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Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme
The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive ex...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Hindawi Publishing Corporation
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3398583/ https://www.ncbi.nlm.nih.gov/pubmed/22844600 http://dx.doi.org/10.1155/2012/951267 |
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author | Wong, Dominic W. S. Chan, Victor J. McCormack, Amanda A. Hirsch, Ján Biely, Peter |
author_facet | Wong, Dominic W. S. Chan, Victor J. McCormack, Amanda A. Hirsch, Ján Biely, Peter |
author_sort | Wong, Dominic W. S. |
collection | PubMed |
description | The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53 kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were K (m) 0.25 mM, V (max) 16.3 μM·min(−1), and k (cat) 9.27 s(−1) with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-β-D-xylopyranoside as the substrate. |
format | Online Article Text |
id | pubmed-3398583 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Hindawi Publishing Corporation |
record_format | MEDLINE/PubMed |
spelling | pubmed-33985832012-07-27 Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme Wong, Dominic W. S. Chan, Victor J. McCormack, Amanda A. Hirsch, Ján Biely, Peter Biotechnol Res Int Research Article The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53 kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were K (m) 0.25 mM, V (max) 16.3 μM·min(−1), and k (cat) 9.27 s(−1) with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-β-D-xylopyranoside as the substrate. Hindawi Publishing Corporation 2012 2012-07-04 /pmc/articles/PMC3398583/ /pubmed/22844600 http://dx.doi.org/10.1155/2012/951267 Text en Copyright © 2012 Dominic W. S. Wong et al. https://creativecommons.org/licenses/by/3.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Wong, Dominic W. S. Chan, Victor J. McCormack, Amanda A. Hirsch, Ján Biely, Peter Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme |
title | Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme |
title_full | Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme |
title_fullStr | Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme |
title_full_unstemmed | Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme |
title_short | Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme |
title_sort | functional cloning and expression of the schizophyllum commune glucuronoyl esterase gene and characterization of the recombinant enzyme |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3398583/ https://www.ncbi.nlm.nih.gov/pubmed/22844600 http://dx.doi.org/10.1155/2012/951267 |
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