Laccase mutants

Abstract

By analyzing the three-dimensional structure of the Coprinus laccase structural parts or specific amino acid residues can be identified, which from structural or functional considerations appear to be important for the oxidative stability of a laccase. When comparing the three-dimensional structure of the Coprinus laccase structure with known amino acid sequences of various laccases, it has been found that several similarities exist between the sequences.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a 35 U.S.C. 371 national application of PCT/DK01/00292 filed Apr. 30, 2001 and claims, under 35 U.S.C. 119, priority or the benefit of Danish application nos. PA 2000 00707 and PA 2001 00327 filed Apr. 28, 2000 and Feb. 28, 2001, respectively, and U.S. application Nos. 60/203,345 and 60/277,817 filed May 10, 2000 and Mar. 21, 2001, respectively, the contents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates to laccase mutants with improved stability properties.

BACKGROUND

[0003] Laccase is a polyphenol oxidase (EC 1.10.3.2) which catalyses the oxidation of a variety It of inorganic and aromatic compounds, particularly phenols, with the concomitant reduction of molecular oxygen to water.

[0004] Because laccases are able to catalyze the oxidation of a variety of inorganic and aromatic compounds, laccases have been suggested in many industrial applications such as lignin modification, paper strengthening, dye transfer inhibition in detergents, phenol polymerization, hair colouring, and waste water treatment. A major problem with the use of laccases is their poor stability against oxidative attack from e.g. radicals formed from the oxidation of mediators (also referred to as “enhancing agents”).

[0005] Accordingly, it is the purpose of the present invention to create laccase variants with improved oxidative stability by using the information of a three-dimensional structure of a Coprinus cinereus laccase.

SUMMARY OF THE INVENTION

[0006] By analysing the three-dimensional structure of the Coprinus laccase structural parts or specific amino acid residues can be identified, which from structural or functional considerations appear to be important for the oxidative stability of a laccase. Furthermore, when comparing the three-dimensional structure of the Coprinus laccase structure with known amino acid sequences of various laccases, it has been found that several similarities exist between the sequences. The present invention is based on these findings.

[0007] Accordingly, as a first aspect the invention provides variants of a Coprinus laccase and of Coprinus-like laccases with improved oxidative stability as compared to the parent Coprinus laccase or Coprinus-like laccase.

[0008] In still further aspects the invention relates to DNA encoding such variants and to the use of the variants for various industrial purposes.

DETAILED DESCRIPTION

[0009] The Coprinus-Like Laccases

[0010] A number of laccases produced by different fungi are homologous on the amino acid level. For instance, when using the homology percent obtained from UWGCG program using the GAP program with the default parameters (penalties: gap weight=3.0, length weight=0.1; WISCONSIN PACKAGE Version 8.1-UNIX, August 1995, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) the following homology was found:

[0011] Coprinus cinereus laccase comprising the amino acid sequence shown in SEQ ID No. 1: 100%;

[0012] Polyporus pinsitus (I) laccase comprising the amino acid sequence shown in SEQ ID No. 2: 74.4%;

[0013] Polyporus pinsitus (II) laccase comprising the amino acid sequence shown in SEQ ID No. 3: 73.8%;

[0014] Phlebia radiata laccase comprising the amino acid sequence shown in SEQ ID No. 4: 69.9%;

[0015] Rhizoctonia solani (I) laccase comprising the amino acid sequence shown in SEQ ID No. 5: 64.8%;

[0016] Rhizoctonia solani (II) laccase comprising the amino acid sequence shown in SEQ ID No.6: 63.0%;

[0017] Rhizoctonia solani (III) laccase comprising the amino acid sequence shown in SEQ ID No. 7: 61.0%;

[0018] Rhizoctonia solani (IV) laccase comprising the amino acid sequence shown in SEQ ID No. 8: 59.7%;

[0019] Scytalidium thermophilum laccase comprising the amino acid sequence shown in SEQ ID No.9: 57.4%;

[0020] Myceliophthora thermophila laccase comprising the amino acid sequence shown in SEQ ID No. 10:56.5%.

[0021] Because of the homology found between the above-mentioned laccases, they are considered to belong to the same class of laccases, namely the class of “Coprinus-like laccases”.

[0022] Accordingly, in the present context, the term “Coprinus-like laccase” is intended to indicate a laccase which, on the amino acid level, displays a homology of at least 50% and less than 100% to the Coprinus cinereus laccase SEQ ID NO.1, or at least 55% and less than 100% to the Coprinus cinereus laccase SEQ ID NO.1, or at least 60% and less than 100% to the Coprinus cinereus laccase SEQ ID NO.1, or at least 65% and less than 100% to the Coprinus cinereus laccase SEQ ID NO.1, or at least 70% and less than 100% to the Coprinus cinereus laccase SEQ ID NO.1, or at least 75% and less than 100% to the Coprinus cinereus laccase SEQ ID NO. 1, or at least 80% and less than 100% to the Coprinus cinereus laccase SEQ ID NO. 1, or at least 85% and less than 100% to the Coprinus cinereus laccase SEQ ID NO. 1, or at least 90% and less than 100% to the Coprinus cinereus laccase SEQ ID NO. 1, or at least 95% and less than 100% to the Coprinus cinereus laccase SEQ ID NO. 1.

[0023] In the present context, “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 organism containing said DNA sequence. Finally, 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.

[0024] The Three-Dimensional Coprinus Laccase Structure

[0025] The Coprinus laccase consists of the 539 amino acids derived from Coprinus cinereus laccase IFO 8371 as disclosed in SEQ ID No.1.

[0026] The three-dimensional structure is believed to be representative for the structure of any Coprinus-like laccase.

[0027] The structure of the laccase was solved in accordance with the principle for X-ray crystallographic methods given in “X-Ray Structure Determination”, Stout, G. K. and Jensen, L. H., John Wiley & Sons, inc. NY, 1989. The structural coordinates for the solved crystal structure of the laccase at 2.2 Å resolution using the isomorphous replacement method are given in a standard PDB format (Brookhaven Protein Data Base) in Appendix 1. It is to be understood that Appendix 1 forms part of the present application. In Appendix 1 the amino acid residues of the enzyme are identified by three-letter amino acid code (capitalized letters).

[0028] The laccase structure is made up of three plastocyanin-like domains. These three domains all have a similar beta-barrel fold.

[0029] Three copper atoms were observed in the three-dimensional structure:

[0030] The so-called type 1 copper ion is coordinated by two histidines and one cysteine.

[0031] The so-called type 2 copper of the trinuclear centre is missing in the structure disclosed in the present application.

[0032] The so-called type 3 copper consists of two type 3 copper atoms (pair of copper atoms) bound to a total of 6 histidine ligands.

[0033] When comparing the amino acid sequence of the crystallized three-dimensional structure with Coprinus cinereus amino acid sequence of SEQ ID No. 1 the following four differences are observed:

[0034] 18 amino acids are missing from the N-terminal of the crystallized protein;

[0035] 17 amino acids are missing from the C-terminal of the crystallized protein;

[0036] Q19 in SEQ ID No. 1 is an A1 in the crystallized protein; and

[0037] Q243 in SEQ ID No. 1 is an E225 in the crystallized protein.

[0038] Generality of Structure

[0039] Because of the homology between the Coprinus laccase and the various Coprinus-like laccases, the solved structure defined by the coordinates of Appendix 1 is believed to be representative for the structure of all Coprinus-like laccases. A model structure of Coprinus-like laccases may be built on the basis of the coordinates given in Appendix 1 adapted to the laccase in question by use of an alignment between the respective amino acid sequences.

[0040] The above identified structurally characteristic parts of the Coprinus laccase structure may be identified in other Coprinus-like laccases on the basis of a model (or solved) structure of the relevant Coprinus-like laccase or simply on the basis of an alignment between the amino acid sequence of the Coprinus-like laccase in question with that of the Coprinus laccase used herein for identifying the amino acid residues of the respective structural elements.

[0041] Furthermore, in connection with Coprinus laccase variants of the invention, which are defined by modification of specific amino acid residues of the parent Coprinus laccase, it will be understood that variants of Coprinus-like laccases modified in an equivalent position (as determined from the best possible amino acid sequence alignment between the respective sequences) are intended to be covered as well.

[0042] Methods of the Invention for Design of Novel Laccase Variants

[0043] The laccase mutants of the present invention may be designed by constructing a variant of a parent Coprinus laccase, which variant has laccase activity and improved stability as compared to the parent laccase, which method comprises:

[0044] i) analysing the three-dimensional structure of the parent Coprinus laccase to identify at least one amino acid residue or at least one structural part of the Coprinus laccase structure, which amino acid residue or structural part is believed to be of relevance for altering the stability of the parent Coprinus laccase (as evaluated on the basis of structural or functional considerations),

[0045] ii) constructing a Coprinus laccase variant, which as compared to the parent Coprinus laccase, has been modified in the amino acid residue or structural part identified in i) so as to alter the stability, and, optionally,

[0046] iii) testing the resulting Coprinus laccase variant with respect to stability.

[0047] The laccase mutants of the invention may also be designed by constructing a variant of a parent Coprinus-like laccase, which variant has laccase activity and improved stability as compared to the parent laccase, which method comprises:

[0048] i) comparing the three-dimensional amino acid structure of the Coprinus laccase with an amino acid sequence of a Coprinus-like laccase,

[0049] ii) identifying a part of the Coprinus-like laccase amino acid sequence which is different from the Coprinus laccase amino acid sequence and which from structural or functional considerations is contemplated to be responsible for differences in the stability of the Coprinus and Coprinus-like laccase,

[0050] iii) modifying the part of the Coprinus-like laccase identified in ii) whereby a Coprinus-like laccase variant is obtained, which has an improved stability as compared to the parent Coprinus-like laccase, and optionally,

[0051] iv) testing the resulting Coprinus-like laccase variant with respect to stability.

[0052] The analysis or comparison performed in step i) of the methods of the invention may be performed by use of any suitable computer programme capable of analysing and/or comparing amino acid sequences.

[0053] The structural part which is identified in step i) of the methods of the invention may be composed of one amino acid residue. However, normally the structural part comprises more than one amino acid residue, typically constituting one of the above mentioned parts of the Coprinus structure such as one of the copper centres.

[0054] The laccase variants of the invention have improved oxidative stability compared to the un-modified parent laccases. Improved oxidative stability means that the laccase variants of the invention have improved tolerance towards oxidative chemical compounds, such as radicals formed from laccase mediated oxidation of radical precursor compounds. The radical precursor compounds may preferably be mediators or “enhancing agents”, such as those described in EP 705327 (compounds containing N—OH, N—O and NR—OH groups), WO9501426 (compounds containing two aromatic rings etc.), WO 96/10079 (methylsyringate type of compounds) and/or WO 99/57360 (N-hydroxyacetanilide type of compounds).

[0055] According to the invention useful laccase variants may be obtained by:

[0056] protection of the active site center by introduction of steric hindrance in the oxygen entry cleft;

[0057] modification of oxidation labile amino acid residues in or near the substrate entry cleft;

[0058] modification of oxidation labile surface exposed amino acid residues.

[0059] Modifications

[0060] The modification of an amino acid residue or structural part is typically accomplished by suitable modifications of a DNA sequence encoding the parent enzyme in question. The term “modified” as used in the methods according to the invention is intended to have the following meaning: When used in relation to an amino acid residue the term is intended to mean replacement of the amino acid residue in question with another amino acid residue. When used in relation to a structural part, the term is intended to mean: replacement of one or more amino acid residues of said structural part with other amino acid residues, or addition of one or more amino acid residues to said part, or deletion of one or more amino acid residues of said structural part.

[0061] The construction of the variant of interest is accomplished by cultivating a microorganism comprising a DNA sequence encoding the variant under conditions which are conducive for producing the variant, and optionally subsequently recovering the variant from the resulting culture broth. This is described in detail further below.

[0062] Variants with Altered Oxidative Stability

[0063] It is contemplated that it is possible to improve the oxidative stability of a parent Coprinus laccase or a parent Coprinus-like laccase, wherein said variant is the result of a mutation, i.e. one or more amino acid residues having been deleted from, replaced or added to the parent laccase, the stability test performed as described below.

[0064] Preferred positions for mutations are the following: 1 CcL: MtL: F21 V52 H91 G121 F112 F141 H133 — H153 Y177 Y176 H206 H230 M260 H309 P336 F335 T365 Y347 I380 S349 I382 Y375 V406 Y416 — F449 — E455 A506 F456 W507 Y490 W543

[0065] wherein

[0066] CcL: Coprinus cinereus laccase comprising the amino acid sequence shown in SEQ ID No. 1;

[0067] MtL: Myceliophthora thermophila laccase comprising the amino acid sequence shown in SEQ ID No. 10.

[0068] The above shown rows are homologous positions. The following variants are preferred:

[0069] A variant of a parent Coprinus laccase, which comprises one or more of the following substitutions in SEQ ID No. 1:

[0070] F21 A, I, L, N, R, S, Q;

[0071] H91 A, I, L, N, R, S, Q;

[0072] F112 A, V, L, I, P, F, M, G, S, T, C, Y, N, Q, D, E, K, R, H;

[0073] H133A, I, L, N, R, S, Q;

[0074] H153A, I, L, N, R, S, Q;

[0075] Y176A, i, L, N, R, S, Q;

[0076] H230A, I, L, N, R, S, Q;

[0077] H309 A, I, L, N, R, S, Q;

[0078] F335 A, I, L, N, R, S, Q;

[0079] Y347 A, V, L, I, P, F, M, G, S, T, C, Y, N, Q, D, E, K, R, H;

[0080] S349 A, V, L, I, P, F, M, G, S, T, C, Y, N, Q, D, E, K, R, H;

[0081] Y375 A, I, L, N, R, S, Q;

[0082] Y416 A, I, L, N, R, S, Q;

[0083] F449 A, V, L, I, P, F, M, G, S, T, C, Y, N, Q, D, E, K, R, H;

[0084] E455 A, V, L, I, P, F, M, G, S, T, C, Y, N, Q, D, E, K, R, H;

[0085] F456 A, I, L, N, R, S, Q;

[0086] Y490 A, V, L, I, P, F, M, G, S, T, C, Y, N, Q, D, E, K, R, H.

[0087] A variant of a parent Myceliophthora thermophila laccase, which comprises a mutation in a position corresponding to at least one of the following positions in SEQ ID No. 10:

[0088] V52 A, I, L, N, R, S, Q;

[0089] G121 A, I, L, N, R, S, Q;

[0090] F141 A, V, L, I, P, F, M, G, S, T, C, Y, N, Q, D, E, K, R, H;

[0091] Y177A, I, L, N, R, S, Q;

[0092] H206 A, I, L, N, R, S, Q;

[0093] M260 A, I, L, N, R, S, Q;

[0094] P336 A, I, L, N, R, S, Q;

[0095] T365 A, I, L, N, R, S, Q;

[0096] I380 A, V, L, I, P, F, M, G, S, T, C, Y, N, Q, D, E, K, R, H;

[0097] I382 A, V, L, I, P, F, M, G, S, T, C, Y, N, Q, D, E, K, R, H;

[0098] V406A, I, L, N, R, S, Q;

[0099] A506 A, V, L, I, P, F, M, G, S, T, C, Y, N, Q, D, E, K, R, H;

[0100] W507 A, I, L, N, R, S, Q;

[0101] W543 A, V, L, I, P, F, M, G, S, T, C, Y, N, Q, D, E, K, R, H.

[0102] Detergent Composition

[0103] The laccase variants of the invention may be added to and thus become a component of a detergent composition.

[0104] The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.

[0105] In a specific aspect, the invention provides a detergent additive comprising the laccase variants of the invention. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.

[0106] In general the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.

[0107] Proteases:

[0108] Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.

[0109] Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.

[0110] Preferred commercially available protease enzymes include Alcalase™, Savinase™, Primase™, Everlase™, Esperase™, and Kannase™ (Novozymes A/S), Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™, FN2™, and FN3™ (Genencor International Inc.).

[0111] Lipases:

[0112] Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

[0113] Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97104079 and WO 97/07202.

[0114] Preferred commercially available lipase enzymes include Lipolase™, Lipolase Ultra™ and Lipoprime™ (Novozymes A/S).

[0115] Amylases:

[0116] Suitable amylases (&agr; and/or &bgr;) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, &agr;-amylases obtained from Bacillus, e.g. a special strain of B. licheniformis, described in more detail in GB 1,296,839.

[0117] Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

[0118] Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ and BAN™ (Novozymes A/S), Rapidase™ and Purastar™ (Genencor International Inc.).

[0119] Cellulases:

[0120] Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178, 5,776,757 and WO 89/09259.

[0121] Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593, 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

[0122] Commercially available cellulases include Celluzyme™, and Carezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor International Inc.), and KAC-500(B) ™ (Kao Corporation).

[0123] Peroxidases/Oxidases:

[0124] Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.

[0125] The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e. a separate additive or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.

[0126] Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216.

[0127] The detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous.

[0128] The detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight.

[0129] When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.

[0130] When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).

[0131] The detergent may contain 0-65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenyisuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).

[0132] The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.

[0133] The detergent may contain a bleaching system which may comprise a H2O2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.

[0134] The enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.

[0135] The detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.

[0136] It is at present contemplated that in the detergent compositions any enzyme, in particular the laccase variants of the invention, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per liter of wash liquor, preferably 0.05-10 mg of enzyme protein per liter of wash liquor, more preferably 0.1-5 mg of enzyme protein per liter of wash liquor, and most preferably 0.1-1 mg of enzyme protein per liter of wash liquor.

[0137] The laccase variants of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference.

[0138] Methods of Preparing Laccase Variants

[0139] Several methods for introducing mutations into genes are known in the art. After a brief discussion of the cloning of laccase-encoding DNA sequences, methods for generating mutations at specific sites within the laccase-encoding sequence will be discussed.

[0140] Cloning a DNA Sequence Encoding a Laccase

[0141] 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. First, 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. Then, if the amino acid sequence of the laccase is known, homologous, labelled oligonucleotide probes may be synthesized and used to identify laccase-encoding clones from a genomic library prepared from the organism in question. Alternatively, 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.

[0142] 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.

[0143] Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method. In the phosphoroamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.

[0144] Finally, 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.

[0145] Site-Directed Mutagenesis

[0146] Once a laccase-encoding DNA sequence has been isolated, and desirable sites for mutation identified, mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis. In a specific method, a single-stranded gap of DNA, bridging the laccase-encoding sequence, is created in a vector carrying the laccase gene. Then 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. A specific example of this method is described in Morinaga et al. (1984). U.S. Pat. Nos. 4,760,025 discloses the introduction of oligonucleotdes encoding multiple mutations by performing minor alterations of the cassette. However, an even greater variety of mutations can be introduced at any one time by the Morinaga method, because a multitude of oligonucleotides, of various lengths, can be introduced.

[0147] Another method of introducing mutations into laccase-encoding DNA sequences is described in Nelson and Long (1989). It involves the 3-step generation of a PCR fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reactions. From the PCR-generated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid.

[0148] Random Mutagenesis

[0149] The random mutagenesis of a DNA sequence encoding a parent laccase may conveniently be performed by use of any method known in the art.

[0150] For instance, 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. Furthermore, the random mutagenesis may be performed by use of any combination of these mutagenizing agents.

[0151] The mutagenizing agent may, e.g., be one which induces transitions, transversions, inversions, scrambling, deletions, and/or insertions.

[0152] 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.

[0153] When such agents are used, 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.

[0154] When the mutagenesis is performed by the use of an oligonucleotide, 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.

[0155] 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 misincorporation of nucleotides (Deshler 1992; Leung et al., Technique, Vol.1, 1989, pp. 11-15).

[0156] 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.

[0157] 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. Alternatively, the DNA sequence 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. Finally, 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.

[0158] In some cases it may be convenient to amplify the mutated DNA sequence prior to the expression step or the screening step being performed. Such amplification 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.

[0159] Subsequent to the incubation with or exposure to the mutagenizing agent, 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. Examples of suitable host cells are fungal hosts such as Aspergillus niger or Aspergillus oryzae.

[0160] The mutated DNA sequence may further comprise a DNA sequence encoding functions permitting expression of the mutated DNA sequence.

[0161] Localized Random Mutagenesis

[0162] 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.

[0163] 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.

[0164] Alternatively, 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.

[0165] With respect to the screening step in the above-mentioned method of the invention, this may conveniently be performed by use of aa filter assay based on the following principle:

[0166] 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. Subsequent to the incubation, 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.

[0167] 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 Durapore™. 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.

[0168] 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.

[0169] 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.

[0170] Laccase Activity

[0171] The laccase activity may be measured using 10-(2-hydroxyethyl)-phenoxazine (HEPO) as substrate. HEPO was synthesized using the same procedure as described for 10-(2-hydroxyethyl)-phenothiazine, (G. Cauquil in Bulletin de la Society Chemique de France, 1960, p. 1049). In the presence of oxygen laccases (E.C. 1.10.3.2) oxidize HEPO to a HEPO radical that can be monitored photometrically at 528 nm.

[0172] The Coprinus cinereus laccase was measured using 0.4 mM HEPO in 50 mM sodium acetate, pH 5.0, 0.05% TWEEN-20 at 30° C. The absorbance at 528 nm was followed for 200 s and the rate calculated from the linear part of the progress curve.

[0173] The Myceliophthora thermophila laccase was measured using 0.4 mM HEPO in 25 mM Tris-HCl, pH 7.5, 0.05% Tween-20 at 30° C. The absorbance at 528 nm was followed for 200 s and the rate calculated from the linear part of the progress curve.

[0174] The Polyporus pinsitus laccase was measured using 0.4 mM HEPO in 50 mM MES-NaOH, pH 5.5. The absorbance at 528 nm was followed for 200 sec. and the rate calculated from the linear part of the progress curve.

[0175] Testing of Variants of the Invention

[0176] The stability against oxidation by radicals (oxidative stability) of Coprinus variants or Coprinus-like variants may be measured as described in the following.

[0177] The enzyme is diluted in 100 mM phosphate pH 5 or 6 (which is closest to the pH optimum for the enzyme with methylsyringate as substrate) to a concentration of 0.1 mg enzyme protein per ml.

[0178] To 0.9 ml enzyme dilution is added 0.1 ml 5 mM methylsyringate (in 50% ethanol). As a reference 0.9 ml enzyme dilution is added 0.1 ml 50% ethanol.

[0179] Both sample and reference are stored at room temperature (approx. 25° C.) for 20 hours. After dilution residual activity of sample and reference is determined by the LACU or LAMU assays using syringaldazine as substrate.

[0180] Conditions for some fungal laccases are: 2 Laccase from Incubation Assay Polyporus/Trametes pH 5 LACU Coprinus cinereus pH 6 LAMU Myceliophthora thermophila pH 6 LAMU Rhizoctonia solani pH 6 LAMU

[0181] Laccase Activity (LACU)

[0182] Laccase activity may be determined from the oxidation of syringaldazin under aerobic conditions. The violet colour produced is photometered at 530 nm. The analytical conditions are 19 mM syringaldazin, 23 mM acetate buffer, pH 5.5, 30° C., 1 min. reaction time.

[0183] 1 laccase unit (LACU) is the amount of enzyme that catalyses the conversion of 1.0 &mgr;mole syringaldazin per minute at these conditions.

[0184] Laccase Activity (LAMU)

[0185] Laccase activity may be determined from the oxidation of syringaldazin under aerobic conditions. The violet colour produced is photometered at 530 nm. The analytical conditions are 19 mM syringaldazin, 23 mM Tris/maleate buffer, pH 7.5, 30° C., 1 min. reaction time.

[0186] 1 laccase unit (LAMU) is the amount of enzyme that catalyses the conversion of 1.0 &mgr;mole syringaldazin per minute at these conditions.

[0187] Expression of Laccase Variants

[0188] According to the invention, a DNA sequence encoding the variant produced by methods described above, or by any alternative 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.

[0189] 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. Thus, 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. Alternatively, 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.

[0190] In the vector, 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. Examples of 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 &agr;-amylase, A. niger acid stable &agr;-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.

[0191] The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation 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.

[0192] The vector may further comprise 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, pUB110, pE194, pAMB1 and pIJ702.

[0193] 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 ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Furthermore, 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.

[0194] The procedures used to ligate the DNA construct of the invention encoding a laccase variant, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al. (1989)).

[0195] The cell of the invention, either comprising a DNA construct or an expression vector of the invention as defined above, 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.

[0196] 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.

[0197] 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.

[0198] In a yet further aspect, 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.

[0199] 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).

[0200] The laccase variant secreted from the host cells may conveniently 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.

INDUSTRIAL APPLICATIONS

[0201] The 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, bleaching of textiles (in particular bleaching of denim as described in WO 96/12845 and WO 96112846) and waste water treatment. Any detergent composition normally used for enzymes may be used, e.g., the detergent compositions disclosed in WO 95/01426.

Claims

1. A variant of a parent Coprinus laccase, which comprises a mutation in a position corresponding to at least one of the following positions in SEQ ID No. 1:

F21,

H91,

F112,

H133,

H153,

Y176,

H230,

H309,

F335,

Y347,

S349,

Y375,

Y416,

F449,

E455,

F456, and/or

Y490.

2. A variant of a parent Myceliophthora thermophila laccase, which comprises a mutation in a position corresponding to at least one of the following positions in SEQ ID No. 10:

V52,

G121,

F141,

Y177,

H206,

M260,

P336,

V406,

T365,

I380,

I382,

A506,

W507, and/or

W543.

3. A DNA construct comprising a DNA sequence encoding a laccase variant of claim 1.

4. A recombinant expression vector which carries a DNA construct of claim 3.

5. A cell which is transformed with a DNA construct of claim 3.

6. A cell of claim 5, which is a microorganism.

7. A cell of claim 6, which is a bacterium or a fungus.

8. A cell of claim 7, which is an Aspergillus niger or an Aspergillus oryzae cell.

9. A method for oxidizing a substrate, comprising contacting the substrate with a laccase variant of claim 1.

10. A method for inhibiting dye transfer during washing of fabrics, comprising adding a laccase variant of claim 9 during washing.

11. A method for bleaching a textile, comprising applying a laccase variant of claim 9 to the textile.

12. A detergent additive comprising a laccase variant of claim 1 in the form of a non-dusting granulate, a stabilised liquid or a protected enzyme.

13. A detergent additive of claim 12, which additionally comprises one or more other enzyme such as a protease, a lipase, an amylase, and/or a cellulase.

14. A detergent composition comprising a laccase variant of claim 1 and a surfactant.

15. A detergent composition of claim 14 which additionally comprises one or more other enzymes such as a protease, a lipase, an amylase and/or a cellulase.