Extraction of acid phosphomonoesterase from soil: testing of various extractants

HOLIK, L., DUNDEK, P., VRANOVA, V., REJSEK, K., JANOUS, D., FORMANEK, P.: Extraction of acid phosphomonoesterase from soil: testing of various extractants. Acta univ. agric. et silvic. Mendel. Brun., 2011, LIX, No. 3, pp. 51–54 The aim of this work was to to investigate the suitability of various types of extractants for extraction of acid phosphomonoesterase from soil using various types of extractants. Succinate-borate buff er of pH 4.8 or 0.1 M K2SO4 were the most effi cient to extract this enzyme compared to 0.1 M glutamic acid or 0.005 M salicylic acid. Extraction using 0.1 M glutamic acid gave signifi cantly (P<0.05) lowest extraction yield. The following extracts were obtained: clear K2SO4 and glutamic acid extracts, succinate-borate buff er extracts were of slight coloration, and in some cases, rose-colored extracts of salicylic acid. The results of this work are in accordance with low extraction yields of phosphomonoesterase reported in other studies. acid phosphomonoesterase, extracts, glutamic acid, K2SO4, salicylic acid, soil Activity of extracellular enzyme acid phosphomonoesterase (orthophosphoric monoester phosphohydrolase, E.C. 3.1.3.2.) in soil is commonly determined to evaluate release of available phosphorus from soil organic matter. Besides the commonly determined total soil acid phosphomonoesterase activity, several attempts were performed to extract some parts of this activity. For this purpose, various types of extractants were used. For example, extraction using sodium pyrophosphate (0.14 M, pH 7.1) is based on solubilization of soil organic matter when enzyme-humic substances complexes are extracted. In this case, activity of acid phosphomonoesterase may be inhibited by humic substances (Rejsek et al., 2011, sent to journal). Sodium pyrophosphate extracts were brown with signifi cant amount of humic substances displaying enzymatic activity mostly below the detection limit, in some cases extraction yield 0.1% of total soil acid phosphomonoesterase activity was reported. Tris-HCl, Tris-HCl with bovine serum albumine (BSA) and Triton X-100 separately or together gave colorless to light yellow extracts indicating very low solubilization of humic substances. Treatment with Tris-HCl buff er (50 mM, pH 7.5) gave acid phosphomonoesterase extraction yield from 0 to approx. 1% (see Fornasier and Margon, 2007). Tris-HCl buff er plus 1% Triton X-100 extraction may have a loosening eff ect of the enzymes and soil organic matter complexes and as in case of extraction using Tris-HCl buff er with 4 % BSA when protein exchange is supposed, 2–8 times higher extraction yield of enzymes compared to Tris-HCl extraction was reported (Fornasier and Margon, 2007). When both Triton X-100 and BSA were added into Tris-HCl buff er, more than additive extraction yield reaching 2–13% was obtained (Fornasier and Margon, 2007). Other types of extractants used are mentioned in works of Batistic et al. (1980), Nannipieri et al. (1980), Hayano (1988), Pascual et al. (2002) etc. Besides extraction of fraction of acid phosphomonoesterase from soil, other types of treatments were tested. For example, sonication was reported to increase acid phosphomonoesterase activity of soils (De Cesare et al., 2000). In this work we have attempted to extract acid phosphomonoesterase from soil using various 52 L. Holik, P. Dundek, V. Vranova, K. Rejsek, D. Janous, P. Formanek type of extractants including 0.1 M K2SO4, 0.1 M glutamic acid, 0.005 M salicylic acid and succinateborate buff er of pH 4.8. The aim of this study was to fi nd the best extractant to extract fraction of acid phosphomonoesterase from soils. The results may be used for various types of ecological studies. MATERIAL AND METHODS The soil used for the experiments was obtained from the “Bilý Křiž” (“White Cross”) experimental research station, located in the Moravian-Silesian Beskids Mts. in the northeastern part of the Czech Republic. The region has a sub continental climate with a typical mean annual air temperature of 4.9 °C and mean annual precipitation of 1100 mm. The number of days with snow cover is 160 per year. The experimental 23-year-old Norway spruce stand is located at 908 m a. s. l. (N 49°30’10”, E 18°32’20”). The stand comprises approximately 99% Norway spruce (Picea abies (L.) Karsten) and 1% fi r (Abies alba Mill.). The fi rst mixed sample composed from 5 sub-samples of H horizon was taken there. The second mixed sample was obtained from Ah horizon of the experimental meadow at the same locality. The moderately mown meadow plant association belongs to the Nardo-Callunetea class (Formanek et al., 2008). A er sieving through a 5 mm sieve, the soil samples were stored at 6 oC until the analyses. Acid phosphomonoesterase was extracted using 0.1 M K2SO4, 0.1 M glutamic acid, 0.005 M salicylic acid or succinate-borate buff er of pH 4.8. In all cases, 5 g of wet soil were treated with 25 mL of each of the solution, shaken (20 min., 300 rpm), and fi ltered. Consequently, 4 mL of fi ltrate were incubated with 6 mL p-NPP in succinate-borate buff er of pH 4.8. Determination of acid phosphomonoesterase was performed according to Rejsek (1991). All data were processed using the Statistica 9 so ware ( = 0.05, n = 3). One Way Anova plus Fischer’s LSD test were used. RESULTS AND DISCUSSION The results of this study showed that succinate-borate buff er of pH 4.8 or 0.1 M K2SO4 were the most effi cient agents to extract acid phosphomonoesterase from soil (Fig. 1). While the extracts using K2SO4 were colorless, succinateborate buff er extracts were of slight coloration, but still virtually colorless. Other types of extractants like salicylic acid produced rose-colored extract, when Ah horizon soils were extracted. In case of H horizon of forest soil, colorless extracts were obtained using salicylic acid. Colorless extracts were produced also by glutamic acid treatment giving the lowest extraction yield (P<0.05). The results obtained in this work correspond with low extraction yields when acid phosphomonoesterase or other types of enzymes were extracted from soils within other works. In various studies, extraction of phosphatase was performed using water, Chelex 100-Na+ active resins and distilled water, 4 mM CaCl2, sodium pyrophosphate (e.g. 0.14 M, pH 7.1), phosphate buff er (e.g. 0.2 M, pH 8) in the presence of EDTA, Tris-HCl buff er (50 mM, pH 7.5), Tris-HCl buff er plus 1% (w/v) Triton X-100 (permeabilizing K2SO4 glutamic acid salicylic acid Succinate-borate buffer 0 1 2 3 4 5 6 g pN P g 1 fre sh so il h 1 Ah horizon H horizon 1: Extraction of acid phosphomonoesterase from soil using various extractants (mean± SE) Extraction of acid phosphomonoesterase from soil: testing of various extractans 53 agent), Tris-HCl buff er plus 4% (w/v) BSA, TrisHCl buff er plus both 1 % Triton X-100 and 4% BSA (Batistic et al., 1980; Nannipieri et al., 1980; Hayano, 1988; Pascual et al., 2002; Fornasier and Margon, 2007). Increased activity of extracted phosphatases appeared a er ultrafi ltration (Nannipieri et al., 1982). Activity of acid phosphomonoesterase in soil extracts obtained in this work corresponds with those reported by e.g. Fornasier and Margon (2007) or Margon and Fornasier (2008). Nevertheless, more research is necessary to better evaluate occurrence of easily extractable acid phosphomonoesterase in various soils.