved, and the immobilized P-glucosidase exhibited wider pH and temperature ranges, higher accessibility of the substrate, better thermal stability, and better storage stability than that of the free enzyme. The enzyme-magnetic nanoparticles could be separated magnetically for easy reuse. Immobilization of β-glucosidase onto the magnetic nanoparticles has the potential for industrial application. Secondary immobilized magnetic nanoparticles P-glucosidase on chitosan possess high activity recovery (88.42%).3. This is the first research about immobilization of P-glucosidase on MCM-41 and SBA-15-1000. The different immobilized carrier and method of immobilization of P-glucosidase was studied, and the results showed that the activity recovery of immobilized enzyme was affected by the spatial structure of mesoporous molecular sieves, In addition, MCM-41 was found to be the better one in two carriers, and the adsorption-crosslinking method was found to be the best one in two different immobilizing methods, indicating that the difference of molecular sieve pore structure caused differences in the efficiency of the immobilized P-glucosidase. Moreover, RSM was employed to obtain the optimal levels of enzyme concentration (41.82 μg/ml), GA concentration (0.63%) and adsorption-crosslinking time (2.55 h), and the predicted maximum activity recovery (134.08%) is in excellent agreement with the experimental value (133.72%), which is 45.76% higher than that before optimization (91.74%).4. Various properties for free and immobilized P-glucosidase on MCM-41 by the optimum coditions such as the pH-activity curve, the temperature-activity curve, thermal stability, storage stability and reusability were evaluated. The free (3-glucosidase and immobilized enzyme showed the same optimum pH and temperature, however, the immobilized β-glucosidase shows broader pH ranges and stronger temperature adaptability than the free form. The immobilized β-glucosidase has a more stable performance in reuse. The immobilized β-glucosidase shows improved enzymatic properties than the free form, such as enhanced thermal stability, higher accessibility of the substrate and better storage stability. The activity recovery of secondary immobilized MCM-41 P-glucosidase on chitosan was basically unchanged. Due to the suitable micro-environment provided by the pore structure of MCM-41, the immobilized β-glucosidase shows improved stability and usability.5. The magnetic immobilized enzyme was characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscope (TEM), and vibrating sample magnetometer (VSM). FTIR revealed that β-glucosidase was successfully immobilized on the magnetic nanoparticles. TEM showed that enzyme-magnetic nanoparticles possessed nano-scale size distribution. VSM confirmed that the enzyme-magnetic nanoparticles were superparamagnetic. In addition, the immobilized β-glucosidase on MCM-41 was characterized by FT-IR, scanning electron microscope (SEM), X-Ray Diffraction (XRD), and so on. Both the FT-IR and XRD analysis proved the success of the β-glucosidase adsorbed on the surface of MCM-41, and the basic structure of MCM-41 after immobilization was not changed. SEM showed that both MCM-41 and the immobilized β-glucosidase presented similar morphology, and the immobilized β-glucosidase showed better dispersibility, which would contribute to the catalytic reaction. The results of N2 adsorption experiment revealed that MCM-41 and the immobilized β-glucosidase presented a narrow pore size distribution of the mesoporous structure. The analysis of specific surface area and pore size distribution showed that the pore diameter, the pore volume and the specific surface area of MCM-41 decreased after immobilization, confirming the existence of β-glucosidase in MCM-41. Elemental analysis further confirmed that β-glucosidase was successfully immobilized in MCM-41.7. The different immobilized β-glucosidase were utilized to the intermittent bioconversion of isoflavones, and the bioconversion rate of daidzin and the yield of daidzein were tested by high performance liquid chromatography. The results showed that the bioconversion rate of daidzin in both magnetic and MCM-41 immobilized enzyme as same as the free β-glucosidase, were over 93%, while the chitosan immobilized enzyme were 71%, and secondary immobilized β-glucosidase showed more than 85%. A boiling-bed reactor was designed and made in the lab, the beads of immobilized β-glucosidase were promoted to stream by nitrogen. The secondary magnetic immobilized enzyme was used to the continuous biotransformation of isoflavones, and the bioconversion rate of daidzin remained above 80% after 10 times biotransformation, indicating that the immobilized P-glucosidase possessed good stability, and was suitable for applications. |
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