Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-01-05
2001-08-28
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S320100, C435S252300, C435S325000, C435S189000, C435S193000, C435S195000, C435S232000, C435S233000
Reexamination Certificate
active
06280976
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to isolated nucleic acid sequences encoding polypeptides having cellobiose dehydrogenase activity. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as recombinant methods for producing the polypeptides.
2. Description of the Related Art
Cellobiose-oxidizing enzymes were first described in the extracellular enzyme system of the white-rot fungus
Phanerochaete chrysosporium
(Westermark and Eriksson, 1974,
Acta Chem. Scand. Ser. B
28: 204-208; Westermark and Eriksson, 1974,
Acta Chem. Scand. Ser. B
28: 209-214; Ayers et al., 1978,
European Journal of Biochemistry
90: 171-181). The cellobiose-oxidizing enzymes from this fungus have been identified: one is a flavoprotein called cellobiose:quinone oxidoreductase (E.C 1.1.5.1) suggested to be involved in lignin degradation, and the other is a haemoflavoprotein called cellobiose dehydrogenase (E.C. 1.1.99.18) proposed to be preferentially involved in cellulose degradation.
Cellobiose dehydrogenases have also been found in the brown-rot fungus
Coniophora putena
(Schmidhalter and Canevascini, 1992,
Applied Microbiology Biotechnology
37: 431-436) and soft-rot fungi such as Monilia sp. (Dekker, 1980,
Journal of General Microbiology
120: 309-316),
Chaetomium cellulolyticum
(Fähnrich and Irrgang,
Biotechnology Letters
4: 775-780),
Myceliophthora thermophila
(Coudray et al., 1982,
Biochemical Journal
203: 277-284),
Sclerotium rolfsii
(Sadana and Patil, 1985,
Journal of General Microbiology
131: 1917-1923), and
Humicola insolens
(Schou et al., 1998,
Biochemical Journal
330: 565-571).
The cloning of the
Phanerochaete chrysosporium
cellobiose dehydrogenase gene has been disclosed (Raices et al., 1995,
FEBS Letters
369: 233-238).
It is an object of the present invention to provide alternative isolated nucleic acid sequences encoding polypeptides having cellobiose dehydrogenase activity.
SUMMARY OF THE INVENTION
The present invention relates to isolated nucleic acid sequences encoding polypeptides having cellobiose dehydrogenase activity, selected from the group consisting of:
(a) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 65% identity with amino acids 22 to 785 of SEQ ID NO. 2;
(b) a nucleic acid sequence having at least 65% homology with nucleotides 135 to 2425 of SEQ ID NO. 1;
(c) a nucleic acid sequence which hybridizes under low stringency conditions with (i) nucleotides 135 to 2425 of SEQ ID NO. 1, (ii) the genomic sequence comprising nucleotides 135 to 2425 of SEQ ID NO. 1, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);
(d) a nucleic acid sequence encoding a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 comprising a substitution, deletion, and/or insertion of one or more amino acids;
(e) an allelic variant of (a), (b), or (c);
(f) a subsequence of (a), (b), (c), or (e), wherein the subsequence encodes a polypeptide fragment which has cellobiose dehydrogenase activity; and
(g) a nucleic acid sequence encoding a polypeptide having cellobiose dehydrogenase activity with a pH optimum at a pH of about 5-9, such as about 7 using DCPIP or benzoquinones as the electron acceptor, about pH 7-9 using cytochrome C as the electron acceptor; and about pH 9 using ferric cyanide as the electron acceptor; a temperature optimum of 55° C. at pH 9.5, a molecular weight of about 92 kDa by SDS-PAGE; an isoelectric point of about 4-5; a relative activity of at least 70% at a pH of about 9 and a temperature of about 50° C.; and stability at a pH of 5-10.
The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as recombinant methods for producing the polypeptides.
REFERENCES:
Subramaniam et al., SPTREMBL Database, Accession No. 074240, Nov. 1998.*
Hillier et al., EST Database, Accession No. H91018, Nov. 1995.*
Westermark & Eriksson. 1974. Acta Chem. Scand. Ser. B28:204-208.
Westermark & Eriksson. 1974. Acta Chem. Scand. Ser. B28:209-214.
Avers et al.. 1978. European Journal of Biochemistry 90:171-181.
Schmidhalter & Canevascini. 1992. Applied Microbiology Biotechnology 37:431-436.
Dekker. 1980. Journal of General Microbilogy 120: 309-316.
Fähnrich & Irrgang. Biotechnology Letters 4: 775-780.
Coudray et al., 1982. Biochemical Journal 203: 277-284.
Sadana & Patil. 1985. Journal of General Microbiology 131: 1917-1923.
Schou et al., 1998. Biochemical Journal 330: 565-571.
Raices et al., 1995. FEBS Letters 369: 233-238.
Brown Kimberly M.
Golightly Elizabeth J.
Novozymes Biotech Inc.
Prouty Rebecca E.
Starnes Robert L.
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