A novel staining method for detecting phytase activity

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Abstract

Differential agar media for the detection of microbial phytase activity use the disappearance of precipitated calcium or sodium phytate as an indication of enzyme activity. When this technique was applied to the study of ruminal bacteria, it became apparent that the method was unable to differentiate between phytase activity and acid production. Strong positive reactions (zones of clearing around microbial colonies) observed for acid producing, anaerobic bacteria, such as Streptococcus bovis, were not corroborated by subsequent quantitative assays. Experimentation revealed that acidic solutions generated false positive results on the selected differential medium. Empirical studies undertaken to find a solution to this limitation determined the false positive results could be eliminated through a two step counterstaining treatment (cobalt chloride and ammonium molybdate/ammonium vanadate) which reprecipitates acid solubilized phytate. This report discusses the application of the developed two step counterstaining treatment for the screening of phytase producing ruminal bacteria as well as its use in phytase zymogram assays.

Introduction

Phytic acid [myo-inositol hexakis(dihydrogen phosphate)], the predominant form of total phosphate found in cereal grains and oilseed meals (Reddy et al., 1982), passes largely intact through the monogastric digestive tract. Swine and poultry diets must be supplemented with inorganic phosphate, while phytate phosphorus is excreted in manure and contributes to eutrophication of surface waters in areas of the world with intensive monogastric livestock production Common, 1989, Wodzinski and Ullah, 1996. Amendment of monogastric animal rations with enzymes is one solution currently employed to overcome the inefficiencies and pollution caused by dietary constituents (Campbell and Bedford, 1992, Wodzinski and Ullah, 1996) and phytase amendment has been adopted recently in areas with intensive monogastric livestock production.

The majority of phytase research has been directed at the characterization, production and application of Aspergillus niger phytases. Although phytate degrading activities have been described for a number of plants, animals, fungi and aerobic bacteria Wodzinski and Ullah, 1996, Howson and Davis, 1983, Rapoport et al., 1941, Shieh and Ware, 1968, Shimizu, 1992, many possible sources of novel phytases remain unexplored. Despite reports of phytase activity in the rumen Raun et al., 1956, Morse et al., 1992, the microbial inhabitants of this ecosystem have been largely ignored in the search for novel phytases with unique biochemical properties.

Suitable enzyme assays are necessary prerequisites for screening large numbers of microbial isolates such as would be found in complex ecosystems like the rumen. Although effective for characterizing individual isolates, standard solution assays for measuring phytase activity Shimizu, 1992, Van Hartingsveldt et al., 1993 are unsatisfactory for the rapid screening of a complex mixture of microbial populations. A more suitable approach is the use of a differential plating medium. One method may be to use non-specific chromogenic phosphatase substrates, such as those used for histochemical and molecular biology applications, including 5-bromo-4-chloro-3-indolyl phosphate (BCIP), naphthol AS phosphates (West et al., 1990) and phenolphthalein diphosphate/methyl green (Riccio et al., 1997). Unfortunately, this approach lacks the desired specificity. A more specific method for detecting phytase activity would rely on the disappearance of precipitated calcium or sodium phytate as an indication of enzyme activity. Microorganisms expressing phytases produce zones of clearing on agar media containing sodium or calcium phytate Howson and Davis, 1983, Shieh and Ware, 1968. However, the solid medium assays described in the literature were found to be unsatisfactory for screening anaerobic bacteria for phytase activity because of the false positive reactions of acid producing bacteria such as Streptococcus bovis. To overcome this problem, a two step counterstaining procedure can be used in which solid agar medium is flooded first with an aqueous cobalt chloride solution and second by an aqueous ammonium molybdate/ammonium vanadate solution. This staining technique has also proven to be useful in the detection of phytase activity in situ in polyacrylamide gels.

Section snippets

Sodium phytate stability

Stabilities of sodium phytate solutions were determined by measuring the release of free phosphate following sterilization through a syringe filter (0.2 μm, cellulose acetate) or in an autoclave (121°C at 15 psi for 20 min). Released orthophosphate (Pi) in the reaction mixture was measured by a modification of the method of Fiske and Subbarow (1925). Colour reagent (750 μl), prepared daily by mixing four volumes of 1.5% (w/v) ammonium molybdate in a 5.5% (v/v) sulfuric acid solution and one

Stability of sodium phytate as a phytate substrate

Preliminary experiments with phytate revealed that orthophosphate was released from phytate salts during autoclaving. This phenomenon is problematic as it not only interferes with the phosphate assay by increasing the background but may also release enough phosphate to inhibit phytase activity or repress phytase expression. Shieh and Ware (1968) observed a decline of 50% in the phytase activity of A. ficuum NRRL 3135 when the phosphorus content of a corn starch medium was increased from 40 to

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Cited by (0)

1

Present address: Department of Dairy Science and Technology, Sung Kyun Kwan University, Suwon 440-746, Seoul, South Korea.

2

Present address: Department of Animal Science, University of British Columbia, Vancouver, BC, Canada V6T 1Z4.

3

Present address: Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, AB, Canada T1K 3M4.

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