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WO1998038286A1 - Mutants de laccase - Google Patents

Mutants de laccase Download PDF

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Publication number
WO1998038286A1
WO1998038286A1 PCT/DK1998/000067 DK9800067W WO9838286A1 WO 1998038286 A1 WO1998038286 A1 WO 1998038286A1 DK 9800067 W DK9800067 W DK 9800067W WO 9838286 A1 WO9838286 A1 WO 9838286A1
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WO
WIPO (PCT)
Prior art keywords
laccase
variant
parent
gly
val
Prior art date
Application number
PCT/DK1998/000067
Other languages
English (en)
Inventor
Allan Svendsen
Original Assignee
Novo Nordisk A/S
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
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to EP98902969A priority Critical patent/EP0972014A1/fr
Priority to AU59830/98A priority patent/AU5983098A/en
Publication of WO1998038286A1 publication Critical patent/WO1998038286A1/fr

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    • 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)
    • C12N9/0061—Laccase (1.10.3.2)

Definitions

  • the present invention relates to laccase mutants with increased oxidation potential and/or changed pH optimum and/or altered mediator pathway and/or altered 0 2 /OH ⁇ pathway.
  • Laccase is a polyphenol oxidase (EC 1.10.3.2) which catalyses the oxidation of a variety of inorganic and aromatic compounds, particularly phenols, with the concomitant reduction of molecular oxygen to water.
  • Laccase belongs to a family of blue copper-containing oxidases which includes ascorbate oxidase and the mammalian plasma protein ceruloplasmin. All these enzymes are multi-copper- containing proteins.
  • laccases are able to catalyze the oxidation of a variety of inorganic and aromatic compounds
  • laccases have been suggested in many potential industrial applications such as lignin modification, paper strengthening, dye transfer inhibition in detergents, phenol polymerization, hair colouring, and waste water treatment .
  • the various applications ask for laccases with specific properties. It is the purpose of the present application to create laccase variants with increased oxidation potential and/or changed pH optimum and/or altered mediator pathway and/or altered 0 2 /OH-pathway.
  • the present invention relates to laccase variants, in particular to
  • a variant of a parent laccase which variant has laccase activity, and increased oxidation potential and comprises a mutation in a position corresponding to at least one of the following positions: G511A,V,P,L,I,F,Y,W;
  • SEQ ID No. 1 or the parent laccase has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1;
  • a variant of a parent laccase which variant has laccase activity and an altered pH optimum and comprises a mutation in a position corresponding to at least one of the following positions :
  • SEQ ID No. 1 or the parent laccase has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1;
  • a variant of a parent laccase which variant has laccase activity and an altered mediator efficiency and comprises a mutation in a position corresponding to at least one of the following positions:
  • a variant of a parent laccase which variant has laccase activity and an altered 0 2 /OH -pathway and comprises a mutation in a position corresponding to at least one of the following positions :
  • SEQ ID No. 1 or the parent laccase has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1.
  • the invention relates to DNA encoding such variants and methods of preparing the variants .
  • the invention relates to the use of the variants for various industrial purposes .
  • Myceliophthora-like laccase is intended to indicate a laccase which, on the amino acid level, displays a homology of at least 80% to the Myceliophthora laccase SEQ ID NO 1, or a laccase which, on the amino acid level, displays a homology of at least 85% to the Myceliophthora laccase SEQ ID NO 1, or a laccase which, on the amino acid level, displays a homology of at least 90% to the Myceliophthora laccase SEQ ID NO 1, or a laccase which, on the amino acid level, displays a homology of at least 95% to the Myceliophthora laccase SEQ ID NO 1, or a laccase which, on the amino acid level, displays a homology of at least 98% to the Myceliophthora laccase SEQ ID NO 1.
  • derived from is intended not only to indicate a laccase produced or producible by a strain of the organism in question, but also a laccase encoded by a DNA sequence isolated from such strain and produced in a host or- ganism containing said DNA sequence.
  • the term is intended to indicate a laccase which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the laccase in question.
  • Myceliophthora thermophila (SEQ ID No. 1): 0.48 Scytalidium thermophilum (SEQ ID No. 2): 0.53 It is contemplated that it is possible to increase the oxidation potential of a parent laccase, wherein said variant is the result of a mutation, i.e. one or more amino acid residues have been deleted from, replaced or added to the parent laccase.
  • Myceliophthora thermophila laccase (SEQ ID No. 1) :
  • G514A,V,L,I,F Preferred variants include any combination of the above mentioned mutations.
  • the desired pH optimum of a laccase depends on which application is of interest, e.g., if the laccase is to be used for denim bleaching the preferred pH optimum will be around pH 5- 8, whereas if the laccase is to be used for washing purposes the preferred pH optimum will be around pH 8-10.
  • Myceliophthora thermophila (SEQ ID No. 1) :
  • Preferred substitutions are the following: E, D, L, I, F, Y, W.
  • Laccases are often used in combination with so called mediators or enhancers, e.g., in combination with phenothiazine or phenothiazine related compounds (see WO 95/01426) or in combination with acetosyringone or acetosyringone related compounds (see WO 96/10079) .
  • Myceliophthora thermophila laccase (SEQ ID No. 1) :
  • substitutions are in particular one or more of the following mutations: N189G,A,S,T; S190G,A;
  • Myceliophthora thermophila (SEQ ID No. 1) :
  • the DNA sequence encoding a parent laccase may be isolated from any cell or microorganism producing the laccase in question, using various methods well known in the art.
  • a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the laccase to be studied.
  • homologous, labelled oligonucleotide probes may be synthesized and used to identify laccase-encoding clones from a genomic library prepared from the organism in question.
  • a labelled oligonucleotide probe containing sequences homologous to a known laccase gene could be used as a probe to identify laccase-encoding clones, using hybridization and washing conditions of lower stringency.
  • a method for identifying laccase-encoding clones involves inserting cDNA into an expression vector, such as a plasmid, transforming laccase-negative fungi with the resulting cDNA library, and then plating the transformed fungi onto agar containing a substrate for laccase, thereby allowing clones expressing the laccase to be identified.
  • the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method.
  • oligonu- cleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
  • the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence) , in accordance with standard techniques .
  • the DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers. Site-directed mutagenesis
  • mutations may be introduced using synthetic oligonucleotides . These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis.
  • a single-stranded gap of DNA, bridging the laccase-encoding sequence is created in a vector carrying the laccase gene.
  • the synthetic nucleotide, bearing the desired mutation is annealed to a homologous portion of the single- stranded DNA. The remaining gap is then filled in with T7 DNA polymerase and the construct is ligated using T4 ligase.
  • Random mutagenesis The random mutagenesis of a DNA sequence encoding a parent laccase may conveniently be performed by use of any method known in the art .
  • the random mutagenesis may be performed by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to
  • PCR generated mutagenesis may be performed by use of any combination of these mutagenizing agents .
  • the mutagenizing agent may, e.g., be one which induces transitions, transversions, inversions, scrambling, deletions, and/or insertions .
  • Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N' -nitro-N-nitrosoguanidine (MNNG) , O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS) , sodium bisulphite, formic acid, and nucleotide analogues .
  • the mutagenesis is typically performed by incubating the DNA sequence encoding the parent enzyme to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions for the mutagenesis to take place, and selecting for mutated DNA having the desired properties .
  • the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions which are to be changed.
  • the doping or spiking may be done so that codons for unwanted amino acids are avoided.
  • the doped or spiked oligonucleotide can be incorporated into the DNA encoding the laccase enzyme by any published technique, using e.g. PCR, LCR or any DNA polymerase and ligase .
  • PCR-generated mutagenesis When PCR-generated mutagenesis is used, either a chemically treated or non-treated gene encoding a parent laccase enzyme is subjected to PCR under conditions that increase the mis- incorporation of nucleotides (Deshler 1992; Leung et al . , Technique, Vol.l, 1989, pp. 11-15).
  • a mutator strain of E. coli (Fowler et al . , Molec . Gen. Genet., 133, 1974, pp. 179-191), S. cereviseae or any other microbial organism may be used for the random mutagenesis of the DNA encoding the laccase enzyme by e.g. transforming a plasmid containing the parent enzyme into the mutator strain, growing the mutator strain with the plasmid and isolating the mutated plasmid from the mutator strain. The mutated plasmid may subsequently be transformed into the expression organism.
  • the DNA sequence to be mutagenized may conveniently be present in a genomic or cDNA library prepared from an organism expressing the parent laccase enzyme.
  • the DNA se- quence may be present on a suitable vector such as a plasmid or a bacteriophage, which as such may be incubated with or otherwise exposed to the mutagenizing agent.
  • the DNA to be mutagenized may also be present in a host cell either by being integrated in the genome of said cell or by being present on a vector harboured in the cell.
  • the DNA to be mutagenized may be in isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence .
  • telomere amplify may be performed in accordance with methods known in the art, the presently preferred method being PCR-generated amplification using oligonucleotide primers prepared on the basis of the DNA or amino acid sequence of the parent enzyme .
  • the mutated DNA is expressed by culturing a suitable host cell carrying the DNA sequence under conditions allowing expression to take place.
  • the host cell used for this purpose may be one which has been transformed with the mutated DNA sequence, optionally present on a vector, or one which was carried the DNA sequence encoding the parent enzyme during the mutagenesis treatment.
  • suitable host cells are fungal hosts such as Aspergillus niger or Aspergillus oryzae .
  • the mutated DNA sequence may further comprise a DNA sequence encoding functions permitting expression of the mutated DNA sequence .
  • the random mutagenesis may advantageously be localized to a part of the parent laccase in question. This may, e.g., be advantageous when certain regions of the enzyme have been identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme.
  • the localized random mutagenesis is conveniently performed by use of PCR-generated mutagenesis techniques as described above or any other suitable technique known in the art .
  • the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g. by being inserted into a suitable vector, and said part may subsequently be subjected to mutagenesis by use of any of the mutagenesis methods discussed above.
  • this may conveniently be performed by use of aa filter assay based on the following principle:
  • a microorganism capable of expressing the mutated laccase enzyme of interest is incubated on a suitable medium and under suitable conditions for the enzyme to be secreted, the medium being provided with a double filter comprising a first protein- binding filter and on top of that a second filter exhibiting a low protein binding capability.
  • the microorganism is located on the second filter.
  • the first filter comprising enzymes secreted from the microorganisms is separated from the second filter comprising the microorganisms.
  • the first filter is subjected to screening for the desired enzymatic activity and the corresponding microbial colonies present on the second filter are identified.
  • the filter used for binding the enzymatic activity may be any protein binding filter e.g. nylon or nitrocellulose.
  • the top filter carrying the colonies of the expression organism may be any filter that has no or low affinity for binding proteins e.g. cellulose acetate or DuraporeTM.
  • the filter may be pretreated with any of the conditions to be used for screening or may be treated during the detection of enzymatic activity.
  • the enzymatic activity may be detected by a dye, fluorescence, precipitation, pH indicator, IR-absorbance or any other known technique for detection of enzymatic activity.
  • the detecting compound may be immobilized by any immobilizing agent, e.g., agarose, agar, gelatine, polyacrylamide, starch, filter paper, cloth; or any combination of immobilizing agents.
  • immobilizing agent e.g., agarose, agar, gelatine, polyacrylamide, starch, filter paper, cloth; or any combination of immobilizing agents.
  • the laccase activity was measured using 10- (2-hydroxyethyl) -phenoxazine (HEPO) as substrate for the various laccases.
  • HEPO was synthesized using the same procedure as described for 10- (2-hydroxyethyl) - phenothiazine, (G. Cauquil in Bulletin de la Society Chemique de
  • the Myceliophthora thermophila laccase was measured using
  • a DNA sequence encoding the variant produced by methods described above, or by any alterna- tive methods known in the art can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
  • the recombinant expression vector carrying the DNA sequence encoding a laccase variant of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e.
  • a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication e.g. a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome (s) into which it has been integrated.
  • the DNA sequence should be operably connected to a suitable promoter sequence .
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the DNA sequence encoding a laccase variant of the invention, especially in a fungal host are those derived from the gene encoding A . oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A . niger neutral ⁇ -amylase, A . niger acid stable ⁇ -amylase, A .
  • niger glu- coamylase Rhizomucor miehei lipase
  • Rhizomucor miehei lipase Rhizomucor miehei lipase
  • a . oryzae alkaline protease A. oryzae triose phosphate isomerase or A . nidulans acetamidase .
  • the expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, poly- adenylation sequences operably connected to the DNA sequence encoding the laccase variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
  • the vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMBl and pIJ702.
  • the vector may also comprise a selectable marker, e.g. a gene, the product of which complements a defect in the host cell, such as one which confers antibiotic resistance such as ampicil- lin, kanamycin, chloramphenicol or tetracyclin resistance.
  • a selectable marker e.g. a gene, the product of which complements a defect in the host cell, such as one which confers antibiotic resistance such as ampicil- lin, kanamycin, chloramphenicol or tetracyclin resistance.
  • the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g. as described in WO 91/17243.
  • the cell of the invention is advantageously used as a host cell in the recombinant production of a laccase variant of the invention.
  • the cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
  • the cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g. a fungal cell.
  • the filamentous fungus may advantageously belong to a species of Aspergillus, e.g. Aspergillus oryzae or Aspergillus niger.
  • Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se .
  • a suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.
  • the present invention relates to a method of producing a laccase variant of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the variant and recovering the variant from the cells and/or culture medium.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the laccase variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. as described in catalogues of the American Type Culture Collection) .
  • the laccase variant secreted from the host cells may con- veniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • laccase variants of this invention possesses valuable properties allowing for various industrial applications, in particular lignin modification, paper strengthening, dye transfer inhibition in detergents, phenol polymerization, hair dyeing, textile dyeing, bleaching of textiles (in particular bleaching of denim as described in WO 96/12845 and WO 96/12846) and waste water treatment .

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  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne des mutants de laccase dotés d'un potentiel d'oxydation accru et/ou un pH optimal modifié et/ou une voie médiatrice modifiée et/ou une voie O2/OH- modifiée.
PCT/DK1998/000067 1997-02-28 1998-02-20 Mutants de laccase WO1998038286A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP98902969A EP0972014A1 (fr) 1997-02-28 1998-02-20 Mutants de laccase
AU59830/98A AU5983098A (en) 1997-02-28 1998-02-20 Laccase mutants

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DK0221/97 1997-02-28
DK22197 1997-02-28

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001021809A3 (fr) * 1999-09-22 2001-10-18 Genencor Int Nouvelles enzymes oxydant le phenol
US6905853B1 (en) 2000-09-07 2005-06-14 Genencor International, Inc. Phenol oxidizing enzyme variants
US7319112B2 (en) 2000-07-14 2008-01-15 The Procter & Gamble Co. Non-halogenated antibacterial agents and processes for making same
EP2431048A2 (fr) 2002-10-08 2012-03-21 Genencor International, Inc. Peptides de liaison phénolique
WO2016090059A1 (fr) 2014-12-02 2016-06-09 Novozymes A/S Variants de laccase et polynucléotides codant pour ceux-ci
CN111690622A (zh) * 2020-07-29 2020-09-22 中国农业科学院北京畜牧兽医研究所 一种真菌来源的漆酶g2589及其基因和应用
CN114958788A (zh) * 2022-06-24 2022-08-30 武汉轻工大学 一种耐高温漆酶及基因与菌株和应用
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

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WO1995011296A1 (fr) * 1993-10-18 1995-04-27 Degussa Aktiengesellschaft Procede de modification controlee d'enzymes, enzymes modifiees et utilisation
WO1995033837A1 (fr) * 1994-06-03 1995-12-14 Novo Nordisk Biotech, Inc. Laccases purifiees de scytalidium et acides nucleiques les codant
WO1995033836A1 (fr) * 1994-06-03 1995-12-14 Novo Nordisk Biotech, Inc. Phosphonyldipeptides efficaces dans le traitement de maladies cardiovasculaires
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URSULA A. GERMANN et al., "Characterization of Two Allelic Forms of Neurospora Crassa Laccase. Amino- and Carboxyl-Terminal Processing of a Precursor", THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 263, No. 2, January 1988, pages 885-896. *
YASUSHI KOJIMA et al., "Cloning, Sequence Analysis and Expression of Ligninolytic Phenoloxidase Genes of the White-Rot Basidiomycete Coriolus Hirsutus", THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol 265, No. 25, Sept. 1990, pages 15224-15230. *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001021809A3 (fr) * 1999-09-22 2001-10-18 Genencor Int Nouvelles enzymes oxydant le phenol
CN100378224C (zh) * 1999-09-22 2008-04-02 金克克国际有限公司 葡萄穗霉属的酚氧化酶
US7319112B2 (en) 2000-07-14 2008-01-15 The Procter & Gamble Co. Non-halogenated antibacterial agents and processes for making same
US6905853B1 (en) 2000-09-07 2005-06-14 Genencor International, Inc. Phenol oxidizing enzyme variants
EP2431048A2 (fr) 2002-10-08 2012-03-21 Genencor International, Inc. Peptides de liaison phénolique
US8293702B2 (en) 2002-10-08 2012-10-23 Danisco Us Inc. Phenolic binding peptides
WO2016090059A1 (fr) 2014-12-02 2016-06-09 Novozymes A/S Variants de laccase et polynucléotides codant pour ceux-ci
US10781428B2 (en) 2014-12-02 2020-09-22 Novozymes A/S Laccase variants and polynucleotides encoding same
CN111690622A (zh) * 2020-07-29 2020-09-22 中国农业科学院北京畜牧兽医研究所 一种真菌来源的漆酶g2589及其基因和应用
CN111690622B (zh) * 2020-07-29 2022-05-31 中国农业科学院北京畜牧兽医研究所 一种真菌来源的漆酶g2589及其基因和应用
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes
CN114958788A (zh) * 2022-06-24 2022-08-30 武汉轻工大学 一种耐高温漆酶及基因与菌株和应用
CN114958788B (zh) * 2022-06-24 2023-04-25 武汉轻工大学 一种耐高温漆酶及基因与菌株和应用

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EP0972014A1 (fr) 2000-01-19
AU5983098A (en) 1998-09-18

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