Immobilization Of β-d-glucuronidase On Pilc And Biosynthesis Of Gamg

Pillared Interlayered Clay (PILC), a porous solid material, has been widely applied in catalysis due to its large specific surface area, characteristics of pore size and acid controlled. The main material preparation for PILC is montmorillonite which have higher content in bentonite. The bentonite of Xiazijie of Xinjiang province is one of the most abundant in the world, and the predicted resource measured is about 5 billion ton. But the composition of bentonite is far from in the difference producting districts. So the study attempts to use the bentonite of Xiazijie of Xinjiang province as a material for preparation of PILC. Absorption method was used to immobilizeβ-D-glucuronidase using PILC as support. The effect of different PILC prepared using different bentonite on the activity of the immobilizedβ-D-glucuronidase was investigated, and the catalytic character of the optimization immobilizedβ-D-glucuronidase for transforming glycyrrhizin to Glycyrrhetinic acid monoglucuronide (GAMG) was studied. The main conclusions as follows:1. The character of support was determined by X-ray diffraction (XRD), N₂-adsorption– desorption, Fourier transform infrared spectroscopy (FTIR) and Scanning electron microscopy (SEM). Compared with purification bentonite, the surface area of PILC was enhanced from 57.99 m²/g to 177.0 m²/g, and BJH desorption pore volume was increased from 0.059mL/g to 0.25 mL/g. The results indicated that PILC was a promising support for enzyme immobilization, and enzyme could be attached to the support by hydrogen bonds.2. The effect of some factors on the activity of the immobilizedβ-D-glucuronidase were investigated. The results showed that the optimum conditions for immobilization were as follows:β-D-glucuronidase dosage was 2700 U/g carrier, immobilizing for 60 min at 40℃under pH 3.6. The maximum activity of immobilizedβ-D-glucuronidase was 1790 U/g carrier under these conditions, and had high immobilization rate.3. The catalytic character and dynamics of immobilized enzyme were investigated using glycyrrhizin as substrate. The results displayed that the optimum catalytic reaction pH value and temperature were 6.0 and 60℃respectively, and K_m was 1.35 mmol·L^-1. The best enzyme activity of 20 U/mL for immobilized enzyme was obtained when the concentration of glycyrrhizin was 2 g/L. Compared with the free enzyme, the optimum catalytic pH of immobilized enzyme was shifted to alkaline, the optimum catalytic temperature was increased 10℃, but the affinity of enzyme to the substrate had decreased. The catalytic specificity of immobilized enzyme has not been changed, and its storage stability was greatly improved, at the same time, it has a recycling performance to some extent. In this study, theβ-D-glucuronidase was immobilized by PILC as support prepared with the bentonite of Xiazijie of Xinjiang province, and the catalytic character of immobilized enzyme was investigated. This study will provide an important application basis for the deep processing of bentonite in Xinjiang.