Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Utility Patent
1999-09-22
2001-01-02
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C536S023100, C435S820000, C435S069100
Utility Patent
active
06168936
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel phenol oxidizing enzymes, in particular, novel phenol oxidizing enzymes derived from strains of
Stachybotrys
and novel strains of the genus Stachybotrys producing these enzymes. The present invention provides methods and host cells for expressing
Stachybotrys
phenol oxidizing enzymes as well as methods for producing expression systems.
BACKGROUND OF THE INVENTION
Phenol oxidizing enzymes function by catalyzing redox reactions, i.e., the transfer of electrons from an electron donor (usually a phenolic compound) to molecular oxygen (which acts as an electron acceptor) which is reduced to H
2
O. While being capable of using a wide variety of different phenolic compounds as electron donors, phenol oxidizing enzymes are very specific for molecular oxygen as the electron acceptor.
Phenol oxidizing enzymes can be utilized for a wide variety of applications, including the detergent industry, the paper and pulp industry, the textile industry and the food industry. In the detergent industry, phenol oxidizing enzymes have been used for preventing the transfer of dyes in solution from one textile to another during detergent washing, an application commonly referred to as dye transfer inhibition.
Most phenol oxidizing enzymes exhibit pH optima in the acidic pH range while being inactive in neutral or alkaline pHs.
Phenol oxidizing enzymes are known to be produced by a wide variety of fungi, including species of the genii Aspergillus, Neurospora, Podospora, Botytis, Pleurotus, Fomes, Phlebia, Trametes, Polyporus, Rhizoctonia and Lentinus. However, there remains a need to identify and isolate phenol oxidizing enzymes, and organisms capable of naturally-producing phenol oxidizing enzymes, which present pH optima in the alkaline range for use in detergent washing methods and compositions.
SUMMARY OF THE INVENTION
The present invention relates to novel phenol oxidizing enzymes. In a preferred embodiment, the present invention relates to phenol oxidizing enzymes obtainable from
Stachybotrys
. In particular, the enzymes of the present invention are capable of modifying the color associated with dyes and colored compounds having different chemical structures, especially at neutral or alkaline pH. Based on their color modifying ability, phenol oxidizing enzymes of the present invention can be used, for example, for pulp and paper bleaching, for bleaching the color of stains on fabric and in detergent and textile applications. In one aspect of the present invention, the phenol oxidizing enzyme is able to modify the color of a dye or colored compound in the absence of an enhancer. In another aspect of the present invention, the phenol oxidizing enzyme is able to modify the color in the presence of an enhancer.
The present invention is based upon the identification and characterization of a genomic sequence (SEQ ID NO:3) encoding a phenol oxidizing enzyme obtainable from
Stachybotrys
and having the deduced amino acid sequence as shown in SEQ ID NO:2.
Accordingly, the present invention provides phenol oxidizing enzymes comprising between at least 68% and 100% identity, that is, at least 68% identity, at least 70%, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity and at least 95% identity to the phenol oxidizing enzyme having the amino acid sequence disclosed in SEQ ID NO:2, as long as the enzyme is capable of modifying the color associated with dyes or colored compounds. In one embodiment, the phenol oxidizing enzyme has the amino acid sequence as shown in SEQ ID NO:2 or as contained in
Stachybotrys chartarum
having MUCL accession number 38898.
In one embodiment, the phenol oxidizing enzyme is obtainable from a
Stachybotrys
species including
Stachybotrys parvispora, Stachybotrys chartarum; S. kampalensis; S. theobromae; S. bisbyi, S. cylindrospora, S. dichroa, S. oenanthes
and
S. nilagerica
. In another embodiment, the
Stachybotrys
includes
Stachybotrys chartarum
MUCL 38898 and
S. chartarum
MUCL 30782.
In yet another embodiment, the present invention provides an isolated polynucleotide encoding a phenol oxidizing enzyme wherein said polynucleotide comprises a nucleic acid sequence having between at least 65% and 100% identity, that is, at least 65% identity, at least 70%, at least 75% identity, at least 80%, at least 85%, at least 90% and at least 95% identity to SEQ ID NO:1, as long as the polynucleotide encodes a phenol oxidizing enzyme capable of modifying the color associated with dyes or colored compounds. The present invention encompasses polynucleotide sequences that hybridize under conditions of high stringency to the polynucleotide shown in SEQ ID NO:1 or SEQ ID NO:3 as long as the sequence is capable of modifying the color associated with dyes or colored compounds. The present invention also encompasses polynucleotides that encode the amino acid sequence as shown in SEQ ID NO:2. In one embodiment, the polynucleotide has the nucleic acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:3 or as contained in
Stachybotrys chartarum
having MUCL accession number 38898. The present invention also provides expression vectors and host cells comprising polynucleotides of the present invention.
The present invention additionally relates to methods for producing a phenol oxidizing enzyme of the present invention. Accordingly, the present invention provides a method for producing a phenol oxidizing enzyme comprising the step of culturing a host cell comprising an isolated polynucleotide encoding a phenol oxidizing enzyme having a sequence comprising between at least 68% and 100% identity, that is, at least 68% identity, at least 70%, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity and at least 95% identity to the phenol oxidizing enzyme having the amino acid sequence disclosed in SEQ ID NO:2 under conditions suitable for the production of said phenol oxidizing enzyme; and optionally recovering said phenol oxidizing enzyme produced. In one embodiment, the polynucleotide comprises the sequence as shown in SEQ ID NO:1. In another embodiment, the polynucleotide comprises the sequence as shown in SEQ ID NO: 3. In an additional embodiment, the polynucleotide hybridizes under conditions of high stringency with the polynucleotide having the sequence as shown in SEQ ID NO:1 or SEQ ID NO:3 or as contained in
Stachybotrys chartarum
having MUCL accession number 38898. In a further embodiment, the polynucleotide has between 65% and 100%, that is, at least 65% identity, at least 70%, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity and at least 95% identity to SEQ ID NO: 1 or SEQ ID NO:3.
The present invention also provides a method for producing a recombinant host cell comprising a polynucleotide encoding a phenol oxidizing enzyme, comprising the steps of obtaining an isolated polynucleotide encoding said phenol oxidizing enzyme said polynucleotide having between at least 65% and 100% identity, that is, at least 65% identity, at least 70%, at least 75% identity, at least 80%, at least 85%, at least 90% and at least 95% identity to SEQ ID NO:3; introducing said polynucleotide into said host cell; and growing said host cell under conditions suitable for the production of said phenol oxidizing enzyme. In one embodiment, the polynucleotide is integrated into the host genome and in another embodiment, the polynucleotide is present on a replicating plasmid. The present invention also encompasses polynucleotide sequences that hybridize under conditions of high stringency to the polynucleotide shown in SEQ ID NO:1 or SEQ ID NO:3. The present invention also provides polynucleotides that encode the amino acid sequence as shown in SEQ ID NO:2. In one embodiment, the polynucleotide has the nucleic acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:3 or as contained in
Stachybotrys chartarum
having MUCL accession number 38898.
In one embodiment of the present invention, the host cell comprising a polynucleotide encoding
Achutamurthy Ponnathapu
Fronda Christian L.
Genencor International Inc.
Genencor International Inc.
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