US 20120028333A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0028333 A1 Piatesi et al. (43) Pub. Date: Feb. 2, 2012

(54) USE OF ENZYMES TO REDUCE ALDEHYDES (30) Foreign Application Priority Data FROMALDEHYDE-CONTAINING PRODUCTS Apr. 7, 2009 (EP) ...... O9157522.5 Publication Classification (76) Inventors: Andrea Piatesi, Mannheim (DE); (51) Int. Cl. Tilo Habicher, Speyer (DE); CI2N 9/02 (2006.01) Michael Bischel, Worms (DE); CI2N I/00 (2006.01) Li-Wen Wang, Mannheim (DE): CI2N 15/63 (2006.01) Jirgen Reichert, Limburgerhof A62D 3/02 (2007.01) (DE); Rainer Packe-Wirth, C7H 2L/04 (2006.01) Trostberg (DE); Kai-Uwe (52) U.S. Cl. ... 435/189: 435/262:536/23.2:435/320.1; Baldenius, Heidelberg (DE); Erich 435/243 Kromm, Weisenheim am Sand (57) ABSTRACT (DE); Stefan Häfner, Speyer (DE); Carsten Schwalb. Mannheim (DE); The invention relates to the use of an enzyme preparation Hans Wolfgang Höffken, which catalyzes the degradation of formaldehyde for reduc Ludwigshafen (DE) ing the formaldehyde content in a formaldehyde-containing formulation. In a preferred embodiment, the enzyme prepa ration contains a formaldehyde dismutase from a Pseudomo (21) Appl. No.: 13/262,662 nas putida Strain. Further, the invention refers to a process for reducing the formaldehyde content in cross-linking agents for textile finishing or in polymer dispersions used, e.g. in con (22) PCT Filed: Mar. 31, 2010 struction chemistry. Further the invention relates to the use of an enzyme preparation which catalyzes the degradation of (86). PCT No.: PCT/EP1OAS4284 aldehydes for reducing the formaldehyde content in an alde hyde-containing formulation. Furthermore, the invention S371 (c)(1), relates to a novel variant of the formaldehyde dismutase from (2), (4) Date: Oct. 3, 2011 Pseudomonas putida. Patent Application Publication Feb. 2, 2012 Sheet 1 of 9 US 2012/0028333 A1

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(A) (B) (C) Patent Application Publication Feb. 2, 2012 Sheet 2 of 9 US 2012/0028333 A1

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00:00 00:30 01:00 01:30 02:00 02:30 reaction time h) -4-301 FDM aSax. F93A assy F93A 301 Patent Application Publication Feb. 2, 2012 Sheet 3 of 9 US 2012/0028333 A1

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Formaldehyde R 1750

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R &OOO- f - Acetaldehyde 15'g 7000 - | \ 8O co

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1R a 2 1 3. M f retention time (min) Patent Application Publication Feb. 2, 2012 Sheet 4 of 9 US 2012/0028333 A1

Figure 4

140 120 100 SS, e 80 2 60 8 40 20 O

temperature (C) Patent Application Publication Feb. 2, 2012 Sheet 5 of 9 US 2012/0028333 A1

Figure 5

8 FDM 301

25 35 45 55 65 temperature (C) Patent Application Publication Feb. 2, 2012 Sheet 6 of 9 US 2012/0028333 A1

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Patent Application Publication Feb. 2, 2012 Sheet 8 of 9 US 2012/0028333 A1

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Patent Application Publication Feb. 2, 2012 Sheet 9 of 9 US 2012/0028333 A1

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US 2012/0028333 A1 Feb. 2, 2012

USE OF ENZYMES TO REDUCE ALDEHYDES finishing of woven and knitted fabrics composed of cotton, FROMALDEHYDE-CONTAINING other cellulosic fibres, and their blends with synthetic fibres. PRODUCTS 0006 Initially, resin-finishing agents were developed to improve the shrinkage of Viscose staple fabrics. These com FIELD OF THE INVENTION pounds were usually derived from formaldehyde and urea. In order to improve the competitiveness of cotton on the textile 0001. The present invention relates to the enzymatic market, heterocyclic cross-linking reagents based on formal removal of formaldehyde from formulations containing dehyde, urea and glyoxal have been developed and are com formaldehyde and from products treated with said formula monly used for easy-care and wrinkle-free finishing. Due to tions. the Suspected harm in humans, FA levels in products and industrial processes have to be kept as low as possible. BACKGROUND OF THE INVENTION 0007. The prior art discloses various technologies for the 0002 Formaldehyde (for the purposes of convenience purpose of removing FA, e.g. air-borne when released from hereinafter often referred to as “FA) is an important chemi products or directly from well-known and widely used resins cal used widely by industry to manufacture building materials as introduced supra. U.S. Pat. No. 5,352.274 discloses air and numerous household products. FA is used for example for filtration utilizing a plurality of corrugated base sheets which crease-resistant finishing in the textile industry, for the pro are stacked or nestled and which have entrapped carbon dust duction and coating of chipboards in the wood processing for adsorption of impurities such as FA, acetaldehyde, and industry, and for the production of synthetic resins like phe acrolein. This technology provides a method to adsorb FA nolic plastics or aminoplasts in the chemical industry. Due to molecules physically but not degradation by a chemical or its high volatility FA is released into the air during the pro biochemical reaction. U.S. Pat. No. 5,830,414 discloses the duction processes and is considered as an important impact treatment of carbon fibers with an active small molecule such on health and environment. as a strong acid, strong base, or strong oxidizing agent. These 0003 FA has four basic uses: as an intermediate in the chemicals can only be used to treat fibers having high chemi production of resins; as intermediate in the production of cal resistances such as activated carbon fibers. Further, fibers industrial chemicals; as a biocide; and as a component in the thus treated are potentially hazardous to handle. The use of formulation of end-use consumer items. The manufacture of formaldehyde degrading enzymes in air filters is described in resins accounts for about 65 percent of total consumption. JP2OO1340436. About one-third is used in the synthesis of high volume 0008. With respect to textile industry and building mate chemical derivatives, including pentaerythritol, hexamethyl rials, FA reducing agents should not adversely affect fabric enetetramine, and butanediol. Two percent is used in textile properties such as hand, shrinkage, strength retention and treating and Small amounts of FA are present as preservatives shade or whiteness or the mechanical properties of the par or bioicides in consumer and industrial products, such as ticleboard. And, of course, it must be economical to use in cosmetics, shampoos and glues. The largest amounts of FA production and efficient at reasonable levels. In the textile are used for producing condensates (i.e. resins) with urea, industry, compounds having active methylene groups have melamine, naphthaline Sulfonate, and phenol and, to a small been used as FA reducing agents to reduce the amount of FA extent, with their derivatives. The main part of these resins is released from durable press-treated fabrics as described in used for the production of adhesives and impregnating resins, Textile Chemist and Colorist, Vol. 16, No. 12, p. 33, Decem which are employed for manufacturing particle boards, ply ber 1984 (published by the American Association of Textile wood, and furniture. These condensates are also employed for Chemists and Colorists). FA reducing agents containing the production of curable molding materials; as raw materials active methylene hydrogens also may be added to coating for Surface coating and as controlled-release nitrogen fertil compositions containing urea/formaldehyde or melamine? izers. They are used as auxiliaries in the textile, leather, rub formaldehyde resin to reduce formaldehyde concentration ber, and cement industries. Further uses include binders for (e.g. described in U.S. Pat. No. 5,795,933). Also the addition foundry sand, rockwool and glasswool mats in insulating of urea and its derivatives is known to Scavenge formalde materials, abrasive paper, and brake linings. Very small hyde. amounts of urea-FA condensates are used in the manufacture 0009. The prior art has not disclosed FA reducing agents of foamed resins that have applications in the mining sector which are effective in reducing released FA to the low levels and in the insulation of buildings and transportation vehicles. which are currently desired without detrimental effects on the 0004 Some products based on FA contain unreacted FA in properties of the materials to be treated with said resins. excess which may be released from the product or released Currently, the FA reducing agents most widely used in through Subsequent hydrolysis. One example is urea-FA durable press finishing compositions are polyhydric alcohols, resin. Urea-FA resin is a generic name that actually represents Such as diethylene glycol and Sorbitol and in the manufacture an entire class of related formulations. About 60 percent of of particleboard nitrogen containing compounds such as urea, urea-FA resin production is consumed by particleboard and melamine, diazine, triazine and amine compounds (U.S. Pat. plywood manufacturing, where the resin is used as glue. No. 4,559,097). Compounds such as these, however, are not Urea-FA resins are also used in decorative laminates, textiles, sufficiently effective in reducing FA levels to produce the low paper, and foundry sand molds. levels which are currently desired. Moreover, they only bind 0005 Finally, FA resins are used to treat textiles to impart FA and do not catalyze its degradation. Also some formalde wrinkle-resistance to clothing. Resin or chemical finishing is hyde scavengers like urea slow down reactivity of textile in most cases the last stage of modern textile production. The cross-linkers, reducing their efficiency. goal is to convert the bleached, dyed or printed fabric by 0010 Formaldehyde Dismutase (hereinafter referred to as mechanical and chemical treatment into Suitable state for “FDM') activity has been first described by Kato and co sale. One of the most important processes is the washfast workers in 1983 (Kato et al., 1983, Agric. Biol. Chem., 47(1), US 2012/0028333 A1 Feb. 2, 2012 pages 39-46) but no corresponding gene has been identified catalyzes the degradation of formaldehyde. Furthermore, it until 1995 (Yanase et al. 1995, Biosci. Biotechnol. Biochem. relates to formulations comprising a cross-linking agent for 59(2), 197-202). The first protocol for recombinant produc textiles or a polymeric dispersant and an enzyme preparation tion and purification of soluble FDM has been published in which catalyzes the degradation of formaldehyde. 2002 (Yanase et al. 2002, Biosci. Biotechnol. Biochem. 0020. Other embodiments of the invention relate to a 66(1), 85-91). A crystal structure for FDM (Hasegawa et al. codon-optimized, dismutase-encoding nucleic acid, vectors 2002, Acta Crystallogr., Sect. A, 58, C102-C102) and other containing said nucleic acid, and expression host. related enzymes are available since 2002 (Tanaka et al. 2002, 0021. Furthermore the invention relates to isolated nucleic Journal of Molecular Biology, 324,519-533). acid encoding novel FDM variants, the amino acid sequence of said FDM variants, as well as the particular uses thereof. SUMMARY OF THE INVENTION 0011. Accordingly, the technical problem underlying the DESCRIPTION OF THE DRAWING present invention is to provide efficient methods and means to 0022 FIG. 1: A few formaldehyde-based cross-linking reduce the FA content from formulations that are used to treat reagents. (A) urea-FA, (B) melamine-FA, (C) dimethy various materials, e.g. in textile or construction industry, loldihydroxyethylene urea (DMDHEU). overcoming the disadvantages of the prior art. The problem is 0023 FIG. 2: Initial rates of acetaldehyde dismutation (20 Solved by the Subject-matter of the present invention, namely, mM in 50 mM. KHPO pH 8, 100 mM KCl) for FDM, FDM the inventors have surprisingly found that the formaldehyde I'L, FDM FA and FDM IL/FA determined by content in resins that are used to treat Such materials, can be HPLC. Enzyme concentration 50 ug/mL. efficiently reduced by using an enzyme that catalyzes the 0024 FIG. 3: Enzymatic conversion of formaldehyde degradation of formaldehyde. (top) and acetaldehyde (bottom) followed overtime by HPLC 0012. Furthermore, the present invention describes the using the FDM I'L variant. The reaction mixture was ana design, characterization and crystal structures of FDM lyzed in 30' intervals over 2 h. The reactions were performed mutants with improved specificity towards formaldehyde, in 50 mM. KHPO (pH 7.3 for formaldehyde and pH 8 for enhanced activity towards acetaldehyde, and increased ther acetaldehyde), 100 mM KCl and 20 mMaldehyde. Enzyme mostability. Particularly, the FDM I'L mutant shows concentration for acetaldehyde was 50 ug/mL and for form increased activity on formaldehyde compared to the wild aldehyde 2.4 ug/mL. Retention time formaldehyde: 16.6 min: type protein and is more thermostable. This enzyme can be formic acid: 17.5 min: methanol: 22.9 min; acetaldehyde: easily produced in high yield by fermentation and formulated 21.2 min; acetic acid: 18.9 min: ethanol: 25.2 min. Product as a spray-dried powder. generation is equimolar. Detection by RI. 0013 Thus, an object of the invention relates to the use of an enzyme preparation which catalyzes the degradation of (0025 FIG. 4: Temperature-activity profile of FDM I'L formaldehyde for reducing the formaldehyde content in a and FDM I'L/FA. Substrate:acetaldehyde formaldehyde-containing formulation. (0026 FIG. 5: Temperature-activity profile of FDM and 0014. In a preferred embodiment, the enzyme preparation FDM I'L. Substrate: formaldehyde contains an enzyme which comprises the amino acid (0027 FIG. 6: The wild-type Phe 93 shown with the elec sequence of SEQ ID NO:2 or variants thereof. In a further tron-density map of the FDM I'L/FA mutant. embodiment, the enzyme is a formaldehyde dismutase (0028 FIG. 7: Close-up of the active site of wildtype FDM (FDM) from a bacterial strain, preferably of the E.C classifi in complex with formaldehyde (FA) cation EC 1.2.99.4. or EC 1.2.1.46., which is derived from a 0029 FIG. 8: Close-up of the active site of FDM I'L in Pseudomonas putida Strain. complex with formaldehyde (FA) 0015. In a particularly preferred embodiment, the enzyme 0030 FIG. 9: Close-up of the active site of FDM IL/ preparation contains an enzyme which comprises the amino FA in complex with acetaldehyde (AA). acid sequence of SEQID NO: 8, or SEQID NO: 10. 0016. In a further preferred embodiment, the formulation DESCRIPTION OF THE SEQUENCES is a resin. The resin can be a cross-linking agent for textile fabrics or a polymeric dispersant, employed to generate poly 0031 SEQID NO:1 mer dispersions. In a preferred embodiment the cross-linking 0032) Nucleic acid sequence of formaldehyde dismutase, agent is used for textile fabrics that contain cellulosic fibers genebank accession number L25862 (CDS 323 ... 1522) such as cotton or viscose or a mixture thereof and blends with 0033 SEQID NO:2 synthetics. 0034 Protein sequence of formaldehyde dismutase, 0017. The resulting polymer dispersions are suitable for genebank accession number L25862 treating materials e.g. Such as building or construction mate 0035) SEQID NO:3 rials, leather and hides, fiberboard, particle board, plywood 0036) Optimized DNA sequence for FDM from and/or carpeting, and Suitable for coating applications or Pseudomonas putida F61 (1197 bp) paper making. 0037 SEQID NO:4 0.018. In another embodiment, the invention refers to a 0038 Nucleotide sequence of pDHE-FDM process for reducing the formaldehyde content in a formal 0039 SEQID NO:5 dehyde-containing formulation comprising contacting the 0040. Nucleotide sequence of pAgro formulation with an enzyme preparation that catalyzes the 0041 SEQID NO:6 degradation of formaldehyde. 0042 Nucleotide sequence of pHSG 0019. The invention relates also to a process for reducing 10043 SEQID NO:7 the formaldehyde content in a textile fabric, comprising con 0044) Nucleic acid sequence of formaldehyde dismutase tacting the textile fabric with an enzyme preparation which Ile-301-Leu US 2012/0028333 A1 Feb. 2, 2012

0045 SEQID NO:8 Smoke. At higher concentrations (up to 1000 ppm), acetalde 0046 Protein sequence of formaldehyde dismutase Ile hyde irritates the mucous membranes. The perception limit of 301-Leu acetaldehyde in air is in the range between 0.07 and 0.25 ppm. 10047 SEQID NO:9 At such concentrations the fruity odor of acetaldehyde is 0048 Nucleic acid sequence of formaldehyde dismutase apparent. Conjunctival irritations have been observed after a Phe-93-Ala/Ile-301-Leu 15-min exposure to concentrations of 25 and 50 ppm, but 0049 SEQID NO:10 transient conjunctivitis and irritation of the respiratory tract 0050 Protein sequence of formaldehyde dismutase Phe have been reported after exposure to 200 ppm acetaldehyde 93-Ala/Ile-301-Leu for 15 min. DEFINITIONS 0054 The term “methylglyoxal refers to a compound of the general formula (CH CO-CH=O), having the CAS 0051. It is to be understood that this invention is not lim number 78-98-8 (Kato et al., 1983, Agric. Biol. Chem., 47(1), ited to the particular methodology, protocols, cell lines, plant pages 39-46). It also known to the skilled artisan as pyruval species or genera, constructs, and reagents described as such. dehyde, 2-oxopopanal, 2-oxopropionaldehyde, and is formed It is also to be understood that the terminology used herein is as a side product of several metabolic pathways. for the purpose of describing particular embodiments only, 0055 “Formulation', as used herein, means a chemical and is not intended to limit the scope of the present invention, composition, manufactured according to a specific formula which will be limited only by the appended claims. It must be and/or recipe. It is, thus, distinguished from naturally occur noted that as used herein and in the appended claims, the ring FA-containing Sources. The formulation is manufactured singular forms “a,” “and” and “the include plural reference by employing the addition of FA. In some instances, the unless the context clearly dictates otherwise. Thus, for formulation is also referred to as “FA condensate'. example, reference to “a vector is a reference to one or more 0056. As used herein, the phrase “resin' means a low vectors and includes equivalents thereof known to those molecular weight substance that will subsequently be reacted skilled in the art, and so forth. The term “about is used herein to form a high molecular weight polymer or to crosslink to mean approximately, roughly, around, or in the region of functional polymer chains like cellulose. Particularly the term When the term “about' is used in conjunction with a numeri resin refers to “synthetic resins” which are defined as resins cal range, it modifies that range by extending the boundaries resulting from controlled chemical reactions such as polyad above and below the numerical values set forth. In general, dition or polycondensation between well-defined reactants the term “about is used herein to modify a numerical value including formaldehyde that do not themselves have the char above and below the stated value by a variance of 20 percent, acteristics of resins. Synthetic resins can mean also resins preferably 10 percent up or down (higher or lower). As used obtained by polymerization of unsaturated monomers. This herein, the word 'or' means any one member of a particular term includes (i) Hydrocarbon resins, i.e., synthetic resins list and also includes any combination of members of that list. from coaltar, petroleum, and turpentine streams, produced by 0052. The term “formaldehyde' or “FA” refers to a com polymerization. These resins are used like natural ones, e.g., pound of the general formula CHO, having the CAS number in combination with other polymers to impart special prop 50-00-0. It also known to the skilled artisan as formalin, erties such as tack, flow, and hardness to a material, and (ii) methylene oxide, methylaldehyde, methanal, HCHO, formic Synthetic resins obtained mainly by addition polymerization aldehyde, oxomethane, formol, oxymethylene, morbicid, and polycondensation in the presence of formaldehyde, Veracur, methylene glycol, formalin 40, BFV, fannoform, formalith, FYDE, HOCH, karsan, lysoform, superlysoform, which are intermediates in the synthesis of higher molecular methan 21. In pure form, formaldehyde is a gas but is often mass plastics. Examples and preferred embodiments of Such used in liquid form after diluting with water as the hydrate resins are disclosed in greater detail hereinafter. HO-(CH2O), H known as methandiol. Aqueous solutions 0057. As used herein, the term “enzyme preparation' is of formaldehyde are referred to as formalin. It is a colorless intended to cover any preparation of enzyme (howsoever highly flammable liquid or gas with a pungent odor that is obtained) at any level of purity (including using the host detectable at 1 part per million (ppm). Formaldehyde mix expressing the enzyme, i.e. E. coli), as long as the preparation tures (e.g. mixtures with water, acetone, benzene, diethyl is enzymatically active. The enzyme preparations of the ether, chloroform and ethanol) are included for the purposes invention include preparations exhibiting a plurality of dif of the present invention as well. Polymers of formaldehyde ferent specific activities, and are conveniently used in the encompassed in the present definition include low and high form of more or less crude enzyme extracts in admixture with molecular mass polymers, in particular paraformaldehyde, as one or more carriers. well as linear and cyclic polyoxymethylenes 0.058 As used herein, the term “cross-linking agent” or 0053. The term “acetaldehyde' or 'AA' refers to a com “crosslinker” means a FA-containing resin, as defined Supra, pound of the general formula CHCHO having the CAS which can be used to cross-link cellulose molecules in textile number 75-07-0. It is also known to the skilled artisan as fabrics for imparting wrinkle resistance and durable press ethanal and is a colorless liquid with a pungent, Suffocating properties, particularly to cellulosic textiles. Examples and odor that is slightly fruity when diluted. Acetaldehyde is an preferred embodiments of Such cross-linking agents are dis intermediate in the metabolism of plant and animal organ closed in greater detail hereinafter. isms, in which it can be detected in Small amounts. Larger 0059. The term “textile fabric' or “fabric' as used herein amounts of acetaldehyde interfere with biological processes. means products and objects made from natural textile fabrics As an intermediate in alcoholic fermentation processes it is Such as jute, sisal, ramie, hemp, and cotton as well as many of present in Small amounts in all alcoholic beverages, such as the synthetic fibers. Such as Viscose, rayon, cellulose esters, beer, wine, and spirits. Acetaldehyde also has been detected in vinyl resin fibers, polyacrylonitrile and copolymers thereof, plant juices and essential oils, roasted coffee, and tobacco polymers and copolymers of olefins such as ethylene, poly US 2012/0028333 A1 Feb. 2, 2012 imide or nylon types, polyester and the like. The fabrics used ponents in reference sequence segment, i.e., the entire refer can be those of a single composition or a mixture of fibers. ence sequence or a smaller defined part of the reference 0060. The term “polymeric dispersants’ refers to poly sequence. “Percent identity” is the identity fraction times electrolytes that are readily soluble in water. The most com 100. Optimal alignment of sequences for aligning a compari mon representatives are alkali-metal polycarbonates, son window are well known to those skilled in the art and may polysulfonates, or polyphosphates, usually sodium salts. Pre be conducted by tools such as the local homology algorithm ferred dispersants are produced by condensation of aromatic of Smith and Waterman, the homology alignment algorithm compounds with formaldehyde. The use of condensation of Needleman and Wunsch, the search for similarity method products of aromatic sulfonic acid with formaldehyde is very of Pearson and Lipman, and preferably by computerized widespread. Typically those are either anionic formaldehyde implementations of these algorithms such as GAP, BESTFIT, resins based on naphthalinsulfonate, melamine Sulfonate or FASTA, and TFASTA available as part of the GCG(R) Wis phenol or derivatives thereof. Other polymeric dispersants consin PackageR (Accelrys Inc. Burlington, Mass.). include graft polymers based on anionic backbones and non 0063. The term “ppm (parts per million) refers to mass ionic side chains. As such typically polycarboxylates are used shares and is equivalent to 'mg/kg. as backbones and polyalkylenglykols are used as sidechains. Said polymeric dispersants allow reducing the water content Polypeptide(s)/Protein(s) in hydraulic binder mixtures as cement and calcium sulfate based systems, without a reduction in workability, rheologi 0064. The terms “polypeptide' and “protein’ are used cal properties respectively. They further can be used to interchangeably herein and refer to amino acids in a poly improve the workability of hydraulic binders or to increase meric form of any length, linked together by peptide bonds. the strength development. Polymeric dispersants are also known in the art as “superplasticizers”. Such polymeric dis Polynucleotide(s)/Nucleic Acid(s)/Nucleic Acid Sequence persants are also used in textile dying liquors in order to (s)/Nucleotide Sequence(s) stabilize dye dispersions. 0065. The terms “polynucleotide(s)”, “nucleic acid 0061 The term “variant' with respect to a sequence (e.g., sequence(s)”, “nucleotide sequence(s)”, “nucleic acid(s)'. a polypeptide or nucleic acid sequence Such as for “nucleic acid molecule' are used interchangeably herein and example—a transcription regulating nucleotide sequence of refer to nucleotides, either ribonucleotides or deoxyribo the invention) is intended to mean Substantially similar nucleotides or a combination of both, in a polymeric sequences. For nucleotide sequences comprising an open unbranched form of any length. reading frame, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical Homologue(s) amino acid sequence of the native protein. Naturally occur ring allelic variants such as these can be identified with the 0.066 “Homologues' of a protein encompass peptides, use of well-known molecular biology techniques, as, for oligopeptides, polypeptides, proteins and enzymes having example, with polymerase chain reaction (PCR) and hybrid amino acid Substitutions, deletions and/or insertions relative ization techniques. Variant nucleotide sequences also include to the unmodified protein in question and having similar synthetically derived nucleotide sequences, such as those biological and functional activity as the unmodified protein generated, for example, by using site-directed mutagenesis from which they are derived. and for open reading frames, encode the native protein, as 0067. A deletion refers to removal of one or more amino well as those that encode a polypeptide having amino acid acids from a protein. substitutions relative to the native protein. Generally, nucle 0068 An insertion refers to one or more amino acid resi otide sequence variants of the invention will have at least 30, dues being introduced into a predetermined site in a protein. 40, 50, 60, to 70%, e.g., preferably 71%, 72%, 73%, 74%, Insertions may comprise N-terminal and/or C-terminal 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., fusions as well as intra-sequence insertions of single or mul 81%-84%, at least 85%, e.g., 86%, 87%, 88%. 89%, 90%, tiple amino acids. Generally, insertions within the amino acid 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% sequence will be smaller than N- or C-terminal fusions, of the nucleotide “sequence identity” to the nucleotide sequence of order of about 1 to 10 residues. Examples of N- or C-terminal SEQ ID NO:1. By “variant” polypeptide is intended a fusion proteins or peptides include the binding domain or polypeptide derived from the protein of SEQ ID NO:2 by activation domain of a transcriptional activator as used in the deletion (so-called truncation) or addition of one or more yeast two-hybrid system, phage coat proteins, (histidine)-6- amino acids to the N-terminal and/or C-terminal end of the tag, glutathione S-transferase-tag, protein A, maltose-binding native protein; deletion or addition of one or more amino protein, dihydrofolate reductase, Tag 100 epitope, c-myc acids at one or more sites in the native protein; or Substitution epitope, FLAG(R)-epitope, lacz, CMP (calmodulin-binding of one or more amino acids at one or more sites in the native peptide), HA epitope, protein C epitope and VSV epitope. protein. Such variants may result from, for example, genetic 0069. A substitution refers to replacement of amino acids polymorphism or from human manipulation. Methods for of the protein with other amino acids having similar proper Such manipulations are generally known in the art. ties (such as similar hydrophobicity, hydrophilicity, antige 0062 “Sequence identity” refers to the extent to which nicity, propensity to form or break C-helical structures or two optimally aligned DNA or amino acid sequences are B-sheet structures). Amino acid Substitutions are typically of invariant throughout a window of alignment of components, single residues, but may be clustered depending upon func e.g., nucleotides or amino acids. An "identity fraction' for tional constraints placed upon the polypeptide and may range aligned segments of a test sequence and a reference sequence from 1 to 10 amino acids; insertions will usually be of the is the number of identical components that are shared by the order of about 1 to 10 amino acid residues. The amino acid two aligned sequences divided by the total number of com Substitutions are preferably conservative amino acid substi US 2012/0028333 A1 Feb. 2, 2012

tutions. Conservative substitution tables are well known in Zyme (as coSubstrate) in typical alcohol dehydrogenases, is the art (see for example Creighton (1984) Proteins. W.H. tightly but not covalently bound to the enzyme and acts as a Freeman and Company (Eds). cofactor. 0070 Amino acid substitutions, deletions and/or inser tions may readily be made using peptide synthetic techniques 0075 "Tightly bound' means that the cofactor is bound to well known in the art, such as Solid phase peptide synthesis the enzyme by interactions such as ionic bonds, intermolecu and the like, or by recombinant DNA manipulation. Methods lar forces, hydrogen bonds, van der Waal's forces, hydropho for the manipulation of DNA sequences to produce substitu bic interactions. Since the cofactor is being recycled during tion, insertion or deletion variants of a protein are well known the disproportionation reaction (reduction of FA to methanol in the art. For example, techniques for making Substitution and oxidation of FA to formic acid by the same enzyme), the mutations at predetermined sites in DNA are well known to process of the present invention bears the particular advan those skilled in the art and include M13 mutagenesis, T7-Gen tage of minimizing costs, because the cofactor does not have in vitro mutagenesis (USB, Cleveland, Ohio), QuickChange to be provided continuously to the reaction mixture, in order Site Directed mutagenesis (Stratagene, San Diego, Calif.), to maintain the enzyme activity. PCR-mediated site-directed mutagenesis or other site-di 0076 Particularly suitable for the use of the present inven rected mutagenesis protocols. tion are oxidoreductases that are acting on the aldehyde or oxo group of donors (E.C. class 1.2.). Preferred are oxi Derivatives doreductases with acceptors other than NAD or NADP, cyto 0071 “Derivatives” include peptides, oligopeptides, chrome, oxygen, a disulfide, an iron-sulfur protein (E.C. class polypeptides which may, compared to the amino acid 1.2.99). Preferably, the enzyme is a formaldehyde dismutase sequence of the naturally-occurring form of the protein, Such (hereinafter often abbreviated as “FDM') from a bacterial as the protein of interest, comprise Substitutions of amino strain, more preferably of the E.C classification EC 1.2.99.4. acids with non-naturally occurring amino acid residues, or or EC 1.2.1.46., which is derived from a Pseudomonasputida additions of non-naturally occurring amino acid residues. strain. Said enzyme is further described in Kato, N., et al. “Derivatives' of a protein also encompass peptides, oligopep (1983) Agric. Biol. Chem., 47(1), 39-46, Yanase, H., et al. tides, polypeptides which comprise naturally occurring (1995) Biosci. Biotechnol. Biochem. 59(2), 197-202 and altered (glycosylated, acylated, prenylated, phosphorylated, Yanase, H., et al. (2002) Biosci. Biotechnol. Biochem. 66(1), myristoylated, Sulphated etc.) or non-naturally altered amino 85-91. acid residues compared to the amino acid sequence of a 0077. In a preferred embodiment, the enzyme preparation naturally-occurring form of the polypeptide. A derivative contains an enzyme which comprises the amino acid may also comprise one or more non-amino acid Substituents or additions compared to the amino acid sequence from sequence of SEQ ID NO:2 or variants thereof. In a further which it is derived, for example a reporter molecule or other preferred embodiment, the enzyme preparation contains an ligand, covalently or non-covalently bound to the amino acid enzyme encoded by the nucleic acid of SEQ ID NO:1, or sequence, such as a reporter molecule which is bound to variants thereof. facilitate its detection, and non-naturally occurring amino 0078. In a particularly preferred embodiment, the enzyme acid residues relative to the amino acid sequence of a natu preparation contains an enzyme which comprises a variant or rally-occurring protein. Furthermore, "derivatives” also derivative of the amino acid sequence of SEQ ID NO:2, as include fusions of the naturally-occurring form of the protein described in more detail hereinafter, wherein the phenylala with tagging peptides such as FLAG, HIS6 or thioredoxin (for nine at position 93 and/or the isoleucine at position 301, a review of tagging peptides, see Terpe, Appl. Microbiol. and/or the methionine at position 337 and/or the phenylala Biotechnol. 60, 523-533, 2003). nine at position 127 have been substituted by any other amino acid. DETAILED DESCRIPTION OF THE INVENTION 007.9 For the use of the present invention, the enzyme preparation can be used either purified having various levels 0072 The present invention relates to the use of an of purity or as unpurified extract, for example as bacterial enzyme preparation which catalyzes the degradation of form extract, i.e. extract from the bacteria which naturally produce aldehyde for reducing the formaldehyde content in a formal the desired enzyme or bacteria which are used as expression dehyde-containing formulation. hosts. Alternatively, it is also possible to use growing cells 0073 Enzymes that catalyze the degradation of formalde which comprise FDM encoding nucleic acids, nucleic acid hyde are known in the art. For example, Bystrykh et al. (1993) constructs or vectors bearing said nucleic acids, without J. Gen. Microbiol. 139, 1979-1985; Sakai, Y. et al (1995) including any protein purification step. It is also possible to FEMS Microbiol. Lett. 127, 229-234; or Ito et al. (1994) J. use quiescent or disrupted cells. Disrupted cells are under Bacteriol. 176,2483-2491 or Gonzalez et al., J. Biol. Chem. stood to mean, for example, cells which have been made Vol. 281, NO. 20, pp. 14514-14522, May 19, 2006 describe permeable by means of a treatment with, for example, Sol enzymes like S-formylglutathione hydrolase, formaldehyde vents, or cells which have been disrupted by means of an dismutase, methylformate synthase, or glutathione-indepen enzyme treatment, by means of a mechanical treatment (for dent formaldehyde dehydrogenase, which can be employed example French Press or ultrasound) or by means of another for degradation of FA. method. The crude extracts thus obtained are suitable in an 0.074 Enzymes belonging to the zinc-containing medium advantageous manner for the use according to the invention. chain alcohol dehydrogenase family are particularly Suitable It is also possible to use purified or partly purified enzymes for for the present invention (see also Tanaka et al., J. Mol. Biol. the process. Immobilized microorganisms or enzymes, which (2002) 324,519-533). In a preferred embodiment, the pyri can advantageously find use in the reaction, are likewise dine nucleotide NAD(H), which is distinct from the co-en suitable. When free organisms or enzymes are used for the US 2012/0028333 A1 Feb. 2, 2012 process according to the invention, they are appropriately a preservative in hundreds of products, for example, Soaps, removed before the extraction, for example by means of a deodorants, shampoos, and nail-hardening preparations. filtration or centrifugation. Formaldehyde solutions are also known in the art to be used 0080 Depending on the FA-containing formulation to be as a preservative for tanning liquors, dispersions, crop pro contacted, the enzyme preparation may be employed in a free tection agents, and wood preservatives. Furthermore, form (soluble or solid) or immobilized form. An immobilized aldehyde is required in the Sugar industry to prevent bacterial enzyme means an enzyme which is fixed to an inert Support. growth during syrup recovery. Suitable support materials and the enzymes immobilized I0083. Accordingly, in a preferred embodiment, the form thereon are disclosed in EP-A-1149849, EP-A-1 069183 and aldehyde-containing formulation to be contacted with an DE-A 100193773 and in the references cited therein. On this enzyme preparation which catalyzes the degradation of form matter, reference is made to the disclosure of these publica aldehyde is a resin. A general definition of a resin, in particu tions in their entirety. Examples of Suitable Support materials lar synthetic resins, is given Supra. are clays, clay minerals such as kaolinite, diatomaceous earth, I0084 Preferably, the present invention can be applied to perlite, silicon dioxide, aluminum oxide, Sodium carbonate, FA resins which are obtained by addition polymerization and calcium carbonate, cellulose powder, anion exchanger mate polycondensation. Examples include furan resins, ketone and rials, synthetic polymers such as polystyrene, acrylic resins, aldehyde resins, such as acetophenone formaldehyde resins phenol-formaldehyde resins, polyurethanes and polyolefins or acetone formaldehyde resins, phenol resins such as novol Such as polyethylene and polypropylene. The Support mate acs and resols, epoxy resins such as liquid epoxy resins rials are usually employed in a finely divided, particulate form (DGEBA), solid epoxy resins based on DGEBA, halogenated for preparing the Supported enzymes, with preference being epoxy resins epoxy novolac resins, Sulfonamide resins or given to porous forms. The particle size of the Support mate aniline resins. rial is usually no more than 5 mm, in particular no more than I0085 For example, phenol resins which are widely 2 mm (sieve grade). Analogously, when using the FDM as employed for gluing woods are condensates of different phe whole cell catalyst, a free or immobilized form may be cho nolic compounds and aldehydes. The phenolic compound can sen. Examples of Support materials are calcium alginate and be phenol itself, polyhydric phenols, and aliphatically or aro carrageenan. Enzymes as well as cells may also be linked matically Substituted phenols. Examples of phenolic com directly with glutaraldehyde (crosslinking to give CLEAS). pounds are alkyl phenols such as resorcinol, alkyl resorcinol, Corresponding and other immobilization processes are cresols, ethyl phenol and Xylenol, and also phenolic com described, for example, in J. Lalonde and A. Margolin pounds of natural origin such as tannins, cardenol, and cardol. “Immobilization of Enzymes' in K. Drauz and H. Waldmann, Formaldehyde based phenol resins in the phenol resin com Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991 position include resorcinol-formaldehyde, phenol-resorci 1032, Wiley-VCH. Weinheim. nol-formaldehyde, and tannin-formaldehyde resins. 0081. The amount of enzyme to be used depends on the I0086. In a particularly preferred embodiment, the FA resin purity level of the enzyme preparation. Typical amounts for is an amino resin, Such as urea resin, urethane resin, melamine the process of the present invention ranges between 0.1 to resin, cyanamide and dicyanodiamide resin 1000 units per gram of treated FA-containing formulation, I0087 Amino resins are commonly used as adhesives: preferably between 1 to 500 units, more preferably between 5 impregnating resins; molding materials; starting materials for to 100 units, even more preferably between 8 to 30 units, most making Surface coatings; auxiliaries for paper, textiles, preferably between 9 to 15 Units per gram of treated FA leather, and flotation; strengtheners for building materials; containing formulation. One Unit is defined as the amount of Superplasticizers; binders for glass fibers and foundry sand enzyme necessary to catalyze the formation of 1 Jumol formic casting; fire lighters; emery papers; flame retardant coatings; acid per minute. Purified FDM has a specific activity of about flameproofed combustible items; foamed resins for many 100-200 U/mg. Enzyme amounts are merely approximate purposes; grinding wheels; ion-exchange resins; sewage floc values that may vary depending on the reaction conditions culants; and microcapsule production. like temperature and incubation period. The optimal enzyme I0088 As used herein, the phrase “adhesive” means a glue amount can easily be determined by performing routine to hold materials together, a laminating resin and a matrix experiments. resin to hold materials together. Glues and impregnating res 0082. The present invention can be applied to all formu ins are aqueous adhesives made from urea, melamine and/or lations which have been furnished with formaldehyde. The phenol with formaldehyde. Glues are known in the art for the largest group of formaldehyde-containing formulations is the manufacturing of wood based panels like e.g. particle board, group of resins containing urea, melamine, naphthaline and medium density fibre-board MDF, oriented strand board phenol and their derivatives like DMDHEU. Spray drying OSB, plywood, core board or sheeting. Impregnating resins additives and rheology modifiers used in the production of are used for impregnating papers, which are used for the dispersions like polycondensates of FA with phenol sulfonic decorative coating of wood based panels, e.g. on the Surface acid ornaphthalene sulfonic acid. Otherformulations include of furniture and laminate flooring. Impregnating resins are binders for foundry sand, rockwool and glasswool mats in used as resin glues in the particle board, plywood, fiberboard, insulating materials, abrasive paper, and brake linings or and furniture industries. Impregnating resins also are used to urea-formaldehyde condensates that are used in the manufac impregnate papers for decorative laminates and for coating ture of foamed resins. Other formulations containing formal wood particle board. dehyde include pentaerythritol (employed chiefly in raw I0089 Textile materials are presently generally rendered materials for Surface coatings and in permissible explosives) wrinkle resistant or non-iron in a process known as pad-dry and hexamethylenetetramine used as a cross-linking agent for cure process, generally to crosslink the cellulose molecules. phenol-formaldehyde condensates and permissible explo Such cross-linking of the cellulose imparts to the fabric a sives. In the cosmetics industry, formaldehyde is employed as tendency to return to its original shape and Smoothness. Some US 2012/0028333 A1 Feb. 2, 2012

classes of DMDHEU resins, as described supra, have been are usually modified by reaction with other compounds. extensively used in the past as the cross-linking agents in Such Especially useful are condensation products based on a process. melamine, formaldehyde and sulfite (see for example EP 0 0090 Accordingly, in another embodiment of the present 336 165). invention, the crosslinker to be contacted with an enzyme 0098. These polymers may be conventionally prepared as preparation which catalyzes the degradation of formaldehyde dispersions by emulsion polymerization, initiated by poly is a cross-linking agent Suitable for finishing textile fabrics. meric dispersants, as defined in the definition section, in the 0091 Cross-linking agents used in the textile-finishing presence of radical initiators, emulsifiers and/or protective industry have been widely described in the art (See, for colloids, where called for regulators and other additives. example, Ullmann IVth Edition Vol 23). Cross-linking agents 0099. Accordingly, in a further preferred embodiment of which are known to the person skilled in the art include the present invention, the resin is a FA-containing polymeric 'self-crosslinking’ (having a reactive hydrogen atom on the dispersant or FA-condensate which is used to prepare poly nitrogenatom) and “reactant crosslinking agents (nitrogenis mer dispersions. part of a heterocyclic ring). 0100. The term “polymer dispersion” refers to raw mate 0092 Multifunctional methylol derivatives of urea, sub rials for the construction chemicals sector. Products include stituted ureas, or melamine produced by reacting formalde acrylic-dispersions as well as acrylic-powders and styrene? hyde for with these compounds are preferred cross-linking butadiene-dispersions. They enhance workability and techni agents for commercial easy care finishes. An important group cal performance of construction chemicals, building adhe is constituted by hydroxymethyl compounds of cyclic urea sives and Sealants. High-class polymer dispersions and derivatives; examples are dihydroxymethylethylene urea, additives are used as architectural coatings raw materials in dihydroxymethylpropylene urea, and dihydroxymethylu combination with other components to produce ready-to-use rone. Acyclic compounds, such as various alkyl carbamates, products such as paints, wood stains or texture finishes. In are also typical finishing agents. In contrast to the pure urea particular, the term is referred to dispersions containing meth formaldehyde compounds, these exhibit little tendency to acrylic acid (MAS), methylolmethacrylamide (MAMol) and/ form self-cross-linked resins and react predominantly with or methylolacrylamide (AMol), homo- and copolymers of cellulose to cross-link the fibers. acrylamide, acrylic acid, acrylonitrile, acrylic acid esters and 0093. The methylol derivatives of urea itselfare used par styrene also used as binders in textile pigment printing and/or ticularly on rayon fabrics. Examples include dimethylol urea, coating. Further examples and preferred embodiments of N,N'-bis(hydroxymethyl)urea, dimethyl ether of trimethy such polymer dispersions are disclosed hereinafter. lolmelamine, urons, i.e., tetrahydro-3,5-bis(hydroxymethyl)- 0101 The term “pigment printing refers to processes for 4H-1,3,5-oxadiaZin-4-one, cyclic urea products, methylol the coloristic patterning of sheetlike textile material which are derivatives of carbamates, especially methyl carbamate, and common knowledge in the art and which have long been methoxyethyl carbamate, practiced all over the world. In pigment printing, the particu 0094 Preferably, methylol derivatives of dihydroxyethyl lar pigments are usually applied to the textile web from aque ene urea, produced by reacting glyoxal with urea, are used as ous print pastes together with a binder system and then dried. cross-linkers in easy care finishes, e.g. dimethylol dihydroxy A Subsequent dry heat treatment for curing the preferably ethyleneureas (DMDHEU), described for example in WO98/ synthetic resin binder system and hence fixing the applied 029393, 1,3-dimethoxymethyl DHEU and the fully methy colorant concludes the printing process. lated product. All of the various modifications of the glyoxal 0102. Furthermore, for use as building materials, the con ureatype products are in commercial use. The products which densation products, such as Sulfite-modified melamine resins, have been both methylated and contain a hydroxy compound naphthaline sulfonates are combined with water-soluble afford the lowest formaldehyde evolution potential of all vinyl- or acryl-based polymers. Examples of appropriate commercially available easy care finishes of the methylol polymers are products of vinylacetate, vinylpropionate, type. A variety of crosslinkers is described in Ullmann IV.th vinyllaurate, vinylchloride, vinylidenechloride, straight Edition Vol 23 Chap 7. chain or branched vinylesters with 3 to 18 C atoms, poly(vinyl 0095 Since the described cross-linking agents are pre alcohol), poly(vinyl Sulfates), acryl- and methacryl-mono dominantly used to cross-link cellulose molecules, the textile mers, in particular esters, also styrene and ethane, maleic fabrics to be treated preferably contain cellulose or cellulosic acid-styrene copolymers which may be present in the form of fibers. their homo-, co-, ter-polymers and as graft polymers. 0096. The present invention is suited to treating fibrous 0103) The preferred fields of application of such polymer cellulosic material containing at least 10%, preferably at least dispersions are paints, decorative and protective coatings and 20%, at least 30%, at least 40%, at least 50%, at least 60%, at lacquers, building chemicals, as additives in cement mortars least 70%, at least 80%, at least 90%, most preferably up to and filling compounds, auxiliary materials for the manufac 100% of cellulosic fibers. Examples include jute, linen, flax, ture of paper and paper coatings, textile coating, and colorants hemp, viscose, regenerated cellulose Such as rayon, and, pref for plastics. erably cotton. The cellulosic material may be woven, non 0104 Furthermore, the inventors of the present invention woven or knit or in the form of fibers, linters, rovings, slivers, have established different methods to apply the enzyme scrims or papers. The fibrous cellulosic material may consist preparation that catalyzes the degradation of FA depending entirely of cotton or cottonblended with a synthetic fiber such on the application. as polyester or nylon. 0105. Accordingly, another object of the present invention 0097. As described supra, resins, particularly melamine relates to a process for reducing the formaldehyde content in resins, or any other amino-s-triazine Such as guanamines, are a formaldehyde-containing formulation comprising contact widely used as building materials or Superplasticizers (also ing the formulation with an enzyme preparation that catalyzes known as concrete liquefiers). For these purposes, said resins the degradation of formaldehyde. US 2012/0028333 A1 Feb. 2, 2012

0106 The “contacting may occur prior or during the acid. Formaldehyde in air can be determined down to con intended use of the formulation. Contacting can mean adding centrations in the ul/m range with the aid of gas sampling to and/or mixing if the FA containing formulation is liquid or apparatus. The quantitative determination of formaldehyde in applying to a Surface, if the formulation is a rather viscous or air by the sulfite/pararosaniline method is described in Verein Solid material. Deutscher Ingenieure (VDI): 1, Messen gasförmiger Immis 0107. In one example, the enzyme preparation may be Sionen, Bestimmender Formaldehydkonzentration nach dem added directly to aurea-formaldehyde resin used in the manu Sulfit-Pararosanilin-Verfahren, Richtlinie VDI 3484, Blatt 1, facture of particleboard or diluted with water and sprayed on Düsseldorf 1979. the surface of the board before it is pressed. The enzyme 0115 There are a number of further test methods that are applied or added depends on the nature of the resin added to used for the formaldehyde release analysis on textiles, such as the particleboard and the curing conditions. However, the Japanese Law 112 (i.e. acetylacetone method), AATCC-1 12 correct amount for any particular case may be determined by (i.e. chromotropic acid method), the Shirley I and II Methods testing various amounts of enzyme and evaluating the amount and others. Preferred methods for the detection of FA in of formaldehyde released by the board. textiles, are the LAW112 and the AATCC112 methods, used 0108. The inventors have also found that the formalde by the “European Committee for Standardization' in EN ISO hyde-content can be reduced in textile fabrics when the 14184 part 1 and part 2. With the process of the present enzyme preparation which catalyzes the degradation of form invention it is possible to reduce the residual formaldehyde aldehyde is directly applied to the textile itself. content to less than 250, preferably less than 100, more pref 0109 Accordingly, another object of the present invention erably less that 50, even more preferably less than 20, and refers to a process for reducing the formaldehyde content in a most preferably less than 10 ppm. textile fabric, comprising contacting the textile fabric with an 0116. The temperature range needed to carry out the pro enzyme preparation which catalyzes the degradation of form cess of the present invention can vary between 10°C. to 100° aldehyde. C., preferably 20° C. to 40°C., more preferably 25°C. to 35° 0110 Preferably, the textile fabrics are “crosslinked fab C., most preferred at 30°C. The process of the present inven rics” which have been imparted with crease and wrinkle tion is usually carried out under pH conditions varying resisting properties under both wet and dry conditions by between 3 and 12, preferably between 5 and 9, more prefer heating, drying, and curing with a finishing agent such as ably between 7 and 8. The optimal pH value can be deter glyoxal resin, formalin, ureaformaldehyde resin, dimethy mined and adjusted by means well-known to the person lolurea, dimethyl ether of ureaformaldehyde, melamine skilled in the art. formaldehyde resins, cyclic ethylene urea formaldehyde res 0117 The incubation period can vary, depending on the ins, e.g. dimethylol urea, triazine-formaldehyde resins, tria chosen amount of enzyme, the reaction temperature and the Zone formaldehyde resins and the like as described Supra. FA content in the formulation, as well as depending on the 0111 Briefly, textile fabrics treated with a cross-linker as nature of the formulation itself. Typically, the incubation defined Supra are impregnated with the enzyme preparation period is in a range of minutes or hours, preferably 5 minutes as described in the example section. The detection of residual to 10 hours, more preferably, 20 minto 5 hours, more pref FA after incubation with the enzyme preparation can be car erably 30 min to 2 hours. The optimal incubation time can ried out with wet or dried textile fabrics. Preferably, it is easily be determined and adjusted by the person skilled in the performed with wet textile fabrics. art depending on the product being treated. 0112 The FA-containing formulations referred to in the 0118. The process of the present invention can be carried present invention exhibit a concentration of formaldehyde of out batch-wise, semi-continuously or continuously in con about 1 to 50.000 ppm. Typical amounts are 10 to 5.000 ppm. ventional bioreactors. Suitable regimes and bioreactors are The process of the present invention allows for a significant familiar to the skilled worker and are described, for example, reduction of the formaldehyde content. in Römpp Chemie Lexikon 9th edition. Thieme Verlag, entry 0113 Suitably, the FA contentis reduced by 10, 20,30,40, header “Bioreactors' or Ullmann's Encyclopedia of Indus 50, 60, to 70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, trial Chemistry, 5th edition, volume B4, page 381 ff, herein 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%- incorporated by reference. The operation of the reactor and 84%, at least 85%, e.g., 86%, 87%, 88%. 89%, 90%, 91%, the process regime can be adapted to the skilled artisan to the 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99%, and particular requirements of the desired FA degradation reac 100% as compared to formulations, end products which were tion. If the process is performed in batch mode the enzyme not contacted with the enzyme preparation. preparation is added directly after synthesis, polymerization, 0114 Methods to determine the residual FA content are isolation, and, optionally, purification of the formulation. well-known in the prior art. Formaldehyde can be quantita After Successful formaldehyde degradation using FA dismu tively determined by either physical or chemical methods. tase the enzyme can be separated from the formulation. Alter Quantitative determination of pure aqueous Solutions of natively, the enzyme can be left in the formulation and inac formaldehyde can be carried out rapidly by measuring their tivated, e.g. by heat or acidification, if desired and provided specific gravity. Gas chromatography and high-pressure liq such inactivation is not deleterious to the formulation. If the uid chromatography (HPLC) can also be used for direct deter process is performed continuously the enzyme preparation is mination. The most important chemical methods for deter preferably immobilized on a carrier which can be packed, mining formaldehyde are summarized in H. Petersen, N. e.g., into a specific column. Typically, the formulation is the Petri, Melliand Textilber. 66 (1985) 217-222, 285-295,363 pumped over the column under suitable conditions. To further 369. The sodium sulfite method is most commonly used. It is enhance the FA reducing effect, it is possible to serially con based on the quantitative liberation of base produced when nect several enzyme reactors one after the other. formaldehyde reacts with excess sodium sulfite. The stoichio 0119 For the purposes of the present invention, the metrically formed base is determined by titration with an enzyme preparation can be added in any form, formulation US 2012/0028333 A1 Feb. 2, 2012 and composition known to the person skilled in the art. Those pounds such as polyamid oligomers or polymeric composi skilled in the art will readily appreciate that the enzyme tions such as lignin, water-soluble vinyl copolymers. Further preparation suitable for the present invention will be depen more, antioxidizing agents such as dithiothreitol (DTT) are dent on several factors, including but not limited to the precise typically used enzyme stabilizers. composition of the FA-containing formulation. They will 0.125 If the enzyme preparation that catalyzes the degra further appreciate that there are several methods for formu dation of formaldehyde shall be directly incorporated into the lating enzyme preparations. The enzyme preparation may be FA containing formulation, it is required in some embodi formulated employing standard approaches of enzyme gran ments that the enzyme is present in dried form. If the enzyme ule and/or liquid formulations. A description of the steps preparation that catalyzes the degradation of formaldehyde is associated with enzyme granule and/or liquid formulation of added only during the application of the FA containing for enzymes is found in “Industrial Enzymes and their Applica mulation, the enzyme preparation can be present in liquid, tion', by Helmut Uhlig, John Wiley and Sons, 1998 gelatinous, or paste-like form. Subsequent to protein purifi 0120 Suitable enzyme preparations are, for example, cation and isolation methods well-known in the art the Solid enzyme preparations obtainable by granulation, extru enzyme preparation can be added as concentrated aqueous Sion, spray drying, or lyophilization of enzyme solutions, as Solution, Suspension or emulsion. Examples of solvents well as preferably concentrated solutions of the enzyme, which can be used to obtain Suitable enzyme preparations optionally containing stabilizers. Alternatively, the enzyme include alcohols, alkanolamines or glycol or glycol ethers, preparation in Solid or liquid form can be adsorbed on a solid glycerol, Sorbitol, glucose, saccharine. For the purposes of carrier and/or being encapsulated. Methods that have been increasing the viscosity, the enzyme preparation can contain proposed for preparation of immobilized enzymes include the one or more thickener, also known as Swelling agents. Suit Substrate-binding method, the cross-linking polymerization able thickeners include, for example, alginates, pectins, method, the gel-inclusion method, and the like. starch, dextrine or synthetic thickener poly carbonic acids, 0121. In some embodiments, when the enzyme prepara polyethylene glycol, polyacryl compositions, polyamides, or tion used for the present invention is employed in a granular polyethers. composition or liquid, it is desirable for the enzyme prepara 0.126 For some formulations, it is desired that the enzyme tion to be in the form of an encapsulated particle to protect preparation is already contained within the formulation keep Such enzyme from other components of the granular compo ing the FA content in these formulations as low as possible. sition during storage. In addition, encapsulation is also a I0127. In a preferred embodiment, the enzyme preparation means of controlling the availability of the enzyme prepara of the present invention comprises (a) 0.1-10% of an FA tion during the FA degrading process and may enhance per degrading enzyme, preferably FDM, and (b) 1-80% of one or formance of the enzyme preparation. It is contemplated that several polyols (glycol, glycerol, Sorbitol, glucose, saccha any suitable encapsulating material will find use in the present rose, polyethylene glycol etc.) and (c) 1-99% water. invention. The encapsulating material typically encapsulates I0128. In another embodiment of the invention, a dry at least part of the enzyme preparation. Typically, the encap (solid) enzyme preparation is added to a solid product formu Sulating material is water-soluble and/or water-dispersible. lation. The FA reducing activity is started upon addition of Briefly, the enzyme preparation is mixed with compounds water to the Solid preparation. Such as Sodium alginate, agarose or Sephadex and Subse I0129. Therefore, another object of the present invention quently precipitated according to methods known in the art. refers to a formulation suitable for textile-finishing, the for Alternatively, the encapsulation can be carried out by spray mulation comprising a cross-linking agent and an enzyme drying or extrusion of the enzyme preparation. preparation which catalyzes the degradation of formalde 0122 Examples of protective coating materials for said hyde. enzyme granules include natural materials such as saccha 0.130. The enzyme preparation may be used in a manner rides, polysaccharides, polypeptides such as collagen, albu analogous to known formaldehyde-reducing agents. For min or gelatine, oils, fatty acids, waxes. Also included as example, the enzyme preparation may be incorporated into a coating materials are semi-synthetic materials such as chemi durable press finishing cross-linking agent comprising an cally modified cellulosic compounds, starch derivatives, or N-methyol cross-linking system, such as DMDHEU. A fabric synthetic coating materials such as polyacrylates, polya composed either entirely or in part of cellulose fibers may be mides. The coating can further comprise polyelectrolyte com padded, foam finished or otherwise impregnated with the plexes that are generated by the interaction of polycations and durable press finishing composition. polyanions. Typical polycations include natural compounds I0131 Preferably, the cross-linking agent is selected from Such as cytosan as well as synthetic polymers. the group consisting of melamine-FA, urea-FA or urea-gly 0123. The enzyme preparation can be granulated together oxal-FA compounds. with a chemically inert carrier material or binding material. I0132) Another object of the present invention refers a for Carrier materials include silicates, carbonates or Sulfates. mulation Suitable for treating construction materials, particu Binding materials are for example non-crosslinked polymer larly hydraulic binders such as cement, gypsum, mortar, or compositions such as polyacrylate, polymethacrylate, poly lean lime, fiberboard, particle board, plywood, wood, leather vinylpyrrolidone, polysaccharides. and/or carpeting, the formulation comprising a polymeric 0.124. Alternatively, in order to protect the enzyme prepa dispersant as described Supra and an enzyme preparation ration against inactivation or denaturation, the addition of which catalyzes the degradation of formaldehyde. stabilizing compounds may be suitable. Examples for stabi I0133. In a preferred embodiment, the polymer dispersant lizing compounds include protease inhibitors, such as is selected from the group consisting of naphthalene formal boronic acid as well as derivatives thereof, amino alcohols, dehyde condensates, phenol formaldehyde condensates, urea and low aliphatic alcohols. The enzyme preparations can be formaldehyde condensates, and melamine formaldehyde protected against physical effects or pH variations with com condensates. US 2012/0028333 A1 Feb. 2, 2012

0134. The cross-linking agent or the polymer dispersant duction, as used in the present context, are intended to com can be provided already mixed with the enzyme preparation prise a multiplicity of prior-art processes for introducing for or can be provided as kit, where the enzyme preparation is eign nucleic acid (for example DNA) into a host cell, added to the cross-linking agent or the polymer dispersion including calcium phosphate, rubidium chloride or calcium prior to its intended uses described Supra, in order to reduce chloride co-precipitation, DEAE-dextran-mediated transfec the FA content of the end product. In some cases incubation tion, lipofection, natural competence, carbon-based clusters, time must be optimized to avoid excessive degradation of the chemically mediated transfer, electroporation or particle end product. bombardment (e.g., "gene-gun'). 0135 For the purposes of the present invention, it is desir 0143. In a preferred embodiment, the vector suitable for able to have the enzyme easily available in large amounts. the present invention is a nucleic acid comprising the Suitably, the enzyme is expressed in bacteria which allows for sequence of SEQID NO:4, 5, and or 6. the large scale production of heterologously expressed pro 0144. Another aspect of the present invention refers to the teins. In order to optimize the expression, i.e. increase the use of an enzyme preparation which catalyzes the degrada yield of the expressed enzyme, the inventors of the present tion of acetdehyde (AA) for reducing the acetdehyde content invention have constructed a nucleic acid coding for an in an acetaldehyde-containing formulation. enzyme that catalyzes the degradation of formaldehyde. 0145 As described in the definition section, AA is present 0136. Accordingly, another object of the present invention in a variety of liquids and compounds, and is harmful to man. relates to an isolated nucleic acid coding for an enzyme that Furthermore, acetaldyde is known to be a residual compound catalyzes the degradation of formaldehyde, wherein the in polyvinylalcohol. Polyvinyl alcohols are polymers of vinyl sequence of the nucleic acid is codon-optimized for expres alcohol. As the latter cannot exist in free form, all polyvinyl sion in an expression host. alcohols have so far been manufactured by polymerization of 0.137 Preferably, the expression host is Escherichia coli. vinyl acetate which, unlike vinyl alcohol, is stable. The poly 0138 Although Escherichia coli is one example of a bac vinyl acetate produced then undergoes alcoholysis. As the terial host cell used commonly to express an enzyme that technical properties of polyvinyl alcohol depend in the first catalyzes the degradation of formaldehyde, other bacterial place on the molar mass and residual acetyl group content, host cells can be used in the present invention to express industrial manufacturing processes are designed to ensure foreign DNA, including for example, Escherichia, Entero exact adherence to these parameters. bacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Borde 0146 In a particularly preferred embodiment, the enzyme tella, Rhodobacter, Xvella, Klebsielia, Proteus, Salmonella, preparation contains an enzyme having aldehyde dismutase Serratia, Shigella, Rhizobium, Vitreoscilla, and Paracoccus, activity which comprises a variant or derivative of the amino as well as fungal host cells, including for example, Aspergil acid sequence of SEQID NO:2, as described in more detail lus, Pichia, Trichoderma, Hansenula, Saccharomyces, hereinafter, wherein the phenylalanine at position 93 and/or Kluyveromyces, Schizosaccharomyces, Chrysosporium, the isoleucine at position 301, and/or the methionine at posi Candida and Torulopsis. tion 337 and/or the phenylalanine at position 127 is substi 0.139. The characteristics and advantages of codon-opti tuted by any other amino acid. mization for expression in E. coli are well-described in Bur 0147 As further illustrated by the attached figures, the gess-Brown et al., Protein Expr Purif., 2008 May: 59(1):94 analysis of the crystal structure of the formaldehyde dismu 102. tase enzyme of Pseudomonasputida (SEQID NO:2) allowed 0140. In a preferred embodiment, the sequence of the the identification of individual amino acid residues or amino nucleic acid comprises the sequence of SEQ ID NO:3 or a acid sequence portions involved in the formation of the reac variant thereof. tivity centre of the enzyme, so that a model system or refer 0141 Another object of the present invention refers to an ence enzyme for suitable further enzymes with aldehyde, in expression vector containing the nucleic acid of the present particular formaldehyde or acetaldehyde dismutase activity invention. could be established. 0142 Suitable vectors encompass phage, plasmid, viral or 0.148. In particular, for said specific reference enzyme cer retroviral vectors as well as artificial chromosomes, such as tain key amino acid residues could be identified, which are bacterial or yeast artificial chromosomes. Moreover, the term predicted to be involved in the formation of functionally also relates to targeting constructs which allow for random or distinct portions of the substrate pocket. Said functionally site-directed integration of the targeting construct into distinct portions are designated catalytic site 1 (CS1), cata genomic DNA. Such target constructs, preferably, comprise lytic site 2 (CS2), catalytic site 3 (CS3), catalytic site 4 (CS4). DNA of sufficient length for either homolgous or heterolo 0149. A first functional portion is CS1 and the key amino gous recombination as described in detail below. The vector acid residue is Ile 301. encompassing the polynucleotides of the present invention, 0150 Moreover, it was found that sequence portions not preferably, further comprises selectable markers for propaga adjacent to each other in the primary amino acid sequence, are tion and/or selection in a host. The vector may be incorpo nevertheless functionally related, by contributing to the same rated into a host cell by various techniques well known in the functional portion of the binding pocket. Thus, it was art. If introduced into a host cell, the vector may reside in the observed that the functional portion CS2 comprises key cytoplasm or may be incorporated into the genome. In the amino acid residue Met337. latter case, it is to be understood that the vector may further 0151. It was also observed that said reference enzyme comprise nucleic acid sequences which allow for homolo forms the binding pocket regions CS3 and CS4, the amino gous recombination or heterologous insertion. Vectors can be acid residues associated therewith may be further subdivided introduced into prokaryotic or eukaryotic cells via conven in accordance with their preferential orientation with respect tional transformation or transfection techniques. The terms to the substrate attached to the enzyme. CS3 and CS4 com “transformation' and “transfection', conjugation and trans prise key amino acid residues Phel27 and Phe93. US 2012/0028333 A1 Feb. 2, 2012

0152 The inventors of the present invention have surpris or no influence on the geometry or mobility of the substrate ingly found that by Substituting one or more of the key amino pocket of the above-mentioned structural elements (CS1,2,3, acid residues the activity of the enzyme could be increased by or 4). at least 5%, preferably by at least 7%, more preferably by at 0160 Examples of mutants potentially having a more pro least 10%, most preferably by 10 to 20% as compared to the nounced influence on the enzymatic activity could be those activity of the wild type Pseudomonas putida enzyme with which a stronger influence on the geometry or mobility of the SEQID NO: 2. In addition, by substituting one or more of the substrate pocket or the above-mentioned structural elements key amino acid residues the thermostability of the enzyme (CS1, 2, 3, or 4). could be increased, resulting in a stability at a temperature 0.161 With respect to the Pseudomonas putida protein of which is at least 1, 2, 3, 4, 5 degree Celsius higher than the SEQ ID NO: 2 non-limiting examples of key amino acid temperature at which the wild type Pseudomonas putida Substitution which may contribute to at least one of said types enzyme with SEQID NO: 2 is stable. This renders the enzyme of mutation are listed below. of the present invention particularly advantageous with respect to purification and expression conditions 0153. Accordingly, another object of the present invention refers to an isolated polypeptide having aldehyde dismutase Structural portion Key amino acid Mutation activity and comprising a variant of SEQID NO:2 wherein CS1 Ile301 Leu, Ala, Val, Gly, Ser the phenylalanine at position 93 of SEQID NO:2, and/or the CS2 Met 337 Ala isoleucine at position 301 of SEQ ID NO:2, and/or the CS3 Phel27 Val, Ala methionine at position 337 of SEQ ID NO:2, and/or the CS4 Pe3 Leu, Ile, Gly, Ala, Val phenylalanine at position 127 of SEQID NO:2 is substituted by any other amino acid. 0162. It is to be understood by the skilled artisan that any 0154 It will be understood by the person skilled in the art amino acid besides the ones mentioned in the above table that amino acids located in a close proximity to the positions could be used as a Substitute. Assays to test for the function of amino acids mentioned above may also be substituted. ality of Such mutants are readily available in the art, and Thus, another embodiment refers to an isolated polypeptide respectively, described in the Example section of the present having aldehyde dismutase activity and comprising a variant invention. of SEQID NO:2, wherein an amino acid +3, +2 or +1 amino 0163 The present invention also relates to a nucleic acid acid positions from the phenylalanine at position 93 of SEQ as further defined hereinafter encoding a protein having alde ID NO:2, and/or an amino acid +3, +2 or +1 amino acid hyde dismutase activity as defined above. positions from the isoleucine at position 301 of SEQ ID NO:2, and/oranamino acid-t3, t2 oritlamino acid positions 0164. The present invention also relates to an expression from the methionine at position 337 of SEQID NO:2, and/or cassette, comprising a nucleic acid as defined above, operably an amino acid it3, t2 or it 1 amino acid positions from the linked with at least one regulatory nucleic acid sequence. phenylalanine at position 127 of SEQID NO:2 is substituted 0.165. The present invention also relates to a recombinant by any other amino acid. expression vector comprising at least one expression cassette 0155 Based on this analysis a highly characteristic or nucleic acid as defined above. sequence pattern could be developed, by means of which 0166 The present invention also relates to a recombinant further candidates of proteins with the desired enzymatic microorganism, carrying at least one expression vector as activity may be searched. defined above. 0156 Searching for further candidate enzymes by apply 0167. The present invention also relates to a bioreactor ing said sequence pattern would also be encompassed by the comprising at least one protein having aldehyde dismutase present invention. It will be understood by a skilled reader activity as defined above or a recombinant microorganism as that the above sequence pattern is not limited by the exact defined above, optionally in immobilized form. distances between two adjacent amino acid residues of said pattern. Each of the distances between two neighbours in the 0.168. The present invention also relates to a method of above patterns may, for example, vary independently of each preparing an enzyme with aldehyde dismutase activity, which other by up to +10, +5, +3, +2 or +1 amino acid positions method comprises cultivating a recombinant microorganism without substantially affecting the desired enzymatic activity. as defined above and optionally isolating the said aldehyde 015.7 Inline with said above functional and spatial analy dismutase from the culture. sis of individual amino acid residues based on the crystallo 0169. The present invention also relates to a crystalline graphic data as obtained according to the present invention, form of a protein having aldehyde dismutase activity, in par unique partial amino acid sequences characteristic of poten ticular those forms, wherein the protein having aldehyde dis tially useful enzymes with aldehyde dismutase activity of the mutase activity is as defined above. invention may be identified. 0170 The present invention also relates to a method of 0158. According to a further preferred embodiment the preparing a crystalline form of a protein having aldehyde dismutation reaction is performed with isolated or purified, dismutase activity as defined above, which method comprises optionally immobilized aldehyde dismutase, or by culturing a adding to a solution containing said protein (in a concentra microorganism expressing aldehyde dismutase enzyme tion of about 1 to 50 or 5 to 20 mg/ml) and a crystallization activity as described in more detail Supra. agent (3 M to 3.5 M ammonium sulfate, preferably 3.2 M 0159. Examples of mutants potentially having only a ammonium sulfate or a polyalkylene glycol, like polyethyl minor influence on the enzymatic activity are those which low ene glycol, in particular PEG 400 to 3500, like PEG 400) US 2012/0028333 A1 Feb. 2, 2012 having a pH in the range of 8.3 to 8.7, as for example 8.5, protein molecules according to the invention. Salts of car preferably buffered with Tris buffer. boxyl groups can be produced in a known way and comprise inorganic salts, for example Sodium, calcium, ammonium, Other Embodiments of the Invention iron and Zinc salts, and salts with organic bases, for example amines, such as triethanolamine, arginine, lysine, piperidine Proteins According to the Invention and the like. Salts of acid addition, for example salts with 0171 The present invention is not limited to the specifi inorganic acids, such as hydrochloric acid or Sulfuric acid and cally disclosed “proteins with aldehyde dismutase activity”. salts with organic acids, such as acetic acid and oxalic acid, but also extends to functional equivalents thereof. are also covered by the invention. 0172 “Functional equivalents’ or analogs of the con 0.178 "Functional derivatives” of polypeptides according cretely disclosed enzymes are, within the scope of the present to the invention can also be produced on functional amino invention, Various polypeptides thereof, which moreoverpos acid side groups or at their N-terminal or C-terminal end sess the desired biological function or activity, e.g. enzyme using known techniques. Such derivatives comprise for activity. example aliphatic esters of carboxylic acid groups, amides of 0173 For example, “functional equivalents’ means carboxylic acid groups, obtainable by reaction with ammonia enzymes, which, in a test used for enzymatic activity, display or with a primary or secondary amine; N-acyl derivatives of at least a 1 to 10%, or at least 20%, or at least 50%, or at least free amino groups, produced by reaction with acyl groups; or 75%, or at least 90% higher or lower activity of an enzyme, as O-acyl derivatives of free hydroxy groups, produced by reac defined herein. tion with acyl groups. 0.174 “Functional equivalents', according to the inven 0179. “Functional equivalents' naturally also comprise tion, also means in particular mutants, which, in at least one polypeptides that can be obtained from other organisms, as sequence position of the amino acid sequences stated above, well as naturally occurring variants. For example, areas of have an amino acid that is different from that concretely homologous sequence regions can be established by stated, but nevertheless possess one of the aforementioned sequence comparison, and equivalent enzymes can be deter biological activities. "Functional equivalents' thus comprise mined on the basis of the concrete parameters of the inven the mutants obtainable by one or more amino acid additions, tion. substitutions, deletions and/or inversions, where the stated 0180 "Functional equivalents' also comprise fragments, changes can occur in any sequence position, provided they preferably individual domains or sequence motifs, of the lead to a mutant with the profile of properties according to the polypeptides according to the invention, which for example invention. Functional equivalence is in particular also pro display the desired biological function. vided if the reactivity patterns coincide qualitatively between 0181 “Functional equivalents' are, moreover, fusion pro the mutant and the unchanged polypeptide, i.e. if for example teins, which have one of the polypeptide sequences stated the same substrates are converted at a different rate. Examples above or functional equivalents derived there from and at least of suitable amino acid substitutions are shown in the follow one further, functionally different, heterologous sequence in ing table: functional N-terminal or C-terminal association (i.e. without Substantial mutual functional impairment of the fusion pro tein parts). Non-limiting examples of these heterologous Original residue Examples of substitution sequences are e.g. signal peptides, histidine anchors or enzymes. Ala Ser Arg Lys 0182 “Functional equivalents’ that are also included ASn Gln: His according to the invention are homologues of the concretely Asp Glu disclosed proteins. These possess percent identity values as Cys Ser Gln ASn stated above. Said values refer to the identity with the con Glu Asp cretely disclosed amino acid sequences, and may be calcu Gly Pro lated according to the algorithm of Pearson and Lipman, His ASn; Glin Proc. Natl. Acad, Sci. (USA) 85(8), 1988,2444-2448. The % Ile Leu; Val Leu Ile: Val identity values may also be calculated from BLAST align Lys Arg: Gln: Glu ments, algorithm blastp (protein-protein BLAST) or by Met Leu: Ile applying the Clustal setting as given below. Phe Met; Leu: Tyr 0183) A percentage identity of a homologous polypeptide Ser Thr Thr Ser according to the invention means in particular the percentage Trp Tyr identity of the amino acid residues relative to the total length Tyr Trp; Phe of one of the amino acid sequences concretely described Wal Ile: Leu herein. 0184. In the case of a possible protein glycosylation, 0175 "Functional equivalents' in the above sense are also “functional equivalents' according to the invention comprise “precursors” of the polypeptides described, as well as “func proteins of the type designated above in deglycosylated or tional derivatives” and “salts' of the polypeptides. glycosylated form as well as modified forms that can be 0176 “Precursors' are in that case natural or synthetic obtained by altering the glycosylation pattern. precursors of the polypeptides with or without the desired 0185. Such functional equivalents or homologues of the biological activity. proteins or polypeptides according to the invention can be 0177. The expression “salts' means salts of carboxyl produced by mutagenesis, e.g. by point mutation, lengthening groups as well as salts of acid addition of amino groups of the or shortening of the protein. US 2012/0028333 A1 Feb. 2, 2012

0186 Such functional equivalents or homologues of the cally disclosed herein. “Identity” between two nucleic acids proteins according to the invention can be identified by means identity of the nucleotides, in each case over the entire screening combinatorial databases of mutants, for example length of the nucleic acid. shortening mutants. For example, a variegated database of 0194 For example the identity may be calculated by protein variants can be produced by combinatorial mutagen means of the Vector NTI Suite 7.1 program of the company esis at the nucleic acid level, e.g. by enzymatic ligation of a Informax (USA) employing the Clustal Method (Higgins D mixture of synthetic oligonucleotides. There are a great many G, Sharp PM. Fast and sensitive multiple sequence align methods that can be used for the production of databases of ments on a microcomputer. Comput Appl. Biosci. 1989 April; potential homologues from a degenerated oligonucleotide 5(2): 151-1) with the following settings: sequence. Chemical synthesis of a degenerated gene sequence can be carried out in an automatic DNA synthesizer, Multiple Alignment Parameter: and the synthetic gene can then be ligated in a suitable expres sion vector. The use of a degenerated genome makes it pos 0195 sible to Supply all sequences in a mixture, which code for the desired set of potential protein sequences. Methods of Syn thesis of degenerated oligonucleotides are known to a person Gap opening penalty 10 Gap extension penalty 10 skilled in the art (e.g. Narang, S.A. (1983) Tetrahedron 39:3: Gap separation penalty range 8 Itakura et al. (1984) Annu. Rev. Biochem. 53:323: Itakura et Gap separation penalty Off al., (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acids % identity for alignment delay 40 Res. 11:477). Residue specific gaps Off 0187. In the prior art, several techniques are known for the Hydrophilic residue gap Off screening of gene products of combinatorial databases, which Transition weighing O were produced by point mutations or shortening, and for the screening of cDNA libraries for gene products with a selected property. These techniques can be adapted for the rapid Pairwise Alignment Parameter: screening of the gene banks that were produced by combina torial mutagenesis of homologues according to the invention. (0196) The techniques most frequently used for the Screening of large gene banks, which are based on a high-throughput analysis, comprise cloning of the gene bank in expression FAST algorithm Oil vectors that can be replicated, transformation of the suitable K-tuple size 1 cells with the resultant vector database and expression of the Gap penalty 3 combinatorial genes in conditions in which detection of the Window size 5 desired activity facilitates isolation of the vector that codes Number of best diagonals 5 for the gene whose product was detected. Recursive Ensemble Mutagenesis (REM), a technique that increases the 0.197 Alternatively the identity may be determined frequency of functional mutants in the databases, can be used according to Chema, Ramu, Sugawara, Hideaki, Koike, in combination with the Screening tests, in order to identify Tadashi, Lopez, Rodrigo, Gibson, Toby J. Higgins, Desmond homologues (Arkin and Yourvan (1992) PNAS 89:7811 G, Thompson, Julie D. Multiple sequence alignment with the 7815; Delgrave et al. (1993) Protein Engineering 6(3):327 Clustal series of programs. (2003) Nucleic Acids Res 31 331). (13):3497-500, the web page: http://www.ebi.ac.uk/Tools/ 0188 According to a further embodiment of the present clustalw/index.html and the following settings invention, there is provided an isolated polypeptide selected from: 0189 (i) an amino acid sequence represented by any one DNA Gap Open Penalty 1S.O of SEQID NO: 8, or 10; DNA Gap Extension Penalty 6.66 DNA Matrix Identity 0.190 (ii) an amino acid sequence having at least 50%, Protein Gap Open Penalty 1O.O 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, Protein Gap Extension Penalty O.2 61%. 62%, 63%, 64%. 65%, 66%, 67%, 68%, 69%, 70%, Protein matrix Gonnet 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, Protein DNAENDGAP -1 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, Protein, DNA GAPDIST 4 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented 0198 All the nucleic acid sequences mentioned herein by any one of SEQID NO: 8, or 10. (single-stranded and double-stranded DNA and RNA 0191 (iii) derivatives of any of the amino acid sequences sequences, for example cDNA and mRNA) can be produced given in (i) or (ii) above. in a known way by chemical synthesis from the nucleotide building blocks, e.g. by fragment condensation of individual Coding Nucleic Acid Sequences overlapping, complementary nucleic acid building blocks of the double helix. Chemical synthesis of oligonucleotides can, 0.192 The invention also relates to nucleic acid sequences for example, be performed in a known way, by the phosphoa that code for enzymes as defined herein. midite method (Voet, Voet, 2nd edition, Wiley Press, New 0193 The present invention also relates to nucleic acids York, pages 896-897). The accumulation of synthetic oligo with a certain degree of “identity” to the sequences specifi nucleotides and filling of gaps by means of the Klenow frag US 2012/0028333 A1 Feb. 2, 2012 ment of DNA polymerase and ligation reactions as well as Strand of a nucleic acid sequence according to the invention or general cloning techniques are described in Sambrook et al. of a corresponding antisense Strand. (1989), see below. 0211. An "isolated nucleic acid molecule is separated 0199 The invention also relates to nucleic acid sequences from other nucleic acid molecules that are present in the (single-stranded and double-stranded DNA and RNA natural source of the nucleic acid and can moreover be Sub sequences, e.g. cDNA and mRNA), coding for one of the stantially free from other cellular material or culture medium, above polypeptides and their functional equivalents, which if it is being produced by recombinant techniques, or can be can be obtained for example using artificial nucleotide ana free from chemical precursors or other chemicals, if it is being logs. synthesized chemically. 0200. The invention relates both to isolated nucleic acid 0212. A nucleic acid molecule according to the invention molecules, which code for polypeptides or proteins according can be isolated by means of standard techniques of molecular to the invention or biologically active segments thereof, and biology and the sequence information Supplied according to to nucleic acid fragments, which can be used for example as the invention. For example, cDNA can be isolated from a hybridization probes or primers for identifying or amplifying suitable cDNA library, using one of the concretely disclosed coding nucleic acids according to the invention. complete sequences or a segment thereof as hybridization 0201 The nucleic acid molecules according to the inven probe and standard hybridization techniques (as described for tion can in addition contain non-translated sequences from example in Sambrook, J., Fritsch, E. F. and Maniatis, T. the 3' and/or 5' end of the coding genetic region. Molecular Cloning: A Laboratory Manual. 2nd edition, Cold 0202 The invention further relates to the nucleic acid mol Spring Harbor Laboratory, Cold Spring Harbor Laboratory ecules that are complementary to the concretely described Press, Cold Spring Harbor, N.Y., 1989). In addition, a nucleic nucleotide sequences or a segment thereof. acid molecule comprising one of the disclosed sequences or a 0203. According to a further embodiment of the present segment thereof, can be isolated by the polymerase chain invention, there is therefore provided an isolated nucleic acid reaction, using the oligonucleotide primers that were con molecule selected from: structed on the basis of this sequence. The nucleic acid ampli fied in this way can be cloned in a suitable vector and can be 0204 (i) a nucleic acid represented by any one of SEQID characterized by DNA sequencing. The oligonucleotides NO: 7, or 9; according to the invention can also be produced by standard 0205 (ii) the complement of a nucleic acid represented by methods of synthesis, e.g. using an automatic DNA synthe any one of SEQID NO: 7, or 9: S17. 0206 (iii) a nucleic acid encoding the polypeptide as rep 0213 Nucleic acid sequences according to the invention resented by any one of SEQID NO: 8, or 10, preferably as or derivatives thereof, homologues or parts of these a result of the degeneracy of the genetic code, said isolated sequences, can for example be isolated by usual hybridization nucleic acid can be derived from a polypeptide sequence as techniques or the PCR technique from other bacteria, e.g. via represented by any one of SEQID NO: 8, or 10; genomic or cDNA libraries. These DNA sequences hybridize 0207 (iv) a nucleic acid having at least 30%, 31%, 32%, in standard conditions with the sequences according to the 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, invention. 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 0214) “Hybridize” means the ability of a polynucleotide or 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%. 62%, oligonucleotide to bind to an almost complementary 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, sequence in standard conditions, whereas nonspecific bind 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, ing does not occur between non-complementary partners in 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, these conditions. For this, the sequences can be 90-100% 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence iden complementary. The property of complementary sequences tity with any of the nucleic acid sequences of SEQID NO: of being able to bind specifically to one another is utilized for 7, or 9: example in Northern Blotting or Southern Blotting or in 0208 (v) a nucleic acid molecule which hybridizes with a primer binding in PCR or RT-PCR. nucleic acid molecule of (i) to (iv) under stringent hybrid 0215 Short oligonucleotides of the conserved regions are ization conditions; used advantageously for hybridization. However, it is also 0209 (vi) a nucleic acid encoding a polypeptide having possible to use longer fragments of the nucleic acids accord aldehyde dismustase activity, the polypeptide having at ing to the invention or the complete sequences for the hybrid least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, ization. These standard conditions vary depending on the 59%, 60%, 61%, 62%, 63%, 64%. 65%, 66%, 67%, 68%, nucleic acid used (oligonucleotide, longer fragment or com 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, plete sequence) or depending on which type of nucleic acid— 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, DNA or RNA is used for hybridization. For example, the 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, melting temperatures for DNA:DNA hybrids are approx. 10° or 99% sequence identity to the amino acid sequence rep C. lower than those of DNA:RNA hybrids of the same length. resented by any one of SEQID NO: 8, or 10. 0216 For example, depending on the particular nucleic 0210. The nucleotide sequences according to the inven acid, standard conditions mean temperatures between 42 and tion make possible the production of probes and primers that 58° C. in an aqueous buffer solution with a concentration can be used for the identification and/or cloning of homolo between 0.1 to 5xSSC (1xSSC=0.15 MNaCl, 15 mM sodium gous sequences in other cellular types and organisms. Such citrate, pH 7.2) or additionally in the presence of 50% forma probes or primers generally comprise a nucleotide sequence mide, for example 42°C. in 5xSSC, 50% formamide. Advan region which hybridizes under “stringent conditions (see tageously, the hybridization conditions for DNA:DNA below) on at least about 12, preferably at least about 25, for hybrids are 0.1 xSSC and temperatures between about 20°C. example about 40, 50 or 75 successive nucleotides of a sense to 45° C., preferably between about 30° C. to 45° C. For US 2012/0028333 A1 Feb. 2, 2012

DNA:RNA hybrids the hybridization conditions are advanta be made to the details given above for the polypeptides). geously 0.1 xSSC and temperatures between about 30° C. to Advantageously, the homologies can be higher over partial 55°C., preferably between about 45° C. to 55°C. These stated regions of the sequences. temperatures for hybridization are examples of calculated 0225. Furthermore, derivatives are also to be understood melting temperature values for a nucleic acid with a length of to be homologues of the nucleic acid sequences according to approx. 100 nucleotides and a G+C content of 50% in the the invention, for example animal, plant, fungal or bacterial absence of form amide. The experimental conditions for homologues, shortened sequences, single-stranded DNA or DNA hybridization are described in relevant genetics text RNA of the coding and non-coding DNA sequence. For books, for example Sambrook et al., 1989, and can be calcu example, homologues have, at the DNA level, a homology of lated using formulae that are known by a person skilled in the at least 40%, preferably of at least 60%, especially preferably art, for example depending on the length of the nucleic acids, of at least 70%, quite especially preferably of at least 80% the type of hybrids or the G+C content. A person skilled in the over the entire DNA region given in a sequence specifically art can obtain further information on hybridization from the disclosed herein. following textbooks: Ausubel et al. (eds), 1985, Current Pro 0226. Moreover, derivatives are to be understood to be, for tocols in Molecular Biology, John Wiley & Sons, New York; example, fusions with promoters. The promoters that are Hames and Higgins (eds), 1985, Nucleic Acids Hybridiza added to the stated nucleotide sequences can be modified by tion: A Practical Approach, IRL Press at Oxford University at least one nucleotide exchange, at least one insertion, inver Press, Oxford; Brown (ed), 1991, Essential Molecular Biol sion and/or deletion, though without impairing the function ogy: A Practical Approach, IRL Press at Oxford University ality or efficacy of the promoters. Moreover, the efficacy of Press, Oxford. the promoters can be increased by altering their sequence or 0217 “Hybridization' can in particular be carried out can be exchanged completely with more effective promoters understringent conditions. Such hybridization conditions are even of organisms of a different genus. for example described in Sambrook, J., Fritsch, E. F., Mania tis, T., in: Molecular Cloning (A Laboratory Manual), 2nd Constructs According to the Invention edition, Cold Spring Harbor Laboratory Press, 1989, pages 0227. The invention also relates to expression constructs, 9.31-9.57 or in Current Protocols in Molecular Biology, John containing, under the genetic control of regulatory nucleic Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. acid sequences, a nucleic acid sequence coding for a polypep 0218 “Stringent hybridization conditions mean in par tide or fusion protein according to the invention; as well as ticular: Incubation at 42°C. overnight in a solution consisting vectors comprising at least one of these expression con of 50% formamide, 5xSSC (750 mM. NaCl, 75 mM tri-so StructS. dium citrate), 50 mM sodium phosphate (pH 7.6), 5xDen 0228 “Expression unit’ means, according to the inven hardt Solution, 10% dextran sulfate and 20 g/ml denatured, tion, a nucleic acid with expression activity, which comprises sheared salmon sperm DNA, followed by washing of the a promoter as defined herein and, after functional association filters with 0.1XSSC at 65° C. with a nucleic acid that is to be expressed or a gene, regulates 0219. The invention also relates to derivatives of the con the expression, i.e. the transcription and the translation of this cretely disclosed or derivable nucleic acid sequences. nucleic acid or of this gene. In this context, therefore, it is also 0220 Thus, further nucleic acid sequences according to called a “regulatory nucleic acid sequence'. In addition to the the invention can be derived from the sequences specifically promoter, other regulatory elements may be present, e.g. disclosed herein and can differ from it by addition, substitu enhancers. tion, insertion ordeletion of individual or several nucleotides, 0229) “Expression cassette' or “expression construct’ and furthermore code for polypeptides with the desired pro means, according to the invention, an expression unit, which file of properties. is functionally associated with the nucleic acid that is to be 0221) The invention also encompasses nucleic acid expressed or the gene that is to be expressed. In contrast to an sequences that comprise so-called silent mutations or have expression unit, an expression cassette thus comprises not been altered, in comparison with a concretely stated only nucleic acid sequences which regulate transcription and sequence, according to the codon usage of a special original translation, but also the nucleic acid sequences which should or host organism, as well as naturally occurring variants, e.g. be expressed as protein as a result of the transcription and splicing variants or allelic variants, thereof. translation. 0222. It also relates to sequences that can be obtained by 0230. The terms “expression” or “overexpression” conservative nucleotide Substitutions (i.e. the amino acid in describe, in the context of the invention, the production or question is replaced by an amino acid of the same charge, increase of intracellular activity of one or more enzymes in a size, polarity and/or solubility). microorganism, which are encoded by the corresponding 0223. The invention also relates to the molecules derived DNA. For this, it is possible for example to inserta gene in an from the concretely disclosed nucleic acids by sequence poly organism, replace an existing gene by another gene, increase morphisms. These genetic polymorphisms can exist between the number of copies of the gene or genes, use a strong individuals within a population owing to natural variation. promoter or use a gene that codes for a corresponding enzyme These natural variations usually produce a variance of 1 to 5% with a high activity, and optionally these measures can be in the nucleotide sequence of a gene. combined. Preferably such constructs according to the inven 0224 Derivatives of nucleic acid sequences according to tion comprise a promoter 5'-upstream from the respective the invention mean for example allelic variants, having at coding sequence, and a terminator sequence 3'-downstream, least 60% homology at the level of the derived amino acid, and optionally further usual regulatory elements, in each case preferably at least 80% homology, quite especially preferably functionally associated with the coding sequence. at least 90% homology over the entire sequence range (re 0231. A “promoter', a “nucleic acid with promoter activ garding homology at the amino acid level, reference should ity' or a “promoter sequence” mean, according to the inven US 2012/0028333 A1 Feb. 2, 2012

tion, a nucleic acid which, functionally associated with a Other advantageous regulatory sequences are contained for nucleic acid that is to be transcribed, regulates the transcrip example in the Gram-positive promoters ace, amy and SPO2, tion of this nucleic acid. in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, 0232 “Functional” or “operative' association means, in CYC1, GAPDH, TEF, rp28, ADH. Artificial promoters can this context, for example the sequential arrangement of one of also be used for regulation. the nucleic acids with promoter activity and of a nucleic acid 0238 For expression, the nucleic acid construct is inserted sequence that is to be transcribed and optionally further regu in a host organism advantageously in a vector, for example a latory elements, for example nucleic acid sequences that plasmidor a phage, which permits optimum expression of the enable the transcription of nucleic acids, and for example a genes in the host. In addition to plasmids and phages, vectors terminator, in Such away that each of the regulatory elements are also to be understood as meaning all other vectors known can fulfill its function in the transcription of the nucleic acid to a person skilled in the art, e.g. viruses, such as SV40, CMV, sequence. This does not necessarily require a direct associa baculovirus and adenovirus, transposons, IS elements, phas tion in the chemical sense. Genetic control sequences, such as mids, cosmids, and linear or circular DNA. These vectors can enhancer sequences, can also exert their function on the target be replicated autonomously in the host organism or can be sequence from more remote positions or even from other replicated chromosomally. These vectors represent a further DNA molecules. Arrangements are preferred in which the embodiment of the invention. nucleic acid sequence that is to be transcribed is positioned 0239 Suitable plasmids are, for example in E. coli, behind (i.e. at the 3' end) the promoter sequence, so that the pLG338, p.ACYC184, pBR322, p OC18, p OC19, pKC30, two sequences are bound covalently to one another. The dis pRep4, pHS1, pKK223-3, pIDHE19.2, pHS2, pPLc236, tance between the promoter sequence and the nucleic acid pMBL24, pI G200, pluR290, pIN-III'-B1.gt11 orpEdCl; sequence that is to be expressed transgenically can be less in nocardioform actinomycetes p.AM2; in Streptomyces than 200 bp (base pairs), or less than 100 bp or less than 50 bp. pIJ101, pIJ364, pIJ702 or plJ361; in bacillus puB110, pC194 0233. Apart from promoters and terminators, examples of or pBD214; in CorynebacteriumpSAT7 or p AJ667; in fungi other regulatory elements that may be mentioned are target pALS1, pIL2 or pBB116; in yeasts 2alphaM, paG-1, YEp6, ing sequences, enhancers, polyadenylation signals, selectable YEp13 or pEMBLYe23 or in plants plGV23, pGHlac", markers, amplification signals, replication origins and the pBIN19, p.AK2004 or plDH51. The aforementioned plasmids like. Suitable regulatory sequences are described for example represent a small selection of the possible plasmids. Other in Goeddel, Gene Expression Technology: Methods in Enzy plasmids are well known to a person skilled in the art and will mology 185, Academic Press, San Diego, Calif. (1990). be found for example in the book Cloning Vectors (Eds. 0234 Nucleic acid constructs according to the invention Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, comprise in particular sequences selected from those, specifi 1985, ISBN 0444904018). cally mentioned herein or derivatives and homologues 0240. In a further embodiment of the vector, the vector thereof, as well as the nucleic acid sequences that can be containing the nucleic acid construct according to the inven derived from amino acid sequences specifically mentioned tion or the nucleic acid according to the invention can be herein which are advantageously associated operatively or inserted advantageously in the form of a linear DNA in the functionally with one or more regulating signal for control microorganisms and integrated into the genome of the host ling, e.g. increasing, gene expression. organism through heterologous or homologous recombina 0235. In addition to these regulatory sequences, the natu tion. This linear DNA can comprise a linearized vector such ral regulation of these sequences can still be present in front of as plasmidorjust the nucleic acid constructor the nucleic acid the actual structural genes and optionally can have been according to the invention. altered genetically, so that natural regulation is Switched off 0241 For optimum expression of heterologous genes in and the expression of the genes has been increased. The organisms, it is advantageous to alter the nucleic acid nucleic acid construct can also be of a simpler design, i.e. sequences in accordance with the specific codon usage without any additional regulatory signals being inserted in employed in the organism. The codon usage can easily be front of the coding sequence and without removing the natu determined on the basis of computer evaluations of other, ral promoter with its regulation. Instead, the natural regula known genes of the organism in question. tory sequence is silenced so that regulation no longer takes 0242. The production of an expression cassette according place and gene expression is increased. to the invention is based onfusion of a suitable promoter with 0236 A preferred nucleic acid construct advantageously a suitable coding nucleotide sequence and a terminator signal also contains one or more of the aforementioned enhancer or polyadenylation signal. Common recombination and clon sequences, functionally associated with the promoter, which ing techniques are used for this, as described for example in T. permit increased expression of the nucleic acid sequence. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: Additional advantageous sequences, such as other regulatory A Laboratory Manual, Cold Spring Harbor Laboratory, Cold elements or terminators, can also be inserted at the 3' end of Spring Harbor, N.Y. (1989) as well as in T. J. Silhavy, M. L. the DNA sequences. One or more copies of the nucleic acids Berman and L. W. Enquist, Experiments with Gene Fusions, according to the invention can be contained in the construct. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. The construct can also contain other markers, such as antibi (1984) and in Ausubel, F. M. et al., Current Protocols in otic resistances or auxotrophy-complementing genes, option Molecular Biology, Greene Publishing Assoc. and Wiley ally for selection on the construct. Interscience (1987). 0237 Examples of suitable regulatory sequences are con 0243 The recombinant nucleic acid constructor gene con tained in promoters such as cos-, tac-, trp-, tet-, trp-tet-, lpp-, struct is inserted advantageously in a host-specific vector for lac-lpp-lac-, lacI. T7- T5-, T3-, gal-, trc-, ara-, rhaF (rhap expression in a suitable host organism, to permit optimum BAD)SP6-, lambda-P- or in the lambda-P, promoter, which expression of the genes in the host. Vectors are well known to find application advantageously in Gram-negative bacteria. a person skilled in the art and will be found for example in US 2012/0028333 A1 Feb. 2, 2012

“Cloning Vectors” (Pouwels P. H. et al., Publ. Elsevier, batchwise or continuously. Nutrients can be supplied at the Amsterdam-New York-Oxford, 1985). start of fermentation or can be supplied Subsequently, either 0244. Hosts that can be used according to the invention semi-continuously or continuously. 0245 Depending on the context, the term “microorgan 0251 Recombinant production proteins with aldehyde ism” means the starting microorganism (wild-type) or a dismutase activity genetically modified microorganism according to the inven 0252. The invention also relates to methods for production tion, or both. of proteins according to the invention by cultivating a micro 0246 The term “wild-type' means, according to the organism which expresses said protein, and isolating the invention, the corresponding starting microorganism, and desired product from the culture. need not necessarily correspond to a naturally occurring 0253) The microorganisms as used according to the inven organism. tion can be cultivated continuously or discontinuously in the 0247. By means of the vectors according to the invention, batch process or in the fed batch or repeated fed batch process. recombinant microorganisms can be produced, which have A review of known methods of cultivation will be found in the been transformed for example with at least one vector accord textbook by Chmiel (Bioprocesstechnik 1. Einführung in die ing to the invention and can be used for production of the Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, polypeptides according to the invention. Advantageously, the 1991)) or in the textbook by Storhas (Bioreaktoren and recombinant constructs according to the invention, described periphere Einrichtungen (Vieweg Verlag, Braunschweig/ above, are inserted in a Suitable host system and expressed. Wiesbaden, 1994)). Preferably, common cloning and transfection methods that 0254 The culture medium that is to be used must satisfy are familiar to a person skilled in the art are used, for example the requirements of the particular strains in an appropriate co-precipitation, protoplast fusion, electroporation, retroviral manner. Descriptions of culture media for various microor transfection and the like, in order to secure expression of the ganisms are given in the handbook “Manual of Methods for stated nucleic acids in the respective expression system. Suit General Bacteriology of the American Society for Bacteri able systems are described for example in Current Protocols ology (Washington D.C., USA, 1981). in Molecular Biology, F. Ausubel et al., Publ. Wiley Inter 0255. These media that can be used according to the inven science, New York 1997, or Sambrooketal. Molecular Clon tion generally comprise one or more sources of carbon, ing: A Laboratory Manual. 2nd edition, Cold Spring Harbor Sources of nitrogen, inorganic salts, vitamins and/or trace Laboratory, Cold Spring Harbor Laboratory Press, Cold elements. Spring Harbor, N.Y., 1989. 0256 Preferred sources of carbon are sugars, such as 0248. In principle, all prokaryotic organisms can be con mono-, di- or polysaccharides. Very good sources of carbon sidered as recombinant host organisms for the nucleic acid are for example glucose, fructose, mannose, galactose, according to the invention or the nucleic acid construct. Bac ribose, Sorbose, ribulose, lactose, maltose, Sucrose, raffinose, teria are used advantageously as host organisms. Although starch or cellulose. Sugars can also be added to the media via Escherichia coli is one example of a bacterial host cell used complex compounds, such as molasses, or other by-products commonly to express an enzyme that catalyzes the degrada from Sugar refining. It may also be advantageous to add tion of formaldehyde, other bacterial host cells can be used in mixtures of various sources of carbon. Other possible sources the present invention to express foreign DNA, including for of carbon are oils and fats such as Soybean oil, Sunflower oil, example, Escherichia, Enterobacter, Azotobacter, Erwinia, peanut oil and coconut oil, fatty acids such as palmitic acid, Bacillus, Pseudomonas, Bordetella, Rhodobacter, Xvella, Stearic acid or linoleic acid, alcohols such as glycerol, metha Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizo nol or ethanol and organic acids such as acetic acid or lactic bium, Vitreoscilla, and Paracoccus. Furthermore, eukaryotic acid. fungal host cells can be used in the present invention to 0257 Sources of nitrogen are usually organic or inorganic express foreign DNA, including for example, Aspergillus, nitrogen compounds or materials containing these com Pichia, Trichoderma, Hansenula, Saccharomyces, Kluyvero pounds. Examples of Sources of nitrogen include ammonia myces, Schizosaccharomyces, Chrysosporium, Candida and gas or ammonium salts, such as ammonium Sulfate, ammo Torulopsis. nium chloride, ammonium phosphate, ammonium carbonate 0249. The host organism or host organisms according to or ammonium nitrate, nitrates, urea, amino acids or complex the invention then preferably contain at least one of the Sources of nitrogen, Such as corn-steep liquor, soybean flour, nucleic acid sequences, nucleic acid constructs or vectors Soybean protein, yeast extract, meat extract and others. The described in this invention, which code for an enzyme activity Sources of nitrogen can be used separately or as a mixture. according to the above definition. Inorganic salt compounds that may be present in the media 0250. The organisms used in the method according to the comprise the chloride, phosphate or Sulfate salts of calcium, invention are grown or bred in a manner familiar to a person magnesium, sodium, cobalt, molybdenum, potassium, man skilled in the art, depending on the host organism. As a rule, ganese, Zinc, copper and iron. Inorganic Sulfur-containing microorganisms are grown in a liquid medium, which con compounds, for example Sulfates, Sulfites, dithionites, tains a source of carbon, generally in the form of Sugars, a tetrathionates, thiosulfates, Sulfides, but also organic Sulfur Source of nitrogen generally in the form of organic sources of compounds, such as mercaptains and thiols, can be used as nitrogen Such as yeast extract or salts such as ammonium sources of sulfur. Sulfate, trace elements such as iron, manganese and magne 0258 Phosphoric acid, potassium dihydrogenphosphate sium salts and optionally vitamins, attemperatures between or dipotassium hydrogenphosphate or the corresponding 0° C. and 100° C., preferably between 10° C. to 60° C. with Sodium-containing salts can be used as sources of phospho oxygen aeration. The pH of the liquid nutrient medium can be rus. Chelating agents can be added to the medium, in order to maintained at a fixed value, i.e. regulated or not regulated keep the metal ions in Solution. Especially suitable chelating during growing. Growing can be carried out batchwise, semi agents comprise dihydroxyphenols, such as catechol or pro US 2012/0028333 A1 Feb. 2, 2012

tocatechuate, or organic acids, Such as citric acid. The fer 0265. The following examples only serve to illustrate the mentation media used according to the invention may also invention. The numerous possible variations that are obvious contain other growth factors, such as vitamins or growth to a person skilled in the art also fall within the scope of the promoters, which include for example biotin, riboflavin, thia invention. mine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts often come from complex compo EXAMPLES nents of the media, Such as yeast extract, molasses, corn-steep liquor and the like. In addition, Suitable precursors can be Example 1 added to the culture medium. The precise composition of the compounds in the medium is strongly dependent on the par FDM Expression & Purification ticular experiment and must be decided individually for each specific case. Information on media optimization can be 0266 The following procedure is derived from a previ found in the textbook Applied Microbiol. Physiology, A ously described protocol (Yanase, H., Moriya, K., Mukai, N., Kawata.Y., Okamoto, K., Kato, N. (2002) Effects of GroESL Practical Approach” (Publ. P. M. Rhodes, P. F. Stanbury, IRL Coexpression on the Folding of Nicotinoprotein Formalde Press (1997) p. 53-73, ISBN 0 199635773). Growing media hyde Dismutase from Pseudomonas putida F61 Biosci. can also be obtained from commercial Suppliers, such as Bitechnol. Biochem. 66(1), 85-91) and has been further opti Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) mized. The codon usage of the gene coding for FDM has been etc. All components of the medium are sterilized, either by optimized for E. coli. The new DNA sequence has been heating (20 min at 1.5 bar and 121°C.) or by sterile filtration. synthesized and cloned into pIHE, a rhamnose-inducible The components can be sterilized either together, or if neces expression vector (SEQID NO:4). The GroEL/S chaperones, sary separately. All the components of the medium can be which are necessary for soluble FDM production, are cloned present at the start of growing, or optionally can be added in the IPTG-inducible pagro vector (SEQID NO:5). The lac continuously or by batch feed. repressor is encoded by the pHSG vector (SEQ ID NO:6). 0259. The temperature of the culture is normally between Expression of FDM has been performed using E. coli TG10. 15° C. and 45° C., preferably 25°C. to 40° C. and can be kept a TG1 derivative with no rhamnose isomerase. TG10 cells constant or can be varied during the experiment. The pH value containing pagro and pHSG (TG10+) were transformed with of the medium should be in the range from 5 to 8.5, preferably pDHE-FDM and cultured in LB for 5 hat 37° C. in presence around 7.0. The pH value for growing can be controlled of amplicillin (pDHE), spectinomycin (pAgro) and chloram during growing by adding basic compounds Such as sodium phenicol (pHSG).5 mL of this culture were transferred in 500 hydroxide, potassium hydroxide, ammonia or ammonia mL of the same medium containing 100LLMIPTG and 0.5g/L rhamnose. Induction was performed at 37°C. for about 18 h. water or acid compounds such as phosphoric acid or Sulfuric Cells were collected by centrifugation and resuspended in acid. Antifoaming agents, e.g. fatty acid polyglycol esters, lysis buffer (10 mM. KHPO, pH 7, 0.5 mM MgCl, 140 uM can be used for controlling foaming. To maintain the stability PMSF). Cell lysis was induced by sonication (<1', 1 S/1 of plasmids, Suitable Substances with selective action, e.g. s>x10, 70% amplitude). After 10' incubation at 37° C. and antibiotics, can be added to the medium. Oxygen or oxygen centrifugation, ammonium sulfate was added to the clear containing gas mixtures, e.g. the ambient air, are fed into the lysate to a saturation of 50%. Unwanted proteins (e.g. chap culture in order to maintain aerobic conditions. The tempera erones) were allowed to precipitate for 2-4 h on ice. ture of the culture is normally from 20° C. to 45° C. Culture 0267. The precipitate was removed by centrifugation and is continued until a maximum of the desired product has the clear FDM-containing solution was applied to a phenyl formed. This is normally achieved within 10 hours to 160 sepharose column (HIC chromatography). The FDM-con hours. taining fractions were eluted using a “40% to 0%' ammo 0260 The cells can be disrupted optionally by high-fre nium sulfate gradient (buffer: 10 mM. KHPO, 0.5 mM quency ultrasound, by high pressure, e.g. in a French pressure MgCl, pH 7.2). The presence of FDM was confirmed by a cell, by osmolysis, by the action of detergents, lytic enzymes quick colorimetric assay: 10 uL of each fraction was trans or organic solvents, by means of homogenizers or by a com ferred to a 96-well MTP plate and incubated with 250 uL of a bination of several of the methods listed. formaldehyde solution (12.5 ppm in 100 mM. KHPO, pH 7. 30"). Unreacted formaldehyde was detected by addition of 50 0261 Particular use of the enzyme having aldehyde dis uL Purpald-reagent (10 mg/mL in 1 M NaOH, Sigma) to 50 mutase activity LL reaction mix. Color development indicates absence of 0262 The invention also relates to the use of an isolated enzyme in the collected fraction. polypeptide having aldehyde dismutase activity and compris 0268. After the phenyl-sepharose purification step, the ing a variant of SEQID NO:2 wherein the phenylalanine at eluted fractions were concentrated by ultrafiltration (10 kDa position 93 of SEQID NO:2, and/or the isoleucine at position cut off) and desalted by size-exclusion chromatography 301 of SEQID NO:2, and/or the methionine at position337 of (HiPrep Desalting 26/10 mL, Amersham). 1xPBS, 0.5 mM SEQ ID NO:2, and/or the phenylalanine at position 127 of MgCl (pH 7.2) was used as running buffer. The protein SEQ ID NO:2 is substituted by any other amino acid, for solution was concentrated by ultrafiltration (10 kDa cut off) reducing the aldehyde content in an aldehyde-containing for and glycerol was added to a final concentration of 50%. FDM mulation. stocks (2-5 mg/mL) were stored at -20°C. Typical yield from 0263. In a preferred embodiment, the aldehyde is acetal shake-flasks was about 100 mg/L, from fermentation about 1 dehyde as defined in the definition section. g/L. This procedure allows protein purification to homogene 0264. In another preferred embodiment, the aldehyde is ity, as judged by Coomassie-stained SDS-PAGE (data not methylglyoxal. shown). US 2012/0028333 A1 Feb. 2, 2012

0269. For FDM mutants the following method is employ acid per minute. FDM expressed in TG10+ had a specific able as well activity ranging between 100-200 U/mg. 0270. The following procedure is derived from a previ ously described protocol (Yanase etal. 2002, Biosci. Biotech Example 3 nol. Biochem. 66(1), 85-91) and has been further optimized. The codon usage of the gene coding for FDM (or mutant) has Detection of Formaldehyde been optimized for E. coli (SEQ ID NO:3). The new DNA sequence has been synthesized and cloned into pHE, a (0274 The detection principle of the LAW 112 method rhamnose-inducible expression vector (SEQID NO:4). The relies on the reaction of formaldehyde with acetylacetone in GroEL/S chaperones, which are necessary for soluble FDM the presence of ammonia to form the yellow compound 3.5- production, are cloned in the IPTG-inducible pAgro vector diacetyl-1,4-dihydrolutidine (Hantzsch reaction) (Nash, T. (SEQ ID NO:5). The lac repressor is encoded by the pHSG vector (SEQ 1N NO:6). Expression of FDM has been per (1953) The Colorimetric Estimation of Formaldehyde by formed using E. coli TG10, a TG1 derivative with no rham Means of the Hantzsch Reaction Biochem. J., 55(3), 416–421 nose isomerase. TG10 cells containing pAgro and pHSG and Fregert, S., Dahlquist, I., Gruvberger, B. (1984) A Simple (TG10+) were transformed with plDHE-FDM and cultured in Method for the Detection of Formaldehyde Contact Derma LB for 5 h at 37° C. in presence of ampicillin (pDHE), titis, 10, 132-134). For a quantitative formaldehyde detection, spectinomycin (pAgro) and chloramphenicol (pHSG).5 mL a reagent solution is prepared as follows: 15 gammonium of this culture were transferred in 500 mL of the same acetate, 0.2 mL acethylacetone, 0.3 mL acetic acid are mixed medium containing 100 uM IPTG and 0.5 g/L rhamnose. in 100 mL distilled water (stable for 1 week at 4°C.). Aqueous Induction was performed at 37°C. for about 18 h. Cells were Solutions of formaldehyde ranging from 5 ppm to 25 ppm are collected by centrifugation and resuspended in lysis buffer prepared for the calibration curve. The reaction is started by (10 mM. KHPO, pH 7, 0.5 mM MgCl). Cell lysis was mixing 0.1 mL of an aqueous sample solution (or standard or induced by sonication (<3', 15 S/15 sex3, 85% amplitude). After 10' incubation at 37°C. and centrifugation, ammonium water as blank) with 0.15 mL of reagent solution and incu sulfate was added to the clear lysate to a saturation of 50%. bated at 60° C. for 10'. After the solution has cooled down to Unwanted proteins (e.g. chaperones) were allowed to precipi RT, distilled water is added to a final volume of 1 mL. For tate for 1-2 h on ice. mation of 3,5-diacetyl-1,4-dihydrolutidine is monitored at 0271 The precipitate was removed by centrifugation and 412 nm. the clear FDM-containing solution was applied to a phenyl sepharose column (HIC chromatography). The FDM-con Example 4 taining fractions were eluted using a “40% to 0%' ammo nium sulfate gradient (buffer: 10 mM. KHPO, 0.5 mM Preliminary Enzyme Characterization MgCl, pH 7.2). The presence of FDM was confirmed by a quick colorimetric assay: 10 LIL of each fraction was trans 0275. In order to determine the efficiency of formaldehyde ferred to a 96-well MTP plate and incubated with 250 uL of a dismutase for textile applications, different enzyme concen formaldehyde solution (12.5 ppm in 100 mMKHPO, pH 7. trations were incubated with 220 ppm formaldehyde in 100 30"). Unreacted formaldehyde was detected by addition of 50 mM KHPO, pH 8, RT, 30'. The samples were analyzed by uL Purpald-reagent (10 mg/mL in 1 M NaOH, Sigma) to 50 the previously described acetylacetone method. Specific LL reaction mix. Color development indicates absence of activity of the enzyme stock solution was 165 U/mg. The enzyme in the collected fraction. results are shown in table 1. 0272. After the phenyl-sepharose purification step, the eluted fractions were concentrated by ultrafiltration (10 kDa cut off) and desalted by size-exclusion chromatography TABLE 1 (HiPrep Desalting 26/10 mL, Amersham). 10 mM. KHPO, Enzymatic formaldehyde removal in solution (220 ppm formaldehyde). 0.5 mMMgCl (pH 7) was used as running buffer. The protein Detection by the acethylacetone method. Values indicate remaining solution was concentrated by ultrafiltration (10 kDa cut off) formaldehyde in solution after 30" incubation at RT. and glycerol was added to a final concentration of 50%. FDM FDM (mg/L) Formaldehyde (ppm) stocks (2-5 mg/mL) were stored at -20°C. This procedure O 230 allows protein purification to homogeneity, as judged by Coo 7 11 massie-stained SDS-PAGE (data not shown). 17 not detectable (<5 ppm) Example 2 0276 Even very low enzyme concentrations are more than sufficient to reduce the formaldehyde content almost com FDM Characterization by pH-Stat Titration pletely. Another interesting property for technical applica 0273 FDM activity was assayed in a standard reaction tions is the tolerance to alcohols (ethanol, isopropanol) or mixture containing 20 mM formaldehyde, 100 mMKCl, 0.25 pure water instead of buffer. To that end, 17 mg/L formalde mM. KHPO (pH 7). The reaction was performed at RT by hyde dismutase were incubated at 30°C. in 100 mMKHPO adding 5ull of enzyme solution to 25 mL of standard mixture. (pH 8), 220 ppm formaldehyde and 10 or 20% V/v solvent Formate formation was monitored over 5' by pH-stattitration (ethanol or isopropanol). Alternatively, ddHO was used with 5 mM NaOH. One unit is defined as the amount of instead of phosphate buffer. The results are summarized in enzyme necessary to catalyze the formation of 1 Jumol formic table 2. US 2012/0028333 A1 Feb. 2, 2012 20

formed on textile cross-linked with an alternative dimethy TABLE 2 loldihydroxyethylene urea crosslinker containing a larger Effect of ethanol, isopropanol (in % w/v) and pure water on amount of FA (641 ppm formaldehyde in the textile before the FDM activity (FDM at 17 mg/L, formaldehyde 220 ppm). Values enzymatic treatment). indicate remaining formaldehyde in solution after 30" incubation at 30° C. Detection by the acethylacetone method. TABLE 4 FDM in 10% EtOH FDM in 20% EtOH 20% EtOH Effect of different FDM concentrations on cotton cross-linked 160 ppm 196 ppm 227 ppm with a dimethyloldihydroxyethylene urea formulation. FDM in 10% isopropanol FDM in 20% isopropanol 20% isopropanol 30' incubation 60' incubation FDM formaldehyde formaldehyde Formaldehyde Formaldehyde 26 ppm 104 ppm 229 ppm (mg/ from wet from dry from wet from dry FDM in ddHO ddH2O L) textile (ppm) textile (ppm) textile (ppm) textile (ppm) O 342 345 363 355 240 ppm 201 ppm 2O 150 188 127 190 (133 ppm after 90") (202 ppm after 90") 50 100 156 69 126 1OO 86 159 59 123 0277 Formaldehyde dismutase from P putida F61 does 150 8O 146 59 116 not effectively react with FA in the presence of large amounts of ethanol. Even at 10% V/V, enzyme activity towards FA is 0280. Due to a “wash-out' effect, the samples with no strongly inhibited. Relatively low concentrations of isopro dismutase show a clear drop in the formaldehyde content. panol are not problematic, but more than 10% V/v have a negative effect on activity. Pure water inactivates the enzyme; Nevertheless, application of increasing amounts of dismutase generation of formic acid induces an acidic pH-shift (pH leads to a clear reduction of released formaldehyde from the drops from 7 to about 4). finished textile. A water uptake of about 70% means that about 100 mg dismutaseper kg textile are more than Sufficient Example 5 to substantially reduce the formaldehyde content. 0281. In order to reduce time and energy costs to dry the Treatment of Finished Textiles with FDM textile after the enzymatic treatment, it would be beneficial to 0278 Cotton fiber finished with a dimethyloldihydroxy reduce the water content in Such applications. To that end, ethylene urea cross-linker has been treated with FDM. After crosslinked textile samples have been treated slightly differ condensation of the cross-linker with the cotton fiber, about ently: a concentrated enzyme solution (1350 mg/L in 1xpBS, 114 ppm of free formaldehyde in the textile could be deter 25% glycerol) was sprayed on the textile. The water uptake mined by the acethylacetone method. This textile sample has was fine-tuned, allowing the range 0-50% humidity to be been Subsequently impregnated with enzyme solutions of analyzed (Table 5). All textile probes were incubated for 60'at different concentrations (100 mM. KHPO, pH 8, 0-150 30° C. mg/L enzyme) and immediately passed through two rotating metal cylinders (FIG. 3). The wet sample was incubated for TABLE 5 60' at 30° C. Formaldehyde extracted from 1 g of textile sample was quantified by the acetylacetone method (LAW Effect of different water uptake on cotton cross-linked with a dimethyloldihydroxyethylene urea formulation 112). Table 3 summarizes the data. (641 ppm formaldehyde in the textile before the enzymatic treatment). FDM was sprayed as a concentrated Stock TABLE 3 Solution (1350 mg/L in IX PBS, 25% glycerol). Effect of different FDM concentrations on cotton cross-linked with water uptake of Formaldehyde a modified dimethy-loldihydroxyethylene urea cross-linker (114 ppm the textile sample from dry textile formaldehyde in the textile before the enzymatic treatment). samples (wfw %) (ppm) formaldehyde Formaldehyde buffer 2O 465 from wet textile from dry textile only FDM (mg/L) (ppm) (ppm) --FDM 10 346 --FDM 2O 222 O 73 95 --FDM 30 18O 2O 33 56 --FDM 40 167 50 22 40 --FDM 50 160 100 23 51 150 23 49 0282 Even by applying the enzyme as a spray, some 0279. The formaldehyde content with no dismutase has “wash-out of formaldehyde was unavoidable (20% buffer decreased to about 70 ppm (95 from dry textile) probably due uptake, 465 ppm released formaldehyde). Nevertheless, the to some “wash-out-effect during the impregnation process. enzyme application with the same water uptake (270 mg In general, formaldehyde dismutase is able to clearly lower enzyme per kg textile) still resulted in more than 50% reduc the amount of extracted formaldehyde to levels between 20 tion in released formaldehyde. Compared to the enzyme and 40 ppm. Differences in the wet vs. dry formaldehyde application through the metal cylinders, where the water con detection are probably due to differences in the textile water tent of the textile reaches 70%, the efficiency of the spray content estimations. Table 4 shows the same experiment per application is less pronounced. US 2012/0028333 A1 Feb. 2, 2012

Example 6 0288 Product formulations were treated as follows: 17.4g of crosslinking reagent (as concentrated aqueous solution) Effect of FDM on the FA-Content of Different Tex and 2 mL of 3M KHPO (pH 7) were gently mixed with 0.6 tile Finishing Products mL of FDM (4.8 mg/mL). The final enzyme concentration 0283 An alternative strategy to reduce the formaldehyde was about 140 mg/L. The enzymatic process was carried out amount in textiles would be to apply the dismutase directly on at RT for about 3 h. The formaldehyde content was measured the cross-linker solution, before the condensation step with as previously described (acetylacetone method) (see Table 7). the textile. To that end, 3 products have been tested with formaldehyde dismutase: a dimethyloldihydroxyethylene TABLE 7 urea formulation (formulation 1), a methoxymethylated crosslinking reagent FA (ppm) with FDM FA (ppm) no FDM melamine formulation (formulation 2) and a polyconden 1 1200 4100 sated methoxymethylated melamine formulation with low 2 500 31 OO FA content (formulation 3). The cross-linker formulation 1 is 3 500 2200 a highly concentrated, slightly acidic (pH 5-6) aqueous solu 4 500 3500 tion which contains between 0.1 and 1% formaldehyde (1000 to 10.000 ppm). Formulation 2, is a concentrated aqueous 0289. The formaldehyde content in the product solutions solution (pH 8-9) containing between 1.5 and 2.5% formal was reduced by 70-80%. Formaldehyde removal in product 1 dehyde. Formulation 3 is a slightly basic aqueous solution (unprotected methylol groups) can lead to product degrada containing between 0.2 and 1% formaldehyde (pH 8-9). tion over time. 0284 Typically, 87 uL of pure product were mixed with 10 0290 The enzyme-treated and non-treated formulations uL of 3 MKHPO (pH 7) and 3 uI of 50% glycerol (negative were used in cotton finishing (textile; CO-Popelin, using the control). In the enzyme-treated samples, a enzyme stock Solu following recipe: 57.5 g/L crosslinking reagent, 10 g/L tion (4.8 mg/mL) was used instead of glycerol. The final MgCl2.6H2O, pH 5.0 (by adding acetic acid). The cotton enzyme concentration in the product solution was ~150 samples (pick-up about 70%) were allowed to dry at 110°C. mg/L. Formaldehyde was detected by the acetylacetone to a final humidity of about 6%. The following curing step method. Table 6 shows the effect of FDM on cross-linker was performed at 150° C. for 3 minutes. The formaldehyde Solutions. content in the cotton sample was measured by LAW 112 (values in ppm). Additional performance indicators were dry TABLE 6 crease recovery angles (dcra) (in), tensile strength (tensile in Effect of FDM (~150 mg/L) on Formulation 1, 2 and 3. N on 40*100 mm), smoothness rating Durable Press (DP The reaction-mix was buffered with ca. 300 mM. KH2PO4 (pH rating), Smoothness rating Monsanto (Monsanto rating), 7) and incubated for 60' at 30° C. Values indicate shrinkage (warp (w) & weft (f) in %). The following table remaining formaldehyde after treatment (in ppm) in the concentrated product. Detection by the acetylacetone method. (Table 8) summarizes the results (1: non-finished cotton 2: formulation 1 3: formulation 1+FDM 4: formulation 2 5: cross-linker -FDM --FDM formulation 2+FDM 6: formulation 3 7: formulation3+FDM formulation 1 S2OO 1700 8: formulation 49: formulation 4+FDM). formulation 3 3500 1700 formulation 2 38.000 29.300 TABLE 8

1 2 3 4 5 6 7 8 9

0285. The best results were obtained on formulation 1 FA 1 196 168 53 25 35 22 42 22 with approximately 70% reduction in the formaldehyde con (LAW tent. The formaldehyde content of formulation 3 was reduced 112) by about 50%. Treatment of formulation 2 with FDM only dcra 123 198 187 187 178 180 187 18O 173 reduced the high formaldehyde content by 20%. Note that a tensile 399 361 356 340 282 304 350 342 324 DP 1 2 2 1.5 1.5 1.5 1.5 1.5 1.5 prolonged exposure to FDM can lead to product degradation. rating 0286. Overall, these preliminary results indicate that FDM Mon- 1 3 3 2 2 2.5 2.5 2.5 2.5 could potentially be applied directly on the product solutions. Santo rating Shrink- 4.0 1.4 1.4 2.0 1.6 1.8 2 2 1.8 Example 7 age W Shrink- O2 (0.4 O.4 O.O O2 O2 O O2 O.2 Use of FDM to Reduce the FA-Content in Modified age f Dimethyloldihydroxyethylene Urea Compounds 0287 Crosslinking agents are used for easy-care finishing of textiles made of cellulosic fibres and their blends with Example 8 synthetic fibers. An important class of these chemicals is the Use of FDM to Reduce the FA-Content in Sulpho class of modified dimethyloldihydroxyethylene urea com nated Melamine-FA Polycondensates Used as Poly pounds. Here we treated 4 different product formulations meric Dispersants/Superplasticizers with formaldehyde dismutase (FDM) and measured the remaining formaldehyde in the product. Moreover, enzyme 0291 Sulfonated melamine-FA polycondensates are treated product was used in cotton-finishing. Formaldehyde especially optimized for plastification and water reduction of content of the textile (cotton) and the performance of finish cement and calcium Sulphate based mixtures. Typically, these ing have been quantified. products are used as a 20% w/v aqueous Solution and the US 2012/0028333 A1 Feb. 2, 2012 22 pH-value is about 9 to 11.4 (sample 1) and 7 to 10 (sample 2). The postulated reaction mechanism for the dismutation cata Here (see Table 9) we treated the 20% w/v working-solutions lyzed by FDM is described and illustrated in Tanaka et al. of both samples with FDM (final concentration 140 mg/L) (2002), Journal of Molecular Biology, 324, 519-533. The and quantified the remaining formaldehyde after different active site of FDM from P. putida F61 at 2.3. A resolution is incubation-time using the previously described acetylacetone described and illustrated by Hasegawa et al. (2002), Acta method (values in ppm). Crystallogr., Sect. A, 58, C102-C102. Formaldehyde is tightly packed in a hydrophobic pocket (Met 337, Ile 301, Phe TABLE 9 93 and Phe 127) and is in close proximity to a zinc ion and to the NAD(H)-cofactor 20% Wiv No FDM FDM (1 h) FDM (3h) FDM (24 h) 0297 FDM from Pputida F61 has been shown to have a sample 1 5700 3200 2SOO 1800 limited degree of Substrate promiscuity (Kato et al Agric. sample 2 1700 1200 1OOO 700 1983, Biol. Chem., 47(1), 39-46). The wild-type enzyme shows a weak activity toward acetyladehyde (5-10%, our 0292 Product performance after 1 h enzymatic treatment results suggest even less) and methyl-glyoxal (22%)—the was not negatively affected with respect to set time, flow and relative activity toward formaldehyde being 100%—, but not strength. Similar results can be obtained by using as much as toward sterically more demanding Substrates such as propi /s of the enzyme concentration. In these products, removal of onaldehyde, butyraldehyde, heptaldehyde, glyceraldehyde, formaldehyde can lead to changes in the molecular weight glycolaldehyde, benzaldehyde, glyoxal or glutaraldehyde. In order to improve substrate specificity towards bulkier alde distribution of the polymer over time. hydes particularly acetaldehyde we decided to modify Example 9 the active pocket of FDM by substituting the large hydropho bic residues lining the active site with Smaller counterparts at Use of FDM to Reduce the FA-Content in a Modi position 337, 301, 127 and 93. The following single-mutants fied Anionic Polyalkylglycol-Phenol-FA Resin have therefore been prepared and characterized: 0293 Modified anionic polyalkyl-phenol-aldehyde resins are especially optimized for plastification and water reduc tion of cement and calcium Sulphate based mixtures. The FDM M7A FDM IOIL FDM F127V FDM F93L product is formulated as a concentrated aqueous Solution (pH FDM IOIA FDM F127A FDM FI 5-8). Enzyme treatment was performed by adding FDM FDMI30IV FDMF93G directly to the product. Non-treated product contains about FDMI30IG FDM F93A 250 ppm formaldehyde, as determined by the acethylacetone FDMI301S FDM F93V method. The following table (Table 10) shows the remaining formaldehyde (in ppm) after 0.5 h, 3 h and 24 h incubation with FDM. Example 11 Activity of FDM Mutants Determined by pH-Stat TABLE 10 Titration & by HPLC FDM (30 min) FDM (3h) FDM (24 h) 0298 Activity of FDM and its variants was assayed by FDM (100 mg/L) 10.2 6.8 3.5 pH-stat titration in a standard reaction mixture containing 20 FDM (10 mg/L) 125.5 26.1 2.4 mM formaldehyde, 100 mM KC1, 0.25 mM. KHPO (pH 7). The reaction was performed at RT by adding 5 uL of enzyme 0294 Product performance after 1 h of enzymatic treat solution to 25 mL of standard mixture (typically 0.1-0.5 ment was not negatively affected with respect to set time, flow ug/mL enzyme, depending on activity). Formate formation and strength. was monitored over 5' by titration with 5 mMNaOH. One unit 0295 Similar results can be obtained using non-purified is defined as the amount of enzyme necessary to catalyze the cell extracts or whole cells producing FDM. formation of 1 umol formic acid perminute. FDMFA/I'L (0.7 ug/mL) was the only mutant tested by pH-stat titration Example 10 with 20 mM acetaldehyde instead of formaldehyde at pH 7. Given that the activity of FDM and of all single mutants on Rational Design of FDM for Expanded Substrate acetaldehyde is too weak to be followed by pH-stat titration, Specificity we decided to compare FDM and its variants by HPLC 0296. The active site of FDM shows the formaldehyde (Aminex HPX-87 H column, in 5 mM HSO detection by molecule bound in a relatively narrow, predominantly hydro RI, 0.5 mL/min). Enzymatic reactions were performed in 50 phobic pocket. A strategically positioned Zinc ion is acting as mMKHPO (pH8), 100 mMKCl and 20 mMacetaldehyde. a Lewis acid and activates the Substrate for a nucleophilic Reactions were started by addition of FDM or of its variants attack by a hydrid ion donated by a tightly bound NADH (50 g/ml). Acetaldehyde, ethanol and acetic acid were quan cofactor molecule. The postulated reaction mechanism of the tified in 30' intervals (retention time: acetaldehyde 21.2 min: dismutation reaction consists of two coupled half-reactions. ethanol 25.2 min; acetic acid 18.9 min). The initial reaction In the first half-reaction one formaldehyde molecule is oxi rates were determined (30 values) and units were calculated dized irreversibly by the enzyme (E)-NAD" complex to form as the amount of enzyme needed to generate 1 umol acetic formic acid and the (E)-NADH complex. In the second half acid in 1 min. reaction another formaldehyde molecule binds to the (E)- 0299 The results clearly show that most mutations have a NADH complex and is irreversibly reduced to methanol, deleterious effect on the specific activity toward both alde leaving behind the (E)-NAD" complex for the next reaction. hydes. Only the I'L and the F.A turned out to have inter US 2012/0028333 A1 Feb. 2, 2012 esting properties. Introduction of leucine at position 300 led the wild-type protein (+5° C., FIG. 5). The F'L and the to a 2.5-fold increase in acetaldehyde activity, while the spe F'L/FA mutants show the same thermostability profile on cific activity on formaldehyde increased about 10 to 20%. The acetyladehyde (Tso ca. 55° C.). F'A variant lost formaldehyde activity almost completely, while the specific activity with acetyladehyde increased Example 13 about 3-fold compared to the wild-type enzyme and is similar Crystal Structures of FDM, FDM IL and FDM to the activity of the FDM I'L mutant (Table 11). I301 L/F93A TABLE 11 (0305 Crystals of FDM IL/FA and FDM IL were Formaldehyde Acetaldehyde measured at the Synchrotron SLS in Villingen, Switzerland pH-stat titration HPLC (Table 12). Umg Umg TABLE 12 FDM 150 O.6 FDMI30L 170 1.4 FDMI30IV 90 0.4 Statistics of X-ray data collection FDMI301A 37 O.1 FDMI30L FDMI30LF93A FDM IOG 8 O.O3 FDMI301S 5 Ila. Space group P2 I4 FDMM37A 2 O.1 Resolution range (A) SO-14 SO-18 FDM F127V 26 O.1 Reflections 274886 (50398) 147396 (21502) FDM F127A 8 O.1 Completeness (%) 96.8 (95.2) 97.5 (97.4) FDM F93L 4 Ila. Rsym 6.6 (66.1) 5.7 (49.6) FDM F93I 2 Ila. I?o 11.3 (2.2) 11.9 (2.0) FDM F93G 2 O.O3 FDM FA O.3 1.7 FDM F93V 1 Ila. (0306 The structure of FDM I'L/FA was determined FDM F93AI30L Ila. 2.5% by the molecular replacement method using the wild-type structure as a structural template (pdb code: 2DPH). As an 0300 Table 11 shows specific activities for formaldehyde example of the data quality, the wild-type phenylalanine resi (FA) and acetaldehyde (AA) of different active site mutants. due at position 93 used for the structure determination is FA activity was determined by pH-stat titration: AA activity shown together with the density map of the FDM 'L/FA was determined by HPLC, as described above. *this value is mutant (FIG. 6). The red difference electron density indicates in agreement with the activity measured by pH-stat titration. that the phenylalanine should be modeled as an alanine and a n.a. not active. water molecule is located at the C-position of the phenyl 0301 The mutations at position 301 and 93 have been ring. The overall structure of the whole enzyme does not combined to generate the FDM I'L/FA variant. This change because of the mutations. The impact of the mutations double-mutant doesn’t have any activity toward formalde is confined to the mutagenized positions and their close hyde, but has a 5-fold increased activity toward acetaldehyde. neighbors. FIG. 7 shows a close-up of the active site of the Both Substitutions have a synergistic effect in boosting activ wild type FDM. The protein is depicted in ribbon style. Fol ity with acetaldehyde (FIG. 2). lowing amino acids are shown as ball and stick: CyS 46. His 0302) While the mutant I'L shows expanded substrate 67 and Asp170 coordinate the catalytic zinc: Ile 301 and Phe specificity, the FDM FA variant and especially the 93 are the sites where the mutations were introduced; Met 337 double mutant exhibit a switch of substrate specificity from and Phe 127 are the amino acids which change their confor formaldehyde towards acetaldehyde. FIG.3 shows the activ mation under the influence of the mutations. The Zinc ion and ity profile of FDM IL by HPLC the cofactor (NADH) also are shown. 0307 Structure of the FDMI'L mutant: Example 12 0308 Mutation of isoleucine 301 to leucine is clearly seen Thermostability of FDM, FDM IL and FDM in the electron density map. The side chain adopts an orien I301 L/F93A tation pointing away from the active site leading to a stronger hydrophobic contact to Val 303, Phe 265 and Phe 57. This 0303. An important property of FDM and of its variants is enhanced interaction could be responsible for the increased the tolerance to increased temperatures both, during enzyme temperature stability. The mutation has also an effect on purification (sterilization) and during a technical application. phenylalanine 127. The whole side chain moves a little bit and We compared thermostability of FDM, FDM I'Land FDM phenyl ring rotates about 70° compared to the wild-type I'L/FA by pre-incubating the enzyme at different tem enzyme (FIG. 8). The phenyl ring is part of the substrate peratures for 20 min (25-65°C.) and, subsequently, by testing binding pocket and the new position of the side chain con the sample with acetaldehyde or formaldehyde. FDM I'L stricts the binding pocket in a way that the carbonyl group of and FDM I'L/F'A samples were tested for acetaldehyde formaldehyde and acetaldehyde is in a more favorable posi activity by HPLC, as described above. Data are shown as tion for hydrid-transfer from the NADH cofactor. Moreover, relative activities compared to the values obtained at RT (FIG. the mutation of isoleucine 301 to leucine opens up the binding 4) pocket on the opposite site allowing the sterically more 0304 FDM and FDM I'L samples were tested for form demanding acetaldehyde to be better accepted as a substrate aldehyde activity by pH-stat titration, as described above. compared to the wild-type enzyme. Data are shown as relative activities compared to the values 0309 Structure of the FDM IL/FA: obtained at RT (see FIG. 5). Interestingly, the FDM I'L 0310. The mutation of isoleucine 301 to leucine has the mutant shows a clear increase in thermostability compared to same impact on Leu 301 and on Phe 127 as in the single US 2012/0028333 A1 Feb. 2, 2012 24 mutant structure. The second mutation (Phe 93 to Ala) gives 0313 The formaldehyde content has been determined by room for a water molecule (HO 339) which is located near the acetylacetone-method, as described in Example 3. the C-position of the phenyl ring of the wild type. The water molecule is hydrogen bonded to the nicotinamide oxygen of Example 15 NADH and to the carboxyl oxygen of Asp170 which is one of Use of a Dry Formulation of the FDM I'L Mutant the ligands of the catalytic zinc. A third weak hydrogen bond to Reduce the FA-Content in a Modified Beta-Naph is formed with the main chain carbonyl oxygen of Ala 338. thaline-FA Sulfonate Resin The strong hydrogen bond of the water molecule to Asp 170 distorts the conformation of the side chain and, therefore 0314. The water soluble polymer solution (modified beta weakens the Zinc coordination. In order to make the hydrogen naphthaline-FA sulfonate resin) was treated with different bond, the side chain of Asp170 has to rotate about 14° (FIG. amounts of a dry formulation of FDM I'L (20 to 200 mg 9). By this rotation the distance between the zinc ion and its formulation/L polymer Solution) and incubated at room tem carboxyl oxygen increases from 2.0 to 2.4A. The loss of the perature for 5 h. At this time the same amount of enzyme was optimal Zinc coordination could explain the overall reduced added and incubation continued for up to 11 days. The fol activity of the double mutant. Moreover, the larger active site lowing table (Table 14) shows the remaining formaldehyde compared to the wild-type enzyme or the I301L mutant, seem (in ppm) after 5h, 24h 4 days, 7 days and 11 days using 2x50 to prevent that formaldehyde can adopt a productive orienta mg/L of dry enzyme formulation. tion for catalysis. This could explain the observed switch in specificity from formaldehyde to acetaldehyde. A second effect of the mutation is the altered conformation of Met 337. Sh 24h 4d 7d 11 d The C-atom of Met 337 is rotated by 90° towards the position No enzyme 395 ppm 397 ppm. 384 ppm 386 ppm 383 ppm of the former phenyl ring. This conformation is a very low FDMI30L 278 ppm 266 ppm 185 ppm 87 ppm 50 ppm energy conformation because it is the most preferred rotamer (2x 50 mg/L) for methionines in proteins. 0311. Overall these results suggest that the FDM I'L single mutant is the most preferable candidate for technical 0315. The formaldehyde content has been determined by applications. the acetylacetone-method, as described in Example 3. Example 16 Example 14 Use of a Dry Formulation of the FDM I'L Mutant Use of a Dry Formulation of the FDM I'L Mutant to Reduce the FA-Content in a Stabilized Anionic to Reduce the FA-Content in a Modified Anionic Acrylate Copolymer Dispersion Polyalkylglycol-Phenol-FA Resin 0316 A stabilized anionic acrylate copolymer dispersion in water (54% solid content) has been treated with 10 mg/L of 0312. A further modified anionic polyalkylglycol-phenol the dry FDM I'L formulation at room temperature. After FA resin with a residual FA content of about 120 ppm was centrifugation (2-4 hat 14.000 rpm, RT), the content of form treated with a dry FDMI'L formulation (specific activity of aldehyde in the clear aqueous phase was determined. The formulation: ca. 50U/mg). The test was performed by adding following table (Table 15) shows the formaldehyde content 50 mg/L of enzyme powder to the resin followed by incuba (in ppm) of the dispersion after 5h, 24 hand 96 hofenzymatic tion at room temperature. The following table (Table 13) shows the remaining formaldehyde (in ppm) after 5 h and treatment. after 42 days.

Sh 24h 96 h after Sh after 42 days No enzyme 370 ppm 310 ppm 260 ppm FDM IL (10 mg/L) 304 ppm 170 ppm 150 ppm No enzyme 126 ppm 119 ppm FDM IL (50 mg/L) 2 ppm 1 ppm 0317. The formaldehyde content has been determined by the acetylacetone-method, as described in Example 3.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 10

<21 Os SEQ ID NO 1 &211s LENGTH: 1740 &212s. TYPE: DNA <213> ORGANISM: Pseudomonas putida 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (323) . . (1522)

US 2012/0028333 A1 Feb. 2, 2012 27

- Continued

Asn Asn Lieu Val Asn Pro Asp Ala Asp Lieu. Gly Ala Phe Gly Phe Asp 115 12 O 125 Lieu Lys Gly Trp Ser Gly Gly Glin Ala Glu Tyr Val Lieu Val Pro Tyr 13 O 135 14 O Ala Asp Tyr Met Lieu Lleu Lys Phe Gly Asp Llys Glu Glin Ala Met Glu 145 150 155 160 Lys Ile Lys Asp Lieu. Thir Lieu. Ile Ser Asp Ile Lieu Pro Thr Gly Phe 1.65 17O 17s His Gly Cys Val Ser Ala Gly Val Llys Pro Gly Ser His Val Tyr Ile 18O 185 19 O Ala Gly Ala Gly Pro Val Gly Arg Cys Ala Ala Ala Gly Ala Arg Lieu 195 2OO 2O5 Lieu. Gly Ala Ala Cys Val Ile Val Gly Asp Glin Asn Pro Glu Arg Lieu. 21 O 215 22O Llys Lieu. Lieu. Ser Asp Ala Gly Phe Glu Thir Ile Asp Lieu. Arg Asn. Ser 225 23 O 235 24 O Ala Pro Lieu. Arg Asp Glin Ile Asp Glin Ile Lieu. Gly Llys Pro Glu Val 245 250 255 Asp Cys Gly Val Asp Ala Val Gly Phe Glu Ala His Gly Lieu. Gly Asp 26 O 265 27 O Glu Ala Asn Thr Glu Thr Pro Asn Gly Ala Lieu. Asn. Ser Lieu. Phe Asp 27s 28O 285 Val Val Arg Ala Gly Gly Ala Ile Gly Ile Pro Gly Ile Tyr Val Gly 29 O 295 3 OO Ser Asp Pro Asp Pro Val Asn Lys Asp Ala Gly Ser Gly Arg Lieu. His 3. OS 310 315 32O Lieu. Asp Phe Gly Lys Met Trp Thr Lys Ser Ile Arg Ile Met Thr Gly 3.25 330 335 Met Ala Pro Val Thr Asn Tyr Asn Arg His Lieu. Thr Glu Ala Ile Leu 34 O 345 35. O Trp Asp Gln Met Pro Tyr Lieu Ser Llys Val Met Asn Ile Glu Val Ile 355 360 365 Thir Lieu. Asp Glin Ala Pro Asp Gly Tyr Ala Lys Phe Asp Llys Gly Ser 37 O 375 38O Pro Ala Lys Phe Val Ile Asp Pro His Gly Met Lieu Lys Asn Lys 385 390 395

<210s, SEQ ID NO 3 &211s LENGTH: 1197 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Codon-optimized DNA sequence for FDM from Pseudomonas putida F61 <4 OOs, SEQUENCE: 3 atggcgggta ataaatcggt ggtgt atcat ggt accc.gcg acctg.cgtgt taalaccgtg 6 O ccgitat coga agctggaaca taacaacct aaactggaac acgcagttat cottaaagtt 12 O gtgagcacca acatttgtgg tagcgatcag cat attt acc gcggcc.gctt tattgttgcc.g 18O aaaggc catgttt taggc.ca caaattacc ggcgaagttcg tdgaaaaagg cagtgatgtt 24 O gaactgatgg at attggcga t ctggitat co gtgcc.gttta acgtc.gc.gtg cggtcgttgc 3OO

US 2012/0028333 A1 Feb. 2, 2012 36

- Continued aaaattaaag atctgaccct gat ct cqgat atcct gccaa cqggittittca tdgctg.cgitt 54 O tctg.ccggtg ttaa.gc.ctgg cagc.catgtc. tat attgcgg gcgcaggtoc agt cq9tcgc 6OO tgtgcagc.cg cgggtgcgcg cctgctgggc gcc.gcatgcg tdatcgtggg ggaccagaac 660 ccggagcgtc. tcaaactgtt gtctgatgcc ggctittgaaa C cattgactt acgcaat agt 72 O gcgc.cgctgc gtgat Cagat catcagatc ttaggcaaac C9gaagtgga citgcggcgtg 78O gatgcggtgg gct tcgaagc acatggctta ggtgatgaag C caat actga gacgc.cgaac 84 O ggcgc.cct ga acagcttgtt cacgtggtg cc.gctgggg gcgct attgg tatt ccgggit 9 OO

Ctgtatgtcg ggtctgat Co ggat.ccggtgaacaaagatg ccggctc.cgg cc.gc.ctgcac 96.O ttggattt cq gcaaaatgtg gaccalaatcc attcgitatica tacggggat ggcgc.cagtt 1 O2O actaattata accgtcatct gacggaag.cg attctgtggg accagatgcc gitatctgtcg 108 O aaagtaatga acatcgaagt tattaccctg gaccaggcgc catggitta togalaattit 114 O gatalaaggca gtc.cggcgala atttgttgatt gatccacatg gCatgttgaa aaataaataa 12 OO

<210s, SEQ ID NO 8 &211s LENGTH: 399 212. TYPE: PRT <213s ORGANISM: Pseudomonas 22 Os. FEATURE: <223> OTHER INFORMATION: formaldehyde dismutase Ile-301-Leu <4 OOs, SEQUENCE: 8 Met Ala Gly Asn Llys Ser Val Val Tyr His Gly Thr Arg Asp Lieu. Arg 1. 5 1O 15 Val Glu Thr Val Pro Tyr Pro Llys Lieu. Glu. His Asn. Asn Arg Llys Lieu 2O 25 3O Glu. His Ala Val Ile Lieu Lys Val Val Ser Thr Asn. Ile Cys Gly Ser 35 4 O 45 Asp Gln His Ile Tyr Arg Gly Arg Phe Ile Val Pro Llys Gly. His Val SO 55 6 O Lieu. Gly His Glu Ile Thr Gly Glu Val Val Glu Lys Gly Ser Asp Wall 65 70 7s 8O Glu Lieu Met Asp Ile Gly Asp Lieu Val Ser Val Pro Phe Asin Val Ala 85 90 95 Cys Gly Arg Cys Arg Asn. Cys Lys Glu Ala Arg Ser Asp Val Cys Glu 1OO 105 11 O Asn Asn Lieu Val Asn Pro Asp Ala Asp Lieu. Gly Ala Phe Gly Phe Asp 115 12 O 125 Lieu Lys Gly Trp Ser Gly Gly Glin Ala Glu Tyr Val Lieu Val Pro Tyr 13 O 135 14 O Ala Asp Tyr Met Lieu Lleu Lys Phe Gly Asp Llys Glu Glin Ala Met Glu 145 150 155 160 Lys Ile Lys Asp Lieu. Thir Lieu. Ile Ser Asp Ile Lieu Pro Thr Gly Phe 1.65 17O 17s His Gly Cys Val Ser Ala Gly Val Llys Pro Gly Ser His Val Tyr Ile 18O 185 19 O Ala Gly Ala Gly Pro Val Gly Arg Cys Ala Ala Ala Gly Ala Arg Lieu 195 2OO 2O5 Lieu. Gly Ala Ala Cys Val Ile Val Gly Asp Glin Asn Pro Glu Arg Lieu. 21 O 215 22O

US 2012/0028333 A1 Feb. 2, 2012 38

- Continued ttggattt cq gcaaaatgtg gaccalaatcc attcgitatica tacggggat ggcgc.cagtt 1 O2O actaattata accgtcatct gacggaag.cg attctgtggg accagatgcc gitatctgtcg 108 O aaagtaatga acatcgaagt tattaccctg gaccaggcgc catggitta togalaattit 114 O gatalaaggca gtc.cggcgala atttgttgatt gatccacatg gCatgttgaa aaataaataa 12 OO

<210s, SEQ ID NO 10 &211s LENGTH: 399 212. TYPE: PRT <213s ORGANISM: Pseudomonas 22 Os. FEATURE: <223> OTHER INFORMATION: formaldehyde dismutase Phe-93-Ala / Ile-301-Leu

<4 OOs, SEQUENCE: 10 Met Ala Gly Asn Llys Ser Val Val Tyr His Gly Thr Arg Asp Lieu. Arg 1. 5 1O 15

Val Glu Thr Val Pro Tyr Pro Llys Lieu. Glu. His Asn. Asn Arg Llys Lieu 2O 25 3O

Glu. His Ala Val Ile Lieu Lys Val Val Ser Thr Asn. Ile Cys Gly Ser 35 4 O 45

Asp Gln His Ile Tyr Arg Gly Arg Phe Ile Val Pro Llys Gly. His Val SO 55 6 O

Lieu. Gly His Glu Ile Thr Gly Glu Val Val Glu Lys Gly Ser Asp Wall 65 70 7s 8O

Glu Lieu Met Asp Ile Gly Asp Lieu Val Ser Val Pro Ala Asn. Wall Ala 85 90 95

Cys Gly Arg Cys Arg Asn. Cys Lys Glu Ala Arg Ser Asp Val Cys Glu 1OO 105 11 O

Asn Asn Lieu Val Asn Pro Asp Ala Asp Lieu. Gly Ala Phe Gly Phe Asp 115 12 O 125

Lieu Lys Gly Trp Ser Gly Gly Glin Ala Glu Tyr Val Lieu Val Pro Tyr 13 O 135 14 O

Ala Asp Tyr Met Lieu Lleu Lys Phe Gly Asp Llys Glu Glin Ala Met Glu 145 150 155 160

Lys Ile Lys Asp Lieu. Thir Lieu. Ile Ser Asp Ile Lieu Pro Thr Gly Phe 1.65 17O 17s

His Gly Cys Val Ser Ala Gly Val Llys Pro Gly Ser His Val Tyr Ile 18O 185 19 O

Ala Gly Ala Gly Pro Val Gly Arg Cys Ala Ala Ala Gly Ala Arg Lieu 195 2OO 2O5

Lieu. Gly Ala Ala Cys Val Ile Val Gly Asp Glin Asn Pro Glu Arg Lieu. 21 O 215 22O

Llys Lieu. Lieu. Ser Asp Ala Gly Phe Glu Thir Ile Asp Lieu. Arg Asn. Ser 225 23 O 235 24 O

Ala Pro Lieu. Arg Asp Glin Ile Asp Glin Ile Lieu. Gly Llys Pro Glu Val 245 250 255

Asp Cys Gly Val Asp Ala Val Gly Phe Glu Ala His Gly Lieu. Gly Asp 26 O 265 27 O US 2012/0028333 A1 Feb. 2, 2012 39

- Continued

Glu Ala Asn Thr Gu Thir Pro Asn Gly Ala Lieu. Asn Ser Lieu. Phe Asp 27s 285

Wall Wall Arg Ala Gly Gly Ala Ile Gly Ile Pro Gly Lieu. Tyr Val Gly 29 O 295 3 OO

Ser Asp Pro Asp Pro Wall Asn Asp Ala Gly Ser Gly Arg Luell His 3. OS 310 315 32O

Lieu. Asp Phe Gly Lys Met Trp Thir Ser Ile Arg Ile Met Thir Gly 3.25 330 335

Met Ala Pro Wall Thr Asn Tyr Asn His Lieu. Thir Glu Ala Ile Lieu. 34 O 35. O

Trp Asp Glin Met Pro Tyr Lieu. Ser Wal Met Asn Ile Glu Wall Ile 355 360 365

Thir Luell Asp Glin Ala Pro Asp Gly Ala Phe Asp Gly Ser 37 O 375

Pro Ala Lys Phe Wall Ile Asp Pro His Gly Met Lell Asn Lys 385 390 395

1. Use of an enzyme preparation that catalyzes the degra (i) an amino acid sequence represented by any one of SEQ dation of formaldehyde for reducing the formaldehyde con ID NO: 8 or 10; tent in a formaldehyde-containing formulation. (ii) an amino acid sequence having at least 50%, 51%, 52%, 2. The use of claim 1, wherein the enzyme preparation 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, contains an enzyme of the classification E.C. 1.2. 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 3. The use of claim 1, wherein the enzyme preparation 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, contains an enzyme which has its co-enzyme tightly bound. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 4. The use of claim 1, wherein the enzyme preparation 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, contains an enzyme which comprises the amino acid 98%, or 99% sequence identity to the amino acid sequence of SEQID NO:2 or a variant thereof. sequence represented by any one of SEQID NO: 8, 10 or 5. The use according to claim 4, wherein the variant com 12; and prises an amino acid sequence having the phenylalanine at (iii) derivatives of any of the amino acid sequences given in position 93 and/or the isoleucine at position 301, and/or the (i) or (ii) above. methionine at position 337 and/or the phenylalanine at posi 23. An isolated nucleic acid coding for a polypeptide as tion 127 substituted by any other amino acid. defined in claim 21. 6. The use of claim 1, wherein the formulation is a resin. 24. The nucleic acid of claim 23, wherein the nucleic acid 7. The use of claim 6, wherein the resin is a cross-linking is selected from agent Suitable for finishing textile fabrics or a fixation agent (i) a nucleic acid represented by any one of SEQID NO: 7 for pigment printing or 9: 8. The use of claim 7, wherein the textile fabric contains (ii) the complement of a nucleic acid represented by any cellulosic fibers. one of SEQID NO: 7 or 9: 9. The use of claim 8, wherein the textile fabric is cotton. (iii) a nucleic acid encoding the polypeptide as represented 10. The use of claim 6, wherein the resin is a polymer by any one of SEQID NO: 8 or 10, preferably as a result dispersant. of the degeneracy of the genetic code, said isolated 11. The use of claim 10, wherein the polymer dispersant is nucleic acid can be derived from a polypeptide sequence selected from the group consisting of naphthalene formalde as represented by any one of SEQID NO: 8 or 10; hyde condensates, phenol formaldehyde condensates, urea (iv) a nucleic acid having at least 30%, 31%, 32%, 33%, formaldehyde condensates, and melamine formaldehyde 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, condensates and mixtures thereof. 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 12-20. (canceled) 52%. 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 21. An isolated polypeptide having aldehyde dismutase 61%. 62%, 63%, 64%. 65%, 66%, 67%, 68%, 69%, activity and comprising a variant of SEQID NO:2 wherein 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, the phenylalanine at position 93 of SEQID NO:2, and/or the 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, isoleucine at position 301 of SEQ ID NO:2, and/or the 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, methionine at position 337 of SEQ ID NO:2, and/or the 97%, 98%, or 99% sequence identity with any of the phenylalanine at position 127 of SEQID NO:2 is substituted nucleic acid sequences of SEQID NO: 7 or 9: by any other amino acid. (v) a nucleic acid molecule which hybridizes with a nucleic 22. The polypeptide of claim 21, wherein the polypeptide is acid molecule of (i) to (iv) under stringent hybridization selected from conditions; US 2012/0028333 A1 Feb. 2, 2012 40

(vi) a nucleic acid encoding a polypeptide having aldehyde 26. A recombinant expression vector comprising the dismutase activity, the polypeptide having at least 50%, expression cassette of claim 25. 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 27. A recombinant microorganism carrying the expression 60%, 61%, 62%, 63%, 64%. 65%, 66%, 67%, 68%, vector of claim 26. 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 28. A method of preparing a polypeptide as defined in 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, claim 21, the method comprising cultivating a recombinant 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, microorganism, and, optionally, isolating said polypeptide 96%, 97%, 98%, or 99% sequence identity to the amino from the culture. acid sequence represented by any one of SEQID NO: 8 29. Use of a polypeptide as defined in claim 21 for reducing or 10. the aldehyde content in an aldehyde-containing formulation. 25. An expression cassette, comprising a nucleic acid of 30. (canceled) claim 23, operably linked with at least one regulatory nucleic acid sequence.