Engineering towards catalytic use of fungal class-II peroxidases for dye-decolorizing and conversion of lignin model compounds

Taina Kristina Lundell, Elodie Bentley, Sari Kristiina Hilden, Johanna Tuulikki Rytioja, Jaana Tuulia Kuuskeri, Usenobong F. Ufot, Paula Annukka Nousiainen, Martin Hofrichter, Matti Per-Vilhelm Wahlsten, Wendy Doyle, Andrew T. Smith

Research output: Contribution to journalArticleScientificpeer-review

Abstract

Background. Manganese peroxidases (MnP) and lignin peroxidases (LiP) are haem-including fungal secreted class-II peroxidases, which are interesting oxidoreductases in protein engineering aimed at design of biocatalysts for lignin and lignocellulose conversion, dye compound degradation, activation of aromatic compounds, and biofuel production. Objective. Recombinant short-type MnP (Pr-MnP3) of the white rot fungus Phlebia radiata, and its manganese-binding site (E40, E44, D186) directed variants were produced and characterized. To allow catalytic applications, enzymatic bleaching of Reactive Blue 5 and conversion of lignin-like compounds by engineered class-II peroxidases were explored. Method. Pr-MnP3 and its variants were expressed in Escherichia coli. The resultant body proteins were lysed, purified and refolded into haem-including enzymes in 6-7% protein recovery, and examined spectroscopically and kinetically.
Results. Successful production of active enzymes was attained, with spectral characteristics of high-spin class-II peroxidases. Recombinant Pr-MnP3 demonstrated high affinity to Mn2+, which was noticeably affected by single (D186H/N) and double (E40H+E44H) mutations. Without addition of Mn2+, Pr-MnP3 was able to oxidize ABTS and decolorize Reactive Blue 5. Pc-LiPH8, its Trp-radical site variants, and engineered CiP-LiP demonstrated conversion of veratryl alcohol and dimeric non-phenolic lignin-model compounds (arylglycerol-β-aryl ethers) with production of veratraldehyde, which is evidence for cation radical formation with subsequent Cα-Cβ cleavage. Pc-LiPH8 and CiP variants were able to effectively oxidize and convert the phenolic dimer (guaiacylglycerol-β–guaiacyl ether).
Conclusion. Our results demonstrate suitability of engineered MnP and LiP peroxidases for dye-decolorizing, and efficiency of LiP and its variants for activation and degradation of phenolic and non-phenolic lignin-like aryl ether-linked compounds.
Original languageEnglish
JournalCurrent Biotechnology
Volume6
Issue number2
Pages (from-to)116-117
Number of pages12
ISSN2211-5501
DOIs
Publication statusPublished - 2017
MoE publication typeA1 Journal article-refereed

Fields of Science

  • 414 Agricultural biotechnology
  • Fungal biotechnology
  • 219 Environmental biotechnology
  • Lignocellulose biodegradation
  • 1183 Plant biology, microbiology, virology
  • Mycology
  • Fungal genetics
  • Heterologous expression
  • 116 Chemical sciences
  • 1182 Biochemistry, cell and molecular biology

Cite this

@article{02b6572843fe4ee38c902e222c47d111,
title = "Engineering towards catalytic use of fungal class-II peroxidases for dye-decolorizing and conversion of lignin model compounds",
abstract = "Background. Manganese peroxidases (MnP) and lignin peroxidases (LiP) are haem-including fungal secreted class-II peroxidases, which are interesting oxidoreductases in protein engineering aimed at design of biocatalysts for lignin and lignocellulose conversion, dye compound degradation, activation of aromatic compounds, and biofuel production. Objective. Recombinant short-type MnP (Pr-MnP3) of the white rot fungus Phlebia radiata, and its manganese-binding site (E40, E44, D186) directed variants were produced and characterized. To allow catalytic applications, enzymatic bleaching of Reactive Blue 5 and conversion of lignin-like compounds by engineered class-II peroxidases were explored. Method. Pr-MnP3 and its variants were expressed in Escherichia coli. The resultant body proteins were lysed, purified and refolded into haem-including enzymes in 6-7{\%} protein recovery, and examined spectroscopically and kinetically. Results. Successful production of active enzymes was attained, with spectral characteristics of high-spin class-II peroxidases. Recombinant Pr-MnP3 demonstrated high affinity to Mn2+, which was noticeably affected by single (D186H/N) and double (E40H+E44H) mutations. Without addition of Mn2+, Pr-MnP3 was able to oxidize ABTS and decolorize Reactive Blue 5. Pc-LiPH8, its Trp-radical site variants, and engineered CiP-LiP demonstrated conversion of veratryl alcohol and dimeric non-phenolic lignin-model compounds (arylglycerol-β-aryl ethers) with production of veratraldehyde, which is evidence for cation radical formation with subsequent Cα-Cβ cleavage. Pc-LiPH8 and CiP variants were able to effectively oxidize and convert the phenolic dimer (guaiacylglycerol-β–guaiacyl ether). Conclusion. Our results demonstrate suitability of engineered MnP and LiP peroxidases for dye-decolorizing, and efficiency of LiP and its variants for activation and degradation of phenolic and non-phenolic lignin-like aryl ether-linked compounds.",
keywords = "414 Agricultural biotechnology, Fungal biotechnology, 219 Environmental biotechnology, Lignocellulose biodegradation, 1183 Plant biology, microbiology, virology, Mycology, Fungal genetics, Heterologous expression, 116 Chemical sciences, 1182 Biochemistry, cell and molecular biology",
author = "Lundell, {Taina Kristina} and Elodie Bentley and Hilden, {Sari Kristiina} and Rytioja, {Johanna Tuulikki} and Kuuskeri, {Jaana Tuulia} and Ufot, {Usenobong F.} and Nousiainen, {Paula Annukka} and Martin Hofrichter and Wahlsten, {Matti Per-Vilhelm} and Wendy Doyle and Smith, {Andrew T.}",
year = "2017",
doi = "10.2174/2211550105666160520120101",
language = "English",
volume = "6",
pages = "116--117",
journal = "Current Biotechnology",
issn = "2211-5501",
publisher = "Bentham Science Publishers, Ltd.",
number = "2",

}

Engineering towards catalytic use of fungal class-II peroxidases for dye-decolorizing and conversion of lignin model compounds. / Lundell, Taina Kristina; Bentley, Elodie; Hilden, Sari Kristiina; Rytioja, Johanna Tuulikki; Kuuskeri, Jaana Tuulia; Ufot, Usenobong F.; Nousiainen, Paula Annukka; Hofrichter, Martin; Wahlsten, Matti Per-Vilhelm; Doyle, Wendy; Smith, Andrew T.

In: Current Biotechnology, Vol. 6, No. 2, 2017, p. 116-117.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Engineering towards catalytic use of fungal class-II peroxidases for dye-decolorizing and conversion of lignin model compounds

AU - Lundell, Taina Kristina

AU - Bentley, Elodie

AU - Hilden, Sari Kristiina

AU - Rytioja, Johanna Tuulikki

AU - Kuuskeri, Jaana Tuulia

AU - Ufot, Usenobong F.

AU - Nousiainen, Paula Annukka

AU - Hofrichter, Martin

AU - Wahlsten, Matti Per-Vilhelm

AU - Doyle, Wendy

AU - Smith, Andrew T.

PY - 2017

Y1 - 2017

N2 - Background. Manganese peroxidases (MnP) and lignin peroxidases (LiP) are haem-including fungal secreted class-II peroxidases, which are interesting oxidoreductases in protein engineering aimed at design of biocatalysts for lignin and lignocellulose conversion, dye compound degradation, activation of aromatic compounds, and biofuel production. Objective. Recombinant short-type MnP (Pr-MnP3) of the white rot fungus Phlebia radiata, and its manganese-binding site (E40, E44, D186) directed variants were produced and characterized. To allow catalytic applications, enzymatic bleaching of Reactive Blue 5 and conversion of lignin-like compounds by engineered class-II peroxidases were explored. Method. Pr-MnP3 and its variants were expressed in Escherichia coli. The resultant body proteins were lysed, purified and refolded into haem-including enzymes in 6-7% protein recovery, and examined spectroscopically and kinetically. Results. Successful production of active enzymes was attained, with spectral characteristics of high-spin class-II peroxidases. Recombinant Pr-MnP3 demonstrated high affinity to Mn2+, which was noticeably affected by single (D186H/N) and double (E40H+E44H) mutations. Without addition of Mn2+, Pr-MnP3 was able to oxidize ABTS and decolorize Reactive Blue 5. Pc-LiPH8, its Trp-radical site variants, and engineered CiP-LiP demonstrated conversion of veratryl alcohol and dimeric non-phenolic lignin-model compounds (arylglycerol-β-aryl ethers) with production of veratraldehyde, which is evidence for cation radical formation with subsequent Cα-Cβ cleavage. Pc-LiPH8 and CiP variants were able to effectively oxidize and convert the phenolic dimer (guaiacylglycerol-β–guaiacyl ether). Conclusion. Our results demonstrate suitability of engineered MnP and LiP peroxidases for dye-decolorizing, and efficiency of LiP and its variants for activation and degradation of phenolic and non-phenolic lignin-like aryl ether-linked compounds.

AB - Background. Manganese peroxidases (MnP) and lignin peroxidases (LiP) are haem-including fungal secreted class-II peroxidases, which are interesting oxidoreductases in protein engineering aimed at design of biocatalysts for lignin and lignocellulose conversion, dye compound degradation, activation of aromatic compounds, and biofuel production. Objective. Recombinant short-type MnP (Pr-MnP3) of the white rot fungus Phlebia radiata, and its manganese-binding site (E40, E44, D186) directed variants were produced and characterized. To allow catalytic applications, enzymatic bleaching of Reactive Blue 5 and conversion of lignin-like compounds by engineered class-II peroxidases were explored. Method. Pr-MnP3 and its variants were expressed in Escherichia coli. The resultant body proteins were lysed, purified and refolded into haem-including enzymes in 6-7% protein recovery, and examined spectroscopically and kinetically. Results. Successful production of active enzymes was attained, with spectral characteristics of high-spin class-II peroxidases. Recombinant Pr-MnP3 demonstrated high affinity to Mn2+, which was noticeably affected by single (D186H/N) and double (E40H+E44H) mutations. Without addition of Mn2+, Pr-MnP3 was able to oxidize ABTS and decolorize Reactive Blue 5. Pc-LiPH8, its Trp-radical site variants, and engineered CiP-LiP demonstrated conversion of veratryl alcohol and dimeric non-phenolic lignin-model compounds (arylglycerol-β-aryl ethers) with production of veratraldehyde, which is evidence for cation radical formation with subsequent Cα-Cβ cleavage. Pc-LiPH8 and CiP variants were able to effectively oxidize and convert the phenolic dimer (guaiacylglycerol-β–guaiacyl ether). Conclusion. Our results demonstrate suitability of engineered MnP and LiP peroxidases for dye-decolorizing, and efficiency of LiP and its variants for activation and degradation of phenolic and non-phenolic lignin-like aryl ether-linked compounds.

KW - 414 Agricultural biotechnology

KW - Fungal biotechnology

KW - 219 Environmental biotechnology

KW - Lignocellulose biodegradation

KW - 1183 Plant biology, microbiology, virology

KW - Mycology

KW - Fungal genetics

KW - Heterologous expression

KW - 116 Chemical sciences

KW - 1182 Biochemistry, cell and molecular biology

U2 - 10.2174/2211550105666160520120101

DO - 10.2174/2211550105666160520120101

M3 - Article

VL - 6

SP - 116

EP - 117

JO - Current Biotechnology

JF - Current Biotechnology

SN - 2211-5501

IS - 2

ER -