Physical and enzymatic properties of lignin peroxidase isoenzymes from Phanerochaete chrysosporium

Roberta L. Farrell, Karen E. Murtagh, Ming Tien, Michael D. Mozuch, T. Kent Kirk

Research output: Contribution to journalArticle

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Abstract

Phanerochaete chrysosporium BKM-1767 secretes multiple lignin peroxidase isoenzymes when grown under nitrogen-limited conditions. Here we report the purification of these heme-containing peroxidases, and their physical and catalytic characterization. Ten hemeproteins, designated H1-H10, were separated by anion exchange HPLC. Six of them, H1, H2, H6, H7, H8, and H10, were lignin peroxidases, oxidizing veratryl alcohol in the presence of H2O2. The other four (three peaks were resolved) exhibited manganese-dependent peroxidase activity, oxidizing vanillylacetone in the presence of H2O2 and Mn+2. The lignin peroxidases have different isoelectric points, between p14.7 and 3.3, and molecular weights between 38 and 43 kDa, determined by SDS-PAGE. All are N- and probably O-glycosylated. Three organic substrates and H2O2 were used to compare their kinetic properties: the organic substrates were veratryl alcohol, 1,4-dimethoxybenzene, and the lignin model compound 1-(3,4-dimethoxyphenyl)-2-(o-methoxyphenoxy)-propane-1,3-diol. KM and TN values for each of these substrates varied significantly; e.g. for veratryl alcohol KM values were from 86 to 480 μm and TN values were from 1.3 to 8.3 s-1. The ranking of the isoenzyme activities differed with the different substrates, suggesting differences in affinities or in active site accessibilities. The KM for H2O2 varied between 13 and 77 μm. Immunological blot analysis and partial proteolytic digestion patterns showed that the isoenzymes have a high degree of homology. The isoenzyme concentrations in extracellular culture fluid were found to vary relatively and absolutely with culture time. A nomenclature scheme for these 10 hemeproteins has been proposed. This scheme should simplify identification of these proteins in the literature as well as be adaptable to others found in Phanerochaete chrysosporium.

Original languageEnglish (US)
Pages (from-to)322-328
Number of pages7
JournalEnzyme and Microbial Technology
Volume11
Issue number6
DOIs
StatePublished - Jan 1 1989

Fingerprint

Isoenzymes
Phanerochaete
Lignin
Hemeproteins
Alcohols
manganese peroxidase
Substrates
Peroxidases
Propane
Extracellular Fluid
Isoelectric Point
Terminology
Heme
Manganese
Purification
Anions
Polyacrylamide Gel Electrophoresis
Digestion
Catalytic Domain
Ion exchange

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Biochemistry
  • Applied Microbiology and Biotechnology

Cite this

Farrell, Roberta L. ; Murtagh, Karen E. ; Tien, Ming ; Mozuch, Michael D. ; Kirk, T. Kent. / Physical and enzymatic properties of lignin peroxidase isoenzymes from Phanerochaete chrysosporium. In: Enzyme and Microbial Technology. 1989 ; Vol. 11, No. 6. pp. 322-328.
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Physical and enzymatic properties of lignin peroxidase isoenzymes from Phanerochaete chrysosporium. / Farrell, Roberta L.; Murtagh, Karen E.; Tien, Ming; Mozuch, Michael D.; Kirk, T. Kent.

In: Enzyme and Microbial Technology, Vol. 11, No. 6, 01.01.1989, p. 322-328.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Physical and enzymatic properties of lignin peroxidase isoenzymes from Phanerochaete chrysosporium

AU - Farrell, Roberta L.

AU - Murtagh, Karen E.

AU - Tien, Ming

AU - Mozuch, Michael D.

AU - Kirk, T. Kent

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N2 - Phanerochaete chrysosporium BKM-1767 secretes multiple lignin peroxidase isoenzymes when grown under nitrogen-limited conditions. Here we report the purification of these heme-containing peroxidases, and their physical and catalytic characterization. Ten hemeproteins, designated H1-H10, were separated by anion exchange HPLC. Six of them, H1, H2, H6, H7, H8, and H10, were lignin peroxidases, oxidizing veratryl alcohol in the presence of H2O2. The other four (three peaks were resolved) exhibited manganese-dependent peroxidase activity, oxidizing vanillylacetone in the presence of H2O2 and Mn+2. The lignin peroxidases have different isoelectric points, between p14.7 and 3.3, and molecular weights between 38 and 43 kDa, determined by SDS-PAGE. All are N- and probably O-glycosylated. Three organic substrates and H2O2 were used to compare their kinetic properties: the organic substrates were veratryl alcohol, 1,4-dimethoxybenzene, and the lignin model compound 1-(3,4-dimethoxyphenyl)-2-(o-methoxyphenoxy)-propane-1,3-diol. KM and TN values for each of these substrates varied significantly; e.g. for veratryl alcohol KM values were from 86 to 480 μm and TN values were from 1.3 to 8.3 s-1. The ranking of the isoenzyme activities differed with the different substrates, suggesting differences in affinities or in active site accessibilities. The KM for H2O2 varied between 13 and 77 μm. Immunological blot analysis and partial proteolytic digestion patterns showed that the isoenzymes have a high degree of homology. The isoenzyme concentrations in extracellular culture fluid were found to vary relatively and absolutely with culture time. A nomenclature scheme for these 10 hemeproteins has been proposed. This scheme should simplify identification of these proteins in the literature as well as be adaptable to others found in Phanerochaete chrysosporium.

AB - Phanerochaete chrysosporium BKM-1767 secretes multiple lignin peroxidase isoenzymes when grown under nitrogen-limited conditions. Here we report the purification of these heme-containing peroxidases, and their physical and catalytic characterization. Ten hemeproteins, designated H1-H10, were separated by anion exchange HPLC. Six of them, H1, H2, H6, H7, H8, and H10, were lignin peroxidases, oxidizing veratryl alcohol in the presence of H2O2. The other four (three peaks were resolved) exhibited manganese-dependent peroxidase activity, oxidizing vanillylacetone in the presence of H2O2 and Mn+2. The lignin peroxidases have different isoelectric points, between p14.7 and 3.3, and molecular weights between 38 and 43 kDa, determined by SDS-PAGE. All are N- and probably O-glycosylated. Three organic substrates and H2O2 were used to compare their kinetic properties: the organic substrates were veratryl alcohol, 1,4-dimethoxybenzene, and the lignin model compound 1-(3,4-dimethoxyphenyl)-2-(o-methoxyphenoxy)-propane-1,3-diol. KM and TN values for each of these substrates varied significantly; e.g. for veratryl alcohol KM values were from 86 to 480 μm and TN values were from 1.3 to 8.3 s-1. The ranking of the isoenzyme activities differed with the different substrates, suggesting differences in affinities or in active site accessibilities. The KM for H2O2 varied between 13 and 77 μm. Immunological blot analysis and partial proteolytic digestion patterns showed that the isoenzymes have a high degree of homology. The isoenzyme concentrations in extracellular culture fluid were found to vary relatively and absolutely with culture time. A nomenclature scheme for these 10 hemeproteins has been proposed. This scheme should simplify identification of these proteins in the literature as well as be adaptable to others found in Phanerochaete chrysosporium.

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