Molecular biology of lignin-degrading enzymes from Phlebia radiata

Dissertation

Research output: ThesisDissertationCollection of Articles

Abstract

The ligninolytic enzyme system of the white-rot fungus Phlebia radiata was studied in this thesis by methods of molecular genetics. Chromosomal and cDNA gene libraries were constructed from the fungus and the genomic and cDNA clones encoding one of the lignin peroxidases and the laccase of Phlebia radiata were isolated. The lignin peroxidase gene of Phlebia radiata is 60 % homologous to corresponding genes isolated from Phanerochaete chrysosporium, a related fungus. Putative active site regions of the lignin peroxidase were revealed by sequence comparison to yeast cytochrome c peroxidase. By hydrophobic cluster analysis (HCA) it was shown that the ligninolytic peroxidases of white rot fungi most probably have tertiary structures similar to the yeast cytochrome c peroxidase structure that has been solved by X-ray crystallography. The Phlebia radiata laccase gene shows a homology of 63 % to the laccase gene of Coriolus hirsutus and 30 % to the zucchini ascorbate oxidase that has a known three-dimensional structure. Thus the Phlebia laccase protein most probably folds into a three times repeated ß-barrel as does the ascorbate oxidase. Hypotheses concerning the copper binding residues and a region potentially carrying the cofactor pyrroloquinoline quinone (PQQ) in the active site of the laccase have been made on the basis of its homology to other oxidases. The lignin peroxidase and laccase genes isolated from Phlebia radiata were expressed in the softrot fungus Trichoderma reesei under the major cellulase gene (cbh1) promoter. No lignin peroxidase protein production from Trichoderma could be detected even though the respective mRNA was clearly observed in the transformant strains. The Phlebia radiata laccase was produced by Trichoderma reesei with peak concentrations of 20 mg/l from laboratory fermentations. Expression constructions prepared with the laccase cDNA or chromosomal gene yielded equal levels of expression. The laccase produced by Trichoderma was purified and shown to have equal molecular weight and specific activity to the laccase produced by Phlebia radiata. The ability of the heterologous laccase to remove soluble aromatic compounds from waste lignin derived from the pulping process was demonstrated.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
Award date31 May 1991
Place of PublicationEspoo
Publisher
Print ISBNs951-38-3945-1
Publication statusPublished - 1991
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

Phlebia
laccase
molecular biology
lignin
lignin peroxidase
enzymes
cytochrome-c peroxidase
ascorbate oxidase
genes
Trichoderma reesei
white-rot fungi
Trichoderma
active sites
fungi
Coriolus hirsutus
yeasts
Phanerochaete chrysosporium
pulping
zucchini
DNA libraries

Keywords

  • ligninolytic enzymes
  • genes
  • molecular structure
  • hydrophobic
  • cluster analysis
  • cloning

Cite this

Saloheimo, Markku. / Molecular biology of lignin-degrading enzymes from Phlebia radiata : Dissertation. Espoo : VTT Technical Research Centre of Finland, 1991. 119 p.
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abstract = "The ligninolytic enzyme system of the white-rot fungus Phlebia radiata was studied in this thesis by methods of molecular genetics. Chromosomal and cDNA gene libraries were constructed from the fungus and the genomic and cDNA clones encoding one of the lignin peroxidases and the laccase of Phlebia radiata were isolated. The lignin peroxidase gene of Phlebia radiata is 60 {\%} homologous to corresponding genes isolated from Phanerochaete chrysosporium, a related fungus. Putative active site regions of the lignin peroxidase were revealed by sequence comparison to yeast cytochrome c peroxidase. By hydrophobic cluster analysis (HCA) it was shown that the ligninolytic peroxidases of white rot fungi most probably have tertiary structures similar to the yeast cytochrome c peroxidase structure that has been solved by X-ray crystallography. The Phlebia radiata laccase gene shows a homology of 63 {\%} to the laccase gene of Coriolus hirsutus and 30 {\%} to the zucchini ascorbate oxidase that has a known three-dimensional structure. Thus the Phlebia laccase protein most probably folds into a three times repeated {\ss}-barrel as does the ascorbate oxidase. Hypotheses concerning the copper binding residues and a region potentially carrying the cofactor pyrroloquinoline quinone (PQQ) in the active site of the laccase have been made on the basis of its homology to other oxidases. The lignin peroxidase and laccase genes isolated from Phlebia radiata were expressed in the softrot fungus Trichoderma reesei under the major cellulase gene (cbh1) promoter. No lignin peroxidase protein production from Trichoderma could be detected even though the respective mRNA was clearly observed in the transformant strains. The Phlebia radiata laccase was produced by Trichoderma reesei with peak concentrations of 20 mg/l from laboratory fermentations. Expression constructions prepared with the laccase cDNA or chromosomal gene yielded equal levels of expression. The laccase produced by Trichoderma was purified and shown to have equal molecular weight and specific activity to the laccase produced by Phlebia radiata. The ability of the heterologous laccase to remove soluble aromatic compounds from waste lignin derived from the pulping process was demonstrated.",
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Molecular biology of lignin-degrading enzymes from Phlebia radiata : Dissertation. / Saloheimo, Markku.

Espoo : VTT Technical Research Centre of Finland, 1991. 119 p.

Research output: ThesisDissertationCollection of Articles

TY - THES

T1 - Molecular biology of lignin-degrading enzymes from Phlebia radiata

T2 - Dissertation

AU - Saloheimo, Markku

N1 - Project code: BIO1022

PY - 1991

Y1 - 1991

N2 - The ligninolytic enzyme system of the white-rot fungus Phlebia radiata was studied in this thesis by methods of molecular genetics. Chromosomal and cDNA gene libraries were constructed from the fungus and the genomic and cDNA clones encoding one of the lignin peroxidases and the laccase of Phlebia radiata were isolated. The lignin peroxidase gene of Phlebia radiata is 60 % homologous to corresponding genes isolated from Phanerochaete chrysosporium, a related fungus. Putative active site regions of the lignin peroxidase were revealed by sequence comparison to yeast cytochrome c peroxidase. By hydrophobic cluster analysis (HCA) it was shown that the ligninolytic peroxidases of white rot fungi most probably have tertiary structures similar to the yeast cytochrome c peroxidase structure that has been solved by X-ray crystallography. The Phlebia radiata laccase gene shows a homology of 63 % to the laccase gene of Coriolus hirsutus and 30 % to the zucchini ascorbate oxidase that has a known three-dimensional structure. Thus the Phlebia laccase protein most probably folds into a three times repeated ß-barrel as does the ascorbate oxidase. Hypotheses concerning the copper binding residues and a region potentially carrying the cofactor pyrroloquinoline quinone (PQQ) in the active site of the laccase have been made on the basis of its homology to other oxidases. The lignin peroxidase and laccase genes isolated from Phlebia radiata were expressed in the softrot fungus Trichoderma reesei under the major cellulase gene (cbh1) promoter. No lignin peroxidase protein production from Trichoderma could be detected even though the respective mRNA was clearly observed in the transformant strains. The Phlebia radiata laccase was produced by Trichoderma reesei with peak concentrations of 20 mg/l from laboratory fermentations. Expression constructions prepared with the laccase cDNA or chromosomal gene yielded equal levels of expression. The laccase produced by Trichoderma was purified and shown to have equal molecular weight and specific activity to the laccase produced by Phlebia radiata. The ability of the heterologous laccase to remove soluble aromatic compounds from waste lignin derived from the pulping process was demonstrated.

AB - The ligninolytic enzyme system of the white-rot fungus Phlebia radiata was studied in this thesis by methods of molecular genetics. Chromosomal and cDNA gene libraries were constructed from the fungus and the genomic and cDNA clones encoding one of the lignin peroxidases and the laccase of Phlebia radiata were isolated. The lignin peroxidase gene of Phlebia radiata is 60 % homologous to corresponding genes isolated from Phanerochaete chrysosporium, a related fungus. Putative active site regions of the lignin peroxidase were revealed by sequence comparison to yeast cytochrome c peroxidase. By hydrophobic cluster analysis (HCA) it was shown that the ligninolytic peroxidases of white rot fungi most probably have tertiary structures similar to the yeast cytochrome c peroxidase structure that has been solved by X-ray crystallography. The Phlebia radiata laccase gene shows a homology of 63 % to the laccase gene of Coriolus hirsutus and 30 % to the zucchini ascorbate oxidase that has a known three-dimensional structure. Thus the Phlebia laccase protein most probably folds into a three times repeated ß-barrel as does the ascorbate oxidase. Hypotheses concerning the copper binding residues and a region potentially carrying the cofactor pyrroloquinoline quinone (PQQ) in the active site of the laccase have been made on the basis of its homology to other oxidases. The lignin peroxidase and laccase genes isolated from Phlebia radiata were expressed in the softrot fungus Trichoderma reesei under the major cellulase gene (cbh1) promoter. No lignin peroxidase protein production from Trichoderma could be detected even though the respective mRNA was clearly observed in the transformant strains. The Phlebia radiata laccase was produced by Trichoderma reesei with peak concentrations of 20 mg/l from laboratory fermentations. Expression constructions prepared with the laccase cDNA or chromosomal gene yielded equal levels of expression. The laccase produced by Trichoderma was purified and shown to have equal molecular weight and specific activity to the laccase produced by Phlebia radiata. The ability of the heterologous laccase to remove soluble aromatic compounds from waste lignin derived from the pulping process was demonstrated.

KW - ligninolytic enzymes

KW - genes

KW - molecular structure

KW - hydrophobic

KW - cluster analysis

KW - cloning

M3 - Dissertation

SN - 951-38-3945-1

T3 - Publications / Technical Research Centre of Finland

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Saloheimo M. Molecular biology of lignin-degrading enzymes from Phlebia radiata: Dissertation. Espoo: VTT Technical Research Centre of Finland, 1991. 119 p.