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Factors affecting glucosidase and galactosidase activities in soils
Sod Bid. Btochrm. Vol. 22. No. 7. pp. W-897. tinted ,n Great Bntarn. All nghts reserved
0038-07t7 90 53.00 + 0.00 Copyright C 1990 Pergamon Press pk
1990
FACTORS AFFECTING GLUCOSIDASE AND GALACTOSIDASE ACTIVITIES IN SOILS* F. ElvAzrt Department
of Agronomy,
and M. A. TABATABAI~
Iowa State University.
Ames,
IA 5001 I. U.S.A.
(Accepfed 5 June 1990) Summary-This study was carried out to assess the effects of soil treatments. the addition of I2 inorganic salts and of 20 trace elements on Z- and B-glucosidase and z-and D-galactosidase activities in soils. Results showed that air-drying of field-moist soils resulted in marked increases in the activity of these enzymes and that steam sterilization of soils completely destroyed the activities. In general. the inorganic salts were stronger inhibitors of x-glucosidase and a-galactosidase activities (range 0 to 70%) than the activities of their b counterparts (range 0 to 44%). The effect of trace elements on the activities of these enzymes varied considerably among the elements, enzymes and soils. The percentage inhibition of a-glucosidase activity in Webster soil ranged from 4% with Mo(VI) to 66% with Ag(1) and in Luton soil ranged from 9% with V(IV) to 66% with Cd(ll). In general, the inhibition values of B-glucosidase activity by trace elements were much lower than those obtained for a-glucosidase activity. The inhibition values of z-galactosidase activity in Webster soil ranged from 10% with Hg(l1) to 13% with Sn(Il). The inhibition values for this enzyme in Luton soil ranged from 12% with Hg(ll) to 73% with Mo(V1). Studies on the distribution of the activities of these enzymes showed that they are concentrated in surface soils and decrease with depth. The activities of these enzymes were significantly correlated with organic C in soil profiles and surface soils and were. in most cases, significantly but negatively correlated with pH.
INTWOIWCTION
The term “tract elcmcnt” is used here to refer to elements that are. when prcscnt in sufficient conccntrations, toxic to living systems. Some of these elements (e.g. Pb. V) are present in fuel oils and gasoline, emitted into the atmosphere upon burning and deposited on soils, especially near urban areas (Lagerwerff, 1972; Tyler, 1976). Although the patterns of distribution of activities of several enzymes in soil profiles have been reported (Tabatabai, 1982), little information is available about the relative factors that affect glucosidasc and galactosidase activities through the profile. We used a method for assaying the activities of glucosidases and galactosidases in soils (Eivazi and Tabatabai. 1988) to study the effects of soil trcatments, inorganic salts and trace elements on the activities of these enzymes in soils. We also determined the distribution of glucosidase and galactosidase activities in a range of agricultural soil profiles and also the relationship between the activities of these enzymes and organic C and pH of both surface and subsurface soil samples.
The cnzymcs acting on glycosyl compounds (EC 3.2). including glycosidc hydrolyses (EC 3.2. I .). arc among the hydrolascs lcast studied in soils. Among thcsc, zand /j-glucosidascs and Q- and /I-galactosidascs arc the most important bccausc their hydrolysis products, sugars, arc important cncrgy sources for microorganisms in soils. Although glucosidascs and galactosidases arc widely distributed in nature (Bahl and Agrawal, 1972; Dey and Pridham, 1972; Wallenfels and Wcil, 1975) and should play a significant role in breakdown of low molecular-weight carbohydrates in soils, producing sugars. littlc information is available about effect of sample handling and addition of inorganic salts and trace elements on the reactions catalyzed by these enzymes. Air-drying of field-moist soils is known to change the activities of a number of enzymes (Tabatabai, 1982). but its effect on the activity of glucosidases and galactosidases is not known. In addition, modern agricultural practices make extensive use of fertilizers added to soils in the form of inorganic compounds. Also. a number of trace elements are added to soils as fertilizers, impurities in fcrtilizcrs or as components of municipal and industrial wastes (Allaway, 1971; Berrow and Webber. 1972: Tabatabai and Frankenberger, 1979).
MATERIALS
AND SIETHODS
The soils used (Table I) were surface samples (0-l 5 cm) selected to include a wide range in pH, texture, organic-matter content and in glucosidasc and galactosidase activities and also to represent important agricultural soils in Iowa. Before use. each soil was sieved (2 mm) in the field-moist condition, and subsamples of the sieved soils were air dried at room temperature (23’C) or steam sterilized. The analyses reported in Table I were performed as described by Dick and Tabatabai (1986). The profile samples were selected to include a
*Journal paper No. J-13857 of the Iowa Agriculture and Home Economics Experiment Station, Ames. Iowa. Project No. 2082. tPresent address: Department of Agriculture, Plant and Soil Science, Lincoln University. Jefferson City, MO 65101. U.S.A. :To whom all correspondence should be addressed. 891
F. EIVAZIand M. A. TABATAILU
892 Table I. Analwes of soils SOll
PH
Organic P/o)
Thurmdn HageM W&r Ida Clanon Webster Nicollet Harps Luton Okobq
7 I 6.4 5.1 80 64 5.8 6. I 7.8 6.8 7.4
0.47 0.92 1.51 I .57 1.77 2.54 3.32 3.74 4.35 5.45
c
Total N (%)
Clay (%)
Sand (Oh)
0.046 0.093 0.131 0.147 0.188 0.210 0.253 0.305 0.388 0.463
5 13 17 27 24 23 30 30 42 34
93 64 I 3 39 38 25 26 3 16
wide range of glucosidase and galactosidase activities in their surface soils and to represent important agricultural soils in Iowa. The inorganic salts and trace elements were Fisher certified reagent-grade chemicals obtained from Fisher Scientific Co., Itasca, Ill. Of the trace elements used, Ag(I), Cd(H), Co(II), Cu(II). Fe(II). Zn(I1) and V(V) were added as the sulfate: Cu(I). Ba(II), Hg(II), Mn(II), Ni(II), Se(I1) AI(II1) and Cr(II1) as the chloride; Pb(I1) as the acetate or nitrate; and As(III), B(II1). Se(IV), As(V). Mo(\‘I) and W(VI) as Na,AsO,, Na,B,O,, H$eO,, Na: HAsO, HLMOO, and Na,WO,, respectively. The assay method (Eivazi and Tabatabai. 1988) involves extraction and calorimetric determination of the p-nitrtiphcnol rclcascd when I g of soil is incubated with 5 ml of butfered p-nitrophcnyl glycoside solution
at 37‘C
for
I h.
In testing the effect of inorganic salts on glucosidase and galactosidasc activities of soils, I g of soil in a 50 ml Erlenmeyer flask was treated with 0.2 ml of tolucne, 4ml of Mod&d Universal Buffer (MUB), pH 6.0 made IO mM with rcspcct to the inorganic salt tcstcd. and I ml of 25 mM substrate made in MUB. pH 6.0. In testing the effects of trace elements on the activity of glucosidases and galactosidases, I g of soil was treated with I ml of solution containing 25 pmol of trace element. After 30 min of equilibration, 3 ml of MUB, pH 6.0, was added, and the enzymes were assayed as described for soils treated with inorganic salts. The results of glycosidase activities of soils treated with inorganic salt and trace elements were compared with those obtained without addition of inorganic salt or trace element. Controls were performed with each soil analyzed to allow for color not derived from p-nitrophenol released by glycosidase activity. All results reported are averages of duplicate determinations expressed on a moisture-free basis, moisture being determined from loss in weight following drying at lO5’C for 24 h.
other factors affecting the glucosidase and galactosidase activities in soils. Similarly, the effect of trace metals added to soils on the activities of several enzymes has been reported, but the effects of such metal on the activity of glucosidases and galactosidases and, consequently on the C cycling in soils are not well understood. For convenience, the results obtained in this study will be discussed according to the factors studied. Effects of pretreatments
Air-drying of field-moist soil resulted in a marked increase in the activity of these enzymes in the five soils studied (Table 2). Steam sterilization of air-dried soils at I2 I “C for I h completely destroyed the activities of these enzymes (not shown in Table 2). Because B-glucosidase activity is dominant among the four enzymes in soils, the effect of pretreatments was more pronounced with the activity of this enzyme. Our results support the available information on the effect of drying on enzyme activities. The effect of pretreatments depended upon the enzyme studied, and no general rule can be established for drying soil samples for assay of enzyme activities (Skujins, 1967). For example, Ross (1965) found that air-drying of soils inactivated the enzyme hydrolyzing sucrose (invertasc) and starch (amylasc), whereas Tabatabai and Bremner (1970) found that it increased soil arylsulfatase activity. This observation was explained by noting that the rcwetting of air-dried soil causes breakdown of aggregates and thus increases the accessibility of soil enzymes to their substrates in assay of soil enzyme activity. Distribution
Enzyme activity in soil-profile samples usually dccrcases with sample depth and is accompanied by a decrease in organic-matter content (Kuprevich and Shchrebakova, 1967). We found that glucosidase and galactosidase activities decreased markedly with depth in each of the five soil profiles examined (Figs I and 2) and was associated with a decrease in organic-C content. Statistical analyses showed that the activities of these enzymes are significantly correlated with organic C in each soil profile sample (Table 3). Also, the activities of a-glucosidase and fi-galactosidase were significantly, but negatively, correlated with pH of soils in the profiles of Clarion, Marna, Table 2. Efiecrs of minor treatment on glucosidaseand galactosidasc activilics of soils Glycosidaw acrivity’ Soil
RESULTS AND DISCUSSION
Reviews about enzymes in soils indicate that studies of origin, location and persistence of enzymes in soils are needed (Skujins, 1967, 1976). The available information indicates that the activities of enzymes such as urease. arylsulfatase. rhodanese. acid and alkaline phosphatases and pyrophosphatase are influenced by methods of handling, storing and treating the sample before enzyme assay (Tabatabai. 1982). but little information is available about these and
in soil projfes
Trcatmcnrb
I-GIU
4.GIU
Z-Gal
B-Gal
FM AD FM AD FM AD FM AD FM
3 7 4 8 IO I3 2 8 I2
62 147 103 169 53 73 90 I26 93
IO I7 23 28 9 22 I2 19 I7
I4 20 IO 13 13 15 8 I5 22
AD
I4
I52
25
28
Clarion Webster Harps Nicollet Okoboii
lpg
ofp-nitrophenol
rclcascd
g-’
soilhe’.
galaclosidase.
“FM = field-moire;
AD
=
ax-dried
Glu = glucosidase;
Gal =
Factors
4.5 r
affecting
6;0
893
glycosidases in soils
7;5
4.5 ,
6;O
7;s
FH
GLYCOSIOASE ACTIVITY
60
CLARION
SOIL
t-
HARPS
SOIL
60
I
MARNA
RSHALL
SOIL
SOIL
. a-GLUCOSIDASE o j3-GLUCOSIOASE A ORGANIC 60
CARBON
A PH
t-
NICOLLET
SOIL
of
Fig. I. Distribution organicC, OI-and /I-glucosidase activities and trends of pH with depth in soil profile samples. Clucosidase activity is expressed as pg of p-nitrophenol released g-’ of soil h- ’ at 37’C.
Marshall and Nicollet soils. /I-Glucosidase and a-galactosidase activities were significantly, but negatively, correlated with soil pH of the profiles of Clarion, Marna and Marshall soils. Statistical analyses, however, showed that, when enzyme activities of the surface soils of the five profiles were pooled with those of IO other surface soils, the activities of only a- and j?-glucosidase and ,9-galactosidase were significantly correlated with organic C. No such correlations were obtained between z-galactosidase and organic C in the surface soils examined. Furthermore, only a-glucosidase activity was significantly correlated with pH of the surface soils tested. Unlike the significant but negative, correlation found for the relationships between glycosidase activities and pH of the soils in the profile studied, the a-galactosidase activity of the surface soils was positively correlated with organic C. Statistical analyses of the pooled data (all soil profile samples) showed that the activities of the four glycosidases studied were significantly correlated with organic C. With the exception of z-glucosidase activity, the activities of the other three glycosidases were significantly, but negatively, correlated with soil pH.
Eflects of inorganic salts
The effects of inorganic salts (the incubation medium made 8 mM with respect to the inorganic salt) on glucosidase and galactosidase activities in the soils are shown in Tables 4 and 5, respcctivcly. Generally, the inorganic salts studied were more effective in inhibiting a-glucosidase and a-galactosidase activities than /I-glucosidase and b-galactosidase activities. With a few exceptions, all the inorganic salts tested partly inhibited the activity of these enzymes in soils. From these results, it is clear that the addition of inorganic salts as fertilizer materials or accumulation from irrigation water could have a profound effect on the activities of glycosidases in soils and, therefore, on C cycling. Eflecrs of trace elements on glJcosidases in soils
Metal ions may inhibit enzyme reactions by complexing the substrate by combining with the protein active group of the enzymes or by reacting with the enzyme-substrate complex. The mode of inhibition is dependent on the tyw of substrate used. Metal ions generally are assumed to inactivate enzymes by reac-
894
F. EIVAZIand M. A. TABATABAI PH
4.5 l
ORGANIC 0 C (%I I 0 GLYCOSIOASE ACTIVITY
6.0 I 1.6 ,
1
I
10
7.5 I 3.2 I
4.5 I 0 I,
20
6.0 I 1.6 I
I
300
7.5 1 3.2 ,
I
10
,
20
30
60
h CLARION
t-
SOIL
HARPS
SOIL
MARSHALL
SOIL
60
c
30
.a o
-GALACTOSIOASE /!?-GALACTOSIDASE
A ORGANIC
60
A
CARBON
P"
90
NICOLLET Fig. 2. Distributionof organic
profile samples. Galactosidase
SOIL
C, I- and @-galactosidase activities and trends of pH with depth in soil activity is expressed as pg ofp-nitrophenol released g-’ of soil h“ at 37’C.
tion with sulfhydryl groups, a reaction analogous to the formation of a metal sulfide. Sulfhydryl groups in enzymes may serve as integral parts of the catalytitally-active sites or as groups involved in maintaining the correct structural relationship of the enzyme protein.
The effect of trace elements on the activities of aand /I-glucosidases and I- and p-galactosidases in soils varied considerably (Tables 6 and 7). The percentage inhibition of a-glucosidase activity ranged from 11% with MO(W) to 66% with A&l). However, only Ag(I) and Zn(II) showed inhibition values
Table 3. Correlation coefficients (I)for pairedrelationships bc~cen glycoridaxactivities and organicC or pH in some Iowa soils lb l-GIU’ Soilsample
org. c
Profile slrmples Clarion Harps Mama Marshall Nicollet Surfacesoils
0.94.. 0.98.. 0.94'. 0.99.. 0.82'
of the above
0.58’
(ail samples)
0.65”
z-Gal’
/!I-au PH
-0.94.' -0.31 -0.89.. -0.93.. -0.91.. 0.55’
org. c 0.W' 0.96.' 0.9v* 0.94.. 0.97.. 0.67’.
PH -0.72. -0.39 -0.71* -0.96'. -0.64 0.07
org. c 0.94" 0.95.' 0.98'. 0.94" 0.93.'
B-Gal PH
-0.89" -0.38 -O.Al'* -0.94.. -0.57
O.?S
0.46
0.91.’
-0.61**
Org. C 0.98" 0.99" 0.9s" 0.94" 0.95" 0.75..
PH -0.87" -0.27 -0.84" -0.94.' -0.92** 0.37
profiles plus IO others Pooled
data
soil profile
'Glu -giucosidax.Gal= galactosidase. b*Significant at P
-0.18
0.94”
-0.37.
0.94..
-0.51**
Factors afiecting glycosidascs in soils Tabk
4. Efforts of inorganic
salts on a- and fi-gh~cosidasc
Pcrccntage
inhibition
of glucosidasc
salt
KCI NaCl CaCI, MBCl, KNO, NaNO, NaN, KrSO, (NH,),SD, KHrPQ NaF LSD P < 0.05
activitks
activity
of soils
in soil opccificd
Webster
Ida’ Inorganic
895
z-Glu
pGlU
49 8 65 42 31 27 32 33 IO 45 41
6 2 8 1 10 3 0 7 0 2 7
0.95
I
Okoboji
-Glu
/J-GlU
l-GIU
,9-Glu
31 5 41 20 26 7 10 20 5 20 25
8 I2 I2 4 IS 4 0 8 IS 4 4
45 0 45 27 23 13 I4 31 0 I? 32
I9 16 9 3 I2 0 9 16 16 I2 8
0.48
I.7
1.5
1.7
I.3
‘Glu = glucosidax.
Table
5. Effects of inorganic
salts on a- and B-galactosidax
Percentage
inhibition
of galactosidase
Ida’ Inorganic
sail
s -Gal
Okoboji
a -Gal
B-Gal
a -Gal
/?-Gal
47 6 54 23 3s I2 4 31 I6 20 26
I7 6 44 33 33 IO 5 II I5 6 0
41 I3 31 II 33 8
I6 20 24 IO 36 0 IO 33 16 24 0
25 I5 6 0 20 5 0 22 5 5 0
6. I
LSD P < 0.05
of soils
in soil specified
Webster g-Gal
50 4 70 42 24 50 4 3x I? 25 17
KCI NaCl CaCI, MgCl, KNO, NaNO, NaN, KJO, (NH,)@, KtI:PO, NaF
activities activity
4.3
I.6
3: I7 25 25
4.5
1.7
1.6
‘Gal = g~lactosidase.
Table 6. Elfccls of trwc elements on z- and ,Y-glucosidau
activities
of soils
Percenlage inhibition of a- and flglucosidase activities in soil specified Trace element a-Glucosidax
g-Glucoridase
Oxidation Element
SlillC
Luton
I
66 23
65 I3
I2 7
26 1
Bz Cd co CU FC HI3 Mn l+ Pb acetate Pb nitrate Sn Zn
II
22 49 21 25 38 32 22 45 41 41 I6 50
28 66 24 49 I4 56 14 65 33 33 28 54
5 I5 4 6 9 9 9 5 3 5 7 6
20 19 IO 16 1 32 I8 I5 29 19 I5 I4
Al As B Ct FC
III
41 25 I2 19 38
23 13 42 59 61
I3 II I5 3 I4
II IO 26 17 23
Se V
IV
I6 37
I4 9
2 20
19 I2
As
V
2s
I3
II
10
MO
VI
II 45
I9 2s
IO I2
I8 I9
W LSD P c 0.05
0.99
I.3
Webster
Lu1on
Webster
Al3 cu
0.83
1.5
F. EIVAZI and M. A. TABATABAI
896
Table 7. Effects of trace elements on I- and B-i!alactosidasc activitiw of soil Percentage inhIbItion of I- and ,9galactosidase a&vi& in soils specified z-Galactosidase
Trace element Element
8-Galactosldaw
Oxidation
Webster
Luron
Webster
Luron
‘Q CU
I
15 49
43 32
64 20
50 17
Ba Cd co Cu FC Hg Mn Ni Pb acetate Pb mtrate so Zn
II
15 52 24 39 53 IO 28 69 23 23 73 53
I5 68 52 52 37 I2 21 73 29 28 71
24 38 14 46 8
4b
32 42 22 22 16 52
26 35 ?I ?I 33 62 19 30 26 26 54 32
Al AS B Cr FC
III
59 54 52 47 65
51 57 57 66 41
22 20 33 41 52
45 60 25 33 32
SC V
IV
67 26
70 24
52 4x
35 47
As
V
46
57
IS
5R
M0 W
VI
73 II
74
52
23
LSD P < 0.05
250% for the activity of this enzyme in Webster soil. The inhibition of this enzyme in Luton soil ranged from 9% with V(IV) to 66% with Cd(II). but only Ag(1). Cd(II). Hg(II). Ni(II), Zn(Il), Cr(lII) and Fc(lll) inhibited the activity of this enzyme by 2 50% (Table 6). In general. the inhibition values of /I-glucosidase by trace elements were much lower than those obtained for z-glucosidasc activity (Table 6). The inhibition of b-glucosidase activity in Webster soil ranged from 2% with Sc(IV) to 20% with V(IV). The inhibition values of this enzyme in Luton soil ranged from 7% with Cu(I) to 32% with Hg(II). The inhibition values of this enzyme in Webster soil by trace elements were much lower than those obtained for Luton soil. The magnitude of inhibition of Z- and /j-glucosidases by trace elements was related to the level of activity present in soils; the higher the activity the lower was the percentage of inhibition. The inhibition values of z-galactosidase in Webster soil ranged from 10% with Hg(lI) lo 73% with Sn(Il) (Table 7). The inhibition values of this enzyme in Luton soil ranged from 12% with Hg(ll) to 74% with Mo(VI). The inhibition values of Q-galactosidase in Webster soil ranged from 8% with Fe(II) lo 88% with Hg(lI) and, for this enzyme in Luton soil, ranged from 15% with W(V1) to 62% with Hg(II). In gcncral. the inhibition values of z-galactosidase activity were greater than those of /3-galactosidase activity in these soils. The pH values of the trace element solution varied considerably. They ranged from 2.1 for the Sn(Il) solution to 9.6 for the As(II) and B(III) solutions. Tests showed that, with the use of buffer pH 6.0, the deviation in pH values resulting from addition of
24
I.1
I.1
88
9
I?
1.5
1.2
tract‘ elements in the prescncc of universal buffer did not exceed 20.2 pH unit. REFERENCES Allaway W. H. (1971) Feed and food quality in relation IO fertilizer use. In FrrfilPer Technology und Use (R. A. Olson, Ed.), pp. 533-557. Soil Science Socicly of America. Madison. Bahl 0. P. and Agrawal K. M. L. (1971) z-Galactosidase. p-glucosidase, and S-N-acetylglucosamidase from Asporgillus niger. In Methods in En:ymology, Vol. 28. (V. Ginsburg, Ed.). pp. 728-734. Academic Press. New York. Berrow M. C. and Webber J. (1972) Trace elrmcnts in sewage sludges. Journul of rhe Science of Food and Agriculrure 23. 93-100. Dey P. M. and Pridham J. B. (1972) Biochemistry of z-galactosidases. In Adwnces in Enzymolog)~. Vol. 36, (A. Meiser, Ed.), pp. 91-130. Wiley, New York. Dick R. P. and Tabatabai M. A. (1986) Hydrolysis of polyphosphates in soils. Soil Science 142, 132-140. Eivazi F. and Tabatabai M. A. (1988) Glucosidases and galactosidases in soils. Soil Biology und Biochemi.rrr.t~ 20,
601406. Kuprevich V. F. and Shchrebakova T. A. (1971) Compararive enzymatic activity in diverse types of soil. In Soil Biochemirrry. Vol. 2. (A. D. McLaren and 1. Skujins. Eds). pp. 167-201. Marcel Dekker, New York. LagerwerfT J. V. (1972) Lead, mercury, and cadmium as environmental contaminants. In Micronurrienrs in Agriculture (J. J. Mortvedt. P. M. Giordano and W. L. Lindsay, Eds). pp. 593-636. Soil Science Society of America. Madison. Ross D. J. (1965) Effect of air-drying, refrigerated. and frozen storage on activities of enzymes hydrolyzing sucrose and starch in soil. Journal of Soil Science 16, 86-94. Skujins J. J. (1967) Enzymes in soil. In Soil Biochemistry. Vol. I (A. D. McLaren and G. H. Peterson. Eds), pp. 371-414. Marcel Dekker. New York.
Factors affecting glycosidases in soils Skujins J. (1976) Extracellular enzymes in soil. Crirical Reviews in Microbiology 4, 383421. Tabatabai M. A. (1982) Soil enzymes. In Merhodc of Soil Anu~ysis, Parr 2 (A. L. Page, R. H. Miller and D. R. Kecney, Eds). pp. 903447, Agronomy 9. Soil Science Society of America. Madison. Tabatabai M. A. and Bremner J. M. (1970) Factors affecting soil arylsulfatase activity. Soil Science Sociery 0fAmerico Proceedings 34, 427-429.
897
Tabatabai M. A. and Frankenbergcr W. T. Jr (1979) Chemical composition of sewage sludges in Iowa. Iowa Agriculture and Home Economics Experimenr Srarion Research Bulferin 586.
Tyler G. (1976) Influence of vanadium on soil phosphatase activity. Journal of &nt.+ronmenral Qualiry 5, 2 16-2 Il. Wallenfels K. and Weil R. (1972) p-Galactosidase. In The Enzymes, 3rd edn, Vol. 7 (P. D. Boyer, Ed.), pp. 617-663. Academic Press, New York.
0038-07t7 90 53.00 + 0.00 Copyright C 1990 Pergamon Press pk
1990
FACTORS AFFECTING GLUCOSIDASE AND GALACTOSIDASE ACTIVITIES IN SOILS* F. ElvAzrt Department
of Agronomy,
and M. A. TABATABAI~
Iowa State University.
Ames,
IA 5001 I. U.S.A.
(Accepfed 5 June 1990) Summary-This study was carried out to assess the effects of soil treatments. the addition of I2 inorganic salts and of 20 trace elements on Z- and B-glucosidase and z-and D-galactosidase activities in soils. Results showed that air-drying of field-moist soils resulted in marked increases in the activity of these enzymes and that steam sterilization of soils completely destroyed the activities. In general. the inorganic salts were stronger inhibitors of x-glucosidase and a-galactosidase activities (range 0 to 70%) than the activities of their b counterparts (range 0 to 44%). The effect of trace elements on the activities of these enzymes varied considerably among the elements, enzymes and soils. The percentage inhibition of a-glucosidase activity in Webster soil ranged from 4% with Mo(VI) to 66% with Ag(1) and in Luton soil ranged from 9% with V(IV) to 66% with Cd(ll). In general, the inhibition values of B-glucosidase activity by trace elements were much lower than those obtained for a-glucosidase activity. The inhibition values of z-galactosidase activity in Webster soil ranged from 10% with Hg(l1) to 13% with Sn(Il). The inhibition values for this enzyme in Luton soil ranged from 12% with Hg(ll) to 73% with Mo(V1). Studies on the distribution of the activities of these enzymes showed that they are concentrated in surface soils and decrease with depth. The activities of these enzymes were significantly correlated with organic C in soil profiles and surface soils and were. in most cases, significantly but negatively correlated with pH.
INTWOIWCTION
The term “tract elcmcnt” is used here to refer to elements that are. when prcscnt in sufficient conccntrations, toxic to living systems. Some of these elements (e.g. Pb. V) are present in fuel oils and gasoline, emitted into the atmosphere upon burning and deposited on soils, especially near urban areas (Lagerwerff, 1972; Tyler, 1976). Although the patterns of distribution of activities of several enzymes in soil profiles have been reported (Tabatabai, 1982), little information is available about the relative factors that affect glucosidasc and galactosidase activities through the profile. We used a method for assaying the activities of glucosidases and galactosidases in soils (Eivazi and Tabatabai. 1988) to study the effects of soil trcatments, inorganic salts and trace elements on the activities of these enzymes in soils. We also determined the distribution of glucosidase and galactosidase activities in a range of agricultural soil profiles and also the relationship between the activities of these enzymes and organic C and pH of both surface and subsurface soil samples.
The cnzymcs acting on glycosyl compounds (EC 3.2). including glycosidc hydrolyses (EC 3.2. I .). arc among the hydrolascs lcast studied in soils. Among thcsc, zand /j-glucosidascs and Q- and /I-galactosidascs arc the most important bccausc their hydrolysis products, sugars, arc important cncrgy sources for microorganisms in soils. Although glucosidascs and galactosidases arc widely distributed in nature (Bahl and Agrawal, 1972; Dey and Pridham, 1972; Wallenfels and Wcil, 1975) and should play a significant role in breakdown of low molecular-weight carbohydrates in soils, producing sugars. littlc information is available about effect of sample handling and addition of inorganic salts and trace elements on the reactions catalyzed by these enzymes. Air-drying of field-moist soils is known to change the activities of a number of enzymes (Tabatabai, 1982). but its effect on the activity of glucosidases and galactosidases is not known. In addition, modern agricultural practices make extensive use of fertilizers added to soils in the form of inorganic compounds. Also. a number of trace elements are added to soils as fertilizers, impurities in fcrtilizcrs or as components of municipal and industrial wastes (Allaway, 1971; Berrow and Webber. 1972: Tabatabai and Frankenberger, 1979).
MATERIALS
AND SIETHODS
The soils used (Table I) were surface samples (0-l 5 cm) selected to include a wide range in pH, texture, organic-matter content and in glucosidasc and galactosidase activities and also to represent important agricultural soils in Iowa. Before use. each soil was sieved (2 mm) in the field-moist condition, and subsamples of the sieved soils were air dried at room temperature (23’C) or steam sterilized. The analyses reported in Table I were performed as described by Dick and Tabatabai (1986). The profile samples were selected to include a
*Journal paper No. J-13857 of the Iowa Agriculture and Home Economics Experiment Station, Ames. Iowa. Project No. 2082. tPresent address: Department of Agriculture, Plant and Soil Science, Lincoln University. Jefferson City, MO 65101. U.S.A. :To whom all correspondence should be addressed. 891
F. EIVAZIand M. A. TABATAILU
892 Table I. Analwes of soils SOll
PH
Organic P/o)
Thurmdn HageM W&r Ida Clanon Webster Nicollet Harps Luton Okobq
7 I 6.4 5.1 80 64 5.8 6. I 7.8 6.8 7.4
0.47 0.92 1.51 I .57 1.77 2.54 3.32 3.74 4.35 5.45
c
Total N (%)
Clay (%)
Sand (Oh)
0.046 0.093 0.131 0.147 0.188 0.210 0.253 0.305 0.388 0.463
5 13 17 27 24 23 30 30 42 34
93 64 I 3 39 38 25 26 3 16
wide range of glucosidase and galactosidase activities in their surface soils and to represent important agricultural soils in Iowa. The inorganic salts and trace elements were Fisher certified reagent-grade chemicals obtained from Fisher Scientific Co., Itasca, Ill. Of the trace elements used, Ag(I), Cd(H), Co(II), Cu(II). Fe(II). Zn(I1) and V(V) were added as the sulfate: Cu(I). Ba(II), Hg(II), Mn(II), Ni(II), Se(I1) AI(II1) and Cr(II1) as the chloride; Pb(I1) as the acetate or nitrate; and As(III), B(II1). Se(IV), As(V). Mo(\‘I) and W(VI) as Na,AsO,, Na,B,O,, H$eO,, Na: HAsO, HLMOO, and Na,WO,, respectively. The assay method (Eivazi and Tabatabai. 1988) involves extraction and calorimetric determination of the p-nitrtiphcnol rclcascd when I g of soil is incubated with 5 ml of butfered p-nitrophcnyl glycoside solution
at 37‘C
for
I h.
In testing the effect of inorganic salts on glucosidase and galactosidasc activities of soils, I g of soil in a 50 ml Erlenmeyer flask was treated with 0.2 ml of tolucne, 4ml of Mod&d Universal Buffer (MUB), pH 6.0 made IO mM with rcspcct to the inorganic salt tcstcd. and I ml of 25 mM substrate made in MUB. pH 6.0. In testing the effects of trace elements on the activity of glucosidases and galactosidases, I g of soil was treated with I ml of solution containing 25 pmol of trace element. After 30 min of equilibration, 3 ml of MUB, pH 6.0, was added, and the enzymes were assayed as described for soils treated with inorganic salts. The results of glycosidase activities of soils treated with inorganic salt and trace elements were compared with those obtained without addition of inorganic salt or trace element. Controls were performed with each soil analyzed to allow for color not derived from p-nitrophenol released by glycosidase activity. All results reported are averages of duplicate determinations expressed on a moisture-free basis, moisture being determined from loss in weight following drying at lO5’C for 24 h.
other factors affecting the glucosidase and galactosidase activities in soils. Similarly, the effect of trace metals added to soils on the activities of several enzymes has been reported, but the effects of such metal on the activity of glucosidases and galactosidases and, consequently on the C cycling in soils are not well understood. For convenience, the results obtained in this study will be discussed according to the factors studied. Effects of pretreatments
Air-drying of field-moist soil resulted in a marked increase in the activity of these enzymes in the five soils studied (Table 2). Steam sterilization of air-dried soils at I2 I “C for I h completely destroyed the activities of these enzymes (not shown in Table 2). Because B-glucosidase activity is dominant among the four enzymes in soils, the effect of pretreatments was more pronounced with the activity of this enzyme. Our results support the available information on the effect of drying on enzyme activities. The effect of pretreatments depended upon the enzyme studied, and no general rule can be established for drying soil samples for assay of enzyme activities (Skujins, 1967). For example, Ross (1965) found that air-drying of soils inactivated the enzyme hydrolyzing sucrose (invertasc) and starch (amylasc), whereas Tabatabai and Bremner (1970) found that it increased soil arylsulfatase activity. This observation was explained by noting that the rcwetting of air-dried soil causes breakdown of aggregates and thus increases the accessibility of soil enzymes to their substrates in assay of soil enzyme activity. Distribution
Enzyme activity in soil-profile samples usually dccrcases with sample depth and is accompanied by a decrease in organic-matter content (Kuprevich and Shchrebakova, 1967). We found that glucosidase and galactosidase activities decreased markedly with depth in each of the five soil profiles examined (Figs I and 2) and was associated with a decrease in organic-C content. Statistical analyses showed that the activities of these enzymes are significantly correlated with organic C in each soil profile sample (Table 3). Also, the activities of a-glucosidase and fi-galactosidase were significantly, but negatively, correlated with pH of soils in the profiles of Clarion, Marna, Table 2. Efiecrs of minor treatment on glucosidaseand galactosidasc activilics of soils Glycosidaw acrivity’ Soil
RESULTS AND DISCUSSION
Reviews about enzymes in soils indicate that studies of origin, location and persistence of enzymes in soils are needed (Skujins, 1967, 1976). The available information indicates that the activities of enzymes such as urease. arylsulfatase. rhodanese. acid and alkaline phosphatases and pyrophosphatase are influenced by methods of handling, storing and treating the sample before enzyme assay (Tabatabai. 1982). but little information is available about these and
in soil projfes
Trcatmcnrb
I-GIU
4.GIU
Z-Gal
B-Gal
FM AD FM AD FM AD FM AD FM
3 7 4 8 IO I3 2 8 I2
62 147 103 169 53 73 90 I26 93
IO I7 23 28 9 22 I2 19 I7
I4 20 IO 13 13 15 8 I5 22
AD
I4
I52
25
28
Clarion Webster Harps Nicollet Okoboii
lpg
ofp-nitrophenol
rclcascd
g-’
soilhe’.
galaclosidase.
“FM = field-moire;
AD
=
ax-dried
Glu = glucosidase;
Gal =
Factors
4.5 r
affecting
6;0
893
glycosidases in soils
7;5
4.5 ,
6;O
7;s
FH
GLYCOSIOASE ACTIVITY
60
CLARION
SOIL
t-
HARPS
SOIL
60
I
MARNA
RSHALL
SOIL
SOIL
. a-GLUCOSIDASE o j3-GLUCOSIOASE A ORGANIC 60
CARBON
A PH
t-
NICOLLET
SOIL
of
Fig. I. Distribution organicC, OI-and /I-glucosidase activities and trends of pH with depth in soil profile samples. Clucosidase activity is expressed as pg of p-nitrophenol released g-’ of soil h- ’ at 37’C.
Marshall and Nicollet soils. /I-Glucosidase and a-galactosidase activities were significantly, but negatively, correlated with soil pH of the profiles of Clarion, Marna and Marshall soils. Statistical analyses, however, showed that, when enzyme activities of the surface soils of the five profiles were pooled with those of IO other surface soils, the activities of only a- and j?-glucosidase and ,9-galactosidase were significantly correlated with organic C. No such correlations were obtained between z-galactosidase and organic C in the surface soils examined. Furthermore, only a-glucosidase activity was significantly correlated with pH of the surface soils tested. Unlike the significant but negative, correlation found for the relationships between glycosidase activities and pH of the soils in the profile studied, the a-galactosidase activity of the surface soils was positively correlated with organic C. Statistical analyses of the pooled data (all soil profile samples) showed that the activities of the four glycosidases studied were significantly correlated with organic C. With the exception of z-glucosidase activity, the activities of the other three glycosidases were significantly, but negatively, correlated with soil pH.
Eflects of inorganic salts
The effects of inorganic salts (the incubation medium made 8 mM with respect to the inorganic salt) on glucosidase and galactosidase activities in the soils are shown in Tables 4 and 5, respcctivcly. Generally, the inorganic salts studied were more effective in inhibiting a-glucosidase and a-galactosidase activities than /I-glucosidase and b-galactosidase activities. With a few exceptions, all the inorganic salts tested partly inhibited the activity of these enzymes in soils. From these results, it is clear that the addition of inorganic salts as fertilizer materials or accumulation from irrigation water could have a profound effect on the activities of glycosidases in soils and, therefore, on C cycling. Eflecrs of trace elements on glJcosidases in soils
Metal ions may inhibit enzyme reactions by complexing the substrate by combining with the protein active group of the enzymes or by reacting with the enzyme-substrate complex. The mode of inhibition is dependent on the tyw of substrate used. Metal ions generally are assumed to inactivate enzymes by reac-
894
F. EIVAZIand M. A. TABATABAI PH
4.5 l
ORGANIC 0 C (%I I 0 GLYCOSIOASE ACTIVITY
6.0 I 1.6 ,
1
I
10
7.5 I 3.2 I
4.5 I 0 I,
20
6.0 I 1.6 I
I
300
7.5 1 3.2 ,
I
10
,
20
30
60
h CLARION
t-
SOIL
HARPS
SOIL
MARSHALL
SOIL
60
c
30
.a o
-GALACTOSIOASE /!?-GALACTOSIDASE
A ORGANIC
60
A
CARBON
P"
90
NICOLLET Fig. 2. Distributionof organic
profile samples. Galactosidase
SOIL
C, I- and @-galactosidase activities and trends of pH with depth in soil activity is expressed as pg ofp-nitrophenol released g-’ of soil h“ at 37’C.
tion with sulfhydryl groups, a reaction analogous to the formation of a metal sulfide. Sulfhydryl groups in enzymes may serve as integral parts of the catalytitally-active sites or as groups involved in maintaining the correct structural relationship of the enzyme protein.
The effect of trace elements on the activities of aand /I-glucosidases and I- and p-galactosidases in soils varied considerably (Tables 6 and 7). The percentage inhibition of a-glucosidase activity ranged from 11% with MO(W) to 66% with A&l). However, only Ag(I) and Zn(II) showed inhibition values
Table 3. Correlation coefficients (I)for pairedrelationships bc~cen glycoridaxactivities and organicC or pH in some Iowa soils lb l-GIU’ Soilsample
org. c
Profile slrmples Clarion Harps Mama Marshall Nicollet Surfacesoils
0.94.. 0.98.. 0.94'. 0.99.. 0.82'
of the above
0.58’
(ail samples)
0.65”
z-Gal’
/!I-au PH
-0.94.' -0.31 -0.89.. -0.93.. -0.91.. 0.55’
org. c 0.W' 0.96.' 0.9v* 0.94.. 0.97.. 0.67’.
PH -0.72. -0.39 -0.71* -0.96'. -0.64 0.07
org. c 0.94" 0.95.' 0.98'. 0.94" 0.93.'
B-Gal PH
-0.89" -0.38 -O.Al'* -0.94.. -0.57
O.?S
0.46
0.91.’
-0.61**
Org. C 0.98" 0.99" 0.9s" 0.94" 0.95" 0.75..
PH -0.87" -0.27 -0.84" -0.94.' -0.92** 0.37
profiles plus IO others Pooled
data
soil profile
'Glu -giucosidax.Gal= galactosidase. b*Significant at P
-0.18
0.94”
-0.37.
0.94..
-0.51**
Factors afiecting glycosidascs in soils Tabk
4. Efforts of inorganic
salts on a- and fi-gh~cosidasc
Pcrccntage
inhibition
of glucosidasc
salt
KCI NaCl CaCI, MBCl, KNO, NaNO, NaN, KrSO, (NH,),SD, KHrPQ NaF LSD P < 0.05
activitks
activity
of soils
in soil opccificd
Webster
Ida’ Inorganic
895
z-Glu
pGlU
49 8 65 42 31 27 32 33 IO 45 41
6 2 8 1 10 3 0 7 0 2 7
0.95
I
Okoboji
-Glu
/J-GlU
l-GIU
,9-Glu
31 5 41 20 26 7 10 20 5 20 25
8 I2 I2 4 IS 4 0 8 IS 4 4
45 0 45 27 23 13 I4 31 0 I? 32
I9 16 9 3 I2 0 9 16 16 I2 8
0.48
I.7
1.5
1.7
I.3
‘Glu = glucosidax.
Table
5. Effects of inorganic
salts on a- and B-galactosidax
Percentage
inhibition
of galactosidase
Ida’ Inorganic
sail
s -Gal
Okoboji
a -Gal
B-Gal
a -Gal
/?-Gal
47 6 54 23 3s I2 4 31 I6 20 26
I7 6 44 33 33 IO 5 II I5 6 0
41 I3 31 II 33 8
I6 20 24 IO 36 0 IO 33 16 24 0
25 I5 6 0 20 5 0 22 5 5 0
6. I
LSD P < 0.05
of soils
in soil specified
Webster g-Gal
50 4 70 42 24 50 4 3x I? 25 17
KCI NaCl CaCI, MgCl, KNO, NaNO, NaN, KJO, (NH,)@, KtI:PO, NaF
activities activity
4.3
I.6
3: I7 25 25
4.5
1.7
1.6
‘Gal = g~lactosidase.
Table 6. Elfccls of trwc elements on z- and ,Y-glucosidau
activities
of soils
Percenlage inhibition of a- and flglucosidase activities in soil specified Trace element a-Glucosidax
g-Glucoridase
Oxidation Element
SlillC
Luton
I
66 23
65 I3
I2 7
26 1
Bz Cd co CU FC HI3 Mn l+ Pb acetate Pb nitrate Sn Zn
II
22 49 21 25 38 32 22 45 41 41 I6 50
28 66 24 49 I4 56 14 65 33 33 28 54
5 I5 4 6 9 9 9 5 3 5 7 6
20 19 IO 16 1 32 I8 I5 29 19 I5 I4
Al As B Ct FC
III
41 25 I2 19 38
23 13 42 59 61
I3 II I5 3 I4
II IO 26 17 23
Se V
IV
I6 37
I4 9
2 20
19 I2
As
V
2s
I3
II
10
MO
VI
II 45
I9 2s
IO I2
I8 I9
W LSD P c 0.05
0.99
I.3
Webster
Lu1on
Webster
Al3 cu
0.83
1.5
F. EIVAZI and M. A. TABATABAI
896
Table 7. Effects of trace elements on I- and B-i!alactosidasc activitiw of soil Percentage inhIbItion of I- and ,9galactosidase a&vi& in soils specified z-Galactosidase
Trace element Element
8-Galactosldaw
Oxidation
Webster
Luron
Webster
Luron
‘Q CU
I
15 49
43 32
64 20
50 17
Ba Cd co Cu FC Hg Mn Ni Pb acetate Pb mtrate so Zn
II
15 52 24 39 53 IO 28 69 23 23 73 53
I5 68 52 52 37 I2 21 73 29 28 71
24 38 14 46 8
4b
32 42 22 22 16 52
26 35 ?I ?I 33 62 19 30 26 26 54 32
Al AS B Cr FC
III
59 54 52 47 65
51 57 57 66 41
22 20 33 41 52
45 60 25 33 32
SC V
IV
67 26
70 24
52 4x
35 47
As
V
46
57
IS
5R
M0 W
VI
73 II
74
52
23
LSD P < 0.05
250% for the activity of this enzyme in Webster soil. The inhibition of this enzyme in Luton soil ranged from 9% with V(IV) to 66% with Cd(II). but only Ag(1). Cd(II). Hg(II). Ni(II), Zn(Il), Cr(lII) and Fc(lll) inhibited the activity of this enzyme by 2 50% (Table 6). In general. the inhibition values of /I-glucosidase by trace elements were much lower than those obtained for z-glucosidasc activity (Table 6). The inhibition of b-glucosidase activity in Webster soil ranged from 2% with Sc(IV) to 20% with V(IV). The inhibition values of this enzyme in Luton soil ranged from 7% with Cu(I) to 32% with Hg(II). The inhibition values of this enzyme in Webster soil by trace elements were much lower than those obtained for Luton soil. The magnitude of inhibition of Z- and /j-glucosidases by trace elements was related to the level of activity present in soils; the higher the activity the lower was the percentage of inhibition. The inhibition values of z-galactosidase in Webster soil ranged from 10% with Hg(lI) lo 73% with Sn(Il) (Table 7). The inhibition values of this enzyme in Luton soil ranged from 12% with Hg(ll) to 74% with Mo(VI). The inhibition values of Q-galactosidase in Webster soil ranged from 8% with Fe(II) lo 88% with Hg(lI) and, for this enzyme in Luton soil, ranged from 15% with W(V1) to 62% with Hg(II). In gcncral. the inhibition values of z-galactosidase activity were greater than those of /3-galactosidase activity in these soils. The pH values of the trace element solution varied considerably. They ranged from 2.1 for the Sn(Il) solution to 9.6 for the As(II) and B(III) solutions. Tests showed that, with the use of buffer pH 6.0, the deviation in pH values resulting from addition of
24
I.1
I.1
88
9
I?
1.5
1.2
tract‘ elements in the prescncc of universal buffer did not exceed 20.2 pH unit. REFERENCES Allaway W. H. (1971) Feed and food quality in relation IO fertilizer use. In FrrfilPer Technology und Use (R. A. Olson, Ed.), pp. 533-557. Soil Science Socicly of America. Madison. Bahl 0. P. and Agrawal K. M. L. (1971) z-Galactosidase. p-glucosidase, and S-N-acetylglucosamidase from Asporgillus niger. In Methods in En:ymology, Vol. 28. (V. Ginsburg, Ed.). pp. 728-734. Academic Press. New York. Berrow M. C. and Webber J. (1972) Trace elrmcnts in sewage sludges. Journul of rhe Science of Food and Agriculrure 23. 93-100. Dey P. M. and Pridham J. B. (1972) Biochemistry of z-galactosidases. In Adwnces in Enzymolog)~. Vol. 36, (A. Meiser, Ed.), pp. 91-130. Wiley, New York. Dick R. P. and Tabatabai M. A. (1986) Hydrolysis of polyphosphates in soils. Soil Science 142, 132-140. Eivazi F. and Tabatabai M. A. (1988) Glucosidases and galactosidases in soils. Soil Biology und Biochemi.rrr.t~ 20,
601406. Kuprevich V. F. and Shchrebakova T. A. (1971) Compararive enzymatic activity in diverse types of soil. In Soil Biochemirrry. Vol. 2. (A. D. McLaren and 1. Skujins. Eds). pp. 167-201. Marcel Dekker, New York. LagerwerfT J. V. (1972) Lead, mercury, and cadmium as environmental contaminants. In Micronurrienrs in Agriculture (J. J. Mortvedt. P. M. Giordano and W. L. Lindsay, Eds). pp. 593-636. Soil Science Society of America. Madison. Ross D. J. (1965) Effect of air-drying, refrigerated. and frozen storage on activities of enzymes hydrolyzing sucrose and starch in soil. Journal of Soil Science 16, 86-94. Skujins J. J. (1967) Enzymes in soil. In Soil Biochemistry. Vol. I (A. D. McLaren and G. H. Peterson. Eds), pp. 371-414. Marcel Dekker. New York.
Factors affecting glycosidases in soils Skujins J. (1976) Extracellular enzymes in soil. Crirical Reviews in Microbiology 4, 383421. Tabatabai M. A. (1982) Soil enzymes. In Merhodc of Soil Anu~ysis, Parr 2 (A. L. Page, R. H. Miller and D. R. Kecney, Eds). pp. 903447, Agronomy 9. Soil Science Society of America. Madison. Tabatabai M. A. and Bremner J. M. (1970) Factors affecting soil arylsulfatase activity. Soil Science Sociery 0fAmerico Proceedings 34, 427-429.
897
Tabatabai M. A. and Frankenbergcr W. T. Jr (1979) Chemical composition of sewage sludges in Iowa. Iowa Agriculture and Home Economics Experimenr Srarion Research Bulferin 586.
Tyler G. (1976) Influence of vanadium on soil phosphatase activity. Journal of &nt.+ronmenral Qualiry 5, 2 16-2 Il. Wallenfels K. and Weil R. (1972) p-Galactosidase. In The Enzymes, 3rd edn, Vol. 7 (P. D. Boyer, Ed.), pp. 617-663. Academic Press, New York.