Assessing phytase activity
Main Article Content
Keywords
inositol phosphatase activity, inositol phosphate, phosphate analysis, phytase activity, phytate
Abstract
Phytases are nutritionally important for increased bioavailability of dietary minerals and phosphate for monogastric animals including humans. Release of minerals and phosphate is accomplished by the enzymatic stepwise degradation of phytate (inositol hexaphosphate, IP6). Activity determinations of phytase is often based on analysis of total released phosphate (Pi), but phytase activity in its purest form represents released product per time from IP6 only. Microbial and plant preparations often also contain mixtures of phosphatases and organic phosphate compounds; hence some released phosphate in enzymatic assays may originate from non-phytase phosphatases degrading non-phytate molecules. Moreover, even purified enzyme extracts assessed via Pi release may result in errors, since commercial IP6 commonly contains contamination of lower inositol phosphates, and further, the products of phytase IP6 hydrolysis are also substrates for the phytase. These facts motivate a quantitative comparative study. We compared enzyme activity determination in phytase assay samples at four different time points, based on analyzing the substrate IP6 versus the product Pi using different selected methods. The calculated activities varied substantially. For example, at 15 min into enzymatic assay, variations from 152 mU/ml (by IP6 analysis on HPIC) to 275-586 mU/ml (by Pi analysis using several methods) was detected. Our work emphasizes the importance of defining the type of activity assessed, showing that phytase activity based on released Pi may yield false positive results and/or overestimations. We propose to differentiate between phytase activity, being the activity by which IP6 is degraded, and total inositol phosphatase activity, corresponding to total released phosphate during the enzymatic reaction.
Downloads
Download data is not yet available.
Metrics
Metrics Loading ...
References
1. Dersjant‐Li Y, Awati A, Schulze H, Partridge G (2014) Phytase in non‐ruminant animal nutrition: a critical review on phytase activities in the gastrointestinal tract and influencing factors. Journal of the Science of Food and Agriculture.
2. Quan C, Zhang L, Wang Y, Ohta Y (2001) Production of phytase in a low phosphate medium by a novel yeast Candida krusei. J Biosci Bioeng 92: 154-160.
3. Hill JE, Kysela D, Elimelech M (2007) Isolation and assessment of phytate-hydrolysing bacteria from the DelMarVa Peninsula. Environ Microbiol 9: 3100-3107.
4. Nuobariene L, Hansen Ã…, Arneborg N (2012) Isolation and identification of phytase-active yeasts from sourdoughs. Lebensmittel-Wissenschaft + [ie und] Technologie Food science + technology Science + technologie alimentaire 48: 190-196.
5. Nuobariene L, Cizeikiene D, Gradzeviciute E, Hansen Ã…S, Rasmussen SK, et al. (2015) Phytase-active lactic acid bacteria from sourdoughs: Isolation and identification. LWT-Food Science and Technology 63: 766-772.
6. Keltti J (2014) Evaluation Report on the Analytical Methods submitted in connection with the Application for Authorisation of a Feed Additive according to Regulation (EC) No 1831/2003. Institute for Reference Materials and Measurements, European Union Reference Laboratory for Feed Additives: Institute for Reference Materials and Measurements. 8 p.
7. Carlsson N-G, Bergman E-L, Skoglund E, Hasselblad K, Sandberg A-S (2000) Rapid analyzes of inositol phosphates. Doktorsavhandlingar vid Chalmers Tekniska Hogskola: 1-10.
8. Nuobariene L, Arneborg N, Hansen Ã…S. Phytase active yeasts isolated from bakery sourdoughs; 2014. Latvia University of Agriculture, Faculty of Food Technology. pp. 223-227.
9. Kumari S, Krishnan V, Jolly M, Sachdev A (2014) 'In vivo'bioavailability of essential minerals and phytase activity during soaking and germination in soybean ('Glycine max'L.). Australian Journal of Crop Science 8: 1168-1174.
10. Caputo L, Visconti A, De Angelis M (2015) Selection and use of a Saccharomyces cerevisae strain to reduce phytate content of wholemeal flour during bread-making or under simulated gastrointestinal conditions. LWT-Food Science and Technology.
11. Sanikommu S, Pasupuleti M, Vadalkonda L (2014) Comparison of Phosphate Estimating Methods in the Presence of Phytic Acid for the Determination of Phytase Activity. Preparative Biochemistry and Biotechnology 44: 231-241.
12. Sandberg AS, Brune M, Carlsson NG, Hallberg L, Skoglund E, et al. (1999) Inositol phosphates with different numbers of phosphate groups influence iron absorption in humans. Am J Clin Nutr 70: 240-246.
13. Turk M, Sandberg AS, Carlsson NG, Andlid T (2000) Inositol hexaphosphate hydrolysis by Baker's yeast. Capacity, kinetics, and degradation products. J Agric Food Chem 48: 100-104.
14. Fiske CH, Subbarow Y (1925) The colorimetric determination of phosphorus. J biol Chem 66: 375-400.
15. Heinonen JK, Lahti RJ (1981) A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase. Anal Biochem 113: 313-317.
16. Greiner R, Alminger ML (1999) Purification and characterization of a phytate-degrading enzyme from germinated oat (Avena sativa). Journal of the Science of Food and Agriculture 79: 1453-1460.
17. Bae HD, Yanke LJ, Cheng KJ, Selinger LB (1999) A novel staining method for detecting phytase activity. J Microbiol Methods 39: 17-22.
18. Dickman SR, Bray RH (1940) Colorimetric Determination of Phosphate. Industrial & Engineering Chemistry Analytical Edition 12: 665-668.
19. Henriksen A (1965) An automatic method for determining low-level concentrations of phosphates in fresh and saline waters. Analyst 90: 29-34.
20. Katewa SD, Katyare SS (2003) A simplified method for inorganic phosphate determination and its application for phosphate analysis in enzyme assays. Analytical Biochemistry 323: 180-187.
21. Hellström AM, Almgren A, Carlsson N-G, Svanberg U, Andlid TA (2012) Degradation of phytate by< i> Pichia kudriavzevii TY13 and< i> Hanseniaspora guilliermondii TY14 in Tanzanian togwa. International journal of food microbiology 153: 73-77.
22. Skoglund E, Carlsson NG, Sandberg AS (1997) Determination of Isomers of Inositol Mono- To Hexaphosphates in Selected Foods and Intestinal Contents Using High-Performance Ion Chromatography. Journal of Agricultural and Food Chemistry 45: 431-436.
23. Peterson GL (1978) A simplified method for analysis of inorganic phosphate in the presence of interfering substances. Analytical Biochemistry 84: 164-172.
24. Lim S (2011) Determination of phosphorus concentration in hydroponics solution. Agilent Technologies Inc. Mulgrave, Australia: Agilent Technologies Inc. pp. 1-6.
25. Central Pollution Control Board I (2011) Guide Manual: Water and wastewater analysis. In: Central Pollution Control Board I, editor. http://cpcbnicin/upload/NewItems/NewItem_171_guidemanualw&wwanalysispdf. http://www.indiawaterportal.org/articles/water-and-wastewater-analysis-guide-manual-central-pollution-control-board: Central Pollution Control Board, India.
26. Vohra A, Satyanarayana T (2002) Purification and characterization of a thermostable and acid-stable phytase from Pichia anomala. World Journal of Microbiology and Biotechnology 18: 687-691.
27. De Angelis M, Gallo G, Corbo MR, McSweeney PL, Faccia M, et al. (2003) Phytase activity in sourdough lactic acid bacteria: purification and characterization of a phytase from< i> Lactobacillus sanfranciscensis CB1. International journal of food microbiology 87: 259-270.
28. Chen C-C, Wu P-H, Huang C-T, Cheng K-J (2004) A< i> Pichia pastoris fermentation strategy for enhancing the heterologous expression of an< i> Escherichia coli phytase. Enzyme and Microbial Technology 35: 315-320.
29. Sandberg A-S, Brune M, Carlsson N-G, Hallberg L, Skoglund E, et al. (1999) Inositol phosphates with different numbers of phosphate groups influence iron absorption in humans. The American journal of clinical nutrition 70: 240-246.
30. Sandberg AS, Andlid T (2002) Phytogenic and microbial phytases in human nutrition. International Journal of Food Science and Technology 37: 823-833.
2. Quan C, Zhang L, Wang Y, Ohta Y (2001) Production of phytase in a low phosphate medium by a novel yeast Candida krusei. J Biosci Bioeng 92: 154-160.
3. Hill JE, Kysela D, Elimelech M (2007) Isolation and assessment of phytate-hydrolysing bacteria from the DelMarVa Peninsula. Environ Microbiol 9: 3100-3107.
4. Nuobariene L, Hansen Ã…, Arneborg N (2012) Isolation and identification of phytase-active yeasts from sourdoughs. Lebensmittel-Wissenschaft + [ie und] Technologie Food science + technology Science + technologie alimentaire 48: 190-196.
5. Nuobariene L, Cizeikiene D, Gradzeviciute E, Hansen Ã…S, Rasmussen SK, et al. (2015) Phytase-active lactic acid bacteria from sourdoughs: Isolation and identification. LWT-Food Science and Technology 63: 766-772.
6. Keltti J (2014) Evaluation Report on the Analytical Methods submitted in connection with the Application for Authorisation of a Feed Additive according to Regulation (EC) No 1831/2003. Institute for Reference Materials and Measurements, European Union Reference Laboratory for Feed Additives: Institute for Reference Materials and Measurements. 8 p.
7. Carlsson N-G, Bergman E-L, Skoglund E, Hasselblad K, Sandberg A-S (2000) Rapid analyzes of inositol phosphates. Doktorsavhandlingar vid Chalmers Tekniska Hogskola: 1-10.
8. Nuobariene L, Arneborg N, Hansen Ã…S. Phytase active yeasts isolated from bakery sourdoughs; 2014. Latvia University of Agriculture, Faculty of Food Technology. pp. 223-227.
9. Kumari S, Krishnan V, Jolly M, Sachdev A (2014) 'In vivo'bioavailability of essential minerals and phytase activity during soaking and germination in soybean ('Glycine max'L.). Australian Journal of Crop Science 8: 1168-1174.
10. Caputo L, Visconti A, De Angelis M (2015) Selection and use of a Saccharomyces cerevisae strain to reduce phytate content of wholemeal flour during bread-making or under simulated gastrointestinal conditions. LWT-Food Science and Technology.
11. Sanikommu S, Pasupuleti M, Vadalkonda L (2014) Comparison of Phosphate Estimating Methods in the Presence of Phytic Acid for the Determination of Phytase Activity. Preparative Biochemistry and Biotechnology 44: 231-241.
12. Sandberg AS, Brune M, Carlsson NG, Hallberg L, Skoglund E, et al. (1999) Inositol phosphates with different numbers of phosphate groups influence iron absorption in humans. Am J Clin Nutr 70: 240-246.
13. Turk M, Sandberg AS, Carlsson NG, Andlid T (2000) Inositol hexaphosphate hydrolysis by Baker's yeast. Capacity, kinetics, and degradation products. J Agric Food Chem 48: 100-104.
14. Fiske CH, Subbarow Y (1925) The colorimetric determination of phosphorus. J biol Chem 66: 375-400.
15. Heinonen JK, Lahti RJ (1981) A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase. Anal Biochem 113: 313-317.
16. Greiner R, Alminger ML (1999) Purification and characterization of a phytate-degrading enzyme from germinated oat (Avena sativa). Journal of the Science of Food and Agriculture 79: 1453-1460.
17. Bae HD, Yanke LJ, Cheng KJ, Selinger LB (1999) A novel staining method for detecting phytase activity. J Microbiol Methods 39: 17-22.
18. Dickman SR, Bray RH (1940) Colorimetric Determination of Phosphate. Industrial & Engineering Chemistry Analytical Edition 12: 665-668.
19. Henriksen A (1965) An automatic method for determining low-level concentrations of phosphates in fresh and saline waters. Analyst 90: 29-34.
20. Katewa SD, Katyare SS (2003) A simplified method for inorganic phosphate determination and its application for phosphate analysis in enzyme assays. Analytical Biochemistry 323: 180-187.
21. Hellström AM, Almgren A, Carlsson N-G, Svanberg U, Andlid TA (2012) Degradation of phytate by< i> Pichia kudriavzevii TY13 and< i> Hanseniaspora guilliermondii TY14 in Tanzanian togwa. International journal of food microbiology 153: 73-77.
22. Skoglund E, Carlsson NG, Sandberg AS (1997) Determination of Isomers of Inositol Mono- To Hexaphosphates in Selected Foods and Intestinal Contents Using High-Performance Ion Chromatography. Journal of Agricultural and Food Chemistry 45: 431-436.
23. Peterson GL (1978) A simplified method for analysis of inorganic phosphate in the presence of interfering substances. Analytical Biochemistry 84: 164-172.
24. Lim S (2011) Determination of phosphorus concentration in hydroponics solution. Agilent Technologies Inc. Mulgrave, Australia: Agilent Technologies Inc. pp. 1-6.
25. Central Pollution Control Board I (2011) Guide Manual: Water and wastewater analysis. In: Central Pollution Control Board I, editor. http://cpcbnicin/upload/NewItems/NewItem_171_guidemanualw&wwanalysispdf. http://www.indiawaterportal.org/articles/water-and-wastewater-analysis-guide-manual-central-pollution-control-board: Central Pollution Control Board, India.
26. Vohra A, Satyanarayana T (2002) Purification and characterization of a thermostable and acid-stable phytase from Pichia anomala. World Journal of Microbiology and Biotechnology 18: 687-691.
27. De Angelis M, Gallo G, Corbo MR, McSweeney PL, Faccia M, et al. (2003) Phytase activity in sourdough lactic acid bacteria: purification and characterization of a phytase from< i> Lactobacillus sanfranciscensis CB1. International journal of food microbiology 87: 259-270.
28. Chen C-C, Wu P-H, Huang C-T, Cheng K-J (2004) A< i> Pichia pastoris fermentation strategy for enhancing the heterologous expression of an< i> Escherichia coli phytase. Enzyme and Microbial Technology 35: 315-320.
29. Sandberg A-S, Brune M, Carlsson N-G, Hallberg L, Skoglund E, et al. (1999) Inositol phosphates with different numbers of phosphate groups influence iron absorption in humans. The American journal of clinical nutrition 70: 240-246.
30. Sandberg AS, Andlid T (2002) Phytogenic and microbial phytases in human nutrition. International Journal of Food Science and Technology 37: 823-833.
Article Sidebar
Published
Jun 4, 2015
Article Details
How to Cite
1.
Qvirist L, Carlsson N-G, Andlid T. Assessing phytase activity. J Biol Methods [Internet]. 2015 Jun. 4 [cited 2024 Apr. 19];2(1):e16. Available from: https://jbmethods.org/index.php/jbm/article/view/58
Issue
Section
Articles
Authors who publish with JBM agree to the following terms:
- Authors retain copyright and grant JBM right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).
