The preparation and characterization of an immobilized L-glutamic decarboxylase and its application for determination of L-glutamic acida

This paper is to study the preparation and characterization of an immobiliz ed L-glutamic decarboxylase (GDC) and develop a sensitive method for the de termination of L-glutamate using a new biosensor, which consists of an enzy me column reactor of GDC immobilized on a novel ion exchange resin (carboxy methyl-copolymer of allyl dextran and N,N'-methylene-bisacrylamide CM-CADB) and ion analyzer coupled with a CO2 electrode. The conditions for the enzy me 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 hi NaAc and 0.10 M sodium citrate-0.10 M citric acid. The initial rate of the enzyme reaction v(0) in this buffer system is 1.76 mol . l(-1) min(-1), mo reover, 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 aci d, the widest range of linearity is obtained with over 30 mg (GDC)/g(resin) . The GDC activity immobilized on CM-CADB reaches a maximum when the immobi lization temperature was kept around 40 degrees 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-CAD B resin). The properties of the immobilized enzyme on CM-CADB were characte rized. No significant improvement can be achieved when the substrate concen tration exceeds 12.00 mmol/l, where the activity of immobilized GDC is equa l 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 degrees C, which is lower than that of free GDC (5 5 degrees C). The critical temperature of the free GDC and the immobilized GDC is approximately 50 degrees C and 45 degrees C, respectively. The half- life of the activity is 127 days when the immobilized enzyme was stored in the cold (4 degrees C). An immobilized GDC enzyme column reactor matched wi th a flow injection system-ion analyzer coupled with CO2 electrode-data col lection system made up the original form of the apparatus of biosensor for determining of L-glutamic acid. The determination conditions are that the b uffer solution is 0.10 M Na2HPO4-0.05 M citric acid at pH 4.4 and t = 37 de grees C. The limit of detection is 1.0 x 10(-5) M. The linearity response i s in the range of 5 x 10(-2) - 5 x 10(-5) M. The equation of linear regress ion of the calibration curve is y = 43.3x + 181.6 (y is the milli-volt of e lectrical potential response, x is the logarithm of the concentration of th e substrate of L-glutamic acid). The correlation coefficient equals 0.99. T he coefficient of variation equals 2.7%. (C) 2000 Elsevier Science Inc. All rights reserved.