Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2000-06-19
2002-04-16
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C530S350000, C435S320100, C435S252300, C435S006120
Reexamination Certificate
active
06372464
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to isolated polypeptides having peroxidase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.
2. Description of the Related Art
Lignin is an aromatic polymer occurring in the woody tissue of higher plants. Due to its hydrophobicity and complex random structure lacking regular hydrolysable bonds, lignin is poorly degraded by most organisms. The best degraders of lignin are white rot fungi that produce extracellular peroxidases and laccases, which are involved in the initial attack of lignin.
Manganese-dependent peroxidase is a frequently encountered peroxidase produced by white rot fungi. The peroxidase has a catalytic cycle involving a 2-electron oxidation of the heme by hydrogen peroxide and subsequent oxidation of compound I via compound II in two 1-electron steps to the native enzyme. The best reducing substrate for compounds I and II is Mn(II), a metal naturally present in wood. The Mn(III) formed oxidizes other substrates.
Organic acids such as oxalate, glyoxylate and lactate are known to have an important role in the mechanism of manganese-dependent peroxidase and lignin degradation. Mn(III) is stripped from the enzyme by organic acids, and the produced Mn(III)-organic acid complex acts as a diffusible mediator in the oxidation of lignin by manganese-dependent peroxidase. Mn(III) can also oxidize organic acids, yielding radicals. The organic acids may also be supplied from the degradation of lignin and by microorganisms.
Field and Mester, 1998,
Journal of Biochemistry
273: 15412-15417, disclose a manganese peroxidase-lignin peroxidase hybrid produced by Bjerkandera sp. strain BOS55, which is able to oxidize various substrates in the absence of manganese.
It is an object of the present invention to provide improved polypeptides having peroxidase activity and nucleic acid encoding the polypeptides.
SUMMARY OF THE INVENTION
The present invention relates to isolated polypeptides having peroxidase activity selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 75% identity with amino acids 22 to 370 of SEQ ID NO: 2, amino acids 22 to 365 of SEQ ID NO: 4, or amino acids 19 to 362 of SEQ ID NO: 6;
(b) a polypeptide encoded by a nucleic acid sequence which hybridizes under high stringency conditions with (i) nucleotides 775 to 2302 of SEQ ID NO: 1, nucleotides 2011 to 3462 of SEQ ID NO: 3, or nucleotides 2851 to 4257 of SEQ ID NO: 5, (ii) the cDNA sequence contained in nucleotides 775 to 2302 of SEQ ID NO: 1, nucleotides 2011 to 3462 of SEQ ID NO: 3, or nucleotides 2851 to 4257 of SEQ ID NO: 5, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);
(c) a variant of the polypeptide having an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 comprising a substitution, deletion, and/or insertion of one or more amino acids;
(d) an allelic variant of (a) or (b); and
(e) a fragment of (a), (b), or (d) that has peroxidase activity.
The present invention also relates to isolated nucleic acid sequences encoding the polypeptides and to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.
REFERENCES:
GeneSeq Database, Accession No. W12392, May 1997.*
Huang et al., J. Agric. Food Chem. 44:3437, 1996.
Agbola et al., J. Agric. Food Chem. 46:84, 1998.
Mester et al., 1998,Journal of Biochemistry273: 15412-15417.
McArdle Barbara
Yaver Debbie S.
Monshipouri M.
Novozymes Biotech Inc.
Prouty Rebecca E.
Starnes Robert L.
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