Site-directed mutagenesis of an alkaline phytase: Influencing specificity, activity and stability in acidic milieu
(2011) In Enzyme and Microbial Technology 49(2). p.177-182- Abstract
- Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding... (More)
- Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant-phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R-K179R) phytase showed higher stability at low pH (pH 2.6-3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3 h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase. (C) 2011 Elsevier Inc. All rights reserved. (Less)
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https://lup.lub.lu.se/record/2161404
- author
- Thi Thuy, Tran LU ; Mamo, Gashaw LU ; Buxo, Laura ; Le, Nhi N. ; Gaber, Yasser LU ; Mattiasson, Bo LU and Hatti-Kaul, Rajni LU
- organization
- publishing date
- 2011
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Phytase, Site-directed mutagenesis, Bacillus sp., Substrate specificity
- in
- Enzyme and Microbial Technology
- volume
- 49
- issue
- 2
- pages
- 177 - 182
- publisher
- Elsevier
- external identifiers
-
- wos:000293439100010
- scopus:79959881355
- pmid:22112406
- ISSN
- 0141-0229
- DOI
- 10.1016/j.enzmictec.2011.05.012
- language
- English
- LU publication?
- yes
- id
- 8b69ffde-91f9-4a42-a996-86632a8e3800 (old id 2161404)
- date added to LUP
- 2016-04-01 10:55:56
- date last changed
- 2022-01-26 03:46:06
@article{8b69ffde-91f9-4a42-a996-86632a8e3800,
abstract = {{Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant-phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R-K179R) phytase showed higher stability at low pH (pH 2.6-3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3 h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase. (C) 2011 Elsevier Inc. All rights reserved.}},
author = {{Thi Thuy, Tran and Mamo, Gashaw and Buxo, Laura and Le, Nhi N. and Gaber, Yasser and Mattiasson, Bo and Hatti-Kaul, Rajni}},
issn = {{0141-0229}},
keywords = {{Phytase; Site-directed mutagenesis; Bacillus sp.; Substrate specificity}},
language = {{eng}},
number = {{2}},
pages = {{177--182}},
publisher = {{Elsevier}},
series = {{Enzyme and Microbial Technology}},
title = {{Site-directed mutagenesis of an alkaline phytase: Influencing specificity, activity and stability in acidic milieu}},
url = {{http://dx.doi.org/10.1016/j.enzmictec.2011.05.012}},
doi = {{10.1016/j.enzmictec.2011.05.012}},
volume = {{49}},
year = {{2011}},
}