Studies On The Preparing Of Novel Composite Support PEI/SiO₂ And Immobilization Of Penicillin Acylase On It

Polyethyleneimine (PEI) was coupled chemically to surface of silica gel particlesthrough the intermedium effect of γ-chlopropyltrimethoxysilane, the novel compositecarrier of PEI/SiO₂ was prepared, and finally immobilized penicillin acylase with higheractivity and stability was obtained. The chemical composition and structure of the surface ofcomposite carrier were characterized by using FITR and conductmetric titration method. Theinfluences of various factors on the preparing of the carrier were examined via measuringcoupled amount of PEI on the composite carrier. The favourable reaction conditions are: thereaction temperature of 90℃⁹4℃, the reaction time of 5h and the concentration of45g/100mL ⁵0g/100mL for PEI solution, the ratio of weight of PEI solution to CP-SIO₂ is5:1. When the penicillin acylase were immobilized on the composite carrier of PEI/SiO₂, theeffects of various immobilizing on the apparent activity and recovery ratio of activity ofimmobilized penicillin acylase were examined, such as the amount of glutaraldehyde,pH,time,temperature and the used amount of enzyme. The appropriate immobilization conditionsfor 0.5g of the composite support PEI/SiO₂ to which 9% of PEI was linked are as follows: 1%of glutaraldehyde, 7.92 of pH, 30℃ of temperature, 14～15h of time and 0.05mL of originalenzyme solution. Under these conditions, the obtained immobilized penicillin acylase possess118U/g of activity and 46% of recovery ratio of activity. we explored the immobilizationmechanism of the composite carrier for penicillin acylase with the zeta electric potentials ofthe carrier before enzyme were determined and the characters of immobilization process. Theexperiment results showed: Owing to a large quantity of amido contained on themacromolecular chains of PEI, there is not only chemical linkage(schiff reaction of amido),but also is a great deal of physical adsorption(electrostatic adsorption between electriferousamido and enzyme molecular)during enzyme immobilization on the surface of PEI/SiO₂. Thecombination of a spot of chemical covalent and a great deal of physical adsorption results infast and stable immobilization and excellent maintenance of the molecular conformation ofenzyme, and leads to high activity of the immobilized enzyme. For examining theproperties of immobilized penicillin acylase, suitable pH and temperature of free enzymeand immobilized enzyme catalysis reaction was examined, continued operation stability andstability in metal ionic solutionan was studied, at the same time, michaelis-menten constant offree enzyme and immobilized enzyme were also measured. The experiment results showed:the optimum pH of enzyme catalysis reaction were 9.32 and 7.34, the optimum temperaturewere 50 ℃ and 40 ℃ for free enzyme and immobilized enzyme, respectively. Theimmobilized enzyme is more stable than free enzyme to the metal ionic solutionan andmichaelis-menten constant of immobilized enzyme and free enzyme were 2.38×10-1mol/Land 5.44×10-2mol/L,respectively.