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Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus
The sequence of a phenylalanine ammonia-lyase (PAL; EC: 4.3.1.24) of the thermophilic and radiotolerant bacterium Rubrobacter xylanophilus (RxPAL) was identified by screening the genomes of bacteria for members of the phenylalanine ammonia-lyase family. A synthetic gene encoding the RxPAL protein wa...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903478/ https://www.ncbi.nlm.nih.gov/pubmed/24475062 http://dx.doi.org/10.1371/journal.pone.0085943 |
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author | Kovács, Klaudia Bánóczi, Gergely Varga, Andrea Szabó, Izabella Holczinger, András Hornyánszky, Gábor Zagyva, Imre Paizs, Csaba Vértessy, Beáta G. Poppe, László |
author_facet | Kovács, Klaudia Bánóczi, Gergely Varga, Andrea Szabó, Izabella Holczinger, András Hornyánszky, Gábor Zagyva, Imre Paizs, Csaba Vértessy, Beáta G. Poppe, László |
author_sort | Kovács, Klaudia |
collection | PubMed |
description | The sequence of a phenylalanine ammonia-lyase (PAL; EC: 4.3.1.24) of the thermophilic and radiotolerant bacterium Rubrobacter xylanophilus (RxPAL) was identified by screening the genomes of bacteria for members of the phenylalanine ammonia-lyase family. A synthetic gene encoding the RxPAL protein was cloned and overexpressed in Escherichia coli TOP 10 in a soluble form with an N-terminal His(6)-tag and the recombinant RxPAL protein was purified by Ni-NTA affinity chromatography. The activity assay of RxPAL with l-phenylalanine at various pH values exhibited a local maximum at pH 8.5 and a global maximum at pH 11.5. Circular dichroism (CD) studies showed that RxPAL is associated with an extensive α-helical character (far UV CD) and two distinctive near-UV CD peaks. These structural characteristics were well preserved up to pH 11.0. The extremely high pH optimum of RxPAL can be rationalized by a three-dimensional homology model indicating possible disulfide bridges, extensive salt-bridge formation and an excess of negative electrostatic potential on the surface. Due to these properties, RxPAL may be a candidate as biocatalyst in synthetic biotransformations leading to unnatural l- or d-amino acids or as therapeutic enzyme in treatment of phenylketonuria or leukemia. |
format | Online Article Text |
id | pubmed-3903478 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-39034782014-01-28 Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus Kovács, Klaudia Bánóczi, Gergely Varga, Andrea Szabó, Izabella Holczinger, András Hornyánszky, Gábor Zagyva, Imre Paizs, Csaba Vértessy, Beáta G. Poppe, László PLoS One Research Article The sequence of a phenylalanine ammonia-lyase (PAL; EC: 4.3.1.24) of the thermophilic and radiotolerant bacterium Rubrobacter xylanophilus (RxPAL) was identified by screening the genomes of bacteria for members of the phenylalanine ammonia-lyase family. A synthetic gene encoding the RxPAL protein was cloned and overexpressed in Escherichia coli TOP 10 in a soluble form with an N-terminal His(6)-tag and the recombinant RxPAL protein was purified by Ni-NTA affinity chromatography. The activity assay of RxPAL with l-phenylalanine at various pH values exhibited a local maximum at pH 8.5 and a global maximum at pH 11.5. Circular dichroism (CD) studies showed that RxPAL is associated with an extensive α-helical character (far UV CD) and two distinctive near-UV CD peaks. These structural characteristics were well preserved up to pH 11.0. The extremely high pH optimum of RxPAL can be rationalized by a three-dimensional homology model indicating possible disulfide bridges, extensive salt-bridge formation and an excess of negative electrostatic potential on the surface. Due to these properties, RxPAL may be a candidate as biocatalyst in synthetic biotransformations leading to unnatural l- or d-amino acids or as therapeutic enzyme in treatment of phenylketonuria or leukemia. Public Library of Science 2014-01-27 /pmc/articles/PMC3903478/ /pubmed/24475062 http://dx.doi.org/10.1371/journal.pone.0085943 Text en © 2014 Kovács et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Kovács, Klaudia Bánóczi, Gergely Varga, Andrea Szabó, Izabella Holczinger, András Hornyánszky, Gábor Zagyva, Imre Paizs, Csaba Vértessy, Beáta G. Poppe, László Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus |
title | Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus
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title_full | Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus
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title_fullStr | Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus
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title_full_unstemmed | Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus
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title_short | Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus
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title_sort | expression and properties of the highly alkalophilic phenylalanine ammonia-lyase of thermophilic rubrobacter xylanophilus |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903478/ https://www.ncbi.nlm.nih.gov/pubmed/24475062 http://dx.doi.org/10.1371/journal.pone.0085943 |
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