Envlron. Sci. Technol. 1987,21, 859-863
Wyman, K. Ecol. Modell. 1982, 15, 29. (13) Benes, P.; Steinnes, E. Water Res. 1974, 8 , 947. (14) Benes, P. Water Res. 1980, 14, 544. (15) Borg, H.; Andersson, P. Verh.-Int. Ver. Theor. Angew. Limnol. 1984, 22, 725. (16) Sakshaug, E.; Jensen, A. Oceanogr. Mar. Biol. 1978,16,81. (17) Jonsson, A. S.; Traghdh, G. Kem. Tidskr. 1984, 12, 41. (18) Jarvinnen, A. W.; Hoffman, M. J.; Thorslund, T. W. J. Fish. Res. Board Can. 1977, 34, 2089.
Luoma, S. N. Sci. Total Environ. 1983,28, 1. Kihlstrom,J. E. Natl. Swed. Environ. Prof. Bd. Rep. PM 1982, 1552, 101.
Sagiura, K.; Ito, N.; Matsumoto, N.; Mihara, Y.; Murata, K.; Tsukakoshi, Y.; Goto, M. Chemosphere 1978, 9, 731. Peters, L. S.; OConnor, J. M. In Environmental Stress and the New York Bight: Science and Management; Mayer, G., Ed.; Estuarine Research Foundation: Charleston, SC, 1982; pp 128-133. Harding, G. C.; Vass, W. P.;Drinckwater,K. F. Can. J. Fish. Aquat. Sci. 1981, 38, 101. Sodergren,A.; Svensson,Bj. Bull. Environ. Contam. Toxicol. 1973, 9, 345. Brown, M. P.; McLaughlin, J. J. A.; O’Connor, J. M.;
Received for review July 2, 1986. Revised manuscript received January 30,1987. Accepted April 24,1987. The investigation was supported by the Swedish Natural Science Research Council.
Enzymatic Hydrolysis of Carbohydrates in Aquatic Fulvic Acid Dorothy J. Bertlno,*st Phillip W. Albro, and J. Ronald Hasst National Institute for Environmental Health Sciences, Research Triangle Park, North Carolina 27709
rn Enzymes were used to investigate the carbohydrate components of fulvic acid samples from Lake Singletary, NC. a-Amylase released 14-19% of the glucose, indicating the presence of glucose polysaccharides containing a-1,4 linkages. Cellulase released 10-14% of the glucose, indicating the presence of glucose polysaccharides with @-1,4 linkages. Smaller amounts of glucose were released by dextranase, laminarinase, a-glucosidase, and acetylesterase. These results show that glucose occurs in a variety of structures in the fulvic acid samples studied. Introduction
Fulvic acid is the fraction of naturally occurring organic matter in soil and water that is soluble in both acid and base, I t is derived from plant material, and therefore its composition is variable and source dependent. Although little is known about the composition of these complex mixtures, fulvic acids are known to be acidic and to contain hydroxyl groups, and attempts to determine the molecular weight range of various samples suggest 500-2000 as typical (1). Better methods for characterizing the organics in water are needed to compare water samples from different sources, to determine seasonal variation, and to observe the changes resulting from pollution and water treatment. Physical and/or chemical interactions of pollutants with humic material may be important in pollutant transport, bioavailability, and degradation. Carbohydrates have been detected in marine, soil, and freshwater humic material. For some marine sedimentary fulvic acids, the phenol-sulfuric acid test indicated 3657% carbohydrates (2). The carbohydrates in soil fulvic acids may be as high as 40% of the fulvic acid carbon (3). Freshwater fulvic acid samples have been reported to contain 2.6-5% carbohydrate ( 4 , 5 ) . By the use of Sephadex gel filtration, dialysis, and ion-exchange chromatography, de Haan and de Boer (6, 7) found that most of the carbohydrate in fulvic acid from Lake Tjeukemeer, The Netherlands, occurred in the high molecular weight fraction (>5000) and that about 20% was not separable from other fulvic acid components by the methods employed. Sweet and Perdue (8) attempted to distinguish between monosaccharides, polysaccharides, and humic-bound sac+ Present address: Triangle Laboratories, Inc., Research Triangle Park, NC 27709.
0013-936X/87/0921-0859$01.50/0
charides in river water. Monosaccharides were determined by gas chromatography as the alditol acetates. XAD-7 resin was used to remove humics before and after acid hydrolysis. Samples from 11 sampling points in the Williamson River system gave variable results for the relative amounts of polysaccharides and humic-bound saccharides, but the monosaccharides were consistently the lowest fraction. The purpose of this study was to investigate the use of enzymes for hydrolysis of fulvic acid. Enzymatic hydrolysis was attempted for three reasons: (1) reactions can be carried out under mild conditions of pH and temperature to minimize artifact formation, (2) the substrate specificity of enzymes may yield structural information about the original components by indicating the types of bonds present, and (3) separation and purification of the substrates prior to hydrolysis are not necessary, which makes it possible to determine compounds present at low concentrations that are not readily isolated. Although this work was concerned with carbohydrates, the approach may also be useful for other types of compounds that occur in fulvic acids. Disadvantages of using enzymes are as follows: (1) results may be negative even if an appropriate substrate is present if there are endogenous enzyme inhibitors present; (2) structures similar to the normal substrate(s) may or may not be hydrolyzed, depending on the specificity of the enzyme; and (3) enzyme reactions do not always go to completion, which is a problem for quantitation. In some cases these problems can be avoided by the appropriate selection of enzymes. Experimental Section
Fulvic acid samples were obtained from Lake Singletary, NC. The procedure for isolation of the fulvic acid from lake water using XAD-8 resins has been published (9). Fulvic acid sample SL103 was obtained on March 27, 1984, and elemental analysis yielded 44 f 1% C, 4.8 f 0.1-70H, and 0.9 f 0.1% N, with an ash content of 6.6 f 1.1% (average and standard deviation for determinations from four laboratories: Huffman, Wheat Ridge, CO; Galbraith, Knoxville, TN; Microtech, Skokie, IL; and MHW Laboratories, Phoenix, AZ). Sample SL109 was obtained on November 20,1984, and had 53% C, 4.15% H, 0.98% N, and 4.23% ash (Huffman, Wheat Ridge, CO). All of the enzymes and reference substrates used were obtained from
0 1987 American Chemical Society
Environ. Sci. Technol., Vol. 21, No. 9, 1987 859
Table I. Substrates Used T o Test Each Enzyme for Appropriate Reaction Conditions a n d Sugars Produced from Enzymatic Hydrolysis
enzyme
source
amyloglucosidase dextranase (Sigma grade I) fi-glucosidase (Sigma type I) a-glucosidase (Sigma type VI) naringinase laminarinase cellulase (Sigma type VII) cellulase (Sigma type I) cellulase (Sigma type VI) a-amylase (Sigma type IA) acetylesterase esterase
Aspergillus oryzae Penicillium sp. almonds brewers' yeast Penicillium sp. Penicillium sp.
P. funiculosum A. niger T. viride hog pancreas orange peel porcine liver
typical substrate (product) amylopectin anthranilate (glucose) dextran (isomaltose) salicin (glucose) maltose (glucose) naringin (glucose and rhamnose) laminarin (glucose) cellulose azure (cellobiose) cellulose azure (glucose) cellulose azure (glucose) cyclodextrin (maltose) glucose pentaacetate
Sigma Chemical Co., St. Louis, MO. All of the reagents were obtained from Fisher Scientific unless otherwise indicated. Total Carbohydrates. The total carbohydrate content of each fulvic acid sample was determined by the 1naphthol/sulfuric acid test according to Dische (10). The fulvic acid samples were 1.0 mg/mL in HPLC-grade water; fulvic acid was added to the reagent blank. The glucose standards were 10, 25, and 50 pg/mL. Acid Hydrolysis. Acid hydrolysis was done with 2 N hydrochloric acid at 100 "C for 4 h in evacuated tubes (11, 12). From 1.4 to 1.8 mg of fulvic acid was used with 1mL of acid. After cooling to room temperature, 15 pg of internal standard was added. The samples were derivatized with 50-100 pL of 1-(trimethylsily1)imidazolein pyridine (Trisil-Z, Pierce Chemical Co.). Gas Chromatography. Gas chromatography of the acid hydrolysate was performed on a Varian 3700 GC with two fused-silica capillary columns. The first column was a bonded-phase methyl silicone column (Alltech, 25 m X 0.25 mm). For identification of monosaccharides, the sample and sugar standards were chromatographed with a temperature program of 150-190 "C at 2 deg/min. For quantitation of glucose and disaccharides,the program was 190-250 "C at 3 deg/min. The helium inlet pressure was 20 psig, injector temperature 250, and hydrogen flame ionization detector (HFID) temperature 280. Peak areas were measured relative to the trimethylsilyl derivative of manno-heptulose or gluco-heptulose with a HewlettPackard Model 3370A chromatographic peak integrator. The chromatography was repeated on a second column, a DB-1701 (J&W Scientific, 30 m X 0.25 mm), to confirm identification of the sugars. The temperature program was 150-190 "C a t 2 deg/min. Mass Spectrometry. The identification of glucose was also verified by gas chromatography/mass spectrometry with a Hewlett-Packard 5890 GC, a DB-5 30-m column (J&W Scientific) programmed from 190 to 250 "C a t 3 deglmin, and a VG 12-250 quadrupole mass spectrometer. The source temperature was 200 "C, electron energy 70 eV, and trap current 200 PA. General Incubation Conditions. The enzymes used in this study were p-D-glucosidase (EC 3.2.1.21), WDglucosidase (EC 3.2.1.20), cellulase (EC 3.2.1.4), amyloglucosidase (EC 3.2.1.3), dextranase (EC 3.2.1.11), naringinase (EC 3.2.1.21 and EC 3.2.1.40), laminarinase (EC 3.2.1.6), a-amylase (EC 3.2.1.1), acetylesterase (EC 3.1.1.6), and carboxylesterase (EC 3.1.1.1). All of the enzyme reactions were carried out in 0.01 M ammonium acetate unless otherwise indicated. Fulvic acid (5.0 0.1 mg/mL) was first dissolved at pH 10 (ammonium hydroxide) and
*
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Environ. Sci. Technol., Vol. 21, No. 9, 1987
linkage hydrolyzed ~ 1 , 4 ~~-1,6
@-glucoside a-1,4 a-glucoside @-glucosidea-rhamnoside
P-L~ 0-1,4 @-1,4 @-L4 (~-1,4 ester ester
then adjusted to the appropriate pH with glacial acetic acid. A few microliters of a stock solution of enzyme (1-2 mg/mL in 0.01 M ammonium acetate) was added with a drop of chloroform to suppress bacterial growth. The samples were incubated in a water bath at 37 "C for 22-23 h, unless otherwise specified. To ensure that reaction conditions were appropriate for the enzyme, known substrates were added to identical fulvic acid/enzyme solutions (Table I). If the known substrate was not hydrolyzed by the enzyme in the presence of fulvic acid, the incubation was done without fulvic acid to be sure the enzyme was active. Each enzyme and fulvic acid were incubated separately as controls. Aliquots of the reaction mixtures (0.5 mL) were freeze-dried with 15 or 20 pg of glucoheptose (internal standard) and 1-2 pL of ethylene glycol to prevent loss of sample. The freeze-dried samples were derivatized with Trisil-Z as for the acid hydrolysates. Freeze-drying effectively removed the ammonium acetate buffer from the reaction products. Incubation Conditions for Specific Enzymes. 8Glucosidase. To 2.0 mL of fulvic acid was added 50 pg (1.5 units) of /3-glucosidase. A known substrate, salicin (100 pg/mL), was added to the fulvic acid solutions and incubated at pH 5.0 for 1, 2, and 3 h and a t pH 5.6 for 3 and 22 h. Additional experiments were done to examine the inhibition of 0-glucosidase by fulvic acid. The salicin concentration was doubled to 200 pg/mL to test for competitive inhibition. Ethylenediaminetetraacetic acid (EDTA), 1mM, was added to test for inhibition by heavy metals, and butylated hydroxyanisole (BHA),0.1 mM, was added to test for inhibition by free radicals. Another fulvic acid sample was incubated at pH 5.4. Cellulase. Three different cellulases were used, one from Penicillium funiculosum, one from Aspergillus niger, and one from Trichoderma uiride. For the first (P. funiculosum), 1unit (0.1 mg) of cellulase was incubated with 2.0 mL of fulvic acid (SL109) a t pH 5.0 in 0.15 M sodium chloride for 2,4, and 24 h. Cellulose azure was added as a known substrate to parallel samples. Since cellulose azure is not very soluble in water at room temperature, a saturated solution was used. The reaction with cellulose azure in the presence of fulvic acid was also done a t pH 5.5 in 0.01 M ammonium acetate. To 0.6 mL of fulvic acid 30 pg of cellulase and an excess of cellulose azure were added, and the mixture was incubated for 22.5 h. Because the first cellulase was severely inhibited by the fulvic acid, the reaction was repeated with the two other cellulases in 0.1 M ammonium acetate, pH 5.1, with incubation a t 38 "C for 22 h. To 0.7 mL of fulvic acid was added 1.27 mg (0.76 unit) of cellulase from A. niger or 1.06 mg (0.58 unit) from T. uiride.
For the second fulvic acid sample, the cellulase from T. viride was purified to remove free glucose before use.
Bio-Gel P-2 (Bio-Rad Laboratories) with an exclusion limit of 1800 was swollen in 0.1 M ammonium acetate/acetic acid, pH 5.1, overnight and washed with the same solution before the 20-mL column was packed. The cellulase solution, 18.0 mg (9.9 units) in 1.0 mL of the pH 5.1 buffer, was centrifuged, and 0.9 mL was applied to the column. The sample was eluted with the same buffer, and 1-mL fractions were collected and measured a t 280 nm on a Beckman DU spectrophotometer. The fraction with the maximum absorbance was also measured a t 260 nm, and the concentration was estimated as 1.07 mg of protein/mL from the equation 1.5[A(280)] - 0.75[A(260)] = mg/mL, where A is absorbance at 280 and 260 nm, respectively (13). No glucose was detected in the purified enzyme. Amyloglucosidase. Fulvic acid was incubated with amyloglucosidase (30 pg or 1.2 units to 0.6 mL) with and without amylopectin anthranilate a t pH 5.5. Amylopectin anthranilate was added in excess because of its limited water solubility a t room temperature. &-Amylase. To 0.5-0.7 mL of fulvic acid 1-5 pL (18-90 units) of a-amylase was added. The samples were incubated a t room temperature (25 "C) for 22 h at pH 7.0-7.1. The known substrate used was a-cyclodextrin. a-Glucosidase. Fulvic acid was incubated with a-glucosidase (50 pg or 1.6 units in 0.5-0.7 mL) a t pH 5.4-5.5, with and without maltose. Dextranase. The experiments with dextranase were carried out at pH 6.2, with 5 pg (3 units) of enzyme added to 0.5-0.7 mL of fulvic acid solution. Dextran (average mol wt 9000) was used for the known substrate. Naringinase. Naringinase, 26-56 pg (0.01-0.02 unit) in 0.5-0.6 mL of fulvic acid solution, was incubated at pH 5.5. Naringin was used as the known substrate. Laminarinase. Fulvic acid and laminarinase, 50-88 pg (0.2-0.4 unit) in 0.5-0.7 mL, were incubated at pH 5.5 with and without laminarin (0.11-0.15 mg in 0.7 mL). Esterase and Acetylesterase. Fulvic acid was incubated with each of the esterases at pH 7.3. Five microliters (8 units) of esterase and 20 pL (1.2 units) of acetylesterase were added to separate samples. Combination Experiments. a-1,6 linkages may occur with a-1,4 linkages in branched polysaccharides (amylopectin, glycogen, and dextrans). /3-1,3 glucans with &1,4 branching occur in lichenin and other glucans. Therefore, some combined enzyme reactions were attempted. Controls were incubated without fulvic acid for each combination of enzymes. Dextranase and Amyloglucosidase. Fulvic acid was incubated with dextranase and amyloglucosidase at pH 5.8. Laminarinase and 8-Glucosidase. Fulvic acid was incubated with laminarinase, 200 pg in 1.2 mL, for 2 h a t pH 5.6 and 37 OC, and then /3-glucosidase (50 pg) was added and the mixture incubated 23.5 h longer. Ester groups may prevent hydrolysis of glucosides by glucosidases. Therefore, combined enzyme experiments were done with esterases followed by the glucosidases. Esterase, Acetylesterase, and a-or @-Glucosidase. The two esterases were incubated with fulvic acid a t pH 7.3. Five microliters of esterase and 20 pL of acetylesterase were added to 2.0 mL of solution. Aliquots were taken for gas chromatographic analysis, the remaining solution was acidified to pH 5.6-5.7, and a- or P-glucosidase was added separately to duplicate solutions. Results and Discussion
All results are given on an ash-free basis. The average of two determinations and the range are given, unless
Table 11. Total Carbohydrate and Glucose Determinations" sample
SL109
SL103
1-naphthol testb 39 f 6 29 f 5 HCl hydrolysis (glucose) 22 f 1 20 free glucose 1.2 f 0.2' 1.07 f 0.07d aResults are expressed as pg of sugar/mg of fulvic acid. bGlucose was used for the standard curve. cAverage of 12 samples and 95% confidence interval. dAverage of 14 samples and 95% confidence interval. The ratio g1ucose:galactose:mannose was 12.8:0.8:1 for SL103 and 3.8:1.3:1 for SL109. ~~
~~
Table 111. Inhibition of b-Glucosidase by Fulvic Acid"
pH 5.0 salicin salicin salicin salicin pH 5.6 salicin salicin
+ FAb + FA + FA + FA + FA
time, h
% of salicin
1.3 1 2 3
52 2 8 11
3 22
hydrolyzed
63 100
"Inhibition is indicated by the decreased rate of hydrolysis of salicin by P-glucosidase in the presence of fulvic acid. The inhibition showed a deDendence on DH.*FA = fulvic acid.
otherwise indicated. For most of the enzyme controls no glucose was detected. Glucose was observed in amyloglucosidase (0-0.49 pg/mL), naringinase (1.1-2.3 pg/mL), cellulase from T. viride (2.6 pg/mL), and cellulase from A. niger (17 pg/mL). The results were corrected for free glucose in the fulvic acid (5-6 pg/mL) and in the enzymes. The t test for comparing the two means was used to verify a nonzero result. The results for the 1-naphthol/sulfuric acid test, the acid hydrolysis, and the glucose content of unreacted fulvic acid are given in Table 11. These results indicate that glucose is the major sugar in the fulvic acid samples and that most of the glucose is bound. Also, the monosaccharides released were bound by hydrolyzable bonds. Therefore, they must occur as polysaccharides, glucosides, or sugar esters, not as sugar ethers or C-glycosides. Preliminary experiments indicated that P-glucosidase and cellulase were inhibited by the fulvic acid (SL109). Salicin was hydrolyzed by the enzyme at a much slower rate in the presence of fulvic acid. A higher pH (5.6) and long incubations times were required for complete hydrolysis of salicin (Table 111). No reaction was observed with the fulvic acid samples. When the salicin concentration was doubled, in the presence of fulvic acid, the glucose released was more than doubled, which suggested competitive inhibition by the fulvic acid. This implies that structures similar to appropriate substrates are present in the fulvic acid since they can bind to the enzyme but are not hydrolyzed. Since EDTA did not overcome the inhibition, heavy metals that chelate with EDTA were not responsible for the inhibition. The free radical trap BHA also was ineffective in overcoming the inhibition. Cellulose azure gave very little hydrolysis product when incubated with cellulase (P.funiculosum) in the presence of fulvic acid. For cellulase, the inhibition problem was avoided by using enzyme preparations from different organisms (A. niger or T. viride) (Table IV). Glucose released from fulvic acid by cellulase indicates the presence of polymers with p-1,4 linkages. The results are shown in Tables V and VI. Amyloglucosidase and a-amylase both hydrolyze a-1,4 linkages. Amyloglucosidase is an ex0 enzyme that removes Environ. Sci. Technol., Vol. 21, No. 9, 1987
861
Table IV. Test for Inhibition of Cellulase (C) by Fulvic Acid (FA)" source of enzyme
P. funiculosum A . niger
T. viride
PH
time, h
5.0 5.5 5.1 5.1
2 23 23 23
C+CA
C+CA+FA
74.6
4.8 5.0 406 505
Cellulose azure (CA) was used for the standard substrate. Results are given as kg of product/mL. Table V. Glucose Released by Enzyme Hydrolysisn enzyme a-amylase ce11u1ase dextranase isomaltose glucose a-glucosidase @-glucosidase esterase acetylesterase amyloglucosidase naringinase laminarinase
SL109
SL103
3.1 f 0.3 2.3 f 0.2b
