EP1066364A2 - Enzymes d'oxydation de phenol et leurs utilisations - Google Patents

Enzymes d'oxydation de phenol et leurs utilisations

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Publication number
EP1066364A2
EP1066364A2 EP99917861A EP99917861A EP1066364A2 EP 1066364 A2 EP1066364 A2 EP 1066364A2 EP 99917861 A EP99917861 A EP 99917861A EP 99917861 A EP99917861 A EP 99917861A EP 1066364 A2 EP1066364 A2 EP 1066364A2
Authority
EP
European Patent Office
Prior art keywords
phenol oxidizing
oxidizing enzyme
stachybotrys
enzyme
detergent composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99917861A
Other languages
German (de)
English (en)
Inventor
Daniel Unilever Res. Vlaardingen CONVENTS
Antoine Amory
Huaming Wang
Patrick Dhaese
Annick Lambrechts-Rongvaux
Cynthia Wang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Unilever PLC
Unilever NV
Original Assignee
Unilever PLC
Unilever NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/218,702 external-priority patent/US6426410B1/en
Application filed by Unilever PLC, Unilever NV filed Critical Unilever PLC
Publication of EP1066364A2 publication Critical patent/EP1066364A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • 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.
  • the present invention also relates to methods for modifying a colored compound and dye transfer prevention during fabric washing.
  • the invention relates to an enzymatic detergent composition for stain bleaching or anti dye transfer.
  • 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 H2O. 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.
  • 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.
  • 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.
  • species of the genii Aspergillus including species of the genii Aspergillus, Neurospora, Podospora, Botytis, Pleurotus, Fomes, Phlebia, Trametes, Polyporus, Rhizoctonia and Lentinus.
  • 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.
  • the present invention relates to detergent composition
  • a novel phenol oxidizing enzymes obtainable from Stachybotrys which are capable of modifying the color associated with dyes and colored compounds having different chemical structures, in particular at neutral or alkaline pH.
  • 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 for anti-dye transfer in detergent and textile applications.
  • the phenol oxidizing enzyme is able to modify the color in the absence of an enhancer.
  • the phenol oxidizing enzyme is able to modify the color in the presence of an enhancer.
  • the phenol oxidizing enzymes are obtainable from Stachybotrys.
  • the Stachybotrys enzymes are selected from strains of the group consisting of S. parvispora, including, in particular, S. parvispora var. hughes MUCL 38996; strains of the species S. chartarum including, in particular, S. chartarum MUCL 38898; S. parvispora MUCL 9485; S. chartarum MUCL 30782; S. kampalensis MUCL 39090; S. theobromae MUCL 39293; and strains of the species S. bisbyi, S. cylindrospora, S.
  • the present invention provides a phenol oxidizing enzyme which has molecular weight of about 38 kD as measured by SDS polyacrylamide gel electrophoresis (PAGE). In another aspect, the present invention provides a phenol oxidizing enzyme which has a molecular weight of about 30.9 kD as measured by SDS polyacrylamide gel electrophoresis.
  • SAGE SDS polyacrylamide gel electrophoresis
  • the present invention provides a phenol oxidizing enzyme which has a molecular weight of about 30.9 kD as measured by SDS polyacrylamide gel electrophoresis.
  • the three molecular weight species were about 70 kD, 45 kD and 22.1 kD.
  • the three ' molecular weight species were about 58.4 kD, 46.1 kD and 19.7 kD.
  • the present invention encompasses any phenol oxidizing enzyme activity inherent to any of these molecular weight species alone or in combination with at least one other of the molecular weight species.
  • the present invention also encompasses any phenol oxidizing enzyme which exhibits an increase in apparent molecular weight after boiling, wherein the molecular weight is determined by SDS- polyacrylamide gel electrophoresis.
  • the present invention also encompasses phenol oxidizing enzymes which are capable of modifying the color associated with dyes or colored compounds and which have at least one antigenic group in common with phenol oxidizing enzyme naturally- produced by S. parvispora MUCL 38996 and/or the phenol oxidizing enzyme naturally- produced by S. chartarum MUCL 38898 as measured by the Ouchterlony technique in which a positive enzyme exhibits an immunoprecipitation line.
  • the immunoprecipitation line is Type 1 line.
  • the phenol oxidizing enzyme having at least one antigenic group in common with phenol oxidizing enzyme naturally produced by S. parvispora MUCL 38996 is obtainable from Stachybotrys.
  • the present invention also encompasses Stachybotrys phenol oxidizing enzyme mutants as long as the mutant is able to modify the color associated with dyes or colored compounds.
  • the present invention provides an isolated polynucleotide encoding a phenol oxidizing enzyme obtainable from Stachybotrys wherein said polynucleotide comprises a nucleic acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and at least 95% identity to SEQ ID NO:1 or SEQ ID NO:3 as long as the polynucleotide encodes a phenol oxidizing enzyme capable of modifying the color associated with dyes or colored compounds.
  • the present invention also encompasses polynucleotide sequences that are capable of hybridizing under conditions of intermediate to high stringency to the polynucleotide shown in SEQ ID NO:1 or SEQ ID NO:3 or which are complementary thereto.
  • the present invention also provides polynucleotides that encode the amino acid sequence as shown in SEQ ID NO:2.
  • the polynucleotide has the nucleic acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:3.
  • 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 obtainable from Stachybotrys. Accordingly, the present invention provides a method for producing said enzyme comprising the steps of obtaining a host cell comprising a polynucleotide encoding said phenol oxidizing enzyme obtainable from Stachybotrys wherein said enzyme has at least 65% identity to the amino acid sequence disclosed in SEQ ID NO:2; growing said host cell under conditions suitable for the production of said phenol oxidizing enzyme; and optionally recovering said phenol oxidizing enzyme produced.
  • the polynucleotide comprises the sequence as shown in SEQ ID NO:1.
  • the polynucleotide comprises the sequence as shown in SEQ ID NO: 3.
  • the polynucleotide is capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 under conditions of intermediate to high stringency or is complementary 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 of the present invention comprising the steps of obtaining a polynucleotide encoding said phenol oxidizing enzyme obtainable from Stachybotrys and having at least 65% identity to the amino acid sequence disclosed in SEQ ID NO:2; introducing said polynucleotide into said host cell; and growing said host cell under conditions suitable for the production of said phenol oxidizing enzyme.
  • 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 a further embodiment, the polynucleotide is capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 under conditions of intermediate to high stringency or is complementary to SEQ ID NO:1 or SEQ ID NO:3.
  • the recombinant host cell comprising a polynucleotide encoding a phenol oxidizing enzyme includes filamentous fungus, yeast and bacteria.
  • the host cell is a filamentous fungus including Aspergillus species, Trichoderma species and Mucor species.
  • the filamentous fungus host cell includes A. niger var. awamori and T. reseei.
  • the host cell is a yeast which includes Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces and Yarrowia species.
  • Saccharomyces species is S. cerevisiae.
  • the host cell is a gram positive bacteria, such as a Bacillus species, or a gram negative bacteria, such as an Escherichia species.
  • the present invention also encompasses methods for purifying the phenol oxidizing enzyme from such host cells.
  • detergent compositions comprising the amino acid having a sequence at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or 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.
  • the amino acid has the sequence as shown in SEQ ID NO: 2.
  • the phenol oxidizing enzyme is encoded by a polynucleotide comprising the sequence as shown in SEQ ID NO: 1.
  • the phenol oxidizing enzyme is encoded by a polynucleotide comprising the sequence as shown in SEQ ID NO:3.
  • the polynucleotide is capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 under conditions of intermediate to high stringency or is complementary to SEQ ID NO:1 or SEQ ID NO:3.
  • the present invention also encompasses methods for modifying the color associated with dyes or colored compounds which occur in stains on fabric, comprising the steps of contacting the fabric with a composition comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or 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.
  • the amino acid has the sequence as shown in SEQ ID NO:2.
  • the pH optimum is between 5.0 and 11.0, in another aspect, the pH optimum is between 7 and 10.5 and in yet another aspect the pH optimum is between 8.0 and 10.
  • the optimum temperature is between 20 and 60 degrees C. and in another aspect between 20 and 40 degrees C.
  • the present invention also provides methods for preventing dye transfer in detergent and textile applications.
  • detergent compositions comprising a Stachybotrys phenol oxidizing enzyme of the present invention alone or in combination with an enhancer and other detergent ingredients, including proteases, amylases and/or cellulases.
  • Enhancers which can be used in detergent compositions of the present invention include but are not limited to phenothiazine-10-propionic acid (PPT), 10- methylphenothiazine (MPT), phenoxazine-10-propionic acid (PPO), 10- methylphenoxazine (MPO), 10-ethylphenothiazine-4-carboxylic acid (EPC) acetosyringone, syringaldehyde, methylsyringate, 2,2'-azino-bis (3- ethylbenzothiazoline-6-sulfonate (ABTS) and 4-Hydroxy-4-biphenyl-carboxylic acid or derivatives thereof.
  • PPT phenothiazine-10-propionic acid
  • MPT 10- methylphenothiazine
  • PPO phenoxazine-10-propionic acid
  • MPO 10- methylphenoxazine
  • EPC 10-ethylphen
  • Figure 2 illustrates the pH profile of Direct Bluel bleaching as a comparison between Stachybotrys parvispora phenol oxidizing enzyme and Myrothecium verrucaria bilirubin oxidase.
  • Figure 3 illustrates the molecular weight of Stachybotrys chartarum phenol oxidizing enzyme as determined by SDS polyacrylamide gel. Lane 1 represents unboiled sample and lane 2 represents boiled sample.
  • Figures 4A-4B is an amino acid alignment of fragments of Stachybotrys chartarum phenol oxidizing enzyme (designated St. ch.) with Myrothecium verracaria bilirubin oxidase (designated biliru oxidas) and LEPTOTHRIX DISCOPHORA manganese oxidizing protein (designated mpf-A).
  • Figure 5 illustrates the nucleic acid (SEQ ID NO:1) and amino acid (SEQ ID NO:2) sequence for a phenol oxidizing enzyme obtainable from Stachybotrys chartarum.
  • Figure 6 illustrates the genomic sequence (SEQ ID NO:3) for a phenol oxidizing enzyme obtainable from Stachybotrys chartarum.
  • Figure 7 is an amino acid alignment of Stachybotrys phenol oxidizing enzyme SEQ ID NO:2 (bottom line) and Bilirubin oxidase (SEQ ID NO:4).
  • Figure 8 provides an illustration of the vector pGAPT which was used for the expression of Stachybotrys phenol oxidizing enzyme in Aspergillus.
  • Base 1 to 1134 contains Aspergillus niger glucoamylase gene promoter.
  • Base 1227 to 1485 and 3079 to 3100 contains Aspergillus niger glucoamylase terminator.
  • Aspergillus nidulans pyrG gene was inserted from 1486 to 3078 as a marker for fungal transformation.
  • the rest of the plasmid contains pUC18 sequences for propagation in E. coli.
  • Nucleic acid encoding the Stachybotrys phenol oxidizing enzyme of SEQ ID NO:1 was cloned into the Bgl II and Xba I restriction sites.
  • Figure 9 shows the nucleic acid sequence of the PCR generated fragment of Stachybotrys described in Example 13 that was expressed in Aspergillus.
  • Figure 10 is an SDS polyacrylamide gel electrophoresis showing the production of phenol oxidizing enzyme produced by Aspergillus niger var. awamori. Detailed Description Definitions
  • phenol oxidizing enzyme refers to those enzymes which catalyze redox reactions and are specific for molecular oxygen and hydrogen peroxide as the electron acceptor.
  • Stachybotrys phenol oxidizing enzymes of the present invention are boiled and subjected to SDS gel electrophoresis, three molecular weight species are observed.
  • enzyme encompasses any molecular weight species which alone or in combination with at least one other molecular weight species is able to modify the color associated with a dye or colored compound.
  • Stachybotrys refers to any Stachybotrys species which produces a phenol oxidizing enzyme capable of modifying the color associated with dyes or colored compounds.
  • the present invention encompasses derivatives of natural isolates of Stachybotrys, including progeny and mutants, as long as the derivative is able to produce a phenol oxidizing enzyme capable of modifying the color associated with dye or colored compounds.
  • the phenol oxidizing enzyme is obtainable from Stachybotrys and is purified by the method disclosed in Examples 4 and 5.
  • the term "obtainable from” means phenol oxidizing enzymes equivalent to those that originate from or are naturally-produced by the particular microbial strain mentioned.
  • phenol oxidizing enzymes obtainable from Stachybotrys refer to those phenol oxidizing enzymes which are naturally-produced by Stachybotrys.
  • the present invention encompasses phenol oxidizing enzymes identical to those produced by Stachybotrys species but which through the use of genetic engineering techniques are produced by non-Stac/jybofrys organisms transformed with a nucleic acid encoding said phenol oxidizing enzyme.
  • Being equivalent means that the phenol oxidizing enzyme has at least one antigenic group in common with phenol oxidizing enzyme obtainable from S. parvispora MUCL 38996 and/or S. chartarum MUCL 38898 as measured by the
  • the phenol oxidizing enzyme is encoded by a polynucleotide capable of hybridizing to the polynucleotide having the sequence as shown in SEQ ID NO:1 or SEQ ID NO:3 under conditions of intermediate to maximum stringency.
  • the phenol oxidizing enzyme comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the phenol oxidizing enzyme having the amino acid sequence disclosed in SEQ ID NO:2.
  • the present invention also encompasses mutants, variants and derivatives of the phenol oxidizing enzymes of the present invention as long as the mutant, variant or derivative phenol oxidizing enzyme is able to retain at least one characteristic activity of the naturally occurring phenol oxidizing enzyme.
  • the term 'colored compound' refers to a substance that adds color to textiles or to substances which result in the visual appearance of stains.
  • a dye is a colored compound that is incorporated into the fiber by chemical reaction, absorption, or dispersion. Examples of dyes include direct Blue dyes, acid Blue dyes, direct red dyes, reactive Blue and reactive Black dyes. A catalogue of commonly used textile dyes is found in Colour Index, 3 rd ed. Vol. 1-8. Examples of substances which result in the visual appearance of stains are polyphenols, carotenoids, anthocyanins, tannins, Maillard reaction products, etc.
  • modify the color associated with a dye or colored compound or “modification of the colored compound” means that the dye or compound is changed through oxidation such that either the color appears modified, i.e., the color visually appears to be decreased, lessened, decolored, bleached or removed, or the color is not affected but the compound is modified such that dye redeposition is inhibited.
  • the present invention encompasses the modification of the color by any means including, for example, the complete removal of the colored compound from stain on a fabric by any means as well as a reduction of the color intensity or a change in the color of the compound.
  • the "anti-dye transfer" or “anti-dye redeposition” effect may be a function of the color modification activity of a phenol oxidizing compound, i.e., soluble dyes or colored components are oxidized or bleached and are not able to be redeposited as a color on the fabric, or a function of substrate modification in the absence of color modification such that a dye or colored component becomes water soluble and is rinsed away.
  • a phenol oxidizing compound used alone or together with an enhancer to oxidize an soluble or dispersed dye or colored compound to a colorless species in a wash solution prevents the color redeposition effect.
  • mutants and variants when referring to phenol oxidizing enzymes, refers to phenol oxidizing enzymes obtained by alteration of the naturally occurring amino acid sequence and/or structure thereof, such as by alteration of the DNA nucleotide sequence of the structural gene and/or by direct substitution and/or alteration of the amino acid sequence and/or structure of the phenol oxidizing enzyme.
  • phenol oxidizing enzyme "derivative" as used herein refers to a portion or fragment of the full-length naturally occurring or variant phenol oxidizing enzyme amino acid sequence that retains at least one activity of the naturally occurring phenol oxidizing enzyme.
  • mutants and variants when referring to microbial strains, refers to cells that are changed from a natural isolate in some form, for example, having altered DNA nucleotide sequence of, for example, the structural gene coding for the phenol oxidizing enzyme; alterations to a natural isolate in order to enhance phenol oxidizing enzyme production; or other changes that effect phenol oxidizing enzyme expression.
  • the term "enhancer” or “mediator” refers to any compound that is able to modify the color associated with a dye or colored compound in association with a phenol oxidizing enzyme or a compound which increases the oxidative activity of the phenol oxidizing enzyme.
  • the enhancing agent is typically an organic compound. Phenol oxidizing enzymes
  • the phenol oxidizing enzymes of the present invention function by catalyzing redox reactions, i.e., the transfer of electrons from an electron donor (usually a phenolic compound) to molecular oxygen or hydrogen peroxide (which acts as an electron acceptor) which is reduced to water.
  • electron donor usually a phenolic compound
  • hydrogen peroxide which acts as an electron acceptor
  • Examples of such enzymes are laccases (EC 1.10.3.2), bilirubin oxidases (EC 1.3.3.5), phenol oxidases (EC 1.14.18.1), catechol oxidases (EC 1.10.3.1).
  • the present invention encompasses Stachybotrys phenol oxidizing enzymes which are capable of modifying the color associated with a dye or colored compounds and which have at least one antigenic group in common with the phenol oxidizing enzyme naturally-produced by S. parvispora MUCL 38996 and/or the phenol oxidizing enzyme naturally-produced by S. chartarum MUCL 38898.
  • One method for measuring the presence of common antigenic determinants is with the double immunodiffusion tests (Ouchterlony technique) following the protocol set forth in, and under the conditions specified in, Clausen, J. ' (1988) Immunochemical Technique for the Identification and Estimation of Macromolecules (3rd revised edition) Burdon, R.H., and P.H.
  • Phenol oxidizing enzyme obtainable from S. parvispora MUCL 38996 and produced according to Examples 4 and 5 has an apparent molecular weight of about 38 kilodaltons (kD's) as determined by an SDS-PAGE analysis method and an apparent isoelectric point of lower than 2.8 as defined in Example 6.
  • Phenol oxidizing enzyme obtainable from S. chartarum MUCL 38898 and produced by the method of Examples 4 and 5 has an apparent molecular weight of about 30.9 kilodaltons as determined by an SDS-PAGE analysis method.
  • the present invention encompasses Stachybotrys phenol oxidizing enzymes comprising at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity to the phenol oxidizing enzyme having the amino acid sequence disclosed in SEQ ID NO:2. Nucleic acid encoding phenol oxidizing enzymes
  • the present invention encompasses polynucleotides which encode phenol oxidizing enzymes obtainable from Stachybotrys species which polynucleotides comprise at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity and at least 95% identity to the polynucleotide sequence disclosed in SEQ ID NO:1 or SEQ ID NO:3 as long as the enzyme encoded by the polynucleotide is capable of modifying the color associated with dyes or colored compounds.
  • the phenol oxidizing enzyme has the polynucleotide sequence as shown in SEQ ID NO:1 or as shown in SEQ ID NO:3 or is capable for hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or is complementary thereto.
  • SEQ ID NO:1 or as shown in SEQ ID NO:3
  • SEQ ID NO:3 or is capable for hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or is complementary thereto.
  • SEQ ID NO:2 As will be understood by the skilled artisan, due to the degeneracy of the genetic code, a variety of polynucleotides can encode the phenol oxidizing enzyme disclosed in SEQ ID NO: 2. The present invention encompasses all such polynucleotides.
  • the nucleic acid encoding a phenol oxidizing enzyme may be obtained by standard procedures known in the art from, for example, cloned DNA (e.g., a DNA "library”), by chemical synthesis, by cDNA cloning, by PCR, or by the cloning of genomic DNA, or fragments thereof, purified from a desired cell, such as a Stachybotrys species (See, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol.
  • Nucleic acid sequences derived from genomic DNA may contain regulatory regions in addition to coding regions. Whatever the source, the isolated nucleic acid encoding a phenol oxidizing enzyme of the present invention should be molecularly cloned into a suitable vector for propagation of the gene. In the molecular cloning of the gene from genomic DNA, DNA fragments are generated, some of which will encode the desired gene. The DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis, PCR and column chromatography.
  • a phenol oxidizing enzyme encoding gene of the present invention or its specific RNA, or a fragment thereof, such as a probe or primer may be isolated and labeled and then used in hybridization assays to detect a generated gene.
  • Those DNA fragments sharing substantial sequence similarity to the probe will hybridize under stringent conditions.
  • the present invention encompasses phenol oxidizing enzymes obtainable from
  • Stachybotrys species which are identified through nucleic acid hybridization techniques using SEQ ID NO:1 or SEQ ID NO:3 as a probe or primer and screening nucleic acid of either genomic of cDNA origin.
  • Nucleic acid encoding phenol oxidizing enzymes obtainable from Stachybotrys species and having at least 65% identity to SEQ ID NO:1 or SEQ ID NO:3 can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes, portions or fragments of SEQ ID NO:1 or SEQ ID NO:3.
  • the present invention provides a method for the detection of nucleic acid encoding a phenol oxidizing enzyme encompassed by the present invention which comprises hybridizing part or all of a nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3 with Stachybotrys nucleic acid of either genomic or cDNA origin.
  • polynucleotide sequences that are capable of hybridizing to the nucleotide sequence disclosed in SEQ ID NO:1 under conditions of intermediate to maximal stringency.
  • Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA) incorporated herein by reference, and confer a defined "stringency” as explained below.
  • Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); “high stringency” at about 5°C to 10°C below Tm; “intermediate stringency” at about 10°C to 20°C below Tm; and “low stringency” at about 20°C to 25°C below Tm.
  • a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate or low stringency hybridization can be used to identify or detect polynucleotide sequence homologs.
  • hybridization shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J (1994) Dictionary of Biotechnology, Stockton Press, New York NY).
  • PCR polymerase chain reaction
  • a preferred method of isolating a nucleic acid construct of the invention from a cDNA or genomic library is by use of polymerase chain reaction (PCR) using degenerate oligonucleotide probes prepared on the basis of the amino acid sequence of the protein having the amino acid sequence as shown in SEQ ID NO:2.
  • PCR polymerase chain reaction
  • the PCR may be carried out using the techniques described in US patent No. 4,683,202.
  • the present invention provides host cells, expression methods and systems for the production of phenol oxidizing enzymes obtainable from Stachybotrys species in host microorganisms, such as fungus, yeast and bacteria. Once nucleic acid encoding a phenol oxidizing enzyme of the present invention is obtained, recombinant host cells containing the nucleic acid may be constructed using techniques well known in the art.
  • Nucleic acid encoding phenol oxidizing enzymes obtainable from Stachybotrys species and having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and at least 95% identity to the nucleic acid of SEQ ID NO:1 or SEQ ID NO:2 or which are capable of hybridizing under conditions of intermediate to high stringency or which is complementary to SEQ ID NO:1 or SEQ ID NO:3 is obtained and transformed into a host cell using appropriate vectors.
  • a variety of vectors and transformation and expression cassettes suitable for the cloning, transformation and expression in fungus, yeast and bacteria are known by those of skill in the art.
  • the vector or cassette contains sequences directing transcription and translation of the nucleic acid, a selectable marker, and sequences allowing autonomous replication or chromosomal integration.
  • Suitable vectors comprise a region 5' of the gene which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination. These control regions may be derived from genes homologous or heterologous to the host as long as the control region selected is able to function in the host cell.
  • Initiation control regions or promoters, which are useful to drive expression of the phenol oxidizing enzymes in a host cell are known to those skilled in the art. Virtually any promoter capable of driving these phenol oxidizing enzyme is suitable for the present invention.
  • Nucleic acid encoding the phenol oxidizing enzyme is linked operably through initiation codons to selected expression control regions for effective expression of the oxidative or reducing enzymes. Once suitable cassettes are constructed they are used to transform the host cell.
  • General transformation procedures are taught in Current Protocols In Molecular Biology (vol. 1 , edited by Ausubel et al., John Wiley & Sons, Inc. 1987, Chapter 9) and include calcium phosphate methods, transformation using PEG and electroporation.
  • PEG and Calcium mediated protoplast transformation can be used (Finkelstein, DB 1992 Transformation. In Biotechnology of Filamentous Fungi. Technology and Products (eds by Finkelstein & Bill) 113-156.
  • Electroporation of protoplast is disclosed in Finkelestein, DB 1992 Transformation. In Biotechnology of Filamentous Fungi. Technology and Products (eds by Finkelstein & Bill) 113-156.
  • Microprojection bombardment on conidia is described in Fungaro et al. (1995) Transformation of Aspergillus nidulans by microprojection bombardment on intact conidia.
  • Agrobacterium mediated transformation is disclosed in Groot et al. (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nature Biotechnology 16 839-842.
  • For transformation of Saccharomyces lithium acetate mediated transformation and PEG and calcium mediated protoplast transformation as well as electroporation techniques are known by those of skill in the art.
  • Host cells which contain the coding sequence for a phenol oxidizing enzyme of the present invention and express the protein may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques which include membrane-based, solution-based, or chip-based technologies for the detection and/or quantification of the nucleic acid or protein.
  • the genomic sequence (SEQ ID NO:3) encoding phenol oxidizing enzyme obtainable from Stachybotrys chartarum (MUCL 38898) was isolated and expressed in Aspergillus niger var. awamori and Trichoderma reesei.
  • the cDNA (SEQ ID NO: 1) obtainable from Stachybotrys chartarum (MUCL 38898) was isolated and expressed in Saccharomyces cerevisiae. Phenol oxidizing enzyme activities
  • the phenol oxidizing enzymes of the present invention are capable of using a wide variety of different phenolic compounds as electron donors, while being very specific for molecular oxygen or hydrogen peroxide as the electron acceptor. Depending upon the specific substrate and reaction conditions, e.g., temperature, presence or absence of enhancers, etc., each phenol oxidizing enzyme oxidation reaction will have an optimum pH.
  • the Stachybotrys parvispora phenol oxidizing enzyme produced as described in Example 4 has a pH optimum of from about 5.0 to about 7.0, as determined by incubation for 2 minutes at 20 degrees C with ABTS as substrate; a pH optimum of from about 6.0 to about 7.5, as determined by incubation for 2 minutes at 20 degrees C with syringaldizin as substrate; and a pH optimum of from about 7.0 to about 9.0, as determined by incubation for 2 minutes at 20 degrees C with 2,6-dimethoxyphenol as substrate, and which is able to oxidize guiacol.
  • Phenol oxidizing enzyme obtained from Stachybotrys chartarum MUCL 38898, produced as described in Examples 4 and 5 and having the amino acid sequence as shown in SEQ ID NO:2 has a pH optimum of about 8.0 at both 20 and 40 degrees C as determined by incubation with DMP as a substrate and in the presence of a total of 17.2 ⁇ g enzyme and a pH optimum of about 5.0 to 7.0 as determined by incubation with ABTS as a substrate and in the presence of a total of 1.7 ⁇ g enzyme.
  • the phenol oxidizing enzymes obtainable from Stachybotrys are capable of oxidizing a wide variety of dyes or colored compounds having different chemical structures, using oxygen or hydrogen peroxide as the electron acceptor. Accordingly phenol oxidizing enzymes of the present invention are used in applications where it is desirable to modify the color associated with dyes or colored compounds, such as in cleaning, for removing the food stains on fabric and anti-dye redeposition; textiles; and paper and pulp applications.
  • a particularly important characteristic of the phenol oxidizing enzymes is their expression of high levels of enzymatic activity, at about 20-40 degrees C, in a broad range of pHs, including a broad range of neutral to alkaline pHs. In particular is their ability to express high levels of enzymatic activity in the pH range of from about 7.0 to about 10.5 in temperatures of about 20- 35 degrees C. Colored compounds
  • colored compounds could be targets for oxidation by phenol oxidizing enzymes of the present invention.
  • colored substances which may occur as stains on fabrics can be a target.
  • Several types or classes of colored substances which may occur in stains are described below. Porphyrin derived structures.
  • Porphyrin structures often coordinated to a metal, form one class of colored substances which occur in stains. Examples are heme or haematin in blood stain, chlorophyll as the green substance in plants, e.g. grass or spinach. Another example of a metal-free substance is bilirubin, a yellow breakdown product of heme.
  • Tannins are polymerised forms of certain classes of polyphenols. Such polyphenols are catechins, leuantocyanins, etc. (P. Ribereau-Gayon, Plant Phenolics, etc.
  • Carotenoids are the coloured substances which occur in tomato (lycopene, red), mango (carotene, orange-yellow) (G.E. Bartley et al., The Plant Cell (1995), Vol 7,
  • Anthocyanins These substance are the highly colored molecules which occur in many fruits and flowers (P. Ribereau-Gayon, Plant Phenolics, Ed. Oliver & Boyd, Edinburgh, 1972,
  • Typical examples, relevant for stains, are berries, but also wine.
  • Anthocyanins have a high diversity in glycosidation patterns.
  • Maillard reaction products Upon heating of mixtures o carbohydrate molecules in the presence of protein/peptide structures, a typical yellow/brown colored substance arises. These substances occur for example in cooking oil and are difficult to remove from fabrics.
  • a phenol oxidizing enzyme of the present invention can act to modify the color associated with dyes or colored compounds in the presence or absence of enhancers depending upon the characteristics of the compound. If a compound is able to act as a direct substrate for the phenol oxidizing enzyme, the phenol oxidizing enzyme can modify the color associated with a dye or colored compound in the absence of an enhancer, although an enhancer may still be preferred for optimum phenol oxidizing enzyme activity. For other colored compounds unable to act as a direct substrate for 5 the phenol oxidizing enzyme or not directly accessible to the phenol oxidizing enzyme, an enhancer is required for optimum phenol oxidizing enzyme activity and modification of the color.
  • Enhancers are described in for example WO 95/01426 published 12 January 1995; WO 96/06930, published 7 March 1996; and WO 97/11217 published 27 March
  • Enhancers include but are not limited to phenothiazine-10-propionic acid (PPT), 10-methylphenothiazine (MPT), phenoxazine-10-propionic acid (PPO), 10- methylphenoxazine (MPO), 10-ethylphenothiazine-4-carboxylic acid (EPC) acetosyringone, syringaldehyde, methylsy ngate, 2,2'-azino-bis (3- ethylbenzothiazoline-6-sulfonate (ABTS) and 4-Hydroxy-4-biphenyl-carboxylic acid or
  • the present invention encompasses Stachybotrys strains and natural isolates, and derivatives of such strains and isolates, such as strains of the species S. parvispora, including, in particular, S.parvispora var. hughes MUCL 38996; strains of
  • the species S. chartarum including, in particular, Stachybotrys chartarum MUCL 38898; S. parvispora MUCL 9485; S. chartarum MUCL 30782; S. kampalensis MUCL 39090; S. theobromae MUCL 39293; and strains of the species S. bisbyi, S. cylindrospora, S. dichroa, S. oenanthes and S. nilagerica which produce phenol oxidizing enzymes of the present invention.
  • the present invention provides substantially biologically-pure cultures of novel strains of the genus Stachybotrys, and, in particular substantially biologically-pure cultures of the strains S. parvispora MUCL 38996 and S. chartarum MUCL 38898 from which phenol oxidizing enzymes can be purified. Purification
  • the phenol oxidizing enzymes of the present invention may be produced by cultivation of phenol oxidizing enzyme-producing Stachybotrys strains (such as S. parvispora MUCL 38996, S. chartarum MUCL 38898) under aerobic conditions in nutrient medium containing assimiable carbon and nitrogen together with other essential nutrient(s).
  • the medium can be composed in accordance with principles well-
  • the phenol oxidizing enzyme-producing strains secrete phenol oxidizing enzyme extracellularly. This permits the isolation and purification (recovery) of the phenol oxidizing enzyme to be achieved by, for example, separation of cell mass from a culture broth (e.g. by filtration or cent fugation).
  • the resulting cell- free culture broth can be used as such or, if desired, may first be concentrated (e.g. by evaporation or ultrafiltration). If desired, the phenol oxidizing enzyme can then be separated from the cell-free broth and purified to the desired degree by conventional methods, e.g. by column chromatography, or even crystallized.
  • the phenol oxidizing enzymes of the present invention may be isolated and purified from the culture broth into which they are extracellularly secreted by concentration of the supernatant of the host culture, followed by ammonium sulfate fractionation and gel permeation chromatography.
  • the phenol oxidizing enzymes of the present invention may be formulated and utilized according to their intended application.
  • the phenol oxidizing enzyme may be formulated, directly from the fermentation broth, as a coated solid using the procedure described in United States Letters Patent No. 4,689,297.
  • the phenol oxidizing enzyme may be formulated in a liquid form with a suitable carrier.
  • the phenol oxidizing enzyme may also be immobilized, if desired.
  • the present invention also encompasses expression vectors and recombinant host cells comprising a Stachybotrys phenol oxidizing enzyme of the present invention and the subsequent purification of the phenol oxidizing enzyme from the recombinant host cell.
  • a Stachybotrys phenol oxidizing enzyme of the present invention may be used in detergent or cleaning compositions.
  • Such compositions may comprise, in addition to the phenol oxidizing enzyme, conventional detergent ingredients such as surfactants, builders and further enzymes such as, for example, proteases, amylases, lipases, cutinases, cellulases or peroxidases.
  • Other ingredients include enhancers, stabilizing agents, bactericides, optical brighteners and perfumes.
  • the detergent compositions may take any suitable physical form, such as a powder, an aqueous or non aqueous liquid, a paste or a gel. Examples of detergent compositions are given in WO 95/01426, published 12 January 1995 and WO 96/06930 published 7 March 1996.
  • a new strain of the species Stachybotrys parvispora var. hughes was isolated from soil samples on an agar-agar nutrient medium and selected by its production of an enzyme having oxidase activity.
  • the new strain was individually cultured on corn meal agar (DIFCO) at 25 degrees C for a period of three weeks.
  • the new strain of S. parvispora was identified by its slow growth in corn meal agar at 25 degrees C, being less than 4 cm in three weeks, its formation of conidia and the morphological characteristics of the formed conidia.
  • Stachybotrys atra var. corda was isolated from soil samples on an agar-agar nutrient medium and selected by its production of an enzyme having oxidase activity.
  • the new strain was individually cultured on corn meal agar (DIFCO) at 25 degrees C for a period of three weeks.
  • the new strain S. chartarum was identified by its rapid growth on corn meal agar at 25 degrees C, being more than 4 cm in three weeks, its formation of conidia and the morphological characteristics of the formed conidia.
  • Stachybotrys parvispora MUCL 38996 obtained as described above in Example 1 , was isolated on PDA (potato dextrose agar) plates (DIFCO).
  • the titer (measured in terms of colony forming units (cfu) per ml) of the resulting suspension was then determined by plating dilutions [in 0.9% (w/v) NaCI] on PDA plates.
  • the titers of the resulting conidial stock suspensions ranged from 10 ⁇ to I0 cfu/ml.
  • a twenty liter fermentor containing glucose and potato extract was prepared by boiling 4.5 kilograms of peeled and diced potatoes for 30 minutes in 15 liters of water (milli-Q quality), filtering the resulting suspension through hydrophilic cotton gauze (STELLA), collecting the resulting filtrate and then supplementing the collected filtrate with 300 grams of glucose.
  • the glucose supplemented filtrate was then placed in the fermentor and sterilized for 30 minutes at 120°C.
  • the sterilized supplemented filtrate had a pH of 5.8.
  • the twenty liter fermentor was then inoculated with 15 ml of the conidial stock suspension, obtained as described above in Example 3, and fermentation was conducted for 144 hours at 37 degrees C.
  • Fermentation was performed under a constant air flow of 4.5 liters/minute and a constant agitation of 100 RPM (revolutions per minute) (diameter 13 cm) without pH control.
  • phenol oxidizing enzyme activity in the supernatant was then measured using the following standard assay procedure, based on the oxidation of ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonate)] by oxygen : a final reaction volume of 1 ml containing Tris [Tris(hydroxymethyl)-aminomethane]/HCI 200 mM (pH 7.0), 0.9 mM ABTS (Diammonium salt from SIGMA) and an appropriate amount of the preparation to be assayed (which, in this example, is the supernatant diluted with water as described below) was prepared.
  • the assay reaction was started by the addition of the preparation to be assayed (which in this example is the supernatant dilution) to form the final 1 ml reaction volume.
  • the greenish-blue color produced by the oxidation of ABTS was then continually measured by recording the optical density (OD) at 420 nm during two minutes, using a spectrophotometer (Ultraspec Plus from Pharmacia). The rate of increase of the optical density per minute ( ⁇ OD/minute) was then calculated from the linear part of the curve during 1 minute.
  • one standard ABTS enzyme unit (hereinafter referred to as one enzyme unit or EU) is defined as the amount of enzyme that produces an increase of one OD 20 p er minute, under these specific conditions.
  • Stachybotrys chartarum phenol oxidizing enzyme production Stachybotrys chartarum was grown on PDA plates (Difco) for about 5 - 10 days. A portion of the plate culture (about 3/4 x 3/4 inch) was used to inoculate 100 ml of PDB (potato dextrose broth) in 500-ml shake flask. The flask was incubated at 26 - 28 degrees C, 150 rpm, for 3 - 5 days until good growth was obtained.
  • the broth culture was then inoculated into 1 L of PDB in a 2.8-L shake flask.
  • the flask was incubated at 26 - 28 degrees C, 150 rpm, for 2 - 4 days until good growth was obtained.
  • a 10-L fermentor containing a production medium was prepared (containing in grams/liter the following components: glucose 15; lecithinl .51 ; t-aconitic acid 1.73; KH 2 PO 4 3; MgSO 4 .7H 2 O 0.8; CaCI 2 .2H 2 O 0.1; ammonium tartrate 1.2; soy peptone 5; Staley 7359; benzyl alcohol 1 ; tween 20 1 ; nitrilotriacetic acid 0.15; MnSO .7H 2 O 0.05; NaCI 0.1 ; FeSO 4 .7H 2 O 0.01 ; CoSO 4 0.01 ; CaCI 2 .2H 2 O 0.01 ; ZnSO 4 .7H 2 O 0.01 ; CuSO 4 0.001; ALK(SO 4 )2.12H 2 O 0.001; H 3 BO 3 0.001 ; NaMoO 4 .2H 2 O 0.001). The fermentor was then inoculated with the 1-L
  • ABTS 2,2'- azino-bis-(3-ethylbenzothiazoline-6-sulfonate)
  • oxygen oxygen
  • ABTS SIGMA, 0.2 ml, 4.5 mM H 2 O
  • NaOAc 1.5ml, 120mM in H 2 O,pH 5.0
  • the color produced by the oxidation of ABTS was then measured every 2 seconds for total period of 14 seconds by recording the optical density (OD) at 420 nm, using a spectrophotometer.
  • One ABTS unit one enzyme unit or EACU in this example is defined as the change in OD measured at 420 per minute/2 (given no dilution to the sample). In this manner a phenol oxidizing enzyme activity of 3.5 EACU/ml of culture supernatant was measured.
  • Stachybotrys parvispora culture broth obtained as described above in Example 4, was then withdrawn from the fermentor and centrifuged for 15 minutes at 4,500 g. Stachybotrys chartarum is purified in a similar fashion. The resulting supernatant was then removed from the pellet and concentrated to 0.6 liters by ultrafiltration using a Amicon ultrafiltration unit equipped with a YMI0 membrane having a 10 kD cutoff.
  • the mixture was centrifuged for 30 minutes at 10,000 g and the resulting pellet was removed from the supernatant. The pellet was then resuspended in a final volume of 800 ml of water.
  • the resulting suspension was then submitted to ammonium sulfate fractionation as follows : crystalline ammonium sulfate (JANSSEN) was added to the suspension to 40% saturation and the mixture incubated at 4 degrees C for 16 hours with gentle magnetic stirring. The mixture was then centrifuged at 10,000 g for 30 minutes and the supernatant removed from the centrifugation pellet for further use. Ammonium sulfate (JANSSEN) was then added to the supernatant to reach 80% saturation, and the mixture incubated at 4 degrees C for 16 hours with gentle magnetic stirring. The suspension was then centrifuged for 30 minutes at 10,000 g and the resulting pellet was removed from the supernatant.
  • JANSSEN crystalline ammonium sulfate
  • the pellet was then resuspended in 15 ml of water and concentrated to 6 ml by ultrafiltration using a CENTRIPREP 3000 (AMICON).
  • the phenol oxidizing enzyme activity of the suspension was then measured using the standard assay procedure, based on the oxidation of ABTS by oxygen, as was described above in Example 4 (but with the exception that the preparation being assayed is the resuspended concentration and not the supernatant dilutions).
  • the phenol oxidizing enzyme activity so measured was 5200 EU/ml.
  • the enzyme was then further purified by gel permeation chromatography.
  • the phenol oxidizing enzyme activity of the suspension was then measured using the standard assay procedure, based on the oxidation of ABTS by oxygen, as was described above in Example 4.
  • the enzyme activity so measured was 390 EU/ml.
  • the isoelectric point (pi) of the enzyme produced by S. parvispora MUCL 38996 was then determined from the purified enzyme, obtained as described above in Example 5.
  • the purified enzyme obtained as described above in Example 5, was submitted to an isoelectric focusing gel (IEF 3-9 from PHARMACIA), as described in the PHARMACIA Technical File IEF No 100.
  • PHARMACIA reference markers were used in this isoelectric focusing: pepsinogen (2.8), amyloglucosidase (3.5), methyl red (3.75), glucose oxidase (4.15), soybean trypsin inhibitor (4.55), b-lactoglobulin A (5.2), bovine carbonic anhydrase B (5.85) and human carbonic anhydrase B (6.55).
  • pepsinogen 2.8
  • amyloglucosidase 3.5
  • methyl red 3.75
  • glucose oxidase 4.15
  • soybean trypsin inhibitor 4.55
  • b-lactoglobulin A 5.2
  • bovine carbonic anhydrase B 5.85
  • human carbonic anhydrase B (6.55).
  • the thirteen buffer samples were then adjusted to the respective pHs noted below in Table 1 A with either HCI or NaOH, as applicable, so that one of the buffer samples possessed each of the pH values noted below in Table 1A. Three 0.9 ml samples were then taken from each of the thirteen buffer samples.
  • Respective substrates were then added to respective mixtures as follows : 0.9 mM ABTS was added to the thirteen mixtures of the first group; 50 ⁇ M DMP (2,6- dimethoxyphenol) (FLUKA) was added to the thirteen mixtures of the second group; and 1mM syringaldizin (SIGMA) was added to the thirteen mixtures of the third group.
  • the respective reactions were started by the addition of 2 EU of purified phenol oxidizing enzyme from S. parvispora MUCL 38996, obtained as described above in Example 5.
  • the final volume of each of the samples assayed was 1 ml.
  • the assays on each of the thirty-nine samples were performed at approximately 20 degrees C with an incubation time of 2 minutes following the protocol set forth above in Example 4.
  • the optical density was recorded during 2 minutes (Ultraspec Plus from
  • DBI Direct Blue No. 1
  • SIGMA Chicago Sky Blue 6B
  • the respective reactions were started by the addition to the respective reaction mixtures of 4.5 EU of phenol oxidizing enzyme from either S. parvispora MUCL 38996 obtained as described above in Example 5, or the bilirubin oxidase from Myrothecium verrucaria (purchased from SIGMA).
  • the final volume of each of the samples assayed was 1 ml.
  • the assays on each of the samples were performed at approximately 20 degrees C with an incubation time of 2 minutes following the protocol set forth above in Example 4.
  • the optical density was recorded during 2 minutes (Ultraspec Plus from Pharmacia), at a wavelength of 620 nm.
  • the rate of decrease of the optical density (- ⁇ OD/min) was calculated from the linear part of the curves.
  • the assay results are summarized below in Table 2.
  • Reaction mixtures (1 ml final volume) were prepared containing 200 tmM Tris/HCI (pH 7.0) and 5 mM quiacol (2-methoxyphenol) (MERCK) as substrate.
  • the reactions were started by the addition of 5 EU of phenol oxidizing enzyme from S. parvispora MUCL 38996, obtained as described above in Example 5, or by the addition of 5 EU of the bilirubin oxidase from Myrothecium verrucaria (purchased from SIGMA).
  • the final volume of each of the samples assayed was 1 ml.
  • the assays on each of the samples were performed at approximately 20 degrees C with an incubation time of 2 minutes following the protocol set forth above in Example 4.
  • the optical density was recorded during 2 minutes (Ultraspec Plus from PHARMACIA), at a wavelength of 440 nm.
  • the rate of increase of the optical density ( ⁇ OD/min) was calculated from the linear part of the curves.
  • the substrate specificity of the phenol oxidizing enzyme from S. parvispora MUCL 38996 was studied versus a number of dyes.
  • the reaction mixtures (1 ml final volume) contained 200 mM Tris/HCI (pH 7.0) and the respective dyes listed below in Table 3, the concentration of which dyes were adjusted by dilution with water, so that an optical density of 1.0 (at the wavelengths listed below in Table 3) was measured therefor.
  • the reactive and dye nomenclature is in accordance with the color index.
  • the bleaching reactions were started by the addition of phenol oxidizing enzyme of S. parvispora MUCL 38996, obtained as described above in Example 5.
  • the amount of phenol oxidizing enzyme was adjusted by dilution with water in order to measure a decrease in OD (at the wavelengths listed in Table 3) in the range of 0.05 to 0.25 - ⁇ OD/minute, in order to obtain a linear curve.
  • the final volume of each of the samples assayed was 1 ml.
  • the assays on each of the samples were performed at approximately 20°C with an incubation time of 2 minutes following the protocol set forth above in Example 4.
  • the optical density was recorded during 2 minutes, at the wavelength indicated in Table 3 (Ultraspec Plus from Pharmacia).
  • the rate of decrease of the optical density (- ⁇ OD/min) was calculated from the linear part of the curve, and multiplied by the enzyme dilution in order to express the final bleaching rate in - ⁇ OD/minute/ml of enzyme solution obtained as described above in Example 5. The results are summarized below in Table 3.
  • the rate of oxygen consumption was measured with each of the dyes, in a magnetically stirred chamber equipped with a Clark electrode (oxygraph K-IC from Gilson).
  • the oxygraph chamber contained, in a final volume of 2 ml, 200 mM Tris/HCI (pH 7.0), 5 mM of each of the dyes, and 100 ml (39 EU) of phenol oxidizing enzyme from S. parvispora MUCL 38996, obtained as described above in Example 5.
  • the reactions were started by the addition of the enzyme, and the dissolved oxygen concentration was recorded during 5 minutes. The slope of the curves were determined from their linear parts.
  • Table 3 The results of this experiment are also summarized below in Table 3.
  • N.D. refers to Not Determined
  • S. chartarum MUCL 38898 (obtained as described above in Example 2) was isolated on Malt Extracted Plates (ME from DIFCO). One colony thereof was then suspended in 5 ml of 0.9% (w/v) NaCI containing about 30 sterile glass beads (diameter 5 mm). The suspension was thoroughly agitated with a vortex mixer until complete homogenization of the mycelium was obtained. 30 grams of TSB (Trypticase Soy Broth from BECTON DICKINSON) powder were dissolved in 1 liter of water and sterilization performed by heating at 120 degrees C for 30 minutes. Respective 500 ml quantities of the sterilized culture medium were then added to two polypropylene shaking flasks (volume 2 liters). The flasks were then inoculated with respective 1 ml samples of the mycelium suspension and run for 96 hours under constant agitation (100 RPM with 1 inch eccentricity) at 37°C.
  • TSB Stepticase Soy Broth from B
  • the culture medium from the respective shaking flasks were centrifuged at 10000 g for 15 minutes.
  • the resulting supernatants were then removed and each was concentrated 20 times by acetone precipitation (1 volume supernatant/ 3 volumes acetone).
  • the mixtures were then incubated at 4°C under magnetic stirring for 45 minutes.
  • the resulting suspensions were then again centrifuged at 10000 g for 15 minutes and the resulting pellets removed therefrom.
  • the removed pellets were then resuspended in 50 ml water (Milli-Q quality).
  • a phenol oxidizing enzyme activity of 0.5 U ABTS was measured on ABTS.
  • the resulting enzymatic solutions were then used for the immunological tests.
  • Respective dilutions of 2X (having 1 volume of enzyme and 1 volume of diluant); 4X (having 1 volume of enzyme sample and 3 volumes of diluant) and 8X (having 1 volume of enzyme sample and 7 volumes of diluant) were prepared using 0.9 % (w/v) NaCI as diluant and of 0.6 EU enzyme samples of S. parvispora MUCL 38996 phenol oxidizing enzyme (obtained as described above in Example 5) the S. chartarum MUCL 38898 phenol oxidizing enzyme (obtained as described below) and the bilirubin oxidase of M. verrucaria (SIGMA).
  • Sample 1 is an undiluted sample of S. parvispora enzyme.
  • Sample 2 is a 2X dilution of S. parvispora enzyme.
  • Sample 3 is a 4X dilution of S. parvispora enzyme.
  • Sample 4 is an 8X dilution of S. parvispora enzyme.
  • Sample 5 is an undiluted sample of S. chartarum enzyme.
  • Sample 6 is a 2X dilution of S. chartarum enzyme.
  • Sample 7 is a 4X dilution of S. chartarum enzyme.
  • Sample 8 is an 8X dilution of S. chartarum enzyme.
  • Sample 9 is an undiluted sample of M. verrucaria bilirubin oxidase.
  • Sample 10 is a 2X dilution of M. verrucaria bilirubin oxidase.
  • Sample 11 is a 4X dilution of M. verrucaria bilirubin oxidase.
  • Sample 12 is an 8X dilution of M. verrucaria bilirubin oxidase.
  • Sample 13 is a 1/1 (v/v) mixture of undiluted samples of S. parvispora phenol oxidizing enzyme and M. verrucaria bilirubin oxidase.
  • the potential of the enzymatic system to prevent dye transfer was assessed by washing a colored swatch in the presence of a white pick-up swatch.
  • the experiments were performed in 25 ml carbonate buffer, pH 9, containing the two swatches of 5x5cm.
  • the enzyme was dosed as ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) units.
  • One ABTS unit is defined as the amount of enzyme which an optical density increase of 1 OD/min at 418 nm in the presence of 2 mM ABTS in 20 mM Tris buffer, pH 9.
  • a phenol oxidizing enzyme of the present invention was assessed by washing cotton swatches soiled with tomato paste in the presence of Stacchybotrys chartarum phenol oxidizing enzyme (which is obtainable by the methods disclosed in Example 4 and 5) and an enhancer.
  • the experiments were performed in 15 ml borate buffer, pH 9, and phosphate buffer, pH 7.
  • the enzyme was dosed as ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) units.
  • One ABTS unit is defined as the amount of enzyme which an optical density increase of 1 OD/min at 418 nm in the presence of 2 mM ABTS, in 20 mM Tris buffer, pH 9. Experiments were performed in the presence of 2.8 units/ml of wash solution.
  • Phenothiazine-10-propionate was added as an enhancer of the enzyme activity. This enhancer was added at concentrations of 250 ⁇ M. The swatches were washed during 30 minutes, at 30 °C. After the wash, the residual color of the stains was measured as in Example 11. In the table below the difference in color measurement is given between the stain before and after the wash.
  • Stachybotrys chartarum Phenol Oxidizing Enzyme Stachybotrys chartarum phenol oxidizing enzyme prepared as disclosed in Example 4 was subjected to SDS polyacrylamide gel electrophoresis and isolated. The isolated fraction was treated with urea and iodoacetamide and digested by the enzyme endoLysC. The fragments resulting from the endoLysC digestion were separated via HPLC (reverse phase monobore C18 column, CH3CN gradient) and collected in a multititer plate. The fractions were analysed by MALDI for mass determination and sequenced via Edman degradation. The following amino acid sequences were determined and are shown in amino terminus to carboxy terminus orientation:
  • Figures 4A-4B is an amino acid alignment of the Stachybotrys chartarum phenol oxidizing enzyme fragments with Myrothecium verrucaria bilirubin oxidase and LEPTOTHRIX DISCOPHORA manganese oxidizing protein.
  • Two degenerated primers were designed based on the peptide sequence.
  • Primer 1 contains the following sequence: TATTACTTTCCNAAYTAYCA where N represents a mixture of all four nucleotides (A, T, C and G) and Y represents a mixture of T and C only.
  • Primer 2 contains the following sequence:
  • DNA isolated from Stachybotrys chartarum was used as a template for PCR.
  • the DNA was diluted 100 fold with Tris-EDTA buffer to a final concentration of 88 ng/ul.
  • Ten microliter of diluted DNA was added to the reaction mixture which contained 0.2 mM of each nucleotide (A, G. C and T), 1x reaction buffer, 0.296 microgram of primer 1 and
  • PCR reaction was performed at 95°C for 1 minute, the primers were annealed to the template at 45°C for 1 minute and extension was done at 68°C for 1 minute. This cycle was repeated 30 times to achieve a gel-visible PCR fragment.
  • the PCR fragment detected by agarose gel contained a fragment of about 1 kilobase which was then cloned into the plasmid vector pCR-ll (Invitrogen). The 1 kb insert was then subjected to nucleic acid sequencing.
  • Stachybotrys chartarum strain (MUCL 38898) was grown in laccase production medium and RNA was extracted from mycellium and used as a template for cDNA isolation.
  • the total cDNA was synthesized by reverse transcriptase using 4.3 microgram of RNA in a 20 microliter reaction containing 0.34 microgram oligo T-
  • the cDNA encoding the Stachybotrys phenol oxidizing enzyme was then cloned by PCR in two steps.
  • the 5 ' cDNA was cloned as a 678 bp fragment using the following two primers: GTCAATATGCTGTTCAAG and CTCGCCATAGCCACTAGG.
  • the 3' cDNA was cloned as a 1301 bp fragment using following two primers: CTTTCGATGGTTGGGCTG and GTTCTAGACTACTCCTCGATTCCAAGATC.
  • the cDNA sequence of 1791 bp is shown in Figure 5.
  • Comparison of the Stachybotrys chartarum phenol oxidizing enzyme genomic DNA and cDNA A comparison of the cDNA with genomic DNA revealed that there were five introns in the genomic DNA.
  • the protein translation start site (ATG) is at nucleotide #1044 to #1046 and the translation stop site is at nucleotide #3093 to #3095. Protein sequence translated from cDNA and genomic DNA contains 594 amino acids.
  • the protein sequence SEQ ID NO:2 was used as query to search GCG (Genetics Computer Group University Research Park, Madison Wisconsin) DNA and protein databases. It showed that Stachybotrys oxidase shared 60 % identity to bilirubin oxidase at the protein sequence level.
  • Figure 7 shows the sequence alignment of the two proteins.
  • the DNA fragment containing nucleic acid encoding the Stachybotrys phenol oxidizing enzyme flanked by two newly introduced restriction enzyme sites (Bgl II and 5 Xba I) was isolated by PCR ( Figure 9). This PCR fragment was first cloned into the plasmid vector pCR-ll and subjected to nucleic acid sequencing to verify the gene sequence. This DNA fragment was then cloned into the Bgl II to Xba I site of vector (pGAPT, see Fig 8).
  • the vector used for expressing the Stachybotrys phenol oxidizing enzyme contains the Aspergillus niger glucoamylase gene promoter (from bases 1 to
  • CSL special medium is CSL medium with the glucose and fructose eliminated.
  • ABTS assays were performed at days 3, 6, and 10. The transformants were also grown in CSL first and then transferred after 1 day's growth to Clofine-special medium. After 6 days growth, these samples were assayed for ABTS activities (>0.2 units). Five best transformants were spore purified and tested again for
  • FIG. 10 shows a SDS-protein polyacrylamide gel indicated the expression level of the recombinant Stachybotrys oxidase in Aspergillus niger var. awamori grown of a 6 day culture grown in CSL special medium.
  • Example 18
  • the expression plasmid for use in transforming Trichoderma reesei was constructed as follows. The ends of the Bglll to Xbal fragment shown in Figure 9 containing the gene encoding the Stachybotrys phenol oxidizing enzyme were blunted by T4 DNA polymerase and inserted into Pmel restriction site of the Trichoderma expression vector, pTrex, which is a modified version of pTEX, see PCT Publication No. WO 96/23928 for a complete description of the preparation of the pTEX vector, which discussion is herein incorporated by reference, which contains a CBHI promoter and terminator for gene expression and a Trichoderma pyr4 gene as a selection marker for transformants.
  • the linear DNA fragment containing only the CBH1 promoter, the Stachybotrys phenol oxidizing gene, the CBH1 terminator and selection marker pyr4 was isolated from a gel and was used to transform a uridine auxotroph strain of Trichoderma reesei (see United States Patent no. 5,472,864) which has the four major cellulase genes deleted. Stable transformants were isolated on Trichoderma minimal plates without uridine. The transformants were grown on 50 ml of Proflo medium in shake flasks for 4 days at 28°C to 30°C and expression of the phenol oxidizing enzyme was assayed by ABTS (> 2 units/ml) and SDS-PAGE protein gel.
  • Proflo medium is composed of (g/l) Proflo 22.5; lactose 30.0; (NH 4 ) 2 SO 4 6.5 KH 2 PO 4 2.0; MgSO 4 .7 H 2 O 0.3; CaCL 2 0.2; CaC0 3 0.72; trace metal stock solution 1.0 ml/l and 10% Tween 80 2.0 ml/l.
  • the trace metal stock solution used had (g/l) FeSO .7H 2 O 5.0; MnSO 4 .H 2 O 1.6; ZnSO 4 .7H 2 O 1.4; CoCI 2 6H 2 O) 2.8.
  • Example 19 Expression of Stachybotrys phenol oxidizing enzyme in Saccharomyces cerevisiae: 1 .
  • the Bglll to Xbal fragment of the cDNA (SEQ ID NO:1) of the phenol oxidizing gene was cloned into yeast expression vector yES2.0 (Invitrogen) which contains the yeast Gal 1 promoter and Cyc 1 terminator, to control expression of the phenol oxidizing gene, and the yeast URA3 gene as a selection marker.
  • the expression plasmid was transformed into a yeast strain (Invitrogen Sc2 strain). The transformants were selected on yeast minimal plate without uridine. Four randomly picked transformants showed activity in plate assay (colored halo formation in yeast minimal plate with 1 mM ABTS) while the control plasmid vector did not show any colored halo formation.

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Abstract

L'invention concerne des enzymes d'oxydation de phénol pouvant être obtenues à partir d'espèces de Stachybotrys et s'utilisant pour modifier la couleur associée aux colorants et composés colorés, ainsi que dans des applications anti-transfert de colorant. L'invention concerne également des cultures biologiquement pures de souches du genre Stachybotrys, désignées ci-après sous le nom de Stachybotrys parvispora MUCL 38996 et Stachybotrys chartarum MUCL 38898, capables de produire naturellement des enzymes d'oxydation de phénol. L'invention concerne aussi la séquence d'acides aminés et la séquence nucléotidique d'enzymes d'oxydation de phénol à partir de Stachybotrys, ainsi que des vecteurs d'expression et des cellules hôtes comprenant l'acide nucléique. L'invention concerne en outre des méthodes de production de l'enzyme d'oxydation de phénol ainsi que des méthodes d'élaboration d'hôtes d'expression. L'invention concerne enfin des compositions détergentes comprenant des enzymes d'oxydation de phénol pouvant être obtenues à partir d'espèces de Stachybotrys.
EP99917861A 1998-03-24 1999-03-23 Enzymes d'oxydation de phenol et leurs utilisations Withdrawn EP1066364A2 (fr)

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US09/218,702 US6426410B1 (en) 1998-12-22 1998-12-22 Phenol oxidizing enzymes
PCT/EP1999/002042 WO1999049010A2 (fr) 1998-03-24 1999-03-23 Enzymes d'oxydation de phenol et leurs utilisations

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US7144717B1 (en) * 1998-03-24 2006-12-05 Genecor International, Inc. Oxidizing enzymes
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DE19962882A1 (de) * 1999-12-24 2001-06-28 Henkel Kgaa Enzymatisches Färbemittel
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