Soil Science and Plant Nutrition

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A method for the determination of β-glucosidase

activity in soil

Koichi Hayano

To cite this article: Koichi Hayano (1973) A method for the determination of β-glucosidase activity
in soil, Soil Science and Plant Nutrition, 19:2, 103-108, DOI: 10.1080/00380768.1973.10432524

To link to this article: https://doi.org/10.1080/00380768.1973.10432524

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 Soil Sci. Plant Nutr., 19 (2), 103-108, 1973

A METHOD FOR THE DETERMINATION OF fi-GLUCOSIDASE

ACfiVITY IN SOIL
Koichi HAY ANO

National Institute of Agricultural Sciences,

Nishigahara, Kita-ku, Tokyo, Japan

Received July 31, 1972

A method is described for the rapid and simple assay of soil fi-glucosidase ac-
tivity. It involves colorimetric estimation of P-nitrophenol released by p-glucosidase
activity when soil is incubated in Mcilvaine buffer (pH 4.8) with P-nitrophenyl ft·
n-glucoside and toluene at 30oC for 1 hr. The method has been applied to three
different soils. The range of fi·glucosidase activity in cultivated soils was from
10.1 to 15.2 mp mole per min per gram of dried soil. K. value for p.nitrophenyl
fi-n-glucoside was 3.3 x 10"' M. Optimum pH was 4.8.

~-Glucosidases constitute one of the common groups of soil enzymes. They help
in the hydrolysis of various ~-glucosides which are frequently supplied to soil from
plant residues. The hydrolysis products may serve as one of the energy sources for
microorganisms in soil. A measure of the ~-glucosidase activity of soil would be of
considerable significance in the study of the soil microflora related to glycoside metab-
olism.
A few methods have been proposed for estimation of the ~-glucosidase activity in
soil ( J-3 ). HOFFMANN and HOFFMANN ( 3) estimated soil ~-glucosidase activity by
determining the saligenin released when soil was incubated with salicin in acetate
buffer (pH 6.2) in the presence of toluene. This method requires a bulky reaction
mixture and consumes 3 hr for incubation and 1 hr for color development. Furthermore
the use of sodium acetate-acetic acid buffer at pH 6.2 is rather inadequate. Their
method showed neither precision nor quantitative recovery of saligenin. The method
proposed here is based on the determination of p-nitrophenol in the reaction mixture
after hydrolysis of p-nitrophenyl ~-D-glucoside by soil enzyme. The present method is
more rapid and simple as compared with the previous ones.

MATERIALS

Soils. The moist soils used were sampled from the surface (0:-20 em) and passed
through a 2 mm sieve. The pH was determined by a glass electrode (soil : water ratio,
1 : 2.5). Moisture content was determined by drying at 105°C for 12 hr. Total carbon
and nitrogen contents were determined by the C.N. corder (Model MT-50, Yanagimoto
MFG., Co., Ltd.). Clay content of each soil was determined by pipette analysis ( 4)
103
104 K. HAYANO

Table 1. Properties of soils.

Moisture content Organic carbon 1> Total nitrogen 1> Clay contentl>
Soil pH (%) (%) (%) (%)

Wakayama 6.8 14.6 0.9 0.10 12

Nishigoshi 5.0 38.5 5.9 0.46 32
Kanoya 5.2 23.7 3.1 0.23 19
ll Percent based on dry soil.

and is shown in Table 1.

METHOD FOR ASSAY OF ,8-GLUCOSIDASE ACTIVITY

Reagents
Toluene. Mcilvaine buffer pH 4.8 ( 5): 98.6 ml of 0.2 M dibasic sodium phosphate
solution was mixed with 101.4 rnl of 0.1 M citric acid solution.
Tris solution, 2M : 242 g of Tris (hydroxyrnethyl) aminomethane was dissolved in
water, and the solution diluted to 200 ml.
p-Nitrophenyl fi-v-glucoside (PNG) solution, Sx JO-Z M: 150 mg of PNG (Sigma
Chern. Co.) was dissolved in 10 ml of distilled water.
Standard p-nitrophenol solution (pmolefml): Ten milliliters of 10·1 M stock solution
(Daiichi Pure Chern. Co.) was diluted to 100 ml.
Procedure
The following procedures gave the results presented in the next section. A 0.5 g
amount of moist soil (<2mm) was placed in a test tube (16mmx175mm) and 0.1 ml
of toluene added. After 10min 0.9ml of distilled water, 1.5rnl of MCILVAINE buffer,.
and 0.6 ml of PNG were added. The test tube was swirled by a vortex mixer for a
few seconds to mix the contents and then was placed in an incubator at 30°C. After
1 hr, 8 ml of ethanol was added, the tube was swirled for about 10 sec, and the soil
suspension was filtered through a Toyo No. 131 folded filter paper. After filtration, 2
rnl of 2 M Tris solution was added to the filtrate and the tube was swirled for a few
seconds. The solution was transferred to a colorimeter cuvette and the intensity of
the yellow color was measured with a Hitachi Model 139 spectrophotometer at 400 m.u _
The p-nitrophenol content of the filtrate was calculated by reference to a calibration
curve plotted from results obtained with standards containing 0, 0.2, 10.4, 0.6, 0.8, and
1.0 pmole of p-nitrophenol. The molar extinction coefficient of p-nitrophenol at 400 m.u
is 16.8 X 108 M-1 • crn- 1 at these experimental conditions. The color was stable for at
least 12 hr. Controls consisted of substrate and each soil analyzed, to correct for color
not derived from p-nitrophenol released by fi-glucosidase activity. One unit of enzyme
was defined as the amount that releases 1 pmole of p-nitrophenol- per min at 30oC and
pH 4.8 in MciLVAINE buffer.
 Estimation of Soil fl-Glucpsidase Activity lOS

RESULTS AND DISCUSSION

Effect of substrate concentration and incubation time on hydrolysis of PNG

The effect of substrate concentration on the reaction rate is shown in Fig. 1.
Maximum reaction rate was obtained between 5 x 10·8 and 10-z M of PNG concentration.
Therefore 10-z M of PNG concentration was used in the present method to give a
maximum velocity. K .. value for PNG was 3.3 x to-• M. Figure 2 shows the propor·
tional relationship between time of incubation and amount of p..nitrophenol released.
Soil p-glucosidase activity was destroyed by autoclaving (130°C, 15 min). The evidence
indicates that enzymatic hydrolysis of PNG can be followed by the method proposed
and that p-glucosidase assay by this method is not complicated by microbial growth
or assimilation of enzymatic reaction products by soil microorganisms.

0.7
.....
......

'1:: 0.6
....1111
~ 0.5
...
-;
E
30.4
g 0.15
il
~
j.
..-; 0.3
0

"'!''
~ 0.10
·g: li 0.2
.... 1..
i .
... 0 05
~ 0.1
....0:: 1>.

1 5 10 30 60 90 120 150 180

PNG {Xl0-3 M) Incubation time (min)

Fig. 1. Effect of substrate concentration on Fig. 2. Effect of incubation time on release

the reaction rate.
Nishigoshi soil was used. p-Glucosidase
was measured by the method described
in the text except for the substrate con-
centration.
of P..nitrophenol in assay of ~-glucosidase
activity of Nishigoshi soil.
fl·Glucosidase activity was measured by
the method described in the text except
for the incubation time. 0, hydrolysis
of PNG by active soil; e, hydrolysis of
PNG by autocluved soil.

Effect oFPII
Figure 3 shows the effects ot pH on the rate ot hydrolysis of PNG by Nishigoshi
soil. The pH of the reaction mixture was almost the same as that of the buffer solu-
tion used. The difference between buffer solution and bufferized soil suspension was
within ±0.1 in the range of pH 3.7 to 7.0. The optimum pH for p-glucosidase activ-
ity was 4.8. MARKOSYAN and GALSTYAN ( 6) have reported that the maximal ac-
tivity of f'·glucosidase .in several soils appears to be at pH .5.9-6.2.
106 K. HAYANO

100

80
!'

.,.
:~ 60
u

•
.:=
;;
~
40

20

oL-~a----.--~s~--~6--~.­
pH

Fig. 3. Effect of pH on hydrolysis of PNG by Nishigoshi soil.

jS-Glucosidase activity was measured by the method de-
scribed in the text except for the buffer pH. e, .MciLVAINE
buffer; O, acetate buffer; X, phosphate buffer. Relative
activity, 100 was defined as 14.4 milliunit/g at 30°C in
MCILVAINE buffer pH 4.8.

A 0.4 M acetate buffer or 0.4 M phosphate buffer could be used in the same manner.
but they both gave a lower value for tl-glucosidase activity than did MCILVAINE buffer.

Recovery of product
p-Nitrophenol (0.3 pmole) was incubated for 30 min in MciLVAINE buffer with 0.5 g
of different soils and assayed as described for PNG hydrolysis. The amount (0.3 pmole)
of p-nitrophenol added was similar to that released from PNG by 0.5 g of soil in present
experimental condition. Table 2 shows that recoveries of p-nitrophenol by ethanol ex-
traction are approximately 90% and vary with soils examined.

Table 2. Recovery of added p.nitrophenol from soil.ll

Percentage recovery from soil

Soil
Mean S.D. 1 >

Wakayama 99.0 2.9

Nishigoshi 91.5 0.8
Kanoya 84.3 2.9

I> Calculated from the net recovery of 0.0. 400 and converted to p.nitrophenol equivalents
by relating to standard. Each soil was assayed eight times.
2
> Standard deviation.

Precision
The high precision of the method described is illustrated by Table 3 whicn gave
 Estimation of Soil jl-Glucosidase Activity 107

Table 3. Precision of method.l)

Milliunit per gram of dried soil

Soil
Mean

Wakayama 15.2 0.6

Nishigoshi 14.4 0.8
Kanoya 10.1 0.5
1> jl-Giucosidase activity was measured by the method described in the text. Each soil
was assayed eight times.
2> Standard deviation.

the results of replicate analyses of different soils. The Jl-glucosidase activities of these
soils are estimated to be 10.1-15.2 milliunit per gram of dried soil. The standard
deviation of the activity determination ranged from 0.5 to 0.8.

Stability of PNG in alkalline solution

To estimate whether non enzymatic degradation of PNG occurred in alkaline solu-
tion, the stability of PNG in alkaline solution was measured. Results are shown in
Table 4. The high pH of a sodium hydroxide solution induced non enzymatic degra·

Table 4. Stability of PNG in alkaline solution.n

O.D .•oo 2)
Addition Final pH
10 min €0 min 390 min
0.5 N NaOH, 0.25 ml 10.0 0.079 0.108 1.78
0.5 N NaOH, 0.10 ml 8.6 0.072 0.081 0.135
2M Tris, 2 ml 10.0 0.064 0.067 0.068
1 > The reaction mixture contained 1.5 ml of MciLVAINE buffer (pH 4.8), 1.5 ml of 2 x 10-z M

PNG, 8 ml of 99% ethanol and each addition. The solution was mixed and its yellow color
intensity at 400 mp was measured.
2 > Optical density per em at 400 mp.

dation of PNG. No degradation of PNG occurred by alkalization with 2M Tris solu-

tion. The discrepancy between the effect of sodium hydroxide and Tris solution on
PNG is obscure.

·. Acknowledgement. The author wishes to thank Dr. N. Hashimoto of The Kyushu Agricultural
Experiment Station, Mr. Z. Ono of The Wakayama Agricultural Experiment Station, and Dr. I.
Tanabe of The Kagoshima Agricultural Experiment Station for the gifts of soil samples, and Dr.
T. Suzuki of this laboratory for his interest and advice.
108 K. HAYANO

REFERENCES

I) HOFFMANN, E. and HoFFMANN, G., Naturwiss., 40, 511 (1953)

2) HoFFMANN, E. and HoFFMANN, G., Biochem. Z., 32.1, 397 (1953)
3) HOFFMANN, G. and DEDEKEN, M., Zeit. Pjlanzenerniihr. Dung. Bode71k., 108, 193 (1965)
4) KILMER, U. J. and ALEXANDER, L. T., Soil·Sd., 68, 15 (1949)
5) MciLVAINE, T. C., J. Bioi. Chern., 49, 183 (1921)
6) MARKOSYAN, L. M. and GALSTYAN, A. SH., lzv. Nauk. Arm. SSR. Bioi. Nauki., 16, 45 (1963)