Cargando…
Characterisation of recombinant pyranose oxidase from the cultivated mycorrhizal basidiomycete Lyophyllum shimeji (hon-shimeji)
BACKGROUND: The flavin-dependent enzyme pyranose 2-oxidase (P2Ox) has gained increased attention during the last years because of a number of attractive applications for this enzyme. P2Ox is a unique biocatalyst with high potential for biotransformations of carbohydrates and in synthetic carbohydrat...
Autores principales: | , , , , , , , |
---|---|
Formato: | Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2010
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2914677/ https://www.ncbi.nlm.nih.gov/pubmed/20630076 http://dx.doi.org/10.1186/1475-2859-9-57 |
_version_ | 1782184778602119168 |
---|---|
author | Salaheddin, Clara Takakura, Yoshimitsu Tsunashima, Masako Stranzinger, Barbara Spadiut, Oliver Yamabhai, Montarop Peterbauer, Clemens K Haltrich, Dietmar |
author_facet | Salaheddin, Clara Takakura, Yoshimitsu Tsunashima, Masako Stranzinger, Barbara Spadiut, Oliver Yamabhai, Montarop Peterbauer, Clemens K Haltrich, Dietmar |
author_sort | Salaheddin, Clara |
collection | PubMed |
description | BACKGROUND: The flavin-dependent enzyme pyranose 2-oxidase (P2Ox) has gained increased attention during the last years because of a number of attractive applications for this enzyme. P2Ox is a unique biocatalyst with high potential for biotransformations of carbohydrates and in synthetic carbohydrate chemistry. Recently, it was shown that P2Ox is useful as bioelement in biofuel cells, replacing glucose oxidase (GOx), which traditionally is used in these applications. P2Ox offers several advantages over GOx for this application, e.g., its much broader substrate specificity. Because of this renewed interest in P2Ox, knowledge on novel pyranose oxidases isolated from organisms other than white-rot fungi, which represent the traditional source of this enzyme, is of importance, as these novel enzymes might differ in their biochemical and physical properties. RESULTS: We isolated and over-expressed the p2ox gene encoding P2Ox from the ectomycorrhizal fungus Lyophyllum shimeji. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in E. coli Rosetta 2. We obtained active, flavinylated recombinant P2Ox in yields of approximately 130 mg per L of medium. The enzyme was purified by a two-step procedure based on anion exchange chromatography and preparative native PAGE, yielding an apparently homogenous enzyme preparation with a specific activity of 1.92 U/mg (using glucose and air oxygen as the substrates). Recombinant P2Ox from L. shimeji was characterized in some detail with respect to its physical and catalytic properties, and compared to the well-characterised enzymes from Phanerochaete chrysosporium and Trametes multicolor. CONCLUSION: L. shimeji P2Ox shows properties that are comparable to those of P2Ox from white-rot fungal origin, and is in general characterised by lower K(m )and k(cat )values both for electron donor (sugar) as well as electron acceptor (ferrocenium ion, 1,4-benzoquinone, 2,6-dichloroindophenol). While L. shimeji P2Ox is the least thermostable of these three enzymes (melting temperature T(m )of 54.9°C; half-life time of activity τ(1/2 )of 0.12 at 50°C and pH 6.5), P. chrysosporium P2Ox showed remarkable thermostability with T(m )of 75.4°C and τ(1/2 )of 96 h under identical conditions. |
format | Text |
id | pubmed-2914677 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-29146772010-08-12 Characterisation of recombinant pyranose oxidase from the cultivated mycorrhizal basidiomycete Lyophyllum shimeji (hon-shimeji) Salaheddin, Clara Takakura, Yoshimitsu Tsunashima, Masako Stranzinger, Barbara Spadiut, Oliver Yamabhai, Montarop Peterbauer, Clemens K Haltrich, Dietmar Microb Cell Fact Research BACKGROUND: The flavin-dependent enzyme pyranose 2-oxidase (P2Ox) has gained increased attention during the last years because of a number of attractive applications for this enzyme. P2Ox is a unique biocatalyst with high potential for biotransformations of carbohydrates and in synthetic carbohydrate chemistry. Recently, it was shown that P2Ox is useful as bioelement in biofuel cells, replacing glucose oxidase (GOx), which traditionally is used in these applications. P2Ox offers several advantages over GOx for this application, e.g., its much broader substrate specificity. Because of this renewed interest in P2Ox, knowledge on novel pyranose oxidases isolated from organisms other than white-rot fungi, which represent the traditional source of this enzyme, is of importance, as these novel enzymes might differ in their biochemical and physical properties. RESULTS: We isolated and over-expressed the p2ox gene encoding P2Ox from the ectomycorrhizal fungus Lyophyllum shimeji. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in E. coli Rosetta 2. We obtained active, flavinylated recombinant P2Ox in yields of approximately 130 mg per L of medium. The enzyme was purified by a two-step procedure based on anion exchange chromatography and preparative native PAGE, yielding an apparently homogenous enzyme preparation with a specific activity of 1.92 U/mg (using glucose and air oxygen as the substrates). Recombinant P2Ox from L. shimeji was characterized in some detail with respect to its physical and catalytic properties, and compared to the well-characterised enzymes from Phanerochaete chrysosporium and Trametes multicolor. CONCLUSION: L. shimeji P2Ox shows properties that are comparable to those of P2Ox from white-rot fungal origin, and is in general characterised by lower K(m )and k(cat )values both for electron donor (sugar) as well as electron acceptor (ferrocenium ion, 1,4-benzoquinone, 2,6-dichloroindophenol). While L. shimeji P2Ox is the least thermostable of these three enzymes (melting temperature T(m )of 54.9°C; half-life time of activity τ(1/2 )of 0.12 at 50°C and pH 6.5), P. chrysosporium P2Ox showed remarkable thermostability with T(m )of 75.4°C and τ(1/2 )of 96 h under identical conditions. BioMed Central 2010-07-14 /pmc/articles/PMC2914677/ /pubmed/20630076 http://dx.doi.org/10.1186/1475-2859-9-57 Text en Copyright ©2010 Salaheddin et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Salaheddin, Clara Takakura, Yoshimitsu Tsunashima, Masako Stranzinger, Barbara Spadiut, Oliver Yamabhai, Montarop Peterbauer, Clemens K Haltrich, Dietmar Characterisation of recombinant pyranose oxidase from the cultivated mycorrhizal basidiomycete Lyophyllum shimeji (hon-shimeji) |
title | Characterisation of recombinant pyranose oxidase from the cultivated mycorrhizal basidiomycete Lyophyllum shimeji (hon-shimeji) |
title_full | Characterisation of recombinant pyranose oxidase from the cultivated mycorrhizal basidiomycete Lyophyllum shimeji (hon-shimeji) |
title_fullStr | Characterisation of recombinant pyranose oxidase from the cultivated mycorrhizal basidiomycete Lyophyllum shimeji (hon-shimeji) |
title_full_unstemmed | Characterisation of recombinant pyranose oxidase from the cultivated mycorrhizal basidiomycete Lyophyllum shimeji (hon-shimeji) |
title_short | Characterisation of recombinant pyranose oxidase from the cultivated mycorrhizal basidiomycete Lyophyllum shimeji (hon-shimeji) |
title_sort | characterisation of recombinant pyranose oxidase from the cultivated mycorrhizal basidiomycete lyophyllum shimeji (hon-shimeji) |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2914677/ https://www.ncbi.nlm.nih.gov/pubmed/20630076 http://dx.doi.org/10.1186/1475-2859-9-57 |
work_keys_str_mv | AT salaheddinclara characterisationofrecombinantpyranoseoxidasefromthecultivatedmycorrhizalbasidiomycetelyophyllumshimejihonshimeji AT takakurayoshimitsu characterisationofrecombinantpyranoseoxidasefromthecultivatedmycorrhizalbasidiomycetelyophyllumshimejihonshimeji AT tsunashimamasako characterisationofrecombinantpyranoseoxidasefromthecultivatedmycorrhizalbasidiomycetelyophyllumshimejihonshimeji AT stranzingerbarbara characterisationofrecombinantpyranoseoxidasefromthecultivatedmycorrhizalbasidiomycetelyophyllumshimejihonshimeji AT spadiutoliver characterisationofrecombinantpyranoseoxidasefromthecultivatedmycorrhizalbasidiomycetelyophyllumshimejihonshimeji AT yamabhaimontarop characterisationofrecombinantpyranoseoxidasefromthecultivatedmycorrhizalbasidiomycetelyophyllumshimejihonshimeji AT peterbauerclemensk characterisationofrecombinantpyranoseoxidasefromthecultivatedmycorrhizalbasidiomycetelyophyllumshimejihonshimeji AT haltrichdietmar characterisationofrecombinantpyranoseoxidasefromthecultivatedmycorrhizalbasidiomycetelyophyllumshimejihonshimeji |