Directed Control Of β-glucuronidase Selectivity In Biocatalysis

Enzymatic reaction has been showing an irreplaceable role in many fields, it can regulate the enzymatic reaction, especially the selective regulation is becoming more and more attention. This paper takes the β-glucuronidases catalytic glycyrrhizic acid to generate single glucuronic acid base glycyrrhetinic acid(GAMG) and glycyrrhetinic acid(GA) as the model reaction, discussing directed control method of PGUS-E, The main results were as follows:(1) PGUS-E is chemically modified by different modification methods, such as macromolecular modifiers and small molecule modification. Study the effects of different modification methods for catalytic selectivity β- glucuronidase enzyme’s selectivity. we found out that after modification of DTNB, the enzyme has the highest selectivity for GAMG which is 65%, and by optimizing the concentration and reaction time of the DTNB during the process, finally we get the result that enzyme PGUS-E-D which is modified by 20 mmol / L DTNB solution modified for 20 minutes has the highest rate of 74%.(2) PGUS-E-D is immobilized by Fe magnetic nanoparticles. we found out that 300μL enzyme solution was immobilized 3h by 10 mg Fe magnetic nanoparticles at pH 4.0, 20 ℃ condition, the immobilized enzyme activity is 212 U / g, which is 6.19% lower compared to the control. We name the immobilized enzyme is PGUS-E-DFe. The Selection Ratio of GAMG is 58%, which is 41% higher than the unmodified PGUS-E and 21.62% lower than PGUS-ED.(3) With research of effects on β-glucuronidases’ activity and selectivity by using organic phase system and two- phase system, we found out that β-glucuronidases’ activity is very low in organic phase system, only about 25% of the control.In two- phase system, when water content is 30% β-glucuronidases’ activity is 60% lower than control; when water content is 50% β-glucuronidases’ activity is 50% lower than control; when water content is 70% β-glucuronidases’ activity loss is less compared with the control. And when the organic phase is ethyl acetate, the optimum temperature is 35℃, the optimum pH is 5.5 and the highest selectivity of GAMG is 56%.