Anal. Chem. 1990, 62, 263-268 (37) Martlnek, K.; Kllbanov, A. M.; Goldmacher, V. S.; Berezln, I. V. Blochlm. Nophys. Acta 1977, 485, 1-12. (38) Martlnek. K.; Kllbanov, A. M.; oddmachef, V. S.; Tchemysheva, A. V.; Mozhaev, V. V.; Berezln, 1. V.; Glotov, B. 0. Blochlm. Blophys. Acta 1977, 485, 13-28. (39) Torchllln, V. P.; Maksimenko, A. V.; Smirnov, V. N.; Berezin, I. V.; Klibanov, A. M.; Martinek, K. Blochim. BlOPhYS. Acta 1978, 522, 277-283. (40) Schnaar, R. L.; Lee, Y. C . B/ochem/stry 1975, 14, 1535-1541. (41) Foulds, N. C.; Lowe, C. R. Anal. Chem. 1988, 60, 2473-2478. (42) Hale, P. D.; Inagakl, T.; Karan, H. I.; Okamoto, Y.; Skothelm, T. A. J . Am. Chem. Soc . 1989, 11 1 , 3482-3484. (43) Bowden, E. F.; Hawkridge, F. M.; Blount, H. N. In Comprehens/ve
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Treatise of Ektrochedstry; Srlnlvasan, S., et el., Eds.; Plenum: New York, 1985; Vol. 10. (44) Murray, R. W. Electroanel. Chem. 1984, 13, 191-388. (45) Castner, J. F.; Wingard, L. B.,Jr. Blochedstry 1984, 23, 2203-2210.
RECEIVED for review June 19,1989. Accepted November 7, 1989. We gratefully acknowledge the financial support for this research from the Office of Naval Research, Contract No. N0014-88-K-0401, the Texas Advanced Research Program, and the Robert A. Welch Foundation.
Flow System for Starch Determination Based on Consecutive Enzyme Steps and Amperometric Detection at a Chemically Modified Electrode J. Emn6us and L. Gorton* Department of Analytical Chemistry, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden
A flow InJectionsystem is described for the determination of the total glucoae content of starch. The starch is sequentially hydrolyzed on-line In two immobilized enzyme reactors for complete degradation to glucose. The first reactor contains a thermostable a-amylase, Termamyi, and is operated at 60 OC. The second reactor contains amyiogiucosidase and is operated at room temperature. The detection is based on the electrochemical oxidation of the hydrogen peroxide formed afler the complete oxidation of the produced glucose in a tMrd reactor contalnlng coimmobiiized mutarotase and glucose oxidase. Rectilinear calibration curves for starch (expressed as glucose) were obtained between 19 pM and 0.6 mM, using an injection volume of 160 pL. the sample throughput was 15 h-'.
INTRODUCTION The determination of the glucose content of starch is a tedious process when performed in the traditional batchwise mode. The various steps are time-consuming, involving the addition of hydrolyzing chemicals (including soluble enzymes), pH regulation, and incubation, followed by the actual glucose analysis. Several attempts have been made to decrease the time necessary for this analysis. Various starch hydrolyzing enzymes, mainly amyloglucosidase but also a-amylase, have been immobilized in reactors and used in flow injection systems, FIA (1-4). In the systems, using only immobilized amyloglucosidase, a range of 90% to complete hydrolysis of soluble starch to glucose has been reported. In the present paper, an extention of previously published systems aimed at a FIA system for on-line, complete hydrolysis and analysis of native starch is reported. A thermostable a-amylase, Termamyl, is contained in one reactor, operated at 60 "C, in which the starch is partially hydrolyzed to a-limit dextrin and oligosaccharide fractions. These are in tum totally hydrolyzed to glucose in a second reactor containing immobilized amyloglucosidase, operating at room temperature. The glucose is then oxidized in a third reactor with coimmobilized glucose oxidase and mutarotase. The hydrogen peroxide
formed is electrocatalytically oxidized at a Pd/Au-modified graphite electrode (5, 6).
EXPERIMENTAL SECTION Enzymes and Enzyme Reactors. One milliliter of a suspension of a thermostable a-amylase, EC 3.2.1.1,from Bacillus licheniformis,Termamyl12OL, Novo Industri A/S, Bagsvaerd, Denmark (7),was added to 1 mL of a 0.1 M citrate buffer at pH 6.7. This solution was transferred to dialysis tubing and dialyzed for 48 h against the same buffer to remove the dark brown compounds present. The activity of the Termamyl suspension is expressed as 120 KNU g-' of solution; 1 KNU (kilo Novo unit) is expressed as the amount of enzyme which breaks down 5.26 g of starch (Merck, Amylum Solubile Erg. B. 6,Batch 9947275) per hour (7). The density of the suspension was found to be 1.22 g mL-', which means that 1 mC should contain 146.4 KNUs. After 48 h the volume in the dialysis tubing containing the enzyme had increased to 4 mL and the dark brown color had virtually disappeared. This solution was immobilized on 330 mg of glutaraldehyde-activated, aminopropyl silanized, controlled pore glass, CPG, Serva catalog no. 44811,particle size 75-125 pm, pore size 2869 A, according to a previously published procedure (8,9). The reaction was allowed to continue for 12 h at reduced pressure and at 4 "C. The immobilization efficiency was 80% expressed as the ratio between the absorbance at 280 nm of the clear enzyme solution after and before immobilization. The enzyme glass was packed into a reactor with a 2.7 mm i.d. and a total length of 8.7 cm. Throughout the experiments a volume of 500 pL was used. When not in use the reactor was filled with 0.1 M acetate buffer at pH 5.0 and kept at 4 "C. A 375-mg portion of amyloglucosidase,AMG, EC 3.2.1.3,from Aspergillus niger, 100 U mg-', Merck catalog no. 1332,was dissolved in 2.0 mL of 0.1 M acetate buffer at pH 5.0, transferred to dialysis tubing, and dialyzed against the same buffer overnight. After dialysis the pH of the solution containing the enzyme, 3.9 mL, was changed to 7.0 and was immobilized on 500 mg of glutaraldehyde-activated, aminopropyl silanized CPG, Serva catalog no. 44770,particle size 125-180 pm, pore size 729 A, as above. The immobilization efficiency, expressed as above, was 99%. The enzyme glass was packed into a reactor with 2.7 mm i.d. and a total length of 13 cm. Throughout the experiments a fied volume of 740 pL was chosen. When not in use it was stored at 4 "C filled with 0.1 M acetate buffer at pH 5.0. A 9.5-mg portion of glucose oxidase, GOx, EC 1.1.3.4,from Aspergillus niger, SERVA catalog no. 22738,270U mg-', and 200
0003-2700/90/0362-0263$02.50/00 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 3, FEBRUARY 1, 1990
carrier 0 5 ml min-'
-------------
I
-0
0 1 M acetate buffer
-
I
TERM.
I
thermostat
I
AMG
GOX 1MUT
PH 7 0
1
make up 02 M
-0
0 5 ml min-I phosphate buffer
pL of a solution of mutarotase, MUT, EC 5.1.3.3, from porcine kidney dissolved in 3.2 M ammonium sulfate, Sigma catalog no. M-4007, were added to 1.4 mL of a 0.1 M phosphate buffer at pH 7.0. This solution was dialyzed overnight against a large surplus of the same phosphate buffer in order to remove the ammonium ions that would otherwise interfere in the immobilization process. The purified enzyme solution, 3.0 mL, was added for coupling with 150 mg of CPG 10, Serva catalog no. 44752, particle size 37-74 pm, pore size 379 A, that had previously been aminopropyl silanized and glutaraldehydeactivated as above. The immobilization efficiency was essentially total (
