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Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from Exiguobacterium sibiricum with Unusual Amino Acid Content

A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium Exiguobacterium sibiricum from Siberian permafrost soil was cloned and expressed in Escherichia coli. The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features c...

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Autores principales: Berlina, Yana Y., Petrovskaya, Lada E., Kryukova, Elena A., Shingarova, Lyudmila N., Gapizov, Sultan Sh., Kryukova, Mariya V., Rivkina, Elizaveta M., Kirpichnikov, Mikhail P., Dolgikh, Dmitry A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392543/
https://www.ncbi.nlm.nih.gov/pubmed/34439895
http://dx.doi.org/10.3390/biom11081229
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author Berlina, Yana Y.
Petrovskaya, Lada E.
Kryukova, Elena A.
Shingarova, Lyudmila N.
Gapizov, Sultan Sh.
Kryukova, Mariya V.
Rivkina, Elizaveta M.
Kirpichnikov, Mikhail P.
Dolgikh, Dmitry A.
author_facet Berlina, Yana Y.
Petrovskaya, Lada E.
Kryukova, Elena A.
Shingarova, Lyudmila N.
Gapizov, Sultan Sh.
Kryukova, Mariya V.
Rivkina, Elizaveta M.
Kirpichnikov, Mikhail P.
Dolgikh, Dmitry A.
author_sort Berlina, Yana Y.
collection PubMed
description A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium Exiguobacterium sibiricum from Siberian permafrost soil was cloned and expressed in Escherichia coli. The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features characteristic for the cold-active enzymes while possessing an unusually high number of proline residues in the loops—a typical feature of thermophilic enzymes. The activity of the purified recombinant protein was tested with p-nitrophenyl α-D-glucopyranoside as a substrate. The enzyme displayed a plateau-shaped temperature-activity profile with the optimum at 25 °C and a pronounced activity at low temperatures (50% of maximum activity at 5 °C). To improve the thermal stability at temperatures above 40 °C, we have introduced proline residues into four positions of EsOgl by site-directed mutagenesis according to “the proline rule”. Two of the mutants, S130P and A109P demonstrated a three- and two-fold increased half-life at 45 °C. Moreover, S130P mutation led to a 60% increase in the catalytic rate constant. Combining the mutations resulted in a further increase in stability transforming the temperature-activity profile to a typical mesophilic pattern. In the most thermostable variant A109P/S130P/E176P, the half-life at 45 °C was increased from 11 min (wild-type) to 129 min.
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spelling pubmed-83925432021-08-28 Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from Exiguobacterium sibiricum with Unusual Amino Acid Content Berlina, Yana Y. Petrovskaya, Lada E. Kryukova, Elena A. Shingarova, Lyudmila N. Gapizov, Sultan Sh. Kryukova, Mariya V. Rivkina, Elizaveta M. Kirpichnikov, Mikhail P. Dolgikh, Dmitry A. Biomolecules Article A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium Exiguobacterium sibiricum from Siberian permafrost soil was cloned and expressed in Escherichia coli. The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features characteristic for the cold-active enzymes while possessing an unusually high number of proline residues in the loops—a typical feature of thermophilic enzymes. The activity of the purified recombinant protein was tested with p-nitrophenyl α-D-glucopyranoside as a substrate. The enzyme displayed a plateau-shaped temperature-activity profile with the optimum at 25 °C and a pronounced activity at low temperatures (50% of maximum activity at 5 °C). To improve the thermal stability at temperatures above 40 °C, we have introduced proline residues into four positions of EsOgl by site-directed mutagenesis according to “the proline rule”. Two of the mutants, S130P and A109P demonstrated a three- and two-fold increased half-life at 45 °C. Moreover, S130P mutation led to a 60% increase in the catalytic rate constant. Combining the mutations resulted in a further increase in stability transforming the temperature-activity profile to a typical mesophilic pattern. In the most thermostable variant A109P/S130P/E176P, the half-life at 45 °C was increased from 11 min (wild-type) to 129 min. MDPI 2021-08-17 /pmc/articles/PMC8392543/ /pubmed/34439895 http://dx.doi.org/10.3390/biom11081229 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Berlina, Yana Y.
Petrovskaya, Lada E.
Kryukova, Elena A.
Shingarova, Lyudmila N.
Gapizov, Sultan Sh.
Kryukova, Mariya V.
Rivkina, Elizaveta M.
Kirpichnikov, Mikhail P.
Dolgikh, Dmitry A.
Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from Exiguobacterium sibiricum with Unusual Amino Acid Content
title Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from Exiguobacterium sibiricum with Unusual Amino Acid Content
title_full Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from Exiguobacterium sibiricum with Unusual Amino Acid Content
title_fullStr Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from Exiguobacterium sibiricum with Unusual Amino Acid Content
title_full_unstemmed Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from Exiguobacterium sibiricum with Unusual Amino Acid Content
title_short Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from Exiguobacterium sibiricum with Unusual Amino Acid Content
title_sort engineering of thermal stability in a cold-active oligo-1,6-glucosidase from exiguobacterium sibiricum with unusual amino acid content
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392543/
https://www.ncbi.nlm.nih.gov/pubmed/34439895
http://dx.doi.org/10.3390/biom11081229
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