Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity
The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylas...
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ftpubmed:oai:pubmedcentral.nih.gov:5478988 2023-05-15T13:44:35+02:00 Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity Yang, Guang Yao, Hua Mozzicafreddo, Matteo Ballarini, Patrizia Pucciarelli, Sandra Miceli, Cristina 2017-06-16 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478988/ http://www.ncbi.nlm.nih.gov/pubmed/28455329 https://doi.org/10.1128/AEM.00449-17 en eng American Society for Microbiology http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478988/ http://www.ncbi.nlm.nih.gov/pubmed/28455329 http://dx.doi.org/10.1128/AEM.00449-17 Copyright © 2017 Yang et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (http://creativecommons.org/licenses/by/4.0/) . CC-BY Enzymology and Protein Engineering Text 2017 ftpubmed https://doi.org/10.1128/AEM.00449-17 2017-07-02T00:08:02Z The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. Text Antarc* Antarctic PubMed Central (PMC) Antarctic The Antarctic Applied and Environmental Microbiology 83 13 |
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PubMed Central (PMC) |
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ftpubmed |
language |
English |
topic |
Enzymology and Protein Engineering |
spellingShingle |
Enzymology and Protein Engineering Yang, Guang Yao, Hua Mozzicafreddo, Matteo Ballarini, Patrizia Pucciarelli, Sandra Miceli, Cristina Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity |
topic_facet |
Enzymology and Protein Engineering |
description |
The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. |
format |
Text |
author |
Yang, Guang Yao, Hua Mozzicafreddo, Matteo Ballarini, Patrizia Pucciarelli, Sandra Miceli, Cristina |
author_facet |
Yang, Guang Yao, Hua Mozzicafreddo, Matteo Ballarini, Patrizia Pucciarelli, Sandra Miceli, Cristina |
author_sort |
Yang, Guang |
title |
Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity |
title_short |
Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity |
title_full |
Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity |
title_fullStr |
Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity |
title_full_unstemmed |
Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity |
title_sort |
rational engineering of a cold-adapted α-amylase from the antarctic ciliate euplotes focardii for simultaneous improvement of thermostability and catalytic activity |
publisher |
American Society for Microbiology |
publishDate |
2017 |
url |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478988/ http://www.ncbi.nlm.nih.gov/pubmed/28455329 https://doi.org/10.1128/AEM.00449-17 |
geographic |
Antarctic The Antarctic |
geographic_facet |
Antarctic The Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_relation |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478988/ http://www.ncbi.nlm.nih.gov/pubmed/28455329 http://dx.doi.org/10.1128/AEM.00449-17 |
op_rights |
Copyright © 2017 Yang et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (http://creativecommons.org/licenses/by/4.0/) . |
op_rightsnorm |
CC-BY |
op_doi |
https://doi.org/10.1128/AEM.00449-17 |
container_title |
Applied and Environmental Microbiology |
container_volume |
83 |
container_issue |
13 |
_version_ |
1766203802282622976 |