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Anal. Chem. 1990, 62, 2688-2691
(18) Robbat, A., Jr.; Xyrafas, G.; Marshall, D. Anal. Chem. 1888, 60, 982. (19) Hale, H. D.; Hileman. F. D.; Mazer, T.; Shell, T. L.; Noble, R. W.; Rook, J. J. Anal. Chem. 1985, 57, 640. (20)Sissons, D.; Wetti, D. J . Chromatogr. 1971, 6 0 , 15-32. (21) Devillers, J. Fresinius' 2.Anal. Chem. 1988, 332,61-62. (22) Hasan, M. N.; Jurs, P. C. Anal. Chem. 1988. 6 0 , 978-982. (23) Randic, M. J . Am. Chem. SOC. 1975, 97, 6609.
(24) Kier, L. E.; Hall, L. H. Molecular ConnecfMty in Chemisfry and Drug Research; Academic Press: New York, 1976. (25) Buser, H. R. Environ. Sci. Technol. 1988, 20,404-408.
RECEIVED for review July 3, 1990. Accepted September 5, 1990.
Determination of Monosaccharides in Cellulosic Hydrolyzates Using Immobilized Pyranose Oxidase in a Continuous Amperometric Analyzer Lisbeth Olsson and Carl Fredrik Mandenius*J Department of Pure and Applied Biochemistry, Chemical Center, University of Lund, Box 124, S-221 00 Lund, Sweden
Jindrich Volc Institute of Microbiology, Czechoslovak Academy of Sciences, Prague 4, Krc, Czechoslovakia
The pyranoses, glucose, xylose, and galactose, were determined in a flow hjectlon syatem using an knmobilred enzyme reactor wlth pyranose oxMase (EC 1.1.3.10). Oxygen consumed by the oxidation was measured wRh an amperometric electrode (Clark type). The electrode response, after partial transfer of the sample through a dialysls membrane, was linear betw6en 0.6-30 mM glucose, 1.0-50 mM xylose, and 2.0-100 mM galactose with an accuracy of f7.0%. The hrfluence of the matrix of a W e medium, spent sumte liquor, a pyranose-contrrhrlng byproduct from the pulp industry, was investlgatted and found negligible with respect to sensitivity and stabMty, at least up to 2000 measurement cycles of the analytical system. Application of the system to continuous monitoring of ethanollc fermentation was also demonstrated.
INTRODUCTION In view of the constantly expressed need for environmentally safe energy conversion processes, analytical methods for the determination and monitoring of analytes, notably metabolites, in such processes are of the utmost importance for their operation. Conversion of lignocellulose into a fermentable monosaccharide hydrolyzate requires continuous determination in order to evaluate the quality, maximal yield, and process condition for a subsequent fermentation ( I , 2). Biosensors, e.g. enzyme electrodes and enzyme thermistors, provide the means for such analysis using specific enzymes (3, 4 ) . Glucose for example, can be determined by using glucose oxidase (EC 1.1.3.4),which has high specificity versus other metabolites (5). Other monosaccharides in lignocellulosic wastes, e.g. xylose, galactose, and mannose, usually require whole cells or enzymes or enzyme sequences that are cofactor dependent or are not stable enough for long-term usage (6-8). This paper describes the analytical use of an enzyme exhibiting specificity for three monosaccharides abundant in lignocellulose (glucose,galactose, and xylose), pyranose oxidase (EC 1.1.3.10)from the fungus Phanerocheate chrysosporium Present address: Kabi Peptide Hormones, Research and De-
velopment, s-11287 Stockholm, Sweden.
(9, 10). The enzyme, which is also referred to as glucose 2-oxidase, oxidizes the hydroxyl group at the C-2 position of the pyranose ring of hexoses and pentoses, thus differing from the extensively employed glucose oxidase which oxidizes at the C-1 position. Immobilized pyranose oxidase (PROD) has recently been shown by us to exhibit high operational stability thereby making it suitable for use in on-line analytical systems, for example FIA systems (11). The on-line analyzer used in this paper is operated with immobilized PROD contained in a reactor which is followed by an amperometric electrode for oxygen determination. A relative measure of the total pyranose concentration in a sample of several analytes in a crude process liquid or an absolute value of individual monosaccharides in a sample without competing analytes could be determined. Stability and reproducibility are evaluated and arguments for process analysis considered.
EXPERIMENTAL SECTION Enzyme Purification. PROD was purified from the basidomycetes P. chrysosporium,Strain K-3, according to a procedure previously described (IO,11). An inoculum of the organism was cultivated in shake flasks for 11 days on a glucose-corn steep medium. After harvesting and disruption of the cells, the resulting homogenate was centrifuged and supplemented with ammonium sulfate to increase the ionic strength of the solution. PROD was then purified by hydrophobic chromatography, anion-exchange chromatography, and gel filtration. Substantially pure PROD was obtained with a specific activity of 12 units/mg protein. Enzyme Immobilization. The purified PROD was immobilized onto controlled pore glass (CPG) with a mean pore diameter of 2215 A and a mesh size of 120/200 (CPG-10-2000 from Electro-Nucleonics,Inc.) by using the glutardialdehydeactivation method (12). The CPG was first boiled in 5% HN03for 45 min and thereafter extensively washed with distilled water. To a solution of 2 g of (y-aminopropy1)triethoxysilane(Sigma Chemical Co.) in 18 mL of water was added 1 g of CPG. The pH was adjusted to 3.5 and the solution heated to 75 "C for 3 h while gently being stirred. The CPG was washed and dried for 4 h at 115 "C. Activation with glutardialdehyde was performed by adding 25 mL of 2.5% glutardialdehyde (grade 11, Sigma Chemical Co.) in 0.1 M sodium phosphate buffer, pH 7.0. The reaction was carried out under reduced pressure for 30 min followed by 30 min at normal pressure. After the activated glass was washed with distilled water, the enzyme was coupled in 50 mM phosphate
0003-2700/90/0362-2688$02.50/0 0 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 82, NO. 24, DECEMBER 15. 1990
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W
. SR
F
Sd~maticdiagram of the insbllmentation in the fermentation experiment: F, fermentor; RF, rough liner: P. pump: UF, ultrafiner; D. dilution device: DL, dilution liquid: W. waste: PA, PROD analyzer. Flgure 2.
1
cs F!gure 1. Schematic diagram of hpseudo Row injection system: SR. signal to recorder: OE, amperometric oxygen electrode in flow cell: ER. enzyme reactor with immobilized pyranose oxidase: FD. flow diverter: MP. microprocessor lor controlling the device: W. waste liquid container; F, flat diilyzer: BS. buffer solulion, 0.1 M sodium phosphate. pH 7.8: SS. Sample solulion: CS. calibration Solution. All flows were conducted through the device by a multichannel peristaltic pump. 8S
ss
buffer (120 units/mL), pH 5.2, with a protein loading of 0.19 mg of protein/mg of CPG overnight at 4 "C. In order to stabilize PROD, coimmobilization with catalase (Reanal, Budapest) in one-tenth of the amount of PROD (w/w) was also carried out. The PROD activity for both of the immobilized preparations was 0.18 units/mg CPG. Solutions and Reagents. calibration solutions were prepared with analytically pure D-glucose, D-xylose, D-galactose, and Dcellobiose in a buffer of 0.1 M sodium phosphate, pH 7.8. Spent sulfite liquor (SSL) was provided hy Mo och Domsjo AB, Domsjo, Sweden, from their pulp process plant. In tests with assimilation of pyranoses, xylose was converted by adding immobilized xylose isomerase (Optisweet H, Miles-Kali-Chemie, Hannover, FRG). Instrumentation. Determination of pyranoses was carried out by using an on-line analyzer (Gambm Blood Glucose Analyzer, Gamhro AB, Lund, Sweden) (Figure 1). The device is based on amperometric monitoring of oxygen using a Clark electrode in connection to a reactor with a volume of 400 r L containing the immobilized enzyme. The analyte is sampled through a double lumen catheter, in the original application connected to a hypodermic needle placed in the patient hut here immersed into the Calibration solution or fermentation broth. A 1:l diluted sample stream is conducted to a flat dialysis membrane, dialyzing the sample stream molecules smaller than the cutoff of 3000 Da at a transfer factor of approximately 0.07. The oxygen content of the solution is measured before and after the reactor by diverting the fluid either through the reactor or to a parallel bypass channel. A time-controlled diverter directs the flow through the enzyme reactor for 60 s and thereafter alters its direction to the bypass loop for 30 s. The oxygen tension is measured with an amperometric oxygen electrode (Clark-type) at the end of each 60- and 30-9 period, respectively. A microprocessor calculates the concentration on the basis of signals from calibration runs with test solutions (13). High-Performance Liquid Chromatography. HPLC determinations of pyranoses in SSL were performed by using a lead column (Bio-Rad Aminex HPX-87P) at 85 "C in a Varian 5000 liquid chromatmraoh (14). The mobile ohase used was distilled water at a tlou ;at; of 0 fi m L min. Saccharides wew detected bv u h g n refmcti.mete-r ITecator Optilnh 5'jOlr. and mmples WQW deicmiztd u ; t h il mixed-bed imi-exchanpr resin. Fermentation Conditions. The anul\lical F r t e m was tested fur hiorenrror mnnituriny t m a lahuratuv wale. The analyer wa9 interface with a I(l-L termentor lCI.C-H-h. C'hcmolcrm. Belach. S t r w k h d m l cimtaining the SSI. medium with Sacchorr,ni)ca cwri twn (strain uhtamed Srom Svenskn .lastbolaget AR, Rotehro. heden!. Yeast cells were inoculated i n t o 5 L uf SSL medium containing 1 m u rrrlium mide (u a suspended s e d I(i(K) g of yeast. wet weipht. waa diluted in ().I M . sodium phnrphnr? buff'er, pH 6.50.IL u.800 1.1, re4tine In an initml rnnreniratiun ~Sccllmaw
of 26 g of dry weight/L of medium. The pyranose content of the medium was determined by Mooch Domsjo AB Research Lahoratory using HPLC (glucose, 4.2 g/L; mannose, 16.4 g/L; xylose, 10.6 g/L; galactose, 6.4 g/L). The fermentation was run under anaerobic conditions with an impeller speed of 200 rpm and a temperature of 30 "C. The fermentation medium was recirculated in a hollow-fiber filter (4001, Pierce AB, Lund, Sweden) at a flow rate of 1000 mL/min by using a perstaltic pump (Masterflex, Cole-Parmer). Samples were withdrawn from the filter by using another peristaltic pump (Miniplus-2:Gilson Ltd., Villiers le Bel, France) and diluted 6-fold with 0.1 M sodium phosphate, pH 7.8, in a T-joint (Figure 2). This diluted sample stream was conducted to the analyzer instrument through its double lumen catheter system to determine the pyranose content. A 23-g sample of glucose (20 mM) was added to the fermented SSL medium after 7.8 h. Samples for comparative HPLC analysis were withdrawn from the filtrate. RESULTS AND DISCUSSION Pyranose oxidase, a flavoprotein with an assumed mechanism similar to glucose oxidase, has only twice been described for analytical use and then with soluble enzyme (15, 16). In a recent study we demonstrated the immobilization of this enzyme with results of significant increased stability at higher temperature and prolongation of operational and storage life (11). In addition, compared to the soluble enzyme certain alterations in the substrate specificity profile as well as in pH optimum were noted. In the present study the enzyme is evaluated with respect to its analytical applicability in a pseudo flow injection system (Figure 1). Dilution and dialysis are performed before conversion in an enzyme reactor containing the immobilized enzyme and amperometric oxygen electrode. The dilution and dialysis system of sampling provides a convenient adaptation to the requirement of presenting a sample in a concentration range suitable for the enzyme. As for all oxidases PROD depends on the limited oxygen solubility in the buffer solution, which restricts the operable range of measurement for linear response (17). The design of the analyzer used here permits an up to 30-fold sample dilution. S u b s t r a t e Specificity. The relative activity of several substrates has been determined and for two of them, glucose and xylose, has been characterized with respect to K,,, and V,, (11). The relative responses for glucose, xylose, galactose, and cellobiose are shown in the calibration graphs in Figure 3. A sensitivity difference of 1:2 between catalase-coimmobilized and noncoimmobilized PROD was observed. This is not surprising since the generation of oxygen by catalase decreases the apparent oxygen consumption per glucose. Table I compares the relative activity data of immobilized PROD for different monosaccharides with the relative response obtained from the measured analytes in Figure 3. Apparently, the conversion is dependent on the activity of the reactor for each monosaccharide, which results in an incomplete conversion for all analytes hut glucose and is thus a non end point analysis. This introduces sensitivity to rate effects such as
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Table 11. Matrix Effect of the Spent Sulfite Liquor after Removal of Pyranoses with Anaerobic Fermentation and Xylose Isomerase Treatment
'ID,
0
20
40
60
80
100
Concentration m M Figure 3. Calibration curves for various pyranoses with an enzyme reactor containing Coimmobillzed pyranose oxidase and catalase. Shown are the relative response versus concentration of glucose (O), xylose (O),galactose (m), and cellobiose (A)for samples injected through the double lumen catheter and the response for glucose of an enzyme reactor containing immobilized pyranose oxidase (without catalase) injected through the double lumen catheter (0). Standard mean deviation was 2 . 5 % for all analytes.
sample
% re1 response
spent sulfite liquor (SSL) reduced SSL reduced SSL containing 5.0 mM glucose 10.0 mM glucose 15.0 mM glucose 20.0 mM glucose 7.5 mM xylose 15.0 mM xylose 22.5 mM xylose 30.0 mM xylose 12.5 mM galactose 25.0 mM galactose 37.5 mM galactose 50.0 mM galactose
100 37 53 72 90 108 54 69 85 100
50 64 77 92
Table 111. Comparison of Slopes (V/mM) in Pure Solution and in SSL analyte
pure soln
SSL soln
glucose xylose galactose
0.048 0.032
0.045 0.029 0.016
0.015
Table I. Relative Response of the Analytes in an Enzyme Reactor with Coimmobilized PROD/Catalase after Dilution in the Dialyzer System analyte
% re1 response
'70 re1 activity
glucose xylose galactose arabinose mannose
100 67 31
100 60 19
