Extraction of Phosphatase, Urease, Proteases, Organic Carbon, and Nitrogen from Soil

1
PAOLO NANNI PI ERI , B RUNELLO CECCANTI, STEFANO CERVELLI, AND EMILIO MATARESE
2
ABSTRACT
Extractions of three soils at different incubation times
demonstrate that 0.14M sodium pyrophosphate at pH 7.1 ex-
tracts phosphatase, urease, casein, and benzoylarginamide hy-
drolyzing proteases. Extraction yields of phosphatase and
casein-hydrolyzing proteases are remarkably high. The most
efficient length of extraction depends both on the type of soil
and on the selected enzyme.
Correlation analyses of extracted enzymes and extracted C
and organic N content generally show a significant correlation
(p = 0.05 and 0.01).
Additional Index Words: soil proteins, humus, organic N
resistance, N mineralization and immobilization.
Nannipieri P., B. Ceccanti, S. Cervelli, and E. Matarese. 1980.
Extraction of phosphatase, urease, proteases, organic carbon,
and nitrogen from soil. Soil Sci. Soc. Am. J. 44:1011-1016.
E
X TRACTION OF SOIL ENZ Y MES in high yields is an
obligatory step that precedes investigations of their
physical and chemical state. B ecause of strong sorptive
and other chemical interactions between proteins and
soil colloids it has been difficult to extract active en-
zymes by using extractants normally employed in bio-
chemistry. Unsuccessful attempts (Conrad, 1940)
(Haig, 1955)
3
or extraction of small amounts of en-
zymes (Briggs and Segal, 1963; McLaren, 1972) have
been reported. Soil enzymes can however be extracted
with high yields under conditions which solubilize the
soil colloids responsible for stabilization of proteins
(Ladd, 1972; Ceccanti et al., 1978) and which are mild
enough to avoid disruption of microorganisms.
Past studies have emphasized the role of inorganic
colloids in stabilizing proteins including enzymes
(McLaren and Estermann, 1956; Estermann et al.,
1959; Aomine and Kobayashi, 1964; Sorensen, 1969).
It is somewhat surprising that only recently the atten-
tion has been directed toward the possible occurrence
of humus-enzymes complexes in soils (Chalvignac and
Mayaudon, 1971; B urns et al., 1972a, 1972b; Skujins,
1967; Ladd and B utler, 1975; Nannipieri et al., 1974,
1975, 1978), especially because a significant proportion
of humic acid-N is accounted for as amino acid-N after
hydrolysis of peptide bonds and since humic com-
pounds have been considered to play an important role
in conferring stability on soil nitrogenous components
(B remner, 1965; Ladd and B utler, 1975).
B remner and Lees (1949) proposed to use 0.1M
sodium pyrophosphate at pH 7 for extracting organic
matter under mild conditions. Recently Nannipieri
et al. (1974, 1975) have reported that this reagent is
effective enough considering currently available me-
thodologies in extracting soil urease and that (under
the employed experimental conditions) ureolytic mi-
1
Contribution from the Laboratory for Soil Chemistry, C.N.R.,
via Corridoni, 78, Pisa, Italy. Scientific Paper no. 125. Partly
based on the junior author's M.S. thesis. Received 13 Aug. 1979.
Approved 3 June 1980.
"Soil Scientists and Graduate Student, respectively.
"A. D. Haig. 1955. Some characteristics of esterase and urease
like activity in the soil. Ph.D. Diss., Univ. of California, Davis.
croorganisms seem to be unaffected by the extractant.
The objective of this research was to ascertain the
efficacy of pyrophosphate for extraction of urease,
phosphatase, and casein- and benzoylarginamide-hy-
drolyzing proteases from three different organic soils,
and to compare the yields of extracted enzymes with
those of C and organic N.
MATERIALS AND METHODS
Soil samples whose characteristics are listed in Table 1 were
air-dried, crushed to pass a 2-mm mesh sieve, and stored in
sealed plastic bags at 2 C until use. All three samples were col-
lected from Al horizons of soils located in Apuan Alps (Italy);
the first was sampled under beech-coppice (Pania Nord-PN);
the second under meadow (Pania Sud-PS), and the third under
chestnut (Alpe S. Antonio-AS).
Enzyme Extraction
Soil samples were extracted with 0.14Af sodium pyrophos-
phate at pH 7.1 (soil/solution ratio 1:10) at 37C for different
times (10, 30, 60 min and 2, 3, 4, 6, 10, 24 hours) in a shaking
water bath. Centrifugation and bacteriological filtration were
carried out as already reported (Nannipieri et al., 1974). Ex-
tracts were stored at 2 C for 1 or 2 days prior to assay.
Soil Enzyme Assays
The procedure reported by Nannipieri et al. (1979) for the
assay of soil urease and phosphatase activities were slightly
modified. Owing to different soil properties, a higher urea
concentration (1.067M) and a higher amount of maleate buffer
(4 ml) were used in these assays. The determination of casein
hydrolysis was carried out according to Nannipieri et al. (1979).
The addition of 7 ml of 3,5 X lO'W Na-jCO., and 1 ml of 4.0
X 10'W CuSOj when assaying PS soil, gave rise to a precipitate
and therefore it was necessary to centrifuge at 5.000 X g for 3
min before adding the Folin reagent. N-benzoyl L-arginine
amide (B AA) hydrolysis was determined by adding 0.5 ml of
0.03M BAA and 1.5 ml of 0.1M pH 7.1 phosphate buffer to
0.5 g of air-dried soil (Ladd and B utler, 1972). After incuba-
tion in a shaking water bath at 40C, 20 ml of 2M KC1 were add-
ed and the mixture was filtered immediately. Ammonium was
determined with an ammonium electrode (Orion Research,
Cambridge, Mass.). Two types of control were prepared: either
substrate or soil were substituted with distilled water.
In all assays soil activity vs. time was determined; the re-
ported values are the' mean of at least three measurements.
Assays of Extracted Enzymes
UreaseReaction mixtures for urea hydrolysis consisted of
0.5 ml of extract, 0.5 ml of 0.1M pH 7.1 phosphate buffer,
and 0.5 ml of 1.067M urea. After 2 hours at 37C, 2.5 ml of
2M KC1 were added to the mixtures. Ammonium was deter-
mined by the colorimetric indophenol method as already re-
ported (Nannipieri et al., 1979). In the control, urea solution
was added to mixtures after the addition of KC1.
PhosphataseOne milliliter of substrate (0.115M p-nitro-
phenylphosphate) and 3.5 ml of 0.1M pH 6.5 maleate buffer
were added to 0.5 ml of soil extract. After incubation at 27 C
for 1 hour, 1 ml of 0.5M CaCl
2
and 4 ml of 0.5M NaOH were
added to the mixture. Samples were filtered and determination
of activities was carried out as described for soil enzyme assay.
In the control, substrate was added after the addition of CaCL
and NaOH and before the filtration.
Casein HydrolysisOne milliliter of soil extract was added
to 2.5 ml of 1% casein dissolved in 0.1M pH 8.1 tris-HCl buffer.
Reaction mixtures were incubated for 2 hours at 50C. At the
end of incubation, 2 ml of 17.5% trichloracetic acid was added
to precipitate proteins and the procedure followed for the colori-
metric determination was the same as that reported for the
soil enzyme essay. Activity was computed after correction for
1011
Published September, 1980
1012
SOIL SCI. SOC. AM. J., VOL. 44, 1980
Table 1Characteristics and activities of soils.
Soil location
PaniaSud(PS)
PaniaNord(PN)
AlpeS. Antonio (AS)
Order
(great soil group)
Mollisol(RendoU)
Histosol(Cryofolist)
Alfisol (Umbraqualf)
pH
7.3
5.3
5.3
C
15.1
53.5
3.6
N
1.20
1.00
0.18
Clay
%
20.3
26.0
33.3
Silt
27.3
55.2
39.5
Sand
52.4
18.8
27.2
Ureaset
65.3
157.1
36.8
Phos-
phateaset
2.3
22.5
5.4
Casein
hydrolysis
2.2
3.0
1.3
B AA
hydrolysis!
29.9
76.4
7.6
t Urease activity is expressed as pmoles of NH,' produced per gram of dry soil and per 2 hours,
t Phosphatase activity is expressed as ftmoles of p-nitrophenol produced per gram of dry soil and per hour.
Casein hydrolysis is expressed as pinoles of tyrosine produced per gram of dry soil and per 2 hours.
1 BAA hydrolysis is expressed as pmoles of N-NH,* produced per gram of dry soil and per 90 min.
the control, in which the substrate was added after trichloracetic
acid.
BAA HydrolysisOne milliliter of 0.1M pH 7.1 phosphate
buffer and 0.5 ml of 0.03M BAA were added to 0.5 ml of soil
extract. Reaction mixtures were incubated for 90 min at 40C
and at the end of incubation diluted with 20 ml of distilled
water. Ammonium was determined by means of ammonium
electrode. In the control, substrate was added after water.
All enzymatic incubations were carried out in a water-shaking
bath; the values reported are the mean of at least three measure-
ments. Standard deviation is 0.18 for urease, 0.2 for phosphatase,
and 0.1 and 0.16 for casein and B AA hydrolyzing proteases, res-
pectively. Activities of extracted enzymes vs. time were linear
under the employed experimental conditions.
Chemical Analyses
Organic C was determined by oxidizing 0.5 g of soil or 2
ml of soil extract with potassium dichromate as already re-
ported by Lotti and Galoppini (1967). Total N analyses (or-
ganic N + ammonium) were conducted as reported (B remner,
1965). Organic N was determined by subtracting ammonium-N
from total N.
RESULTS
Pania Nord soil showed the highest enzymatic acti-
vities (Table 1). Differences among values of urease,
phosphatase, and activities hydrolyzing B AA were fair-
ly large while those among casein-hydrolyzing activi-
ties were less marked.
Lafleur (1969) reported that alkaline extraction of
organic matter by sodium pyrophosphate is strongly
time dependent. On prolonging extraction time, the
percentage of extracted organic C increased up to 10
hours in AS soil and up to 24 hours in PS and PN
soils (Table 2). However while yields in PS and AS
soils accounted for about 40%, in PN soil only 11%
of organic C was brought into solution by sodium py-
rophosphate. The organic matter in PN soil consists
mostly of partially decomposed plant fragments (soil
C/N ratio =53.5).
The highest percentage of organic N was extracted
from PN and PS soils during the 0 to 24 hours incuba-
tion time (Table 2). However longer extraction pe-
riods were not tested because it had been previously
shown that total microbial number increased marked-
ly as the incubation period was raised above 24 hours
(Arcara et al., 1974). The percentage of organic N
extracted from PN and AS soils was twice as much
as that from PS soil. Consequently the C/N ratios in
PS extracts were generally highest. During the first
10 min the pyrophosphate preferably extracted or-
ganic N because C/N ratios were lowest (Table 2).
The percentage of phosphatase extracted from PS
soil increased fourfold from 10 min to 2 hours, after
which activity extracted remained constant (Table
3). Additional extraction of phosphatase from AS and
PS soils continued up to 3 and up to 6 hours, respec-
tively. Phosphatase activities of the three extracts
ranged from 31 to 66% of those of soils and were thus
considerably higher than those of extracted urease
(5-9%).
In terms of urease extracted the chemical process
proposed by Burns et al. (1972), has proven the most
effective since extracted enzyme averaged 16% of the
total soil extracellular activity (Pettit et al., 1976).
B y using neutral sodium pyrophosphate Nannipieri et
al. (1974) extracted only 13% of the extracellular
fractions while Lloyd (1975) reported an efficiency of
extraction of 8-11%. Additional extraction of soil
urease continued up to 24 hours in PS and PN soils
and up to 4 hours in AS soil (Table 3).
The highest extraction of casein-hydrolyzing pro-
teases on the basis of the relative activities of extract
and soil occurred in PN soil (Table 3). The propor-
tion of enzyme solubilized by pyrophosphate from AS
and PS soils was half of that extracted from PN soil.
After 3 hours no further enzyme was extracted; acti-
vities in PN extracts decreased and probably precipita-
tion or inhibition of enzyme occurred.
The poorest extraction of enzyme-hydrolyzing BAA,
which was only 15%, was observed in PS soil (Table
3). Pyrophosphate extraction was slightly more effec-
tive (about 24%) in PN soil. Markedly higher ex-
traction percentages were observed in AS soil. A
decrease in extracted activities occurred after 3 and 4
hours in PN and AS soils, respectively.
Specific activities were referred both to organic
C and N, as already reported (Ceccanti et al., 1978;
B atistic et al., 1980). The lowest values were observed
in PS extracts (Fig. 1, 2, 3, and 4). Examination of
Fig. 1 reveals that phosphatase/C ratios peaked at 10
min and 2 to 3 hours in PN and AS extracts, respec-
tively. At 10 min preferential extraction of organic N
with respect to phosphatase occurred in all three soils.
After this, enzyme/N ratios increased. In PN extracts
the ratio peaked at 4 hours and thereafter decreased
rather markedly, so at longer extraction times organic
N may be again extracted preferentially (Fig. 1).
Table 2Percentage of C and organic N extracted by
_____pyrophosphate and C/N ratios in the extracts._____
Carbon Nitrogen C/N
Extraction
time PS PN AS PS PN AS PS PN AS
10 min
30 min
60 min
2 hours
3 hours
4 hours
5 hours
10 hours
24 hours
7.9
15.6
25.5
28.8
30.0
32.2
33.5
37.2
42.8
4.9
7.3
8.1
8.3
8.9
9.1
9.4
10.2
11.0
14.8
20.7
25.1
27.4
30.7
37.7
37.7
41.1
41.1
10.2
11.2
12.2
12.2
12.7
25.5
15.5
17.0
19.5
17.0
17.2
18.1
21.1
21.1
21.9
23.8
30.5
38.1
24.5
25.1
28.0
30.9
33.8
33.8
35.0
36.1
36.1
9.8
17.7
26.6
30.1
30.0
26.3
27.4
27.9
27.9
15.9
23.4
24.7
21.7
23.3
22.9
21.6
18.3
15.9
12.6
17.2
18.7
18.5
19.1
23.3
22.5
23.7
23.7
NANNIPIERI ET AL. I EXTRACTION OF PHOSPHATASE, UREASE, PROTEASES, ORGANIC C, AND N 1013
Table 3Percentage of soil phosphatase, urease, casein and BAA hydrolyzing activities extracted by pyrophosphate.
time
lOmin
30min
60mln
2 hours
3 hours
4 hours
6 hours
10 hours
24 hours
Phosphatase
PS
15.2
30.0
43.0
65.6
65.6
65.6
65.6
65.6
65.6
PN
29.4
33.5
38.5
42.4
44.6
47.5
50.4
52.4
52.4
AS
13.7
16.3
23.1
27.2
30.7
30.7
30.7
30.7
30.7
PS
1.5
1.6
2.4
3.3
3.3
4.5
4.5
5.2
8.1
Urease
PN
1.0
1.5
1.8
2.6
2.8
2.8
2.8
3.8
5.4
Casein hydrolysis
AS
1.8
2.6
3.1
3.9
4.3
8.6
8.6
8.6
8.6
PS
0
49.1
55.4
55.4
61.4
61.4
61.4
61.4
61.4
PN
76
111
123
129
140
111
111
111
111
AS
30.0
45.4
45.4
53.1
56.9
56.9
56.9
56.9
56.9
BAA hydrolysis
PS
5.9
6.5
7.4
9.0
10.4
11.7
13.6
14.7
14.7
PN
9.0
17.1
17.5
19.7
23.9
22.2
22.2
21.4
21.0
AS
0
65.5
65.5
65.5
67.8
72.0
67.8
67.8
67.8
Urease specific activities from AS were the highest
(Fig. 2). As a rule urease/carbon ratios increased on
prolonging extraction time and consequently prefer-
ential extraction of enzymes may occur at longer time.
On the contrary, examination of Fig. 3 reveals that
ratios between casein-hydrolyzing activities and C or
N decreased at longer extraction time; the highest
ratios were observed in PN extracts.
The highest specific activities hydrolyzing B AA were
obtained in AS extracts (Fig. 4). Only slight changes
were observed in ratios associated with enzymes ex-
tracted from PS soil. In AS extracts both ratios peaked
at 30 rain and thereafter decreased rather markedly.
Correlation analyses of the data obtained at dif-
ferent times during the extraction of the three soils
showed that a significant correlation at the 1 and 5%
level was the rule while noncorrelation was the ex-
ception. Table 4 shows that activities hydrolyzing
B AA were not significantly correlated with organic
N and urease in PN and AS extracts. It also shows
that casein-hydrolyzing activities were not significant-
ly correlated with organic N and urease in PS and PN
extracts, nor with C and phosphatase in PN extracts or
with activities hydrolyzing BAA in PS extracts.
DISCUSSION
Our results indicate that 0.14M sodium pyrophos-
phate at pH 7.1 extracted phosphatase, urease, casein
and benzoylarginamide hydrolyzing proteases, general-
ly with appreciable yields. They also show that the
most efficient extraction time varied according to soil
type and enzyme.
Significant correlations of activities with extracted
C and N suggest that a major proportion of the humic
compounds was removed from soils with the enzymes.
The results however do not prove that in the extracts
all enzymes are intimately associated with humic
molecules to form humus-enzymes complexes or that
all humus contains active enzyme. The possibility still
remains that enzyme-clay complexes, free enzymes and
enzyme-free humic molecules may also exist in the
extracts. More direct evidence based on the proper-
ties of isolated humus-enzyme preparations is required.
4 6 8 10
Extraction time (hours)
24
Fig. 1Phosphatase/C and phosphatase/N ratios in the extracts
at different extraction times.
AS extract
AS extract
\
PN extract
PS extract
Vr-
4 6 8 10
Extraction time (hours)
24
Fig. 2Urease/ C and Urease/N ratios in the extracts at differ-
ent extraction times.
1014 SOIL SCI. SOC. AM. J., VOL. 44, 1980
Table 4Correlation coefficients between soil
extracted parameters.
Organic Phos- BAA
Parameters Carbon N phatase Urease hydrolysis
PS extracts
Organic N
Phosphatase
Urease
BAA hydrolysis
Casein hydrolysis
Organic N
Phosphatase
Urease
BAA hydrolysis
Casein hydrolysis
Organic N
Phosphatase
Urease
BAA hydrolysis
Casein hydrolysis
0.90**
0.92*
0.90**
0.92**
0.83**
0.81**
0.95**
0.90**
0.86**
0.52~
0.97**
0.92**
0.96**
0.68*
0.90**
0.70*
0.97** 0.72*
0.94** 0.82**
0.60 0.85**
PN extracts
0.80**
0.97** 0.87**
0.46 0.84**
0.06 0.39
AS extracts
0.97**
0.90** 0.82**
0.61 0.72*
0.91** 0.95**
0.91**
0.56 0.64
0.64
0.26 0.76*
0.53
0.78* 0.89**
* and ** Significant atp =0.05 and 0.01, respectively.
This would not only define the physical and chemical
state of enzymes but also eventually explain the ob-
served differences in extraction percentages. In some
cases, proteases were not correlated significantly with
extracted C and N; in part such enzymes may become
closely associated with humic compounds in the ex-
tracts to form humus-enzyme complexes.
The changes occurring in enzyme/C and enzymes/N
ratios may indicate that activities and organic matter
are not always extracted proportionally from soil and
therefore enzymes may not be uniformly associated in
soil with the organic matter. Evidence that enzyme-
A
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PS extract
ft
4 6 8 10
Extraction time ( hours )
24
free humic molecules exist in soil has been reported
(Nannipieri et al., 1974; Cervelli et al., 1975; McLaren
et al., 1975). Urease/N ratios were low during the
first extraction hours, increasing later. This is con-
sistent with the finding of Nannipieri et al. (1974)
and McLaren et al. (1975) that extraction of enzyme-
free organic matter precedes and may facilitate the
subsequent extraction of active urease-organo com-
plexes.
The poorest extraction of enzymes, on the basis of
the relative activities of extract and soil, occurred
for urease. However the "percent of control" should
be considered an equivocal measurement used here
merely as an attempt to define the efficiency of ex-
traction; for example, the low percents of extracted
urease may depend on the presence of low levels of
soil extracellular activity rather than on poor effi-
ciency of pyrophosphate as extractant. Soil activity
results from enzymes which function both extracellu-
larly, the so-called abiotic factor (Skujins, 1978), and
intracellularly; with the present methodology it is
impossible to separate accurately microbial activities
from the various extracellular enzymatic activities
(Skujins, 1978). Moreover, during the extraction,
changes in the structure of enzyme-humus complex
may occur and there is also the possibility that some
inhibitors are liberated which interact with enzymes.
Urease is inhibited by catechol and by some derivates
of p-benzoquinone which are possible constituents
of soil organic matter (Bremner and Douglas, 1971;
B undy and Bremner, 1973).
A significant proportion of soil phosphatase and
casein-hydrolyzing protease was extracted by pyro-
phosphate from the three soils. The apparent effi-
ciency of extraction was higher than that for soil pro-
Fig. 3Casein-hydrolyzing activities/C and casein-hydrolyzing
activities/N ratios in the extracts at different extraction
times.
4 6 8 10
Extraction time ( hours)
24
Fig. 4Activities hydrolyzing BAA/C and activities hydrolyzing
BAA/N ratios in the extracts at different extraction times.
NANNI PI ER I ET AL.: EX TRACTION OF PHOSPHATASE, UREASE, PROTEASES, ORGANIC C, AND N 1015
tease and phosphatases reported by B atistic et al.
(1980) but similar to that for protease extracted from
an Australian Rendzina (Ladd, 1972). By using 0.1M
NaHCO
3
at pH 8.1, 14.4% of soil casein-hydrolyzing
activities, 4.0% of soil enzymes hydroly/ing BAA, and
2.6% of soil phosphatase were located in the extract
(Ladd and Paul, 1973). Casein-hydrolyzing proteases,
which split high molecular weight substrates, are
supposed to be short-lived in soil (Ladd and B utler,
1975). Strong bonding of the enzyme to soil colloids
may protect the enzyme protein but may render it
inaccessible to and inactive towards high molecular
weight substrates. Thus measurable activity would
result from relatively free enzymes which would be
vulnerable to proteolysis and would be more easily ex-
tractable by extractant. PN soil is characterized by the
presence of plant fragments whose degradation re-
quires the synthesis of extracellular enzymes like pro-
teinases. Specific activities of soluble enzymes are gen-
erally higher than those of the same enzymes in soil
(Skujins, 1967; Ladd, 1972), or bound to solid support
(Crook, 1968). The results obtained with PN soil for
casein-hydrolyzing activities may be also similarly ex-
plained.
Microbial lysis may occur during pyrophosphate ex-
tractipn. Nannipieri et al. (1974) using the same con-
ditions employed in this work, demonstrated that the
number of soil ureolytic microorganisms was unaffect-
ed by pyrophosphate. On the other hand, Arcara et
al. (1975) reported that whereas total microbial num-
ber determined by plate counts were unchanged dur-
ing the early hours of incubation, after 12 hours an
increase was observed. After 10 hours, no additional
enzyme extraction occurs, with the exception of urease
from PS and PN. The fact that microbial number de-
termined by plate counts was unaffected during the
early hours of extraction does not eliminate the pos-
sibility that some lyses may occur. While lysis of
protozoa occurs by osmotic shock and hand homogeni-
zation, the disruption of bacteria, fungi, and yeast
requires violent breakage methods such as sonication
(Coakley et al., 1977). Since protozoa constitute a
small percentage (0.20%) of total soil biomass (Gray
and Williams 1971) their eventual lysis contributes
very little to the extracted enzymes. Y et Nannipieri
et al. (1975) did not succeed in extracting urease by
methods whose mechanical and osmotic stress was high-
er than that of the solution of 0.14M pyrophosphate
at pH 7.1. Such pyrophosphate extraction is regard-
ed as being sufficiently mild to remove only very small
amounts of enzymes from intact cells. Extraction of
enzymes from soil gives good yields because sodium
pyrophosphate is an effective extractant of organic
matter.
1016 SOIL SCI. SOC. AM. J., VOL. 44, 1980