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 enzyme which hydrolyze starch by randomly cleaving the 1,4-α-D-glucosidic linkages between the adjacent glucose units in linear amylose chain. It has significant advantages in a wide range of applications, in particular in food industry. The eukaryotic α-amylase isolat...
Published in: | Applied and Environmental Microbiology |
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Online Access: | http://hdl.handle.net/11581/396978 https://doi.org/10.1128/AEM.00449-17 https://aem.asm.org/content/83/13/e00449-17.long |
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ftuncamerinoiris:oai:pubblicazioni.unicam.it:11581/396978 2024-04-14T08:02:59+00:00 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 Yang, Guang Yao, Hua Mozzicafreddo, Matteo Ballarini, Patrizia Pucciarelli, Sandra Miceli, Cristina 2017 ELETTRONICO http://hdl.handle.net/11581/396978 https://doi.org/10.1128/AEM.00449-17 https://aem.asm.org/content/83/13/e00449-17.long eng eng info:eu-repo/semantics/altIdentifier/wos/WOS:000403495700009 volume:83 issue:13, Article number e00449-17 firstpage:1 lastpage:14 numberofpages:14 journal:APPLIED AND ENVIRONMENTAL MICROBIOLOGY info:eu-repo/grantAgreement/EC/H2020/MSCA-RISE metable 645693 http://hdl.handle.net/11581/396978 doi:10.1128/AEM.00449-17 info:eu-repo/semantics/altIdentifier/scopus/2-s2.0-85020488405 https://aem.asm.org/content/83/13/e00449-17.long info:eu-repo/semantics/openAccess info:eu-repo/semantics/article 2017 ftuncamerinoiris https://doi.org/10.1128/AEM.00449-17 2024-03-21T20:37:40Z The α-amylases are endo-acting enzyme which hydrolyze starch by randomly cleaving the 1,4-α-D-glucosidic linkages between the adjacent glucose units in linear amylose chain. It has significant advantages in a wide range of applications, in particular in food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, differently from most of the α-amylases characterized so far. Furthermore, EfAmy shows the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at 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 into Thr residues 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 thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for changes in the biochemical properties are discussed by analyzing the three-dimensional structural model.IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted ... Article in Journal/Newspaper Antarc* Antarctic CAMPUS Pubblicazioni Scientifiche Unicam (Università di Camerino) Antarctic The Antarctic Applied and Environmental Microbiology 83 13 |
institution |
Open Polar |
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CAMPUS Pubblicazioni Scientifiche Unicam (Università di Camerino) |
op_collection_id |
ftuncamerinoiris |
language |
English |
description |
The α-amylases are endo-acting enzyme which hydrolyze starch by randomly cleaving the 1,4-α-D-glucosidic linkages between the adjacent glucose units in linear amylose chain. It has significant advantages in a wide range of applications, in particular in food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, differently from most of the α-amylases characterized so far. Furthermore, EfAmy shows the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at 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 into Thr residues 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 thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for changes in the biochemical properties are discussed by analyzing the three-dimensional structural model.IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted ... |
author2 |
Yang, Guang Yao, Hua Mozzicafreddo, Matteo Ballarini, Patrizia Pucciarelli, Sandra Miceli, Cristina |
format |
Article in Journal/Newspaper |
author |
Yang, Guang YAO, HUA MOZZICAFREDDO, MATTEO BALLARINI, Patrizia PUCCIARELLI, Sandra MICELI, Cristina |
spellingShingle |
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 |
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 |
publishDate |
2017 |
url |
http://hdl.handle.net/11581/396978 https://doi.org/10.1128/AEM.00449-17 https://aem.asm.org/content/83/13/e00449-17.long |
geographic |
Antarctic The Antarctic |
geographic_facet |
Antarctic The Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_relation |
info:eu-repo/semantics/altIdentifier/wos/WOS:000403495700009 volume:83 issue:13, Article number e00449-17 firstpage:1 lastpage:14 numberofpages:14 journal:APPLIED AND ENVIRONMENTAL MICROBIOLOGY info:eu-repo/grantAgreement/EC/H2020/MSCA-RISE metable 645693 http://hdl.handle.net/11581/396978 doi:10.1128/AEM.00449-17 info:eu-repo/semantics/altIdentifier/scopus/2-s2.0-85020488405 https://aem.asm.org/content/83/13/e00449-17.long |
op_rights |
info:eu-repo/semantics/openAccess |
op_doi |
https://doi.org/10.1128/AEM.00449-17 |
container_title |
Applied and Environmental Microbiology |
container_volume |
83 |
container_issue |
13 |
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1796319186602950656 |