a method for the determination of β-glucosidase activity in soil

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This article was downloaded by: [Eindhoven Technical University] On: 16 November 2014, At: 14:11 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Soil Science and Plant Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tssp20 A method for the determination of β- glucosidase activity in soil Koichi Hayano a a National Institute of Agricultural Sciences , Nishigahara, Kita- ku, Tokyo , Japan Published online: 29 Mar 2012. 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: http://dx.doi.org/10.1080/00380768.1973.10432524 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/ terms-and-conditions

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Page 1: A method for the determination of β-glucosidase activity in soil

This article was downloaded by: [Eindhoven Technical University]On: 16 November 2014, At: 14:11Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Soil Science and Plant NutritionPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/tssp20

A method for the determination of β-glucosidase activity in soilKoichi Hayano aa National Institute of Agricultural Sciences , Nishigahara, Kita-ku, Tokyo , JapanPublished online: 29 Mar 2012.

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: http://dx.doi.org/10.1080/00380768.1973.10432524

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoeveror howsoever caused arising directly or indirectly in connection with, in relation to orarising out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden. Terms& Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Page 2: A method for the determination of β-glucosidase activity in soil

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 ac­tivity. 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 metab­olism.

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)

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Page 3: A method for the determination of β-glucosidase activity in soil

104 K. HAYANO

Table 1. Properties of soils.

Soil pH Moisture content Organic carbon1> Total nitrogen1> Clay contentl> (%) (%) (%) (%)

Wakayama 6.8 Nishigoshi 5.0 Kanoya 5.2

ll Percent based on dry soil.

and is shown in Table 1.

14.6 38.5 23.7

0.9 5.9 3.1

0.10 0.46 0.23

METHOD FOR ASSAY OF ,8-GLUCOSIDASE ACTIVITY

Reagents

12 32 19

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.

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Page 4: A method for the determination of β-glucosidase activity in soil

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.

g 0.15 ~ 0

"'' ~ 0.10 !' ·g: .... ... 0 05 i .

.... 0::

1 5 10 PNG {Xl0-3 M)

Fig. 1. Effect of substrate concentration on the reaction rate. Nishigoshi soil was used. p-Glucosidase was measured by the method described in the text except for the substrate con­centration.

Effect oFPII

0.7 ...... ... .. '1:: 0.6

1111 .... ~ 0.5 ... -; E 30.4 il j 0.3 .. .. -; li 0.2 1 .. ~ 0.1 1>.

30 60 90 120 150 180 Incubation time (min)

Fig. 2. Effect of incubation time on release 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.

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 solu­tion 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 activ­ity was 4.8. MARKOSYAN and GALSTYAN ( 6) have reported that the maximal ac­tivity of f'·glucosidase .in several soils appears to be at pH .5.9-6.2.

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Page 5: A method for the determination of β-glucosidase activity in soil

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 de­scribed 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 ex­traction are approximately 90% and vary with soils examined.

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

Soil

Wakayama Nishigoshi Kanoya

Percentage recovery from soil

Mean

99.0 91.5 84.3

S.D.1>

2.9 0.8 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

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Page 6: A method for the determination of β-glucosidase activity in soil

Soil

Wakayama Nishigoshi Kanoya

Estimation of Soil jl-Glucosidase Activity

Table 3. Precision of method.l)

Milliunit per gram of dried soil

Mean

15.2 14.4 10.1

0.6 0.8 0.5

107

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 .• oo2)

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 solu­tion. 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.

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Page 7: A method for the determination of β-glucosidase activity in soil

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)

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