Solution-stable enzyme composition

Abstract

To provide solution-stable enzyme compositions that are suitable for use in industrial applications such as in the manufacture of pulp and paper.SOLUTION: Provided is a solution-stable enzyme composition, which is suitable for use in industrial applications such as in the manufacture of pulp and paper, the enzyme composition comprising an enzyme component having a specific sequence, a stabilizer component comprising an organic sugar alcohol, an optional antimicrobial preservative component that prevents the growth of microorganisms, and water. Also provided is its use in the manufacture of pulp and paper.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to solution-stable enzyme compositions that are suitable for use in industrial applications such as, for example, in pulp and paper manufacturing. [Background technology]

[0002] Solution-stable enzyme compositions intended for commercial use advantageously have sufficient stability and good shelf life during storage. Compositions containing sterol esterases are useful for the hydrolysis and synthesis of esters, such as esters of phytosterols, cholesterol and glycerol with carboxylic acids, especially fatty acids. Such compositions can be applied industrially as processing aids, for example in pulp and paper production and recycling, so long as the enzymes are active under conditions typically encountered in these processes.

[0003] Many methods have been pursued to increase the stability of enzymes, and proteins in general, in aqueous solutions, as reviewed in Gianfreda et al., Molecular and Cellular Biochemistry 1991, pp. 97-128. These methods include glycosylation, PEGylation, cross-linking, protein modification by site-directed mutagenesis, and chemical modification of amino acids. Another method is the addition of additives, such as pH buffers, salts, detergents, amino acids, sugars, organic solvents, polymers, and cyclodextrins.

[0004] For example, hydroxypropyl-β-cyclodextrin, sorbitol, and the surfactant polyethylene glycol sorbitan monolaurate, known under the trade name Tween 20, were evaluated for their stabilizing effects on porcine growth hormone protein by Charman et al., Pharmaceutical Research 1993, pp. 954-962, and it was concluded that, in contrast to hydroxypropyl-β-cyclodextrin and Tween 20, sorbitol showed only a minimal effect and did not lead to any advantage against precipitation.

[0005] No additive is known that is effective in stabilizing all protein and enzyme solutions. Nevertheless, certain additives have been found that stabilize specific enzymes. For example, serine protease inhibitors have been found to stabilize lipases (EP 2521732). Lipolytic enzymes in lipid products for pulp and paper applications, such as Resinase HT and Stickaway from Novozymes, as disclosed in the relevant data sheets, are stabilized with propylene glycol. The pH of Stickaway is between 6.5 and 9.5. At pH values ​​greater than 8, sterol esterase from Melanocarpus albomyces is apparently unstable and is more stable within the pH range between pH 3 and 7, as established by Kontkanen et al., Applied Microbiology and Biotechnology 2006, pp. 696-704.

[0006] Pleiss et al., Journal of Molecular Catalysis B 2000, 491-508, established that nearly all lipases, many esterases, and all known cutinases, as well as serine proteases, have a catalytic triad composed of serine, histidine, and aspartate.

[0007] Instead of aspartate, glutamine is present in the catalytic triad of sterol esterase, where the catalytic triad is composed of serine, histidine and glutamine.

[0008] General lipases, also called triacylglycerol lipases and classified as EC 3.1.1.3 according to the IUBMB enzyme nomenclature, such as those from Candida antarctica, Thermomyces lanuginosus and Rhizomucor miehei studied by Kontkanen et al., Journal of Biochemistry 2004, 51-59, are highly active on glycerol esters but show no measurable activity on sterol esters.

[0009] Sterol esterases, classified as EC 3.1.1.13, hydrolyze many different esters, including esters of sterols and glycerol with short- and long-chain carboxylic acids.

[0010] Acetylcholinesterases, classified as EC 3.1.1.7, hydrolyze acetylcholine and other choline esters. Their distinction between esterases and lipases due to their sequence and three-dimensional structure is reviewed in Fojan et al., Biochimie 2000, pp. 1033-1041.

[0011] The enzyme activity on a mixture of plant and wood sterol esters was compared with that on cholesterol esters by Kontkanen et al., Journal of Biochemistry 2004, pp. 51-59, and it was concluded that pure cholesterol esters can be used as model substrates for sterol esterases. Examples of sterols include cholesterol, phytosterols, and ergosterol, which are naturally occurring compounds in animals, plants, and fungi, respectively. Cholesterol esterases from animals are generally the true cholesterol esterases. Cholesterol esterases from microorganisms are, in general terms, sterol esterases.

[0012] In any case, sterol esterases and cholesterol esterases are in one enzyme class, EC 3.1.1.13.

[0013] To maintain the stability of aqueous solutions of sterol esterase from Melanocarpus albomyces, Kontkanen et al., Enzyme and Microbial Technology 2006, pp. 265-273, used 1% polyethylene glycol p-isooctylphenyl ether, a non-ionic surfactant also known by the trade name Trion X-100.

[0014] Therefore, no stabilizing compound effective against all enzymes has been reported yet, and there is a need to provide a liquid composition in which enzymes are stably present. Summary of the Invention

[0015] Although previously unexpected, the inventors have discovered that certain organic polyhydroxy compounds stabilize aqueous compositions containing enzymes having a WGESAG sequence motif and a catalytic triad composed of S, H and E / D. If the organic polyhydroxy compounds are present in appropriate concentrations and have a sufficient number of hydroxyl groups per molecule, the stabilizing effect can be achieved even without the addition of a surfactant.

[0016] According to a first aspect of the present invention, (a) an enzyme component comprising an enzyme characterized by having the amino acid sequence WGESAG and a catalytic triad composed of S, H and E / D; (b) a stabilizer component comprising an organic sugar alcohol having three or more hydroxyl groups per molecule, said stabilizer component being at least one-fifth by weight of the total enzyme component; (c) an optional antimicrobial preservative component to prevent microbial growth; and (d) water dissolved in said components (a), (b) and (c); A solution-stable enzyme composition comprising: A solution-stable enzyme composition is provided, in which the enzyme components of the solution-stable enzyme composition remain in solution for at least four weeks upon storage at 37°C.

[0017] In accordance with another aspect of the present invention, there is provided a solution-stable enzyme composition comprising: (a) an enzyme component comprising an enzyme characterized by having the amino acid sequence WGESAG and a catalytic triad consisting of S, H and E / D; (b) a stabilizer component comprising an organic polyhydroxy compound having three or more, four or more hydroxyl groups per molecule; (c) an optional antimicrobial preservative component which prevents the growth of microorganisms; and (d) water dissolved in components (a), (b) and (c).

[0018] In one embodiment, the concentration of the stabilizer component in the composition is 20 to 75% by weight, preferably 25 to 70% by weight, more preferably 30 to 65% by weight, and most preferably 33 to 60% by weight. In one embodiment, the lower limit of the concentration of the stabilizer component is 20% by weight, preferably 22% by weight, 24% by weight, 25% by weight, more preferably 26% by weight, 28% by weight, 30% by weight, and most preferably 33% by weight. In one embodiment, the upper limit of the concentration of the stabilizer component is 75% by weight, preferably 74% by weight, 72% by weight, 70% by weight, more preferably 68% by weight, 66% by weight, 65% by weight, and most preferably 60% by weight.

[0019] In certain embodiments, the amount of water in the composition is at least 10% by weight, preferably at least 12%, 14%, 15%, more preferably at least 16%, 18%, 20%, and most preferably at least 25% by weight.

[0020] In one embodiment, the pH of the composition is between 2 and 10, preferably between 2.5 and 9, more preferably between 3 and 8, and most preferably between 3.5 and 7.5.

[0021] In a second aspect of the invention, there is provided the use of a solution-stable enzyme composition according to the above aspect in the manufacture of pulp, and a method of use. In one embodiment, the solution-stable enzyme composition is used by adding it in such a way that it is contacted with the material used in the manufacture of pulp that requires enzyme treatment.

[0022] In a third aspect of the invention there is provided the use of a solution-stable enzyme composition according to the above aspect in the manufacture of paper, and a method of use. In one embodiment the solution-stable enzyme composition is used by adding it in such a way that it is contacted with materials used in the manufacture of paper that require enzyme treatment.

[0023] Sequence Listing The sequence below is the mature protein excluding the signal peptide.

[0024] The sequences are written from the amino to the carboxyl terminus. The actual enzyme molecule present in aqueous solution can be truncated from both ends of the sequence (amino and / or carboxyl terminus) without losing the enzyme function. For example, in comparison with the sequence of SEQ ID NO: 1, Kontkanen et al., Enzyme and Microbial Technology 2006, pp. 265-273, found that the first 13 amino acids at the amino terminus of the sequence of SEQ ID NO: 1 were deleted in preparations of sterol esterase from Melanocarpus albomyces, since their sequences start with AAPXVEISTG. Thus, in one embodiment, an N-terminally truncated enzyme having enzymatic activity is disclosed.

[0025] SEQ ID NO:1 is the sequence of a sterol esterase from Melanocarpus albomyces.

[0026] SEQ ID NO:2 is the sequence of a sterol esterase from Chaetomium thermophilum.

[0027] SEQ ID NO:3 is the sequence of a synthetic enzyme obtained from an alignment of the sequences of sterol esterases from Scytalidium thermophilum, Myceliophthora thermophila, Thielavia terestri, Corynascus thermophilus, Myriococcum thermophilum, Melanocarpus albomyces and Chaetomium thermophilum.

[0028] SEQ ID NO:4 is the sequence of a sterol esterase from Myceliophthora thermophila.

[0029] SEQ ID NO:5 is the sequence of a sterol esterase from Corynascus thermophilus.

[0030] SEQ ID NO:6 is the sequence of a sterol esterase from Myriococcum thermophilum.

[0031] SEQ ID NO: 7 is the sequence of a sterol esterase from Thielavia australiensis.

[0032] SEQ ID NO:8 is the sequence of a sterol esterase from Thielavia terestri.

[0033] SEQ ID NO:9 is the sequence of a sterol esterase from Scytalidium thermophilum.

[0034] SEQ ID NO: 10 is the sequence of a sterol esterase from Malbranchea cinnamomea.

[0035] SEQ ID NO:11 is the sequence of a sterol esterase from Thermomyces stellatus.

[0036] SEQ ID NO: 12 is the sequence of a sterol esterase from Chaetomium globosum.

[0037] SEQ ID NO: 13 is the sequence of a sterol esterase from Madurella mycetomatis.

[0038] SEQ ID NO: 14 is the sequence of a sterol esterase from Sordaria macrospora.

[0039] SEQ ID NO: 15 is the sequence of a sterol esterase from Podospora anserina.

[0040] SEQ ID NO: 16 is the sequence of a sterol esterase from Neurospora tetrasperma.

[0041] SEQ ID NO: 17 is the sequence of a sterol esterase from Neurospora crassa.

[0042] SEQ ID NO: 18 is the sequence of a sterol esterase from Coniochaeta ligniaria.

[0043] SEQ ID NO: 19 is the sequence of a sterol esterase from Cutaneotrichosporon oleaginosum.

[0044] SEQ ID NO: 20 is the sequence of a sterol esterase from Sporothrix schenckii.

[0045] SEQ ID NO:21 is the sequence of a sterol esterase from Stachybotrys chlorohalonata.

[0046] SEQ ID NO: 22 is the sequence of a sterol esterase from Colletotrichum orchidophilum.

[0047] SEQ ID NO: 23 is the sequence of a sterol esterase from Colletotrichum incanum.

[0048] SEQ ID NO: 24 is the sequence of a sterol esterase from Colletotrichum tofieldiae.

[0049] SEQ ID NO: 25 is the sequence of a sterol esterase from Hypoxylon sp. EC38.

[0050] SEQ ID NO:26 is the sequence of a sterol esterase from Aspergillus glaucus.

[0051] SEQ ID NO: 27 is the sequence of a sterol esterase from Eutypa lata.

[0052] SEQ ID NO: 28 is the sequence of a sterol esterase from Fusarium oxysporum.

[0053] SEQ ID NO: 29 is the sequence of a sterol esterase from Fusarium avenaceum.

[0054] SEQ ID NO: 30 is the sequence of a sterol esterase from Diaporthe ampelina.

[0055] SEQ ID NO:31 is the sequence of a sterol esterase from Ophiostoma piceae.

[0056] SEQ ID NO:32 is the sequence of a sterol esterase from Pleurotus sapidus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Sterol esterases are enzymes that act on sterol ester bonds, in particular they catalyze the hydrolysis, alcoholysis, acidolysis, transacylation, transesterification and / or synthesis of sterol esters. In one embodiment, the sterol esterase of the present invention belongs to the class EC 3.1.1.13.

[0058] Enzymes are catalytic proteins.

[0059] A protein is a polypeptide.

[0060] A polypeptide is a long chain of amino acids joined by amino bonds. In one embodiment, a peptide is a molecule made up of up to 20 amino acids, and a polypeptide is a molecule made up of more than 20 amino acids.

[0061] The generally accepted IUPAC single letter abbreviations for amino acids and polypeptide side chains are used, specifically, S is serine, H is histidine, A is alanine, G is glycine, E is glutamic acid and glutamate, D is aspartic acid and aspartate, and W is tryptophan.

[0062] A fragment of an enzyme characterized by a specific amino acid sequence is an enzymatically active fragment of a polypeptide that lacks one or more amino acids from the amino and / or carboxyl termini of the sequence, e.g., by alternative splicing, and / or has one or more deletions and / or one or more insertions of amino acids in the sequence. In one embodiment, an enzyme fragment has the same or similar enzymatic activity, and optionally stability, as the unfragmented enzyme.

[0063] Propylene glycol is 1,2-propanediol, an organic polyhydroxy compound with two hydroxyl groups per molecule. 1,2-propanediol exists as two optical isomers and mixtures thereof, including racemic mixtures, which are included in the definition of propylene glycol in this invention.

[0064] Sugar alcohols are organic compounds that can be produced by hydrogenation of carbohydrates, especially monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides. Hydrogenation causes the reduction of aldehyde or ketone groups to hydroxyl groups. Monosaccharides are aldehydes or ketones with two or more hydroxyl groups per molecule. Sugar alcohols therefore have three or more, or four or more hydroxyl groups per molecule.

[0065] Cyclitols are cycloalkanes with hydroxyl groups on three or more, or four or more ring atoms. Cyclitols have three or more, or four or more hydroxyl groups per molecule.

[0066] Pulp is a moist mass of material originally obtained from plants, such as wood, cotton, papyrus, wheat, fruit, paper and rags.

[0067] The ester of cholesterol with linoleic acid is cholesterol linoleate.

[0068] Thermophilic fungi are fungi which grow at temperatures of 45°C, preferably 50°C or higher.

[0069] A thermostable enzyme is an enzyme that retains at least 50% enzymatic activity after incubation in an aqueous composition or aqueous environment or solution at 50° C., preferably at 60° C., more preferably at 70° C., and most preferably at 75° C. for at least 5 minutes, preferably at least 10 minutes, more preferably at least 30 minutes, and most preferably at least 1 hour. Preferably, a thermostable enzyme retains at least 50% enzymatic activity after incubation in an aqueous solution at 50° C. for at least 5 minutes. Enzyme activity may be measured according to Example 1 below.

[0070] The words "a", "an" and "another" mean one or more than one, i.e. at least one, including one or several of the grammatical objects "a", "an" or "another".

[0071] The term "comprise" and variations thereof, such as "comprises" and "comprising," are inclusive and include possible additional components. These terms may also, in certain embodiments, include their narrower meaning of "consisting of."

[0072] In one embodiment of this enzyme, the catalytic triad is composed of serine, histidine and glutamate / aspartate, and the serine of the catalytic triad is incorporated in the sequence WGESAG.

[0073] The catalytic triad is a complex of three amino acid residues involved in catalysis. Such residues of the catalytic triad function as nucleophiles, bases and acids during catalysis. In addition to the catalytic triad, the oxoanion hole and hydrophobic substrate binding site are also formed by the active site residues of the enzyme. Residues of the catalytic triad can be identified by mutation experiments, by structure determination, or by sequence alignment with homologs having known catalytic residues. Examples of sequence alignment tools include, for example, Clustal Omega, PfamScan, Muscle and Emboss Needle using the Needleman-Wunsch algorithm, etc. These tools are available online, for example, at https: / / www.ebi.ac.uk / services / all and at https: / / www.ebi.ac.uk / Tools / pfa / .

[0074] Catalytic triads can be found in hydrolases and transferases. The positions of the catalytic triad in SEQ ID NOs: 1-9 are S221, H466, and E353. Examples of enzymes of the present invention include, but are not limited to, enzymes comprising the amino acid sequences disclosed in SEQ ID NOs: 1-32 or fragments thereof. Further examples of enzymes of the present invention include, but are not limited to, sterol esterases from Melanocarpus albomyces, Thielavia terestri, Thielavia australiensis, Corynascus thermophilus, Myriococcum thermophilum, Malbranchea cinnamomea, Thermomyces stellatus and other thermophilic fungi. Such thermophilic fungi include, but are not limited to, thermophilic ascomycetes (e.g., Canariomyces thermophila, Chaetomium pingtungium, Chaetomium britannicum, Chaetomium mesopotamicum, Chaetomium senegalensis, Chaetomium thermophile, Chaetomium virginicum, Corynascus sepedonium, Corynascus thermophilus, Crassicarpone thermophilum, etc.) thermophilum), Coonemeria aegyptiacaaegyptiaca, Coonemeria crustacea, Dactylomyces thermophilus, Melanocarpus albomyces, Melanocarpus thermophilus, Myriococcum thermophilum, Crassicarpon hotsonii, Talaromyces byssochlamydioides, Talaromyces duponti, Talaromyces emersonii, Talaromyces thermophilus, Thermoascus aurantiacus aurantiacus, Thermomyces stellatus, Thielavia australiensis, Thielavia minor, Thielavia terricola, etc.), thermophilic zygomycetes (e.g. Rhizomucor miehei, Rhizomucor nainitalensis, Rhizomucor pusillus, Rhizopus microspores, Rhizopus rhizopodiformis, etc.), and thermophilic zygomycetes (e.g. Rhizomucor miehei, Rhizomucor nainitalensis, Rhizomucor pusillus, Rhizopus microspores, Rhizopus rhizopodiformis, etc.). deuteromycetes (e.g., Acremonium alabamense, Acremonium thermophilum, Arthrinium pterospermum)pterospermum, Chrysosporium tropicum, Malbranchea cinnamomea, Myceliophthora fergusi, Myceliophthora hinnulea, Myceliophthora thermophila, Scytalidium indonesicum, Scytalidium thermophilum, Remersonia thermophila, Thermomyces ibadanensis, Thermomyces lanuginosus, etc.

[0075] Genes and polypeptides from thermophilic microorganisms expressing thermostable enzymes are of interest as enzyme compositions that are stable during storage and application. In particular, thermophilic fungi that lead to high yields of enzymes in fungal expression hosts are of particular interest for enzyme production and stability during storage and application.

[0076] Maheshwari et al., Microbiology and Molecular Biology Reviews 2000, pp. 461-488, reported that among eukaryotes, only a few fungi have the ability to grow at temperatures between 45 and 55 °C. These fungi are less extreme thermophiles than the thermophilic species of bacteria and archaea. Maheshwari et al. estimated that out of approximately 50,000 known fungal species, only about 30 species exceed this upper temperature limit of eukaryotes. Salar et al., Journal of Agricultural Technology 2007, pp. 77-107, reported 42 species that are thermophilic fungi.

[0077] Further examples of enzymes of the present invention include, but are not limited to, sterol esterases from fungi, particularly from Basidiomycota, in particular from Pleurotus species. Such examples of enzymes of the present invention also include, but are not limited to, sterol esterases from Ascomycota, in particular from the genera Melanocarpus, Chaetomium, Chaetomidium, Corynascus, Crassicarpon, Canariomyces, Colletotrichum, Coonemeria, Crassicarpon, Included are sterol esterases from species such as Dactylomyces, Malbranchea, Myriococcum, Neurospora, Ophiostoma, Talaromyces, Thermoascus, Thermomyces, Thielavia, Fusarium, and Aspergillus.

[0078] One way to show relationships between enzymes is by sequence comparison.

[0079] The percentage of sequence identity was calculated using the Clustal Omega algorithm, available online at https: / / www.ebi.ac.uk / Tools / msa / .

[0080] [Table 1]

[0081] [Table 2]

[0082] TIFF2025063167000003.tif63147

[0083] The serine in the catalytic triad of sterol esterases is incorporated into the conserved sequence WGESAG, which has a glutamate in the catalytic triad and is absent in related but distinct enzymes, such as the acetylcholinesterase from Torpedo californica, the para-nitrobenzyl esterase from Bacillus subtilis, and the lipases from Geotrichum candidum and Candida rugosa, which contain the sequence FGESAG instead. The absence of the WGESAG sequence, and indeed of any GXSXG motif, in an unrelated family of cholesterol esterases from actinomycetes bacteria was demonstrated by Xiang et al., Biochimica et Biophysica Acta 2007, pp. 112-120. Unlike common lipases that do not show activity towards sterol esters, lipase from Candida rugosa is active towards both substrates, glycerol esters and sterol esters. Sequence features other than substrate specificity determine the characteristics for classifying enzymes. In some cases, sequence features are more accurate than specific activity as a means of classification. Due to sequence-structure-function relationships, sequence features and substrate specificity are linked. Also, other enzyme properties such as stability and receptivity to specific stabilizer moieties have their roots in specific sequences.

[0084] Thus, in one embodiment, the stabilizing effect discovered by the inventors for a particular polyhydroxy compound is characteristic of an enzyme that preferably has both a WGESAG motif and a catalytic triad composed of S, H and E / D, the S residue being part of said motif. In the examples below, this stabilizing effect is seen for different enzymes containing said motif and catalytic triad, and for different polyhydroxy compounds. The above parameters therefore define a limited set of enzymes and specific polyhydroxy compounds for which the stabilizing effect is seen.

[0085] The enzymes of the present invention may be isolated from their original biological source, or may be produced in a cell-free system, or may be produced as secreted or intercellular proteins in their original host or in an expression host, such as a heterologous expression host, including, but not limited to, filamentous fungi, yeast, bacteria, plants and seaweeds, such as Trichoderma reesei, Aspergillus oryzae, Pichia pastoris, Bacillus subtilis and Escherichia coli, and reviewed by Fernandez et al., Advanced Technologies for Protein Complex Production and Characterization 2016, pp. 15-24, and Yin et al., Journal of Biotechnology 2007, pp. 335-347. Kontkanen et al., Applied Microbiology and Biotechnology 2006, pp. 696-704, described the expression of a sterol esterase from Melanocarpus albomyces in Trichoderma reesei.

[0086] The enzymes of the invention, and the genes encoding the enzymes of the invention, can be derived from polypeptides and nucleic acids found in nature, or from synthetic polypeptides or synthetic diffusion having sequence information from nucleic acids or polypeptides found in nature. Such sequence information can be derived from two or more sequences found in nature, for example by calculating a common ancestral sequence, calculating a synthetic sequence from an alignment of known homologous sequences, and in particular by calculating the most frequent amino acid at each position and deviations from the consensus sequence.

[0087] Furthermore, the enzymes of the present invention and the genes encoding the enzymes of the present invention may contain one or more modifications without loss of function. Examples of such modifications are insertions, deletions, and / or substitutions, preferably conservative substitutions, to naturally occurring polypeptides or nucleic acids. The experimental interchangeability of amino acids in proteins has been reviewed by Yampolsky and Stoltzfus, Genetics 2005, pp. 1459-1472. Amino acid modifications are indicated by their single letters separated by a slash, e.g. E / D means E is D. A specific example of such a conservative substitution is the exchange between E and D, since E and D are both acidic amino acids and differ only by one methylene spacer in their side chains. Further examples of conservative substitutions are substitutions within the groups of basic amino acids (e.g. R / K / H), acidic amino acids and their amides (e.g. E / D / N / Q), aromatic amino acids (e.g. G / A / S), neutral amino acids (e.g. T / S / A / V / M / C), charged amino acids (e.g. E / D / R / K / H) and other polar amino acids (e.g. S / T / N / Q / H). In addition to substitutions with the 20 standard amino acids, other genetically encoded amino acids, such as selenocysteine ​​and pyrrolysine, and so-called unnatural amino acids, can also be incorporated into proteins. Examples of such unnatural amino acids are reviewed in Wang et al., Chemistry & Biology 2009, pp. 323-336. By stepwise solid-phase peptide synthesis, any available amino acid can be incorporated into peptides, which can be chemically coupled to larger peptides and polypeptides to produce proteins and enzymes.

[0088] Furthermore, the enzyme of the present invention may be a fusion polypeptide in which another polypeptide and / or oligopeptide is attached to the amino-terminus and / or carboxy-terminus of the polypeptide characterized by enzymatic activity against sterol esters. Examples of such oligopeptides and polypeptides include, but are not limited to, polyhistidine tags, signal peptides, linkers, binding domains, antibodies, chaperones, fluorescent proteins and enzymes, such as those having cholesterol oxidase activity.

[0089] The concentration of the enzyme in the composition of the present invention is selected from 0.0001-10% by weight and any range therebetween. Such percentages are meant as active enzyme protein weight per total weight of the composition. In another embodiment, the enzyme concentration is 0.01-10% by weight, preferably 0.1-8% by weight, and even more preferably 1-5% by weight. Such percentages are meant on a dry weight basis. In another embodiment, the enzyme concentration is specified as enzyme activity per weight of the composition and is selected from 10-100,000 SEU / g, preferably 100-50,000 SEU / g, more preferably 200-30,000 SEU / g, and most preferably 400-10,000 SEU / g. The enzyme activity can be measured using the method described in Example 1 below.

[0090] In another embodiment, the enzyme is a sterol esterase. Sterol esterase is a versatile enzyme with a broad substrate specificity. In addition to sterol and glycerol esters of carboxylic acids (e.g., short and long chain carboxylic acids, saturated and unsaturated fatty acids, etc.), other natural and artificial esters, such as polyesters (e.g., polyethylene terephthalate, etc.), polymers containing vinyl acetate monomers (e.g., polyvinyl acetate, etc.) and para-nitrophenyl esters, can also be substrates and / or products of the enzymes of the present invention. Thus, such enzymes are useful in many industrial applications, particularly in pulp and paper, food, feed, textiles, detergents, personal care, and diagnostic applications. Specific examples within such industries include, but are not limited to, cleaning applications, particularly for laundry and / or contact lenses, processing aids in the manufacture of polyester fibers and / or wool, in the manufacture and / or recycling of paper and / or pulp, in biosensors and / or diagnostic reagents for the measurement of total cholesterol (e.g., in blood), and in the synthesis of sterol esters. Examples of uses of such sterol esters, particularly esters of cholesterol and phytosterols, more preferably stigmasterol oleate, include use in food (e.g. added to margarine), feed, cosmetics and pharmaceutical formulations, as well as in technical applications such as in liquid crystal displays containing cholesterol esters.

[0091] Examples of sterols of the present invention include, but are not limited to, cholesterol, ergosterol, lanosterol, phytosterol, sitosterol, stigmasterol, campesterol, sitostanol, stigmasterol, campestalol, brassicasterol, fucosterol, and cycloartenol.

[0092] Examples of sterol esters of the present invention include, but are not limited to, esters of cholesterol, esters of ergosterol, esters of lanosterol, esters of phytosterol, esters of sitosterol, esters of stigmasterol, esters of campesterol, esters of sitostanol, esters of stigmasterol, esters of campestalol, esters of brassicasterol, esters of fucosterol, and esters of cycloartenol.

[0093] Examples of cholesterol esters of the present invention include, but are not limited to, cholesterol linoleate, cholesterol linolenate, cholesterol myristoleate, cholesterol palmitoleate, cholesterol oleate, cholesterol sapienate, cholesterol arachidonic acid, cholesterol erucate, cholesterol crotonate, cholesterol phenylpropionate, cholesterol phenylacetate, cholesterol cinnamate, cholesterol benzoate, cholesterol nitrobenzoate, cholesterol dichlorobenzoate, cholesterol chloroformate, cholesterol formate, cholesterol acetate, cholesterol propionate, cholesterol butyrate, cholesterol valerate (cholesterol pentanoate), and the like. Cholesterol caproate, cholesterol heptanoate, cholesterol octanoate (cholesterol caprylate), cholesterol nonanoate (cholesterol pelargonate), cholesterol decanoate (cholesterol caprate), cholesterol laurate, cholesterol myristate, cholesterol palmitate, cholesterol stearate, cholesterol eicosanoate (cholesterol arachidate), cholesterol docosanoate (cholesterol behenate), methylcholesterol tetracosanoate (cholesterol lignocerate), cholesterol hexacosanoate (cholesterol cerotate), cholesterol acetoacetate, cholesterol hemisuccinate, and cholesterol isobutyrate.

[0094] The activity of the enzymes of the present invention can be measured using sterol esters as enzyme substrates and measuring the pH change or by applying the pH-stat method to measure the release of carboxylic acids from the corresponding esters or by the method described in Example 1 below to measure the release of cholesterol from cholesterol linoleate. In the methods detailed here, cholesterol linoleate may be replaced by other esters of sterols, in particular esters of cholesterol, more in particular the esters of cholesterol exemplified above.

[0095] In one embodiment, the enzyme component is spent fermentation broth containing the enzyme. The spent fermentation broth may be obtained, for example, by recombinant production of the enzyme. The spent fermentation broth may be concentrated and / or clarified after enzyme production. In another embodiment, a mixture of spent fermentation broth from several fermentations may be used.

[0096] In one embodiment, the solution-stable enzyme composition and enzyme components remain in solution at 37° C. for at least 4 weeks, preferably at least 24 weeks of storage. If the enzyme remains in solution, no enzyme turbidity or precipitation occurs. A solution-stable enzyme composition that can keep the enzyme in solution at 37° C. for at least 4 weeks, preferably at least 24 weeks, means that the enzyme remains soluble and can function catalytically. If the enzyme loses its catalytic properties, it is no longer an enzyme, but is, for example, a denatured protein.

[0097] Microbial growth or other reasons may cause the development of turbidity or precipitation during storage at 37° C. In conditions potentially suitable for microbial growth, it may be preferable to include an antimicrobial preservative in the composition.

[0098] In one embodiment, the solution-stable enzyme composition remains clear after 4 weeks of storage at 4° C., preferably after 8 weeks of storage at 4° C., more preferably after 24 weeks of storage at 4° C. In one embodiment, the solution-stable enzyme composition remains clear after 4 weeks of storage at 20° C., preferably after 8 weeks of storage at 20° C., more preferably after 24 weeks of storage at 20° C. In one embodiment, the solution-stable enzyme composition remains clear after 4 weeks of storage at 37° C., preferably after 8 weeks of storage at 37° C., more preferably after 24 weeks of storage at 37° C. Thus, a solution-stable enzyme composition is one that does not undergo significant turbidity or precipitation during storage.

[0099] In one embodiment, the solution-stable enzyme composition has a residual enzyme activity that is greater than one-third, preferably greater than one-half, after 4 weeks of storage at 4° C., preferably greater than one-half, compared to the enzyme activity immediately after preparation of such enzyme composition. In one embodiment, the solution-stable enzyme composition has a residual enzyme activity that is greater than one-third, preferably greater than one-half, after 4 weeks of storage at 20° C., preferably greater than one-half, compared to the enzyme activity immediately after preparation of such enzyme composition. In one embodiment, the solution-stable enzyme composition has a residual enzyme activity that is greater than one-third, preferably greater than one-half, after 4 weeks of storage at 37° C., preferably greater than one-half, compared to the enzyme activity immediately after preparation of such enzyme composition.

[0100] The polyhydroxy compound of the present invention is an organic polyhydroxy compound having 3 or more, or 4 or more, preferably 4 or more, more preferably 5 or more hydroxyl groups per molecule. Examples of organic polyhydroxy compounds include, but are not limited to, pentaerythritol, trimethylolpropane, polyvinyl alcohol, certain carbohydrates, cyclitols, and sugar alcohols. Examples of sugar alcohols of the present invention include, but are not limited to, sorbitol, mannitol, xylitol, glycerol, erythritol, threitol, arabitol, ribitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, polyglycitol, and hydrogenated starch hydrolysates. Inositol is an example of a sugar alcohol and at the same time an example of a cyclitol. Natural cyclitols have six ring atoms and hydroxyl groups on four or more ring atoms. Examples of such cyclitols include inositol, bornesitol, conduritol, ononitol, pinitol, pinpollitol, quebrachitol, valienol, biscumitol, and cyclitol.

[0101] In certain embodiments, the stabilizer component comprises an organic polyhydroxy compound having 4 or more hydroxyl groups, or 5 or more hydroxyl groups per molecule. In a preferred embodiment, the stabilizer component comprises a sugar alcohol having 4 or more hydroxyl groups per molecule.

[0102] In certain embodiments, the stabilizer component does not include sugar.

[0103] In certain embodiments, the stabilizer component does not include a simple sugar.

[0104] In certain embodiments, the stabilizer component does not include a polysaccharide.

[0105] In certain embodiments, the stabilizer component does not include a sugar.

[0106] In certain embodiments, the stabilizer component does not include starch.

[0107] In some embodiments, the organic polyhydroxy compound consists only of carbon, hydrogen, and oxygen atoms. In other embodiments, the organic polyhydroxy compound consists of carbon, hydrogen, oxygen, and other elements. Examples of such elements include nitrogen, sulfur, phosphorus, boron, fluorine, chlorine, bromine, and iodine.

[0108] In certain embodiments, the stabilizer component comprises a synthetic organic polyhydroxy compound that is preferably added to the composition, and thus is not naturally present in the composition in significant amounts.

[0109] In one embodiment, the stabilizer component comprises a mixture of sorbitol and glycerol.

[0110] In another embodiment, the stabilizer component comprises a mixture of maltitol and sorbitol, a mixture of maltitol and glycerol, or a mixture of sorbitol, maltitol and glycerol. In another embodiment, the stabilizer component comprises a mixture of mannitol and sorbitol, a mixture of mannitol and glycerol, or a mixture of sorbitol, mannitol and glycerol. In another embodiment, the stabilizer component comprises a mixture of xylitol and sorbitol, a mixture of xylitol and glycerol, or a mixture of sorbitol, xylitol and glycerol.

[0111] In one embodiment, the stabilizer component comprises a mixture of two or more sugar alcohols in any combination selected from the group consisting of sorbitol, glycerol, maltitol, mannitol, and xylitol.

[0112] In one embodiment, a mixture of sugar alcohols is used instead of a single sugar alcohol. Any suitable mixture of sugar alcohols can be used so long as the sugar alcohols are not incompatible with each other, with the enzyme components, or with any other components present in the enzyme composition.

[0113] The upper limit of the concentration of sugar alcohols in the composition of the present invention is their solubility limit. Aqueous sorbitol solutions are commercially available at a concentration of 70% by weight, and maltitol at a concentration of 75% by weight. The lower limit of the concentration of sugar alcohols in the composition of the present invention is their effectiveness as a stabilizing excipient in the composition of the present invention. Effectiveness can be measured using the method described in Example 2 below.

[0114] In one embodiment, the solution-stable enzyme composition comprises 20%, 24%, 25%, 28%, 30%, 33%, 35%, 40%, or 50% by weight of the sugar alcohol. Such embodiments with higher concentrations of the sugar alcohol are advantageous because such compositions remain liquid at temperatures below 0° C. Such compositions can be stored at temperatures below 0° C., for example, outdoors in the winter, without freezing. The freezing points of compositions with high concentrations of sorbitol, maltitol, lactitol, and hydrogenated corn syrup were reported by Uraji et al., Food Science Technology International 1996, pp. 38-42.

[0115] In one embodiment, the solution-stable enzyme composition comprises a mixture of at least two different sugar alcohols. Different sugar alcohols in various ratios are effective to provide storage stability, as shown in the examples below. The first sugar alcohol may be present, for example, at about 20%, 25%, 30%, 40%, or 50% by weight, while the second sugar alcohol may be present, for example, at about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or 30% by weight.

[0116] Antimicrobial preservative measures are particularly important during long-term storage at ambient temperatures of aqueous compositions containing organic compounds, especially when such compositions are not sterile. Sterilization of liquid products is less common when such products are used in technical applications than in medical applications. Thus, in the present invention, the solution-stable enzyme composition includes an antimicrobial preservative to prevent microbial growth.

[0117] The antimicrobial preservative of the present invention is preferably an antimicrobial and / or antifungal chemical compound, more preferably a chemical compound against mold, yeast, and / or acid-resistant bacteria, such as, for example, a moldicide, a fungistatic, a bactericide, and / or a bacteriostatic. Examples of antimicrobial preservatives of the present invention include, but are not limited to, 1,2-benzisothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-octylisothiazolin-3-one and 4,5-dichloro-2-octylisothiazolin-3-one, phenoxyethanol, benzoic acid, hydroxybenzoic acid, particularly 4-hydroxybenzoic acid and 2-hydroxybenzoic acid (also known as salicylic acid), sorbic acid, propionic acid, lactic acid, hexanoic acid, octanoic acid, sulfur dioxide, as well as the salts of these acids, particularly the sodium, potassium and calcium salts of benzoic acid, hydroxybenzoic acid, salicylic acid derivatives, sorbates, propionates, hexanoates, octanoates, sulfites, bisulfites and pyrosulfites. Further examples of antimicrobial preservatives are esters of hydroxybenzoic acid, such as methyl 4-hydroxybenzoate, ethyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate, butyl 4-hydroxybenzoate, heptyl 4-hydroxybenzoate, isobutyl 4-hydroxybenzoate, isopropyl 4-hydroxybenzoate, phenyl 4-hydroxybenzoate, and benzyl 4-hydroxybenzoate (which are also referred to as methylparaben, ethylparaben, propylparaben, butylparaben, heptylparaben, isobutylparaben, isopropylparaben, phenylparaben, and benzylparaben, respectively), and salts thereof, such as sodium methyl 4-hydroxybenzoate, sodium ethyl 4-hydroxybenzoate, sodium propyl 4-hydroxybenzoate, and the like.

[0118] In further embodiments, instead of or in addition to antimicrobial preservatives, high concentrations of stabilizer components are used to prevent microbial growth. A sufficiently high concentration is one that has antimicrobial effect due to high osmotic pressure and / or low water activity. Thus, the stabilizer components of the present invention can simultaneously function as antimicrobial preservatives of the present invention. Examples of such compounds at high concentrations include sucrose at a concentration of at least about 50% by weight, sorbitol at a concentration of at least about 45% by weight, glycerin at a concentration of at least about 25% by weight, and mixtures of glycerin and sorbitol at various high concentrations as measured by Barr and Tice, Journal of the American Pharmaceutical Association, 1957, pp. 217-223.

[0119] Compounds having antimicrobial preservative effect are used according to the invention in concentrations of 0.002-75% by weight, preferably 0.005-10% by weight, more preferably 0.01-5% by weight, even more preferably 0.02-2% by weight, most preferably 0.04-0.5% by weight. The lower limit of the concentration of antimicrobial preservative in the compositions of the invention is its effectiveness for preventing the growth of microorganisms in such compositions.

[0120] The antimicrobial efficacy may be determined by counting viable cells (e.g., colony forming units) in an aqueous composition (e.g., a composition of the present invention) after inoculation with a microorganism (e.g., the acid-tolerant bacterium Lactobacillus buchneri, the mold Aspergillus oryzae, or the yeast Pichia pastoris) and incubation at storage temperatures (e.g., 8°C, 25°C, or 37°C) for one week or more, particularly several weeks, and comparing with a similar aqueous composition without the antimicrobial preservative and / or comparing with the inoculated aqueous composition before incubation.

[0121] Acid-tolerant bacteria, molds, and yeasts are food spoilage microorganisms.

[0122] No protease inhibitors, antioxidants, or surfactants are required to achieve long term storage stability of the compositions of the present invention.

[0123] The invention will be further described with reference to the following embodiments.

[0124] In one embodiment, the solution stable enzyme composition is for industrial use, hi another embodiment, it is for use in the manufacture of paper and / or pulp.

[0125] In one embodiment, the pH of the solution-stable enzyme composition is selected from the range of strong acidity to neutral to weak alkali. In one embodiment, the pH of the solution-stable enzyme composition is selected from the acidic region. In one embodiment, the pH of the solution-stable enzyme composition is selected from the range of pH 3.0 to 6.2, which is buffered with citrate. In one embodiment, the pH of the solution-stable enzyme composition is selected from the range of pH 3.5 to 5.8, which is buffered with acetate. In one embodiment, the pH of the solution-stable enzyme composition is selected from the range of pH 5.8 to 8.0, which is buffered with phosphate. In one embodiment, the pH of the solution-stable enzyme composition is selected from the unbuffered neutral range. In one embodiment, the pH of the solution-stable enzyme composition is selected from the pH range of 3.7 to 8.1, which is buffered with a pH buffer, preferably a citrate buffer, an acetate buffer, or a phosphate buffer. In one embodiment, the pH of the solution-stable enzyme composition is selected from the range of pH 3 to 8. These ranges are particularly advantageous because within these ranges good stability over long periods of time can be achieved for certain polyhydroxy compounds of the invention, as demonstrated in the examples. Furthermore, within these ranges good stability can be achieved for buffer substances that are compatible with all components of the compositions of the invention during extended storage periods.

[0126] In one embodiment the enzyme is a hydrolase characterized by having enzymatic activity towards sterol esters, preferably esters of sterols with fatty acids, more preferably esters of cholesterol with linoleic acid.

[0127] In one embodiment, the solution-stable enzyme composition comprises a fermentation broth or a clarified and optionally concentrated fermentation broth.

[0128] In one embodiment, the enzyme is a thermostable enzyme. This embodiment is advantageous because thermostability is associated with other types of stability, particularly long-term stability at ambient temperatures. Moreover, this embodiment is also advantageous for use in pulp and paper manufacturing, where temperatures above 50°C, preferably above 60°C, more preferably above 70°C, and most preferably above 75°C are often applied. In one embodiment, the use in pulp and paper manufacturing is at an acidic pH, preferably at pH 5.0. In another embodiment, the use in pulp and paper manufacturing is at a pH between 3.0 and 8.0.

[0129] In one embodiment, the enzyme is a sterol esterase from a fungus, preferably a thermophilic fungus.

[0130] In one embodiment the enzyme is a fungal sterol esterase, preferably a thermophilic fungal sterol esterase.

[0131] In one embodiment, the enzyme is a sterol esterase and the catalytic triad is composed of S, H and E.

[0132] In one embodiment, the enzyme is a sterol esterase from a thermophilic fungus selected from the group consisting of Scytalidium thermophilum, Myceliophthora thermophila, Thielavia terestri, Corynascus thermophilus, Myriococcum thermophilum, Thermomyces stellatus, Thielavia australiensis, Malbranchea cinnamomea, Melanocarpus albomyces, and Chaetomium thermophilum, as well as fragments and conservative modifications of such sterol esterases. In another embodiment, the enzyme is within the group of thermophilic fungi mentioned above and is a sterol esterase close homologue of an enzyme having an amino acid sequence according to any one of SEQ ID NOs: 1-32.

[0133] In one embodiment, the enzyme is a sterol esterase encoded by a gene and fragments thereof and conservative modifications thereof from the genome of a thermophilic fungus selected from the group consisting of Scytalidium thermophilum, Myceliophthora thermophila, Thielavia terestri, Corynascus thermophilus, Myriococcum thermophilum, Thermomyces stellatus, Thielavia australiensis, Malbranchea cinnamomea, Melanocarpus albomyces, and Chaetomium thermophilum.

[0134] In one embodiment, the enzyme has at least 60%, preferably at least 70%, more preferably at least 75%, and most preferably at least 80% sequence identity to a sterol esterase from Melanocarpus albomyces or Chaetomium thermophilum.

[0135] In one embodiment, the enzyme has at least 60%, preferably at least 70%, more preferably at least 75%, and most preferably at least 80% sequence identity to the sterol esterase that is SEQ ID NO:1 or SEQ ID NO:2.

[0136] In another embodiment, the enzyme has at least 50, 60, 70, 75, 80, 85, 90, 95 or 99% sequence identity to the corresponding amino acid sequence of SEQ ID NO:1 or 2.

[0137] In another embodiment, the enzyme has at least 50, 60, 70, 75, 80, 85, 90, 95 or 99% sequence identity to the corresponding sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and is preferably a thermostable sterol esterase, more preferably a fungal thermostable sterol esterase.

[0138] In another embodiment, the enzyme has at least 50, 60, 70, 75, 80, 85, 90, 95 or 99% sequence identity to the corresponding sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32.

[0139] In one embodiment, the enzyme does not exhibit 100% sequence homology with any of the sequences SEQ ID NOs: 1-32.

[0140] In one embodiment, the polyhydroxy compound has 3 or more, preferably 4 or more, more preferably 6 or more hydroxyl groups per molecule.

[0141] In one embodiment, the polyhydroxy compound has 4 or more hydroxyl groups per molecule, preferably 5 or more hydroxyl groups, more preferably 6 or more hydroxyl groups. The hydroxy compound is particularly advantageous in that it can stabilize various enzymes of the present invention, since the stabilizing effect of the polyhydroxy compound increases with increasing concentration and with increasing number of hydroxyl groups. More hydroxyl groups per molecule leads to higher molecular mass and improved water-related properties and water-protein interaction.

[0142] In one embodiment, the polyhydroxy compound is a sugar alcohol.

[0143] In one embodiment, the sugar alcohol is selected from the group consisting of sorbitol, mannitol, maltitol, xylitol, and glycerol.

[0144] In one embodiment, the sugar alcohol is selected from the group consisting of sorbitol, mannitol, maltitol, and xylitol. This embodiment is advantageous in applications where the presence of glycerol is undesirable, for example, where glycerol interferes with the function of the enzyme or is incompatible with its use. Since glycerol is a hydrolysis product of glycerol esters, glycerol may cause a decrease in enzyme activity due to product inhibition. Therefore, the use of a sugar alcohol having four or more hydroxyl groups per molecule, preferably selected from the group consisting of sorbitol, mannitol, maltitol, and xylitol, instead of glycerol, may be desirable when the composition of the present invention is applied for the hydrolysis of glycerol esters. An example of an industrial application of the enzymatic hydrolysis of glycerol esters is in the production of pulp and paper. Glycerol esters and sterol esters are present in wood resins that produce sticky deposits during the production of wood pulp and paper, and are preferably hydrolyzed quickly before deposits occur.

[0145] In one embodiment, the stabilizer component comprises at least one-fifth, preferably at least one-fourth, and more preferably at least one-third of the enzyme composition by weight. These amounts are preferred because they provide long-term stabilizing effects.

[0146] In one embodiment, the stabilizer component comprises at least one-fifth, preferably at least one-fourth, more preferably at least one-third, of the total weight of the enzyme component. Depending on the particular stabilizer component, the stabilizing effect increases with increasing concentration.

[0147] In one embodiment, the solution-stable enzyme composition comprises an antimicrobial preservative selected from the group consisting of 1,2-benzisothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-octylisothiazolin-3-one and 4,5-dichloro-2-octylisothiazolin-3-one. These preservatives are preferred because they are compatible with the enzyme composition and provide antimicrobial efficacy over an extended period of time.

[0148] In one embodiment, the preservative is an isothiazolinone, preferably selected from the group consisting of 1,2-benzisothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-octylisothiazolin-3-one and 4,5-dichloro-2-octylisothiazolin-3-one.

[0149] In one embodiment, the preservative is selected from the group consisting of benzoic acid, 4-hydroxybenzoic acid, 2-hydroxybenzoic acid, and their salts and esters. Such an embodiment, which includes a preservative that is permitted to be added to food, is advantageous as a necessary prerequisite for the food grade registration of the composition of the present invention. Approval as a food and food contact agent may also be necessary or advantageous for use in food, feed, pulp and paper manufacturing.

[0150] In one embodiment, the preservative is active against at least one microorganism selected from the group consisting of acid-tolerant bacteria, molds and yeasts.

[0151] In one embodiment, the solution-stable enzyme composition is essentially free of polyethylene glycol p-alkylphenyl ethers, preferably free of all nonionic surfactants, and more preferably free of all surfactants, which is particularly beneficial in applications where the presence of surfactants is undesirable, for example where foam formation by surfactants is undesirable.

[0152] In one embodiment, the solution-stable enzyme composition is essentially free of serine protease inhibitors, preferably free of all protease inhibitors, which is advantageous when simultaneous activity of the protease and the enzyme composition of the invention is required, such as in detergent compositions for laundry washing and other cleaning applications.

[0153] The present invention is further illustrated by the following examples, which do not limit the scope of the invention, which is defined by the appended claims and equivalents thereof. EXAMPLES

[0154] Example 1 - Measurement of hydrolase activity towards sterol esters and cholesterol linoleate The method is a modification of the method described by Stepien et al., Acta Biochimica Polonica 2013, pp. 401-403, and Sigma-Aldrich, http: / / www.sigmaaldrich.com / technical-documents / protocols / biology / assay-procedure-for-cholesterol-esterase.html. The following chemicals were purchased from Sigma-Aldrich (now Merck) using the relevant catalog numbers: cholesterol linoleate C0289, cholesterol oxidase C8649, horseradish peroxidase 77332, lyophilized bovine serum albumin A2153, 4-aminoantipyrine A4382, sodium 3,5-dichloro-2-hydroxybenzenesulfonate D4645, Triton X-100 X100. The following solutions were freshly prepared:

[0155] The AA solution was 1.76 g 4-aminoantipyrine dissolved in 100 mL water. The DHBS solution was 6 g sodium 3,5-dichloro-2-hydroxybenzenesulfonate dissolved in 100 mL water. The CL solution was 9.8 mg cholesterol linoleate dissolved in 0.5 mL isopropanol to which was added 1% Triton X-100 solution at 75°C, cooled to 25°C, and made up to 25 mL with 1% Triton X-100. Peroxidase powder was dissolved in 0.1 M potassium dihydrogen phosphate buffer at pH 7.0 and diluted to 150 PU / mL. Cholesterol oxidase powder was dissolved in ice-cold water and diluted to 30.3 U / mL. The buffer used for diluting the samples was 0.095 g magnesium chloride, 0.0695 g disodium dihydrogen ethylenediaminetetraacetate dihydrate, and 1 g lyophilized bovine serum albumin dissolved in 500 mL 0.2 M potassium hydrogen phosphate buffer, pH 7.5. The substrate solution was 4.36 mL 0.2 M potassium hydrogen phosphate buffer, pH 7.0, 2.91 mL CL solution, 0.145 mL AA solution, 0.291 mL DHBS solution, and 0.291 mL peroxidase solution. From this substrate solution, 0.917 mL was pipetted into a cuvette and incubated at 37°C. Then, 0.033 mL of cholesterol oxidase solution was added. Then, 0.033 mL of the sample to be measured was quickly mixed into the cuvette. Immediately afterwards, the absorbance change per minute (abbreviated as ΔOD / min) was measured at 37 °C and 512 nm by a Perkin Elmer Lambda 25 spectrophotometer. The enzyme activity, sterol esterase units (abbreviated as SEU), was calculated based on the formula SEU / L = ΔOD / min·3.819, considering a sample volume of 0.033 mL in a total volume of 0.983 mL and a light path through the cuvette of 1 cm. If the sample was diluted before being added to the cuvette, the result was also multiplied by the appropriate dilution factor. Samples were diluted such that ΔOD / min values ​​between 0.06 and 0.22 were measured.

[0156] Significant enzyme activity was measured using the above-mentioned method in samples prepared by expression in Trichoderma reesei of SEQ ID NOs: 1, 2, 3, 5, 6, 9, and 10. Expression in Trichoderma reesei was performed according to known methods, e.g., Kontkanen et al., Applied Microbiology and Biotechnology 2006, pp. 696-704.

[0157] Example 2 - Experimental evaluation of stability of liquid enzyme compositions In all experiments, the enzyme materials used to prepare the test compositions were either clarified fermentation broths or their concentrates, containing preservatives to prevent microbial growth, from several different fermentations of enzyme proteins. The enzyme proteins tested were sterol esterase from Melanocarpus albomyces, sterol esterase from Chaetomium thermophilum, and an enzyme with the synthetic sequence of SEQ ID NO: 3 expressed in Trichoderma reesei. In the test compositions, the enzyme liquids were standardized to activity levels ranging from 1200 to 1900 SEU / g and stabilized using various stabilization conditions as shown in the table below. All test compositions were subjected to accelerated storage stability testing. The total storage time was 24 weeks. For the stability testing, each liquid was dispensed into 15 mL screw-cap tubes with 5 mL of liquid per tube. One tube was prepared for each storage time point. The starting sample tube was placed in the freezer immediately after preparation for visual inspection. All other sample tubes were placed in a 37°C climate chamber. One sample tube was removed from each time point under visual inspection and placed in the freezer prior to activity analysis. The appearance of the liquid from each storage time point was compared to the appearance at the starting point. Activity from the various storage time points was also compared to the starting point. Physical stability results are shown in Tables 3-11.

[0158] [Table 3]

[0159] As shown by the data in Table 3, compositions containing sterol esterase from Melanocarpus albomyces without a stabilizer or with propylene glycol as the polyhydroxy compound at acidic pH buffered with citric acid are not physically stable. The data also show that the 15% sorbitol concentration is not high enough to prevent physical instability in the presence of 15% propylene glycol. However, 35% sorbitol in the presence of 15% propylene glycol is already sufficient to maintain a physically stable liquid for 4 weeks. Compositions containing 30% sorbitol maintain a physically stable liquid for at least 4 weeks. 40-50% sorbitol or glycerol or mixtures thereof (20%+20% or 25%+25%) are stable over the long term. According to the 4-week data, maltitol is an equally good stabilizer as sorbitol and glycerol. Maltitol also works in mixtures with sorbitol or glycerol at higher concentrations.

[0160] [Table 4]

[0161] As shown by the data in Table 4, compositions containing sterol esterase from Chaetomium thermophilum with 40% propylene glycol at acidic pH buffered with citrate are not physically stable. Physically stable compositions were achieved using similar high concentrations of sorbitol or mixtures of mannitol with sorbitol or glycerol as used in the physically stable compositions of sterol esterase from Melanocarpus albomyces in Table 3. This also indicates that mannitol can be an equally good stabilizer as sorbitol, glycerol and maltitol, but due to its solubility limitations, mannitol works in mixtures with other polyhydroxy compounds to maintain good physical stability.

[0162] [Table 5]

[0163] The results shown in Table 5 demonstrate the considerable physical stability of the composition containing the enzyme having the synthetic sequence of SEQ ID NO:3 as the other two sterol esterases in the same composition shown in Tables 3 and 4, respectively.

[0164] [Table 6]

[0165] The physical stability data presented in Tables 3 and 6 reveal similar trends in the presence of the two preservatives tested, sodium benzoate and 1,2-benzisothiazolin-3-one.

[0166] [Table 7]

[0167] [Table 8]

[0168] The physical stability data presented in Tables 3, 6, 7 and 8 reveal similar trends in the presence of the two buffers tested, sodium acetate and sodium citrate. Also, xylitol, mannitol and maltitol are equivalent when used in mixtures with sorbitol.

[0169] [Table 9]

[0170] The physical stability data presented in Table 9 shows the trends shown in the tables above for unbuffered sterol esterase compositions at pHs close to 5 and also in the higher pH range of 7.5 to 8.1.

[0171] [Table 10]

[0172] [Table 11]

[0173] The physical stability data presented in Tables 10 and 11 show the trends shown in the tables above for sodium / potassium phosphate buffered compositions at pH 7.4-8.1.

[0174] Example 3 - Experimental evaluation of increasing pH on the stability of enzyme compositions In all experiments, the enzyme material used to prepare the test compositions was a concentrate of clarified fermentation broth, containing a preservative to prevent microbial growth, from several different fermentations of the same three enzyme proteins as in the previous experiment in Example 2. In the test compositions, the enzyme liquid was not standardized for a specific activity, but was only stabilized using various stabilization conditions shown in the table below. All test compositions were stored at 20° C. in a climate chamber for 4 weeks. Other storage stability tests were performed in the same manner as described in Example 2. Physical stability results are shown in Table 12.

[0175] [Table 12]

[0176] As the examples in Table 12 show, increasing the pH was not sufficient for physical stability. In one experiment with a composition containing sterol esterase from Melanocarpus albomyces and 1,2-benzisothiazolin-3-one as a preservative, the appearance of the liquid was improved by adjusting the pH from 5.6 to 7.5, but the composition was clear only upon preparation and was not physically stable. Also, a high pH such as 7.5 has an overall stability-reducing effect, as can be seen in Table 13 below, which shows a decrease in activity of standardized enzyme compositions at pH 5 and pH 7.5.

[0177] Example 4 - Experimental evaluation of stability of liquid enzyme compositions measured as residual activity The materials and methods are the same as those described in Examples 1 and 2.

[0178] [Table 13]

[0179] A stable enzyme composition is indicated by greater than 50% remaining enzyme activity after 4 weeks of storage at 37° C. Also, a stable enzyme composition is indicated by greater than 40% remaining enzyme activity after 8 weeks of storage at 37° C.

[0180] The above disclosure provides a complete and informative description of the best mode currently understood by the inventors for carrying out the invention as a non-limiting example of specific implementations and embodiments of the invention. However, it will be apparent to those skilled in the art that the invention is not limited to the details of the embodiments shown above, and that it may be carried out in other embodiments using equivalent means without departing from the characteristics of the invention.

[0181] Moreover, some of the features of the above-described embodiments of the invention may be used to advantage without the corresponding use of other features. Accordingly, the foregoing description should be considered merely as illustrative of the principles of the invention, and not as in limitation thereof. The scope of the invention is therefore limited only by the appended claims.

Claims

1. 1. A solution-stable enzyme composition comprising: (a) an enzyme component comprising an enzyme characterized by having the amino acid sequence WGESAG and a catalytic triad composed of S, H and E / D; (b) a stabilizer component comprising an organic sugar alcohol having three or more hydroxyl groups per molecule, said stabilizer component being at least one-fifth of the total weight of said enzyme composition; (c) an optional antimicrobial preservative component that prevents microbial growth; and (d) water dissolved in said components (a), (b) and (c); Including, A solution-stable enzyme composition, wherein the enzyme component of the solution-stable enzyme composition remains in solution for at least 4 weeks upon storage at 37°C.

2. 2. The solution-stable enzyme composition of claim 1, wherein the enzyme is a hydrolase characterized by having enzymatic activity towards sterol esters, preferably esters of sterols with fatty acids, more preferably esters of cholesterol with linoleic acid.

3. 3. The solution-stable enzyme composition of claim 1 or 2, wherein the enzyme is a thermostable enzyme that retains at least 50% enzymatic activity after incubation in an aqueous composition or in an aqueous environment or in an aqueous solution at 50°C, preferably at 60°C, more preferably at 70°C, and most preferably at 75°C for at least 5 minutes.

4. A solution-stable enzyme composition according to any one of claims 1 to 3, wherein the enzyme is a fungal sterol esterase, preferably a thermophilic fungal sterol esterase.

5. 5. The solution-stable enzyme composition of claim 1, wherein the enzyme is a sterol esterase and the catalytic triad is composed of S, H and E.

6. 6. The solution-stable enzyme composition of any one of claims 1 to 5, wherein the enzyme is a sterol esterase encoded by a gene and fragments thereof and conservative modifications thereof from the genome of a thermophilic fungus selected from the group consisting of Scytalidium thermophilum, Myceliophthora thermophila, Thielavia terestris, Corynascus thermophilus, Myriococcum thermophilum, Thermomyces stellatus, Thielavia australiensis, Malbranchea cinnamomea, Melanocarpus albomyces and Chaetomium thermophilum.

7. 7. The solution-stable enzyme composition according to any one of claims 1 to 6, wherein the enzyme has at least 60%, preferably at least 70%, more preferably at least 75%, and most preferably at least 80% sequence identity with a sterol esterase having SEQ ID NO:1 or SEQ ID NO:

2.

8. 8. The solution-stable enzyme composition according to claim 1, wherein the sugar alcohol has four or more, preferably five or more, more preferably six or more hydroxyl groups per molecule.

9. The solution-stable enzyme composition of any one of claims 1 to 8, wherein the sugar alcohol is selected from the group consisting of sorbitol, mannitol, maltitol, xylitol, and glycerol.

10. 10. The solution-stable enzyme composition according to any one of claims 1 to 9, wherein the antimicrobial preservative is selected from isothiazolinones, preferably selected from the group consisting of 1,2-benzisothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-octylisothiazolin-3-one and 4,5-dichloro-2-octylisothiazolin-3-one.

11. A solution-stable enzyme composition according to any one of claims 1 to 10, wherein the stabiliser component comprises at least one quarter, preferably at least one third of the total weight of the enzyme composition.

12. 12. The solution-stable enzyme composition of any one of claims 1 to 11, further comprising an antimicrobial preservative selected from the group consisting of benzoic acid, 4-hydroxybenzoic acid, 2-hydroxybenzoic acid, and salts and esters thereof.

13. 13. The solution-stable enzyme composition of any one of claims 1 to 12, wherein the preservative is active against at least one microorganism selected from the group consisting of acid-tolerant bacteria, molds and yeasts.

14. 14. The solution-stable enzyme composition according to any one of claims 1 to 13, wherein the composition is essentially free of polyethylene glycol p-alkylphenyl ethers, preferably free of all non-ionic surfactants, more preferably free of all surfactants.

15. A solution-stable enzyme composition according to any one of claims 1 to 14, wherein the composition is essentially free of serine protease inhibitors, preferably free of all protease inhibitors.

16. Use of a solution-stable enzyme composition according to any one of claims 1 to 15 in the manufacture of pulp.

17. Use of a solution-stable enzyme composition according to any one of claims 1 to 15 in the manufacture of paper.