The preparation and characterization of an immobilized l-glutamic decarboxylase and its application for determination of l-glutamic acid

This paper is to study the preparation and characterization of an immobilized l-glutamic decarboxylase (GDC) and develop a sensitive method for the determination of l-glutamate using a new biosensor, which consists of an enzyme column reactor of GDC immobilized on a novel ion exchange resin (carboxymethyl-copolymer of allyl dextran and N.N′-methylene-bisacrylamide CM-CADB) and ion analyzer coupled with a CO2 electrode. The conditions for the enzyme immobilization were optimized by the parameters: buffer composition and concentration, adsorption equilibration time, amount of enzyme, temperature, ionic strength and pH. The dynamic response of Na2HPO4-citric acid buffer system selected is much better than that of the others, 0.10 M HAc-0.10 M NaAc and 0.10 M sodium citrate-0.10 M citric acid. The initial rate of the enzyme reaction v0 in this buffer system is 1.76 mol · l−1 min−1, moreover, the rate of the enzyme reaction appears linear in the first 4 min. The optimum adsorption equilibrium time is around 6 h. The amount of enzyme adsorbed on CM-CADB resin affects the response to substrate l-glutamic acid, the widest range of linearity is obtained with over 30 mg (GDC)/gresin. The GDC activity immobilized on CM-CADB reaches a maximum when the immobilization temperature was kept around 40°C. pH was kept at 4.4 when measuring the activity of the immobilized GDC. No variation of the activity of immobilized GDC is observed when the capacity is over 2.5 meq/g.CM-CADB resin. The properties of the immobilized enzyme on CM-CADB were characterized. No significant improvement can be achieved when the substrate concentration exceeds 12.00 mmol/l, where the activity of immobilized GDC is equal to 1.58 mmol/l.min.g. The optimum pH is found to be 5.2, which changes 0.2 unit, comparing with that of the free GDC (5.0). The optimum temperature is found to be around 48°C, which is lower than that of free GDC (55°C). The critical temperature of the free GDC and the immobilized GDC is approximately 50°C and 45°C, respectively. The half-life of the activity is 127 days when the immobilized enzyme was stored in the cold (4°C). An immobilized GDC enzyme column reactor matched with a flow injection system-ion analyzer coupled with CO2 electrode-data collection system made up the original form of the apparatus of biosensor for determining of l-glutamic acid. The determination conditions are that the buffer solution is 0.10 M Na2HPO4-0.05 M citric acid at pH 4.4 and t = 37°C. The limit of detection is 1.0 × 10−5 M. The linearity response is in the range of 5 × 10 −2 − 5 × 10 −5 M . The equation of linear regression of the calibration curve is y = 43.3x + 181.6 (y is the milli-volt of electrical potential response, x is the logarithm of the concentration of the substrate of l-glutamic acid). The correlation coefficient equals 0.99. The coefficient of variation equals 2.7%.