A simple and fast kinetic assay for phytases using phytic acid-protein complex as substrate.
(2011) In Analytical Biochemistry 410(2). p.177-184- Abstract
- Phytase (EC 3.1.3.-) hydrolyzes phytate (IP6) present in cereals and grains to release inorganic phosphate (Pi), thereby making it bioavailable. The most commonly used method to assay phytase, developed nearly a century ago, measures the Pi liberated from IP6. This traditional end point assay is time consuming and well known for its cumbersomeness in addition to requiring extra caution for handling the toxic regents used. This paper reports a simple, fast and nontoxic kinetic method adaptable for high through-put for assaying phytase using IP6-lysozyme as a substrate. The assay is based on the principle that IP6 forms stable turbid complexes with positively charged lysozyme in a wide pH range and hydrolysis of the IP6 in the complex is... (More)
- Phytase (EC 3.1.3.-) hydrolyzes phytate (IP6) present in cereals and grains to release inorganic phosphate (Pi), thereby making it bioavailable. The most commonly used method to assay phytase, developed nearly a century ago, measures the Pi liberated from IP6. This traditional end point assay is time consuming and well known for its cumbersomeness in addition to requiring extra caution for handling the toxic regents used. This paper reports a simple, fast and nontoxic kinetic method adaptable for high through-put for assaying phytase using IP6-lysozyme as a substrate. The assay is based on the principle that IP6 forms stable turbid complexes with positively charged lysozyme in a wide pH range and hydrolysis of the IP6 in the complex is accompanied with a decrease in turbidity monitored at 600 nm. The turbidity decrease correlates well to the released Pi from IP6. This kinetic method was found useful in assaying histidine acid phytases, including 3- and 6-phytases, a class representing all commercial phytases, and alkaline ß-propeller phytase from Bacillus sp. The influences of temperature, pH, phosphate and other salts on the kinetic assay were examined. Salts including NaCl, CaCl2, and phosphate all showed a concentration-dependent interference. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1732322
- author
- Thi Thuy, Tran LU ; Hatti-Kaul, Rajni LU ; Dalsgaard, Søren and Yu, Shukun LU
- organization
- publishing date
- 2011
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Phytate-protein interaction, microbial phytases, kinetic assay for phytases
- in
- Analytical Biochemistry
- volume
- 410
- issue
- 2
- pages
- 177 - 184
- publisher
- Elsevier
- external identifiers
-
- wos:000287103900003
- pmid:21050837
- scopus:79551522610
- pmid:21050837
- ISSN
- 1096-0309
- DOI
- 10.1016/j.ab.2010.10.034
- language
- English
- LU publication?
- yes
- id
- 7f8db40f-33dd-4071-9599-3621dd0f75b1 (old id 1732322)
- date added to LUP
- 2016-04-01 11:15:44
- date last changed
- 2022-01-26 06:35:40
@article{7f8db40f-33dd-4071-9599-3621dd0f75b1,
abstract = {{Phytase (EC 3.1.3.-) hydrolyzes phytate (IP6) present in cereals and grains to release inorganic phosphate (Pi), thereby making it bioavailable. The most commonly used method to assay phytase, developed nearly a century ago, measures the Pi liberated from IP6. This traditional end point assay is time consuming and well known for its cumbersomeness in addition to requiring extra caution for handling the toxic regents used. This paper reports a simple, fast and nontoxic kinetic method adaptable for high through-put for assaying phytase using IP6-lysozyme as a substrate. The assay is based on the principle that IP6 forms stable turbid complexes with positively charged lysozyme in a wide pH range and hydrolysis of the IP6 in the complex is accompanied with a decrease in turbidity monitored at 600 nm. The turbidity decrease correlates well to the released Pi from IP6. This kinetic method was found useful in assaying histidine acid phytases, including 3- and 6-phytases, a class representing all commercial phytases, and alkaline ß-propeller phytase from Bacillus sp. The influences of temperature, pH, phosphate and other salts on the kinetic assay were examined. Salts including NaCl, CaCl2, and phosphate all showed a concentration-dependent interference.}},
author = {{Thi Thuy, Tran and Hatti-Kaul, Rajni and Dalsgaard, Søren and Yu, Shukun}},
issn = {{1096-0309}},
keywords = {{Phytate-protein interaction; microbial phytases; kinetic assay for phytases}},
language = {{eng}},
number = {{2}},
pages = {{177--184}},
publisher = {{Elsevier}},
series = {{Analytical Biochemistry}},
title = {{A simple and fast kinetic assay for phytases using phytic acid-protein complex as substrate.}},
url = {{http://dx.doi.org/10.1016/j.ab.2010.10.034}},
doi = {{10.1016/j.ab.2010.10.034}},
volume = {{410}},
year = {{2011}},
}