Means and methods for cracking zearalenone

Abstract

The present invention relates to a method for increasing the stability of an α / β-hydrolase. In addition, the present invention also relates to an α / β-hydrolase obtainable by the method of the present invention. The present invention also provides an α / β-hydrolase having a reduced total average hydrophilicity (GRAVY) value and / or comprising a specific mutation. In addition, the present invention relates to the use of the α / β-hydrolase of the present invention for degrading zearalenone (ZEN).

Description

Technical Field

[0001] The present invention relates to a method for increasing the stability of an α / β-hydrolase. In addition, the present invention relates to an α / β-hydrolase obtainable by the method of the present invention. The present invention also provides an α / β-hydrolase having a reduced total average hydrophilicity (GRAVY) value and / or comprising a specific mutation. In addition, the present invention relates to the use of the α / β-hydrolase of the present invention for degrading zearalenone (ZEN). Background Art

[0002] Mycotoxins are secondary metabolites produced by filamentous fungi. An important representative of mycotoxins is zearalenone (ZEN) (formerly known as F-2 toxin), which is produced by many Fusarium fungi and can be found all over the world. These fungi infect cultivated plants in particular, such as various cereals, where fungal infection usually occurs before harvest, when fungal growth and / or mycotoxin production can occur before storage or even after harvest, i.e. before storage or under inappropriate storage conditions. The Food and Agriculture Organization of the United Nations (FAO) estimates that 25% of agricultural products are contaminated with mycotoxins worldwide, which leads to huge economic losses. In an international study spanning 8 years, a total of 19,757 samples were analyzed from January 2004 to December 2011, of which 72% tested positive for at least one mycotoxin, 39% were found to be contaminated, and 37% tested positive for ZEN (Schatzmayr and Streit (2013) 'Global occurrence of mycotoxins in the food and feed chain: Facts and figures.' World Mycotoxin Journal 6(3): 213-222). ZEN was found at an incidence as high as 100% in all regions of the world and in all categories of cereals and feed crops tested, such as corn, soybean meal, wheat, wheat bran, DDGS (dispersants dried grains with solubles), and in finished animal feed mixtures.

[0003] ZEN binds to estrogen receptors and can cause hormone disturbances, is absorbed immediately after oral ingestion, and is converted into two stereoisomeric metabolites by mammals, namely α-zearalenol (α-ZEL) and / or β-zearalenol (β-ZEL). For example, α-ZEL and α-zearalenol (α-ZAL) and / or zearalenone (ZAN) have stronger estrogenic effects than ZEN. Although the conjugated ZEN derivatives have lower estrogenic activity than ZEN itself, ZEN can be released again from these conjugated ZEN derivatives in the digestive tract, thereby restoring its full estrogenic activity.

[0004] ZEN has an oral LD50 of up to 20,000 mg / kg body weight, and subacute and / or subchronic toxic effects such as teratogenicity, carcinogenicity, estrogenicity, and immunosuppressive effects may occur in animals or humans with long-term exposure. Feed contaminated with ZEN causes developmental disorders in mammals. Among them, pigs, especially piglets, are extremely sensitive to ZEN. ZEN concentrations exceeding 0.5 ppm in feed cause developmental disorders, and concentrations exceeding 1.5 ppm can cause hyperestrogenicity in pigs. In cattle, 12 ppm ZEN concentrations can cause spontaneous abortion.

[0005] Since ZEN is rapidly absorbed through the mucosa, especially through the gastric mucosa and oral mucosa, immediate and quantitative inactivation is necessary. ZEN can already be detected in the blood 30 minutes after oral administration. Due to the harmful effects of ZEN, the European Union has a legal upper limit for ZEN in food and a recommendation for an upper limit for ZEN in feed (EC No. 1881 / 2006).

[0006] The initial strategy for reducing ZEN contamination of food and feed products is to limit fungal growth, for example by following "good agricultural practices". This includes, in particular, ensuring that seeds are free of pest and fungal infestations, or ensuring that agricultural waste is removed from the fields in a timely manner. In addition, fungal growth in the field can also be reduced by the use of fungicides. After harvesting, harvested material should be stored with a residual moisture of less than 15% and at low temperatures to prevent fungal growth. Similarly, material contaminated by fungal infestations should be removed before further processing. Despite this series of preventive measures, even in regions with the highest agricultural standards (such as North America and Central Europe), up to 37% of tested maize samples were found to be contaminated with ZEN between 2004 and 2011 (Schatzmayr and Streit (2013)). Summary of the invention

[0007] In order to overcome the above problems and defects, it is necessary to develop another α / β-hydrolase that can detoxify ZEN and is suitable for use as a food or feed additive or a food or feed product.

[0008] Aspects of the present invention are described below, illustrated in the examples, shown in the drawings, and reflected in the claims.

[0009] The present invention relates to a method for increasing the stability of an α / β-hydrolase, the α / β-hydrolase comprising a sequence corresponding to positions 145 to 218 of SEQ ID NO: 1, or comprising a sequence having a sequence identity of 58% or more to the sequence corresponding to positions 145 to 218 of SEQ ID NO: 1 (CAP-domain; 58% identity to the CAP-domain of SEQ ID NO: 1), the method comprising:

[0010] Substitute at least one amino acid at the following positions:

[0011] - a position corresponding to positions 160 to 205 of SEQ ID NO: 1, or

[0012] - a position corresponding to positions 159 to 204 of SEQ ID NO: 2, or

[0013] - a position corresponding to position 160 to 205 of SEQ ID NO: 3, 4 or 5, or

[0014] - a position corresponding to positions 176 to 222 of SEQ ID NO: 6,

[0015] wherein the amino acid is replaced with an amino acid having a more negative hydropathic index than the replaced amino acid, wherein the hydropathic index is determined by the Kyte and Doolittle hydropathic index, thereby obtaining an α / β-hydrolase with increased stability.

[0016] Furthermore, the present invention relates to an α / β-hydrolase obtainable by the method of the present invention.

[0017] The present invention also provides an α / β-hydrolase having a polypeptide sequence, wherein the polypeptide sequence comprises a sequence corresponding to positions 145 to 218 of SEQ ID NO: 1, or comprises a sequence having a sequence identity of greater than 58% with the sequence corresponding to positions 145 to 218 of SEQ ID NO: 1,

[0018] Wherein, the polypeptide sequence comprises at least one amino acid substitution at the following position:

[0019] - a position corresponding to positions 160 to 205 of SEQ ID NO: 1, or

[0020] - a position corresponding to positions 159 to 204 of SEQ ID NO: 2, or

[0021] - a position corresponding to position 160 to 205 of SEQ ID NO: 3, 4 or 5, or

[0022] - a position corresponding to positions 176 to 222 of SEQ ID NO: 6,

[0023] The α / β-hydrolase has a global average hydrophilicity (GRAVY) value that is at least 0.6% negative compared to the GRAVY value of the α / β-hydrolase having the polypeptide sequence of SEQ ID NO:1.

[0024] The present invention also relates to an α / β-hydrolase having a polypeptide sequence, wherein the polypeptide sequence comprises a sequence corresponding to positions 145 to 218 of SEQ ID NO: 1, or comprises a sequence having a sequence identity of greater than 58% with the sequence corresponding to positions 145 to 218 of SEQ ID NO: 1,

[0025] Wherein, the polypeptide sequence comprises at least one amino acid substitution at the following position:

[0026] - a position corresponding to positions 185 to 191 of SEQ ID NO: 1, or

[0027] - a position corresponding to positions 184 to 190 of SEQ ID NO: 2, or

[0028] - a position corresponding to position 185 to 191 of SEQ ID NO: 3, 4 or 5, or

[0029] - a position corresponding to positions 201 to 208 of SEQ ID NO: 6,

[0030] wherein the amino acid substitution is selected from V→A, G→R, G→S, A→P, A→R, A→D, A→H, A→N, A→G, S→D, P→H, M→D, G→E, I→A, I→V, H→N and Q→K, and / or

[0031] The amino acid is replaced by an amino acid selected from P, R, D, H, G or N. Preferably, the amino acid used is selected from R, D, H, G or N. More preferably, the amino acid used is selected from R or N.

[0032] The present invention also relates to an α / β-hydrolase having a polypeptide sequence, wherein the polypeptide sequence comprises a sequence corresponding to positions 161 to 235 of SEQ ID NO: 6, or comprises a sequence having a sequence identity of greater than 58% with the sequence corresponding to positions 161 to 235 of SEQ ID NO: 6,

[0033] Wherein, the polypeptide sequence comprises at least one amino acid substitution at the following position:

[0034] - a position corresponding to positions 160 to 205 of SEQ ID NO: 1, or

[0035] - a position corresponding to positions 159 to 204 of SEQ ID NO: 2, or

[0036] - a position corresponding to position 160 to 205 of SEQ ID NO: 3, 4 or 5, or

[0037] - a position corresponding to positions 176 to 222 of SEQ ID NO: 6,

[0038] The α / β-hydrolase has a GRAVY value that is at least 0.6% negative compared to the GRAVY value of the α / β-hydrolase having the polypeptide sequence of SEQ ID NO:6.

[0039] Furthermore, the present invention relates to a use of the α / β-hydrolase of the present invention for degrading ZEN.

[0040] Furthermore, the present invention relates to a composition comprising the α / β-hydrolase of the present invention, preferably, the composition is a food or feed additive or a food or feed product.

[0041] In addition, the present invention relates to the α / β-hydrolase or the composition of the present invention for use in the treatment or prevention of a disease.

[0042] Furthermore, the present invention relates to a kit comprising the α / β-hydrolase or composition of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 : Position of the CAP-domain, VI-domain and CAP-loop. Amino acid positions of the CAP-domain, VI-domain and CAP-loop of SEQ ID NOs: 1-6.

[0044] Figure 2A-2G: Different mutations in the VI-domain and / or CAP-loop and their effect on the GRAVY value. 2A: Effect of modifications in the VI-domain of SEQ ID NO: 1 on the GRAVY value of SEQ ID NO: 1 variants. 2B: Effect of modifications in the VI-domain of SEQ ID NO: 1 on the GRAVY value of the CAP-domain of SEQ ID NO: 1 variants. 2C: Effect of modifications in the VI-domain of SEQ ID NO: 1 on the GRAVY value of the VI-domain of SEQ ID NO: 1 variants. 2D: Effect of modifications in the CAP-loop of SEQ ID NO: 1 on the GRAVY value of the CAP-loop of SEQ ID NO: 1 variants. 2E: Effect of modifications in the VI-domain of SEQ ID NO: 6 on the GRAVY value of SEQ ID NO: 6 variants. 2F: Effect of modifications in the VI-domain of SEQ ID NO: 6 on the GRAVY value of the VI-domain of SEQ ID NO: 6 variants. 2G: Effect of modifications in the VI-domain of SEQ ID NO: 6 on the GRAVY values ​​of the VI-domain of SEQ ID NO: 6 variants.

[0045] Figure 3A-3B : The percentage increase in temperature stability of the polypeptide that degrades ZEN relative to the polypeptide SEQ ID NO: 1 or SEQ ID NO: 6. 3A: The percentage increase in temperature stability of the polypeptide that degrades ZEN relative to the polypeptide SEQ ID NO: 1 (T(50%)). 3B: The percentage increase in temperature stability of the polypeptide that degrades ZEN relative to the polypeptide SEQ ID NO: 6 (T(50%)).

[0046] Figure 4 : Activity of ZEN-degrading polypeptide variants after incubation at pH 4.0 compared to activity after incubation at pH 7.5 (= pH stability). Percent residual activity of ZEN-degrading polypeptide variants after incubation at pH 4.0 compared to the same polypeptide variants after incubation at pH 7.5. The residual activity (pH stability) of the parent polypeptide SEQ ID NO: 1 was 2.5%.

[0047] Figure 5 : Selected reaction monitoring parameters on 6500 QTrap for analysis of samples from pig feeding trials. Analysis of samples from pig feeding trials was performed on an Agilent 1290 series UHPLC system connected to a 6500 QTrap mass spectrometer. Selected reaction monitoring parameters are shown. Product ions are given as quantifiers / qualifiers.

[0048] Figure 6 : Results of analysis of urine samples from pig feeding trials compared to SEQ ID NO: 1. The total amount of ZEN plus α-ZEL in each group of urine samples was determined on an Agilent 1290 Series UHPLC system connected to a 6500QTrap mass spectrometer (mean for each group; n=3). The control group was fed a diet containing ZEN, but without a polypeptide that degrades ZEN. The SEQ ID NO: 1 group, the variant A group, and the variant B group were fed the same diet as the control group, but additionally contained 2.5 U / kg diet, 5 U / kg diet, 10 U / kg diet, or 20 U / kg diet of the designated polypeptide that degrades ZEN. The change in the amount of ZEN plus α-ZEL in urine compared to SEQ ID NO: 1 is shown as a percentage.

[0049] Figure 7 : Results of analysis of fecal samples from a pig feeding trial compared to SEQ ID NO: 1. The total amount of ZEN plus α-ZEL per gram of freeze-dried feces was determined on an Agilent 1290 Series UHPLC system connected to a 6500QTrap mass spectrometer (mean for each group; n=3). The control group was fed a diet containing ZEN, but without a polypeptide that degrades ZEN. The SEQ ID NO: 1 group, the variant A group, and the variant B group were fed the same diet as the control group, but additionally contained 2.5 U / kg diet, 5 U / kg diet, 10 U / kg diet, or 20 U / kg diet of the specified polypeptide that degrades ZEN. The change in the amount of ZEN plus α-ZEL in feces compared to SEQ ID NO: 1 is shown as a percentage.

[0050] Figure 8 : Selected reaction monitoring parameters on 6500 QTrap for analysis of samples from chicken feeding trials. Analysis of samples from chicken feeding trials was performed on an Agilent 1290 series UHPLC system connected to a 6500 QTrap mass spectrometer. Selected reaction monitoring parameters are shown. Product ions are given in quantitative / qualitative terms.

[0051] Fig. 9 : Results of analysis of crop samples from a chicken feeding trial compared to SEQ ID NO: 1. The concentration of ZEN per kg of freeze-dried crop samples was determined on an Agilent 1290 series UHPLC system connected to a 6500QTrap mass spectrometer (mean for each group; n=8). The control group was fed a diet containing ZEN, but without a polypeptide that degrades ZEN. The other groups were fed the same diet as the control group, but additionally contained the indicated amount of enzymatically active ZEN-degrading polypeptide variant B. The change in the concentration of ZEN in the crop compared to the control group is shown in percentage. DETAILED DESCRIPTION

[0052] Surprisingly, it was found that the α / β-hydrolases containing the mutations described herein in specific regions, i.e. the VI-domain and the CAP-loop, exhibited higher temperature stability and / or pH stability. Without being bound by theory, it is believed that the VI-domain and the CAP-loop play an important role in the enzymatic activity, for example, for the substrate to enter the active site of the enzyme. The high flexibility of this part of the enzyme can have a positive impact on the activity, however, this flexibility can also have a negative impact on the stability.

[0053] The inventors of the present invention identified the CAP domain of SEQ ID NO: 1 as amino acids at positions 145 to 218, the CAP domain of SEQ ID NO: 2 as positions 144-217, the CAP domain of SEQ ID NO: 3, 4 and 5 as positions 145-218, and the CAP domain of SEQ ID NO: 6 as positions 161-235. In addition, the inventors of the present application identified the VI domain of SEQ ID NO: 1 as amino acid positions 160 to 205, the VI domain of SEQ ID NO: 2 as amino acid positions 159-204, the VI domain of SEQ ID NO: 3, 4 and 5 as amino acid positions 160-205, and the VI domain of SEQ ID NO: 6 as amino acid positions 176-222. In particular, the combination of dynamic simulations of variants of, for example, SEQ ID NO: 1 or 6 with X-ray diffraction data was generated by Phenix ensemble refinement (https: / / www.phenix-online.org / ; Burnley and Gros (2012) 'phenix.ensemble_refinement: a test study of apo and holo BACE1' Computational crystallography newsletter, volume 4, pp. 51-58) to reflect the flexible loop by generating 65 structures. The region of SEQ ID NO: 1 defined by amino acid positions 185 to 191 (defined herein as the CAP-loop) is part of this flexible loop (and the region defined by the equivalent positions in SEQ ID NO: 2-6 as described herein is also).

[0054] The mutations as described herein introduced into the CAP-domain, in particular into the VI-domain as defined herein, or more particularly into the CAP-loop as defined herein, provide sufficient temperature stability without loss of activity properties and / or pH stability, such that the enzyme can be used in processes at elevated temperatures.

[0055] This is particularly important since in the food and feed industry thermal treatments such as pelleting are often used to produce hygienic products with a reduced microbial load.

[0056] The pelleting of feed is a particularly common standardized method to enhance flowability, reduce dust formation and lower the microbial load (especially Salmonella). During pelleting, the product is usually moistened by hot steam, heated and then pressed through a matrix under pressure. This heat treatment of enzymes or polypeptides often leads to a reduction in their enzymatic activity and / or their irreversible denaturation.

[0057] Furthermore, when enzymes are used in food or feed, they are often subject to inactivation by the conditions in the gastrointestinal tract of animals. In particular, low pH conditions can cause the enzymatic activity of enzymes or polypeptides to be temporarily or permanently reduced or even eliminated.

[0058] However, enzymes that degrade ZEN generally have low temperature stability and / or pH stability and thus cannot be mixed into feed or food. Therefore, using polypeptides or enzymes as additives for pelleted food or feed has become a considerable technical challenge.

[0059] The α / β-hydrolases described herein have increased stability, especially with respect to temperature and / or pH stability, and are therefore very suitable for use in food and feed production processes.

[0060] Therefore, the present invention relates to a method for increasing the stability of an α / β-hydrolase comprising a sequence corresponding to positions 145 to 218 of SEQ ID NO: 1, or comprising a sequence having 58% or more sequence identity with the sequence corresponding to positions 145 to 218 of SEQ ID NO: 1 (CAP-domain; 58% identity with the CAP-domain of SEQ ID NO: 1), the method comprising:

[0061] Substitute at least one amino acid at the following positions:

[0062] - a position corresponding to positions 160 to 205 of SEQ ID NO: 1, or

[0063] - a position corresponding to positions 159 to 204 of SEQ ID NO: 2, or

[0064] - a position corresponding to position 160 to 205 of SEQ ID NO: 3, 4 or 5, or

[0065] - a position corresponding to positions 176 to 222 of SEQ ID NO: 6,

[0066] wherein the amino acid is replaced with an amino acid having a more negative hydropathic index than the replaced amino acid,

[0067] The hydropathic index is determined by the Kyte and Doolittle hydropathic index.

[0068] Thereby an α / β-hydrolase with increased stability is obtained, preferably, the α / β-hydrolase has an increased stability compared to the α / β-hydrolase before replacing the amino acid and / or the α / β-hydrolase has an increased stability compared to the α / β-hydrolase without the amino acid replacement.

[0069] The present invention also relates to a method for increasing the stability of an α / β-hydrolase comprising a sequence corresponding to positions 161 to 235 of SEQ ID NO: 6, or comprising a sequence having 58% or more sequence identity with the sequence corresponding to positions 161 to 235 of SEQ ID NO: 6 (CAP-domain; 58% identity with the CAP-domain of SEQ ID NO: 6), the method comprising:

[0070] Substitute at least one amino acid at the following positions:

[0071] - a position corresponding to positions 160 to 205 of SEQ ID NO: 1, or

[0072] - a position corresponding to positions 159 to 204 of SEQ ID NO: 2, or

[0073] - a position corresponding to position 160 to 205 of SEQ ID NO: 3, 4 or 5, or

[0074] - a position corresponding to positions 176 to 222 of SEQ ID NO: 6,

[0075] wherein the amino acid is replaced with an amino acid having a more negative hydropathic index than the replaced amino acid,

[0076] The hydropathic index is determined by the Kyte and Doolittle hydropathic index.

[0077] Thereby an α / β-hydrolase with increased stability is obtained, preferably, the α / β-hydrolase has an increased stability compared to the α / β-hydrolase before replacing the amino acid and / or the α / β-hydrolase has an increased stability compared to the α / β-hydrolase without the amino acid replacement.

[0078] Increased stability as used herein may mean that, compared to an α / β-hydrolase comprising a sequence corresponding to position 145 to 218 of SEQ ID NO: 1 (and SEQ ID NO: 3, 4, 5), the α / β-hydrolase of the present invention has higher stability. Alternatively or additionally, increased stability as used herein means that, compared to an α / β-hydrolase comprising a sequence corresponding to position 144 to 217 of SEQ ID NO: 2, the α / β-hydrolase of the present invention has higher stability. Alternatively or additionally, the α / β-hydrolase comprises a sequence corresponding to position 161 to 235 of SEQ ID NO: 6.

[0079] Increased stability as used herein may also mean that the α / β-hydrolase of the present invention has a higher stability compared to the α / β-hydrolase of SEQ ID NO: 1. Alternatively or additionally, increased stability as used herein may also mean that the α / β-hydrolase of the present invention has a higher stability compared to the α / β-hydrolase of SEQ ID NO: 2. Alternatively or additionally, increased stability as used herein may also mean that the α / β-hydrolase of the present invention has a higher stability compared to the α / β-hydrolase of SEQ ID NO: 6.

[0080] This includes, compared to an α / β-hydrolase without the substitutions described herein, the α / β-hydrolase with increased stability obtained, for example, by the method of the present invention or the α / β-hydrolase of the present invention has increased stability. Similarly, compared to the α / β-hydrolase before substitution of the amino acids disclosed herein, the α / β-hydrolase with increased stability obtained, for example, by the method of the present invention or the α / β-hydrolase of the present invention has increased stability.

[0081] Various α / β-hydrolases are known to those skilled in the art, which are described in particular in Lenfant et al., (2013) 'ESTHER, the database of the α / β-hydrolase fold superfamily of proteins: tools to explore diversity of functions' Nucleic Acids Research, Volume 41, Issue D1, D423-D429 and Mindrebo et al., (2016) 'Unveiling the functional diversity of the Alpha-Beta hydrolase fold in plants' Curr Opin Struct Biol. 233-246. In short, all α / β-hydrolases have a specific folding feature, called the α / β-fold (alpha / beta-fold). This α / β-hydrolase fold is common to many hydrolases with very different systematic origins and catalytic functions. The core of each enzyme is an α / β-structure (not a barrel) containing 8 β-strands (b1-b8) connected by α-helices (aA-aF) (Ollis et al., (1992) 'The alpha / betahydrolase fold' Protein Eng. 5(3): 197-211). Therefore, the α / β-hydrolase described herein may contain an α / β-fold. Preferably, the α / β-hydrolase described herein comprises an α / β-hydrolase core domain consisting of 8 β-strands (b1-b8) arranged in a central β-fold and additionally comprises 6 cross α-helices (aA-aF).

[0082] In most family members, the β-strands are parallel oriented, but some members have an inversion of the first strand, resulting in a trans-parallel orientation. The prototype of the folded enzyme has a catalytic triad consisting of a nucleophilic residue at the top of the γ-turn between the fifth β-strand and the subsequent α-helix (nucleophile elbow), an acidic amino acid residue (glutamic acid or aspartic acid) and a histidine residue. Some other members lack one or all catalytic residues. Therefore, some members are inactive; some members are involved in surface recognition. Preferably, the α / β-hydrolase described herein comprises the catalytic triad.

[0083] The members of the different classes of α / β-hydrolases and their structural features are described inter alia in Kourist et al. (2010) 'The alpha / beta-hydrolase fold 3DM database (ABHDB) as a tool for protein engineering.' Chembiochem. 11(12): 1635-43).

[0084] As enzyme, α / β-hydrolase is usually described as responsible for the hydrolysis of ester bond and peptide bond. However, α / β-hydrolase also participates in the fracture of carbon-carbon bond, decarboxylation reaction and the cofactor-independent dual oxidation of heteroaromatic ring. Therefore, α / β-hydrolase can include the catalytic member (enzyme) in this superfamily. Non-limiting examples are hydrolases (acetylcholinesterase, carboxylesterase, diene lactone hydrolase, lipase, cutinase, thioesterase, serine carboxypeptidase, proline iminopeptidase, proline oligopeptidase, epoxide hydrolase) and enzymes (haloalkane dehalogenase, haloperoxidase, hydroxynitrile lyase) that require HCN, H2O2 or O2 rather than H2O activation for reaction mechanism. Non-catalytic members can include neuroligin, glutactin, neurochemokine, C-terminal domain of thyroglobulin, yolk protein, CCG1-interacting factor-B and dipeptide aminopeptidase VI.

[0085] The ESTHER database collects and annotates public information related to the gene and protein sequences of this superfamily. Therefore, those skilled in the art can also obtain α / β-hydrolases from ESTHER (http: / / bioweb.supagro.inra.fr / ESTHER / general?what=index), a database of α / β-hydrolase-fold protein superfamily.

[0086] A person skilled in the art can also determine whether an α / β-hydrolase contains a CAP-domain. One method for achieving this is described in the Examples or as follows:

[0087] 1. Search for α / β-hydrolases in online enzyme databases or by comparing a given sequence with SEQ ID NOs: 1-6.

[0088] 2. Determine whether the α / β-hydrolase comprises a CAP-domain, a VI-domain or a CAP-loop, preferably by using the procedure described in Example 2.

[0089] 3. Preferably by using the procedure described in Example 4 determine whether an α / β-hydrolase comprising a CAP-domain, a VI-domain or a CAP-loop can hydrolyze ZEN.

[0090] The α / β-hydrolase as described herein comprises a sequence corresponding to positions 145 to 218 of SEQ ID NO: 1, or comprises a sequence having 58% or higher sequence identity with the sequence corresponding to positions 145 to 218 of SEQ ID NO: 1 (CAP-domain). Therefore, any α / β-hydrolase comprising this sequence is encompassed within the term α / β-hydrolase. This sequence corresponds to the CAP-domain of the α / β-hydrolase of SEQ ID NO: 1.

[0091] Additionally or alternatively, the α / β-hydrolase as described herein may also comprise a sequence corresponding to positions 144 to 217 of SEQ ID NO: 2, or a sequence having 58% or higher sequence identity to the sequence corresponding to positions 144 to 217 of SEQ ID NO: 2 (CAP-domain). Thus, any α / β-hydrolase comprising this sequence is encompassed within the term α / β-hydrolase. This sequence corresponds to the CAP-domain of the α / β-hydrolase of SEQ ID NO: 2.

[0092] Additionally or alternatively, the α / β-hydrolase as described herein may also comprise a sequence corresponding to positions 145 to 218 of SEQ ID NO: 3, 4 or 5, or a sequence having 58% or higher sequence identity to the sequence corresponding to positions 145 to 218 of 3, 4 or 5 (CAP-domain). Thus, any α / β-hydrolase comprising this sequence is encompassed within the term α / β-hydrolase. This sequence corresponds to the CAP-domain of the α / β-hydrolase of 3, 4 or 5.

[0093] Additionally or alternatively, the α / β-hydrolase as described herein may also comprise a sequence corresponding to positions 161 to 235 of SEQ ID NO: 6, or a sequence having 58% or higher sequence identity to the sequence corresponding to positions 161 to 235 of SEQ ID NO: 6 (CAP-domain). Thus, any α / β-hydrolase comprising this sequence is encompassed within the term α / β-hydrolase. This sequence corresponds to the CAP-domain of the α / β-hydrolase of SEQ ID NO: 6.

[0094] For example, the α / β-hydrolase may comprise a sequence that is at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identical to the sequence of SEQ ID NO: 1. Additionally or alternatively, the α / β-hydrolase may comprise a sequence that is at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identical to the sequence of SEQ ID NO: 2. Additionally or alternatively, the α / β-hydrolase may comprise a sequence having at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to the sequence of SEQ ID NO: 3, 4 and / or 5. Additionally or alternatively, the α / β-hydrolase may comprise a sequence having at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to the sequence of SEQ ID NO: 6.

[0095] As used herein, the term "polypeptide" means a peptide, protein or polypeptide, which can be used interchangeably and encompasses an amino acid chain of a given length in which the amino acid residues are linked by covalent peptide bonds. The present invention also encompasses amino acids other than the 20 proteinogenic amino acids of the standard genetic code known to those skilled in the art, such as selenocysteine. Such polypeptides include any one of SEQ ID NO: 1-6.

[0096] The term polypeptide also refers to, and does not exclude, modifications of the polypeptide. Modifications include glycosylation, acetylation, acylation, phosphorylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination; see, e.g., PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. Creighton, W. H. Freeman and Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, BC Johnson, Ed., Academic Press, New York (1983), pgs.1-12; Seifter, Meth.Enzymol.182 (1990); 626-646, Rattan, Ann.NY Acad.Sci.663 (1992); 48-62.

[0097] According to the present invention, in the case of two or more polypeptide sequences (such as SEQ ID NO: 1-6), the term "identical" or "percentage of identity" refers to two or more sequences or subsequences that are identical or have the same specific percentage of nucleotides (e.g., at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity) when comparing and comparing the maximum correspondence using a sequence comparison algorithm known in the art or by manual alignment and visual inspection over a comparison window or over a specified area of ​​measurement. Sequences with, for example, 80% to 95% or higher sequence identity are considered to be substantially identical. This definition can also be applied to the complementary sequence of the test sequence. Those skilled in the art will know how to use, for example, those based on the CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245) as known in the art to determine the percentage of identity between / in sequences.

[0098] Also available to those skilled in the art are BLAST and BLAST 2.6 algorithms (Altschul Nucl. Acids Res. 25 (1977), 3389-3402). The BLASTP program for amino acid sequences uses a default word length (W) of 6, an expected threshold of 10, and both chains are compared. In addition, the BLOSUM62 scoring matrix (Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915; Henikoff and Henikoff (1992) 'Amino acid substitution matrices from protein blocks.' Proc Natl Acad Sci US A. 1992 Nov 15; 89 (22): 10915-9) can also be used.

[0099] For example, BLAST2.6 (Altschul, Nucl. Acids Res. 25 (1997), 3389-3402; Altschul, J. Mol. Evol. 36 (1993), 290-300; Altschul, J. Mol. Biol. 215 (1990), 403-410), which stands for Basic Local Alignment Search Tool, can be used to search local sequence alignments.

[0100] As used herein, a "CAP-domain" relates to the CAP-domain of an alpha / beta-hydrolase, which is described, for example, in Kourist et al., (2010) 'The alpha / beta-hydrolase fold 3DM database (ABHDB) as a tool for protein engineering.' Chembiochem. 11(12): 1635-43. Figure 1or described in Carr and Ollis (2009) 'α / βHydrolase Fold: An Update.' Protein & Peptide Letters, 2009, 16(10): 1137-1148. It is also envisaged that the CAP-domain may be located between the β-sheet and the α-helix, for example, in the excursion between b6 and aD of the α / β-hydrolase, as described, for example, in Ollis et al., (1992) 'The alpha / beta hydrolase fold' Protein Eng. 5(3): 197-211. For example, the CAP-domain may start shortly after the C-terminus of the b6 β-strand of the α / β-hydrolase core domain and may continue to the N-terminal start of the aD α-helix of the α / β-hydrolase core domain. However, the CAP-domain may also contain a β-sheet or other protein structure.

[0101] The method of the present invention requires replacing at least one amino acid at the following positions of the α / β-hydrolase:

[0102] - a position corresponding to positions 160 to 205 of SEQ ID NO: 1, or

[0103] - a position corresponding to positions 159 to 204 of SEQ ID NO: 2, or

[0104] - a position corresponding to position 160 to 205 of SEQ ID NO: 3, 4 or 5, or

[0105] - positions corresponding to positions 176 to 222 of SEQ ID NO: 6. These positions are all located in the VI-domain.

[0106] As used herein, 'VI-domain' is part of the CAP-domain. Thus, the CAP-domain comprises the VI-domain. The VI-domain may start at the first amino acid after the QXAGP motif (SEQ ID NO: 7) present in the CAP-domain and may continue to the last amino acid before the EYDPE motif (SEQ ID NO: 8), wherein the EYDPE motif is not part of the VI-domain. These motifs are underlined in the sequences described in Table 2 herein.

[0107] For example, the VI-domain may comprise a sequence corresponding to positions 160 to 205 of SEQ ID NO: 1, or a sequence having at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to the sequence corresponding to positions 160 to 205 of SEQ ID NO: 1. Additionally or alternatively, the VI-domain may comprise a sequence corresponding to positions 159 to 204 of SEQ ID NO: 2, or a sequence having at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to the sequence corresponding to positions 159 to 204 of SEQ ID NO: 2. Additionally or alternatively, the VI-domain may comprise a sequence corresponding to positions 160 to 205 of SEQ ID NO: 3, 4 and / or 5, or a sequence having at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to a sequence corresponding to positions 160 to 205 of SEQ ID NO: 3, 4 and / or 5. Additionally or alternatively, the VI-domain may comprise a sequence corresponding to positions 176 to 222 of SEQ ID NO: 6, or a sequence having at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to a sequence corresponding to positions 176 to 222 of SEQ ID NO: 6.

[0108] When used according to the present invention, the term "position" means the position of an amino acid in an amino acid sequence described herein. The term "corresponding" as used herein also includes that the position is not only determined by the number of preceding amino acids. The position of a given amino acid that can be replaced according to the present invention can be changed due to a missing or added amino acid, or the position of a given amino acid that can be replaced can be changed due to a missing or added amino acid elsewhere in a (mutant or wild-type) α / β-hydrolase.

[0109] Therefore, with respect to the "corresponding positions" according to the present invention, it is preferably understood that the amino acids may be different in the designated number, but may still have similar adjacent amino acids. The term "corresponding positions" also includes the amino acids that may be exchanged, deleted or added. Specifically, when comparing a reference sequence (subject sequence), such as any one of SEQ ID NOs: 1-6, preferably SEQ ID NO: 1, with a target amino acid sequence (query sequence), a person skilled in the art may, for example, check the target sequence to find the sequence of SEQ ID NO: 1 (or the corresponding amino acid sequence encoding the protein) when searching for the amino acid positions described herein (i.e., positions corresponding to positions 185 and / or 188 of the amino acid sequence shown in SEQ ID NO: 1).

[0110] In the methods of the present invention, the amino acid is replaced with an amino acid having a more negative hydropathic index than the replaced amino acid, wherein the hydropathic index is determined by the Kyte and Doolittle hydropathic index.

[0111] As used herein, "amino acid substitution" means the replacement of an amino acid relative to the corresponding position of the identified SEQ ID NO (e.g., any one of the positions specified herein of SEQ ID NOs: 1-6). For example, in one embodiment, the replacement is an amino acid replacement of an amino acid relative to the amino acid at a position corresponding to position 160 to 205 of SEQ ID NO: 1.

[0112] "Hydrophilic index", also referred to herein as "hydrophilic value", is a number representing the hydrophobicity or hydrophilicity of an amino acid side chain. In particular, for the hydropathic index, each amino acid has been assigned a value reflecting its relative hydrophilicity. Therefore, the hydrophilicity of an amino acid can be determined by the hydropathic index. Jack Kyte and Russell F. Doolittle (Kyte and Doolittle (1983) "A simple method for displaying the hydropathic character of a protein". J. Mol. Biol. 157 (1): 105–32) proposed this hydropathic index for amino acids. The amino acids with the least negative hydropathic index are isoleucine (4.5) and valine (4.2). According to Kyte and Doolittle, the amino acids with the most negative hydropathic index are arginine (-4.5) and lysine (-3.9). The hydropathic index is considered important in protein structure. Amino acids with less negative hydropathic indices tend to be internal (in terms of the three-dimensional shape of the protein) while amino acids with more negative hydropathic indices are more common on the surface of the protein. The hydropathic indices of Kyte and Doolittle have been summarized in Table 1 herein:

[0113] Table 1: Hydrophilicity index of Kyte and Doolittle

[0114]

[0115]

[0116] It is further envisioned that at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more amino acids are substituted.

[0117] The method also contemplates substituting at least one amino acid at the following positions:

[0118] - positions corresponding to positions 185 to 191 of SEQ ID NO: 1, and / or

[0119] - a position corresponding to positions 184 to 190 of SEQ ID NO: 2, and / or

[0120] - a position corresponding to position 185 to 191 of SEQ ID NO: 3, 4 or 5, and / or

[0121] - positions corresponding to positions 201 to 208 of SEQ ID NO: 6.

[0122] All of these positions are located within the CAP-loop.

[0123] In this context, it should be noted that the CAP-domain and the VI-domain may further comprise a loop (sequence / domain). This 'loop' is also referred to herein as the 'CAP-loop', which may start after the first amino acid after the G(F / Y)XXAA (SEQ ID NO: 9) motif present in the VI-domain and may continue to the last amino acid before the ARXF motif (SEQ ID NO: 10) (or the QLFP motif (SEQ ID NO: 11) for SEQ ID NO: 6), wherein the ARXF motif (SEQ ID NO: 10) (or the QLFP motif (SEQ ID NO: 11) for SEQ ID NO: 6) is not part of the CAP-loop. These motifs are underlined in the sequences described in Table 2 below.

[0124] For example, the CAP-loop may comprise a sequence corresponding to positions 185 to 191 of SEQ ID NO: 1, or a sequence that is at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identical to the sequence corresponding to positions 185 to 191 of SEQ ID NO: 1. Additionally or alternatively, the CAP-loop may comprise a sequence corresponding to positions 184 to 190 of SEQ ID NO: 2, or a sequence that is at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identical to the sequence corresponding to positions 184 to 190 of SEQ ID NO: 2. Additionally or alternatively, the CAP-loop may comprise a sequence corresponding to positions 185 to 191 of SEQ ID NO: 3, 4 and / or 5, or a sequence having at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to the sequence corresponding to positions 185 to 191 of SEQ ID NO: 3, 4 and / or 5. Additionally or alternatively, the CAP-loop may comprise a sequence corresponding to positions 201 to 208 of SEQ ID NO: 6, or a sequence having at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to the sequence corresponding to positions 201 to 208 of SEQ ID NO: 6.

[0125] Thus, an α / β-hydrolase as described herein may comprise a VI-domain and / or a CAP-loop as described herein.

[0126] It is also contemplated that the amino acid is replaced with an amino acid selected from R, K, N, Q, D, E, H, P, Y, W, S, T, G, A, M, C, F, L or V.

[0127] It is further envisaged that the amino acid is replaced with an amino acid selected from R, K, N, Q, D, E, H, P, Y, W, S, T or G.

[0128] Substitution of the amino acid with an amino acid selected from R, K, N, Q, D, E, H or P is also contemplated.

[0129] It is further envisaged that the amino acid is replaced with an amino acid selected from R, K, D, Q, D, N, E, P, G, T, S or H.

[0130] It is also contemplated that the amino acid is replaced with an amino acid selected from S, P, R, D, H, G or N. The amino acid may also be replaced with an amino acid selected from R, D, H, G or N.

[0131] It is also contemplated that the amino acid is replaced with an amino acid selected from P, S, R or H. The amino acid may also be replaced with an amino acid selected from R or N.

[0132] It is further envisioned that the amino acid substitution is selected from one or more of the following: V→A, G→R, G→S, A→P, A→R, A→D, A→H, A→N, A→G, S→D, P→H, M→D, G→E, I→A, I→V, H→N, Q→K, F→Y and / or V→C.

[0133] The amino acid substitutions may also be selected from one or more of the following: V160A, G185R, G185S, A186P, A186R, A188D, A188H, A188N, A188G, A188R, S189D, P190H, M191D, G199E, I200A, I200V, H203N, Q205K, F183Y and / or V197C.

[0134] It is also contemplated that the amino acid substitutions are selected from one or more of the following: V→A, G→R, G→S, A→P, A→R, A→D, A→H, A→N, A→G, S→D, P→H, M→D, G→E, I→A, I→V, H→N, and / or Q→K. The amino acid substitutions may also be selected from one or more of the following: V160A, G185R, G185S, A186P, A186R, A188D, A188H, A188N, A188G, A188R, S189D, P190H, M191D, G199E, I200A, I200V, H203N, and / or Q205K.

[0135] The amino acid substitutions may also be selected from G185R, A186R, A188R, A188D, A188H, A188N and / or M191D.

[0136] It is further envisaged that the amino acid is replaced with an amino acid selected from R, D, H, G, N or P.

[0137] It is also contemplated that the methods of the invention comprise substituting at least one amino acid at the following positions:

[0138] - positions corresponding to positions 185 to 191 of SEQ ID NO: 1, and / or

[0139] - a position corresponding to positions 184 to 190 of SEQ ID NO: 2, and / or

[0140] - a position corresponding to position 185 to 191 of SEQ ID NO: 3, 4 or 5, and / or

[0141] - positions corresponding to positions 201 to 208 of SEQ ID NO: 6.

[0142] And wherein the amino acid is replaced by an amino acid selected from R, D, H, G, N or P.

[0143] The present invention relates to a method for increasing the stability of α / β-hydrolase. The increased stability can be a decrease in GRAVY value, an increase in pH stability and / or an increase in temperature stability.

[0144] As used herein, the "GRAVY value" of a protein is a measure of its relative hydrophobicity or hydrophilicity. The two measures are combined in a hydrophilicity scale or hydropathic index. According to Kyte and Doolittle (Kyte J, Doolittle RF (May 1983). "A simple method for displaying the hydropathic character of a protein". J. Mol. Biol. 157 (1): 105–32), the GRAVY value is calculated by adding the hydropathic value (hydropathy index, see Table 1 above) of each residue and dividing it by the number of residues in the sequence. Therefore, the GRAVY value can be calculated by dividing the sum of the hydropathic values ​​(index) of all amino acids by the number of amino acid residues in the sequence (calculated according to Kyte and Doolittle). As used herein, the term "temperature stability" refers to the property of an enzyme maintaining its catalytic activity after temporary exposure to an elevated temperature. Temperature stability is determined by measuring and comparing the enzyme activity of an enzyme or polypeptide solution before and after 10 minutes of heat treatment or without heat treatment under the same defined conditions.

[0145] In particular, temperature stability can be measured in the following manner. The polypeptide is diluted to a concentration of 0.001526923U / ml using a sample buffer (Teorell Stenhagen buffer (Stenhagen & Teorell. (1938) Nature 141, 415) of pH 7.5, containing 0.1mg / ml bovine serum albumin) and placed on ice for standby use. 40 aliquots of 50μl diluted polypeptide solutions are transferred to 4 groups of 12-tubes (e.g., purchased from starlab), wherein the first and last tubes of each group of tubes are left vacant. The tubes are sealed with 12-caps (e.g., purchased from starlab). As a positive control, 4 aliquots of 50μl diluted enzyme solutions are transferred to 4 PCR tubes. All PCR tubes and tubes are placed on ice until the temperature incubation step is started. As a negative control, 4 aliquots of 50μl sample buffer are transferred to 4 PCR tubes. These tubes are stored at 25°C.

[0146] At a selected temperature of + / -10°C, 4 sets of 12-tube strips are incubated in a preheated PCR cycler with a gradient function (e.g., Eppendorf Mastercycler gradient). The PCR cycler automatically calculates the temperature gradient along the PCR cycler heating block (selected temperature of + / -10°C). The PCR tubes containing the positive control are incubated on ice, and the PCR tubes containing the negative control are incubated at 25°C. After 0, 5, 10, and 20 minutes, 1 set of PCR strips and 1 negative control tube are transferred to be placed on ice until the end of incubation, which is 20 minutes after the start of incubation. After all incubation steps are completed and all strips and tubes are placed on ice, the ZEN degradation analysis is started.

[0147] ZEN degradation assay buffer (Teorell Stenhagen buffer, pH 7.5, containing 0.1 mg / ml bovine serum albumin and 5.3 ppm ZEN) was prepared and aliquots of 660 μl of the assay buffer were transferred to 48 reaction tubes. The tubes were sealed and stored at 25°C until the start of the ZEN degradation assay. For the degradation assay, 40 μl of each of the 40 temperature treated samples from the PCR strip tubes, 40 μl of each of the 4 negative controls and 40 μl of each of the 4 positive controls were added to the tube containing 660 μl of the assay buffer, thereby achieving a final ZEN concentration of 5 ppm in the assay reaction. In addition, a final concentration of the polypeptide was thereby achieved to effectively degrade ZEN within three hours (i.e., 90%-100% ZEN degradation).

[0148] By adding the temperature treated sample, positive or negative control to the analysis buffer, the degradation analysis is started. The ZEN degradation reaction is incubated in a preheated water bath at 37°C. Immediately after the degradation reaction begins, vortex mix for about 2 seconds, and 100 μl of the 0h sample is transferred to a new reaction tube. After 0.5, 1.0, 2.0 and 3.0 hours, other samples are extracted from the ZEN degradation analysis reaction. Once the sample is extracted from the degradation reaction, the enzyme heat inactivation in the sample is made by incubation at 99°C for 10 minutes. Subsequently, the tube is centrifuged (2 minutes, 25°C, 14674xg) and 90 μl of supernatant is transferred to the HPLC bottle with an insert. These HPLC bottles are stored at 4°C until the HPLC-DAD measurement is performed as described in Example 4.

[0149] The enzyme activity was calculated in units / liter (U / l) using the linear decrease in ZEN concentration determined by HPLC-DAD analysis of ZEN degradation samples. One unit was defined as the amount of enzyme activity that degrades 1 μmol of ZEN in 1 minute under the conditions described. The residual activity after 0, 5, 10 and 20 minutes of incubation at different temperatures was calculated as follows: the enzyme activity in the temperature treated samples divided by the average of the enzyme activity of the positive controls, multiplied by 100.

[0150] The temperature stability (T(50%)) is defined as the temperature at which the polypeptide has 50% residual activity compared to the positive control after incubation for 10 minutes at this temperature. The following example is used for illustration: the parent enzyme has an enzyme activity of 50 U / ml after incubation on ice for 10 minutes and an activity of 25 U / ml after incubation at 59.3°C for 10 minutes, then the T(50%) value is 59.3°C. If the enzyme variant has a T(50%) value of 61.0°C, the relative increase in temperature stability (T(50%)) compared to the parent enzyme is 2.9%. This is obtained by dividing the difference between the two T(50%) values, 1.7°C, by the T(50%) value of the parent enzyme, 59.3°C, and multiplying by 100.

[0151] Thus, temperature stability as used herein is a measure of the resistance of an enzyme activity to inactivation when temporarily exposed to a temperature selected from 20° C. to 85° C. The temperature at which the residual activity of the heat-treated enzyme after 10 minutes of incubation is 50% can be compared to a positive control. An increase in T(50%) of a polypeptide variant relative to its parent polypeptide is defined herein as increased temperature stability, and this increase can be expressed relatively as a percentage value or absolutely in degrees Celsius.

[0152] As used herein, the term "pH stability" refers to the property that a polypeptide maintains its catalytic activity after temporary incubation at a specific pH, and thus "pH stability" is reflected by the residual activity of the polypeptide after temporary incubation at a specific pH. The residual activity after incubation at a specific pH is determined by comparing the enzyme activity of a polypeptide solution degrading ZEN after incubation for 60 minutes in different pH buffers with the enzyme activity of the same polypeptide solution after incubation for 60 minutes in a buffer at pH 7.5. The pH stability is a measure of the resistance of an enzyme to temporary exposure to a specific pH environment. An increase in pH stability is defined as an increase in the residual activity of a polypeptide variant after incubation at pH 4.0 (= pH treatment) compared to the residual activity of the parent enzyme variant after incubation at pH 4.0.

[0153] The pH stability can be measured as follows. The polypeptide of degraded ZEN is incubated in buffer solutions of different pH values ​​for 1 hour. The aliquot containing the polypeptide variant is transferred to 8 sample tubes containing 8 incubation buffers of different pH values. The incubation buffer is a semi-concentrated Fed State Simulated Gastric Fluid intermediate buffer (Jantratid et al., (2008) 'Dissolution media simulating conditions in the proximal human gastrointestinal tract: an update.' Pharm Res. 2008 Jul; 25 (7): 1663-76). The pH values ​​of the incubation buffers in the 8 sample tubes are set to 3.5, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0 and 6.0 respectively. One aliquot of the polypeptide variant is also transferred to a tube containing sample buffer (Teorell Stenhagen buffer, pH 7.5, containing 0.1 mg / ml bovine serum albumin) as a positive control. As a negative control, 100 μl of sample buffer was incubated in a preheated water bath at 37° C. for 1 hour. After incubation, the ability of the samples to degrade ZEN was determined in an assay buffer solution similar to that described elsewhere herein or described in the Examples (e.g., Example 4). The addition of ZEN degradation assay buffer ensured a constant pH of pH 7.5 in all samples. Samples were taken throughout the ZEN degradation assay reaction and analyzed for concentrations of ZEN, hydrolyzed zearalenone (HZEN), and decarboxylated hydrolyzed zearalenone (DHZEN) using HPLC-DAD measurements, as described, for example, in Example 4. Activity was calculated, for example, as described in Example 4.

[0154] The increase in pH stability is defined as the increase in the residual activity of the polypeptide solution after incubation at pH 4.0 compared to the residual activity of the unmutated parent enzyme solution after the same treatment. The residual activity is defined by comparing the activity of the polypeptide solution treated by pH- with the activity of the same polypeptide variant solution after incubation at pH 7.5. The residual activity is calculated as follows: the enzyme activity of the pH-treated sample is divided by the enzyme activity of the control incubated at pH 7.5, and then multiplied by 100. The following example is used to illustrate: if the enzyme activity of the polypeptide sample after incubation at pH 4.0 is 0.5U / l and the enzyme activity of the polypeptide after incubation at pH 7.5 is 2.7U / l, then the residual activity is 18.5%. If the residual activity of the parent polypeptide SEQ ID NO:1 after incubation at pH 4.0 is measured to be 2.5%, then the pH stability of the polypeptide variant is increased by 7.4 times compared to the parent polypeptide.

[0155] The present invention also relates to an α / β-hydrolase obtainable by the method described herein.

[0156] The present invention also relates to an α / β-hydrolase having a polypeptide sequence, wherein the polypeptide sequence comprises a sequence corresponding to positions 145 to 218 of SEQ ID NO: 1, or comprises a sequence having a sequence identity of greater than 58% with the sequence corresponding to positions 145 to 218 of SEQ ID NO: 1,

[0157] Wherein, the polypeptide sequence comprises at least one amino acid substitution at the following position:

[0158] - a position corresponding to positions 160 to 205 of SEQ ID NO: 1, or

[0159] - a position corresponding to positions 159 to 204 of SEQ ID NO: 2, or

[0160] - a position corresponding to position 160 to 205 of SEQ ID NO: 3, 4 or 5, or

[0161] - a position corresponding to positions 176 to 222 of SEQ ID NO: 6,

[0162] The α / β-hydrolase has a GRAVY value that is at least 0.6% negative compared to the GRAVY value of the α / β-hydrolase having the polypeptide sequence of SEQ ID NO:1.

[0163] The present invention also relates to an α / β-hydrolase having a polypeptide sequence, wherein the polypeptide sequence comprises a sequence corresponding to positions 161 to 235 of SEQ ID NO: 6, or comprises a sequence having a sequence identity of greater than 58% with the sequence corresponding to positions 161 to 235 of SEQ ID NO: 6,

[0164] Wherein, the polypeptide sequence comprises at least one amino acid substitution at the following position:

[0165] - a position corresponding to positions 160 to 205 of SEQ ID NO: 1, or

[0166] - a position corresponding to positions 159 to 204 of SEQ ID NO: 2, or

[0167] - a position corresponding to position 160 to 205 of SEQ ID NO: 3, 4 or 5, or

[0168] - a position corresponding to positions 176 to 222 of SEQ ID NO: 6,

[0169] The α / β-hydrolase has a GRAVY value that is at least 0.6% negative compared to the GRAVY value of the α / β-hydrolase having the polypeptide sequence of SEQ ID NO:6.

[0170] It is also contemplated that the α / β-hydrolase has a GRAVY value that is at least 3.0%, 4.2%, 4.8%, 6.0%, 6.6%, 7.8%, 10.2%, 12.0% or more lower than the GRAVY value of the α / β-hydrolase having the polypeptide sequence of SEQ ID NO:1.

[0171] It is also contemplated that the α / β-hydrolase has a GRAVY value that is at least 1.0%, 2.0%, 2.5%, 2.6%, 3.0%, 4.0%, 5.0%, 6.0%, 6.8% or more lower than the GRAVY value of the α / β-hydrolase having the polypeptide sequence of SEQ ID NO:6.

[0172] The present invention also relates to an α / β-hydrolase having a polypeptide sequence, wherein the polypeptide sequence comprises a sequence corresponding to positions 145 to 218 of SEQ ID NO: 1, or comprises a sequence having a sequence identity of greater than 58% with the sequence corresponding to positions 145 to 218 of SEQ ID NO: 1,

[0173] Wherein, the polypeptide sequence comprises at least one amino acid substitution at the following position:

[0174] - a position corresponding to positions 185 to 191 of SEQ ID NO: 1, or

[0175] - a position corresponding to positions 184 to 190 of SEQ ID NO: 2, or

[0176] - a position corresponding to position 185 to 191 of SEQ ID NO: 3, 4 or 5, or

[0177] - a position corresponding to positions 201 to 208 of SEQ ID NO: 6,

[0178] wherein the amino acid substitution is selected from V→A, G→R, G→S, A→P, A→R, A→D, A→H, A→N, A→G, S→D, P→H, M→D, G→E, I→A, I→V, H→N and Q→K, and / or

[0179] Wherein, the amino acid is replaced by an amino acid selected from P, R, D, H, G or N, preferably, the amino acid used is selected from R, D, H, G or N, more preferably, the amino acid used is selected from R or N. Such α / β-hydrolases may have a higher stability than the same α / β-hydrolases without or before the introduction of these replacements. For example, such α / β-hydrolases may have a higher stability than the α / β-hydrolases of SEQ ID NO: 1.

[0180] The present invention also relates to an α / β-hydrolase having a polypeptide sequence, wherein the polypeptide sequence comprises a sequence corresponding to positions 161 to 235 of SEQ ID NO: 6, or comprises a sequence having a sequence identity of greater than 58% with the sequence corresponding to positions 161 to 235 of SEQ ID NO: 6,

[0181] Wherein, the polypeptide sequence comprises at least one amino acid substitution at the following position:

[0182] - a position corresponding to positions 185 to 191 of SEQ ID NO: 1, or

[0183] - a position corresponding to positions 184 to 190 of SEQ ID NO: 2, or

[0184] - a position corresponding to position 185 to 191 of SEQ ID NO: 3, 4 or 5, or

[0185] - a position corresponding to positions 201 to 208 of SEQ ID NO: 6,

[0186] Wherein, the amino acid substitution is selected from F→Y or V→C. Such α / β-hydrolase may have a higher stability than the same α / β-hydrolase without or before the introduction of these substitutions. For example, such α / β-hydrolase may have a higher stability than the α / β-hydrolase of SEQ ID NO: 6.

[0187] It is envisaged that the α / β-hydrolase as described herein comprises the following amino acid substitutions:

[0188] - G→R and A→N, preferably G185R and A188N;

[0189] - G→S and A→R, preferably G185S and A188R;

[0190] - G→R, A→R, A→H, S→D, P→H and M→D, preferably G185R, A186R, A188H, S189D, P190H and M191D;

[0191] - V→A, G→R, A→N, G→E, I→A, H→N and Q→K, preferably V160A, G185R, A188N, G199E, I200A, H203N and Q205K;

[0192] - V→A, G→S, A→R, G→E, I→A, H→N and Q→K, preferably V160A, G185S, A188R, G199E, I200A, H203N and Q205K;

[0193] - V→A, G→E, I→A, H→N and Q→K, preferably V160A, G199E, I200A, H203N and Q205K;

[0194] - V→A, G→R, A→R, A→H, G→E, I→V, H→N and Q→K, preferably V160A, G185R, A186R, A188H, G199E, I200V, H203N and Q205K; and / or

[0195] F→Y and V→C, preferably F183Y and V197C.

[0196] The present invention also relates to nucleic acid molecules encoding α / β-hydrolases as described herein. The nucleic acid molecules can be introduced into or inserted into expression vectors. The term "expression vector" refers to a nucleic acid molecule construct capable of expressing a gene in vivo or in vitro. In particular, it encompasses DNA constructs that are suitable for transferring a nucleotide sequence encoding a polypeptide to a host cell, thereby integrating the nucleotide sequence into the genome or being present in the extrachromosomal space freely, and expressing the nucleotide sequence encoding the polypeptide in the cell and optionally extracting the polypeptide from the cell.

[0197] The expression vector described herein can be expressed in a host cell. The term "host cell" refers to all such cells: it comprises a nucleotide sequence to be expressed, or comprises an expression vector, and can produce an enzyme or polypeptide according to the present invention. In particular, the term refers to prokaryotic cells and / or eukaryotic cells, preferably Pichia pastoris (Pichiapastoris), Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces), Hansenula (Hansenula), Trichoderma (Trichoderma), Lactobacillus (Lactobacillus), Aspergillus (Aspergillus), plant cells and / or Bacillus (Bacillus), Trichoderma (Trichoderma) or Aspergillus (Aspergillus) spores. The name Pichia pastoris used herein has the same meaning as the name Komagataella pastoris, wherein Pichia pastoris is the older name and K. pastoris is the systematic newer name (Yamada et al., (1995) 'The Phylogenetic Relationships of Methanol-assimilating Yeasts Based on the Partial Sequences of 18S and 26S Ribosomal RNAs: The Proposal of Komagataella Gen. Nov. (Saccharomycetaceae)' Bioscience, Biotechnology and Biochemistry, Vol. 59, issue 3, pp. 439-444). Of note, the species Komagataella pastoris has recently been reassigned as Komagataella phaffii (Kurtzman (2009) “Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis.” J Ind Microbiol Biotechnol. 36(11):1435-8).Komagataella phaffii as used herein may, for example, relate to the strain Komagataella phaffii CBS 7435, Komagataella phaffii GS115 or Komagataella phaffii JC308.

[0198] The present invention also relates to the use of the α / β-hydrolase described herein for degrading zearalenone (ZEN).

[0199] ZEN is a nonsteroidal estrogenic macrolide with the following structural formula, which is synthesized via the polyketide metabolic pathway:

[0200]

[0201] And, according to the IUPAC nomenclature, its name is (2E,11S)-15,17-dihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraene-7,13-dione.

[0202] However, there are also many ZEN derivatives in nature, which can be formed by enzymatic or chemical modification of ZEN. Examples include glycoside ZEN conjugates or those containing sulfate esters formed by fungal, plant or mammalian metabolism, as well as ZEN metabolites formed in the human or animal body, etc. In the following, ZEN derivatives are understood to be ZEN conjugates or ZEN metabolites that occur naturally or are synthesized by chemical or biochemical synthesis, but in particular refer to α-zearalenol (α-ZEL; (2E,7R,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraen-13-one), β-zearalenol (β-ZEL; (2E,7S,11S)-7, 15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraen-13-one), α-zearalanol (α-ZAL; (7R,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),14,16-trien-13-one), β-zearalanol (β-ZAL; (7S,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),14,16-trien-13-one), 1-methyl-12-oxabicyclo[12.4.0]octadeca-1(14),15,17-trien-13-one), zearalenone-14-sulfate (Z14S; [(2E,11S)-15-hydroxy-11-methyl-7,13-dioxo-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraen-17-yl] hydrogen sulfate), zearalenone-14-glycoside (Z14G; (2E,11S)-15-hydroxy-11-methyl-7,13-dioxo-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraen-17-yl] hydrogen sulfate), -11-methyl-17-[(3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-tetrahydropyran-2-yl]oxy-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraene-7,13-dione) and zearalanone (ZAN; (11S)-15,17-dihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),14,16-triene-7,13-dione).

[0203] Due to their high chemical and physical stability, ZEN and ZEN derivatives, in particular α-ZEL, β-ZEL, Z14S, α-ZAL, β-ZAL, Z14G and ZAN, can also be detected in processed food and animal feed products such as bread or beer.

[0204] ZEN and ZEN derivatives were successfully hydrolyzed using any of the polypeptides with sequence ID numbers 1 to 6. According to the following reaction mechanism, it is believed that the hydrolysis of ZEN or its derivatives occurs at the ester group:

[0205]

[0206] The hydrolysis of ZEN to form non-toxic hydrolyzed zearalenone (HZEN) and / or hydrolyzed ZEN derivatives is carried out by the α / β-hydrolases of the present invention. Further decarboxylation of HZEN to decarboxylated hydrolyzed ZEN (DHZEN) and / or decarboxylated hydrolyzed ZEN derivatives is believed to occur spontaneously.

[0207] The α / β-hydrolases described herein are capable of and suitable for degrading ZEN. For example, the α / β-hydrolases can be suitable for hydrolytically cleaving ester groups of ZEN and / or its derivatives independently of oxygen and without cofactors.

[0208] ZEN degradation can be measured by adding the α / β-hydrolase of the invention to Teorell Stenhagen buffer (pH 7.5) with 0.1 mg / ml bovine serum albumin at a temperature of 37° C. The initial substrate concentration in the reaction is 15.71 μM ZEN. ZEN, HZEN, DHZEN and / or other derivatives can be detected from samples extracted from the reaction using HPLC or other methods well known to those skilled in the art.

[0209] Specifically, ZEN degradation can be measured as follows in a sample buffer (Teorell Stenhagen buffer (Stenhagen & Teorell. (1938) Nature 141, 415) containing 0.1 mg / ml bovine serum albumin at pH 7.5 at 37°C for 3 hours. The polypeptide / enzyme is diluted with sample buffer and placed on ice until use. As a negative control, a sample buffer containing 5 μg / ml ZEN is incubated. For the degradation approach, a sample buffer containing 5 μg / ml The sample buffer of ZEN is mixed with the peptide / enzyme solution to reach a final enzyme concentration that degrades the available ZEN to 90% to 100% within 3 hours. The reaction is started by adding peptides or enzymes to the degradation group. No enzyme is added to the negative control. Vortex for about 2 seconds immediately after the start of each reaction, and transfer the sample (100 μl) of 0h to a new reaction tube. The reaction is incubated in a preheated water bath at 37°C, the sample is heat-inactivated by incubation at 99°C for 10 minutes, centrifuged (2 minutes, 25°C, 14674xg) and 90 μl of the supernatant is transferred to an HPLC vial with an insert. The sample is stored at 4°C until HPLC-DAD measurement. Sampling is repeated after 0.5, 1.0, 2.0 and 3.0 hours.

[0210] The concentrations of ZEN, HZEN and DHZEN can be analyzed by HPLC-DAD as described in Vekiru et al. (Vekiru et al., (2016) 'Isolation and characterization of enzymatic zearalenone hydrolysis reaction products' World Mycotoxin Journal 9:353-363). The analysis was performed on an Agilent 1100 series HPLC equipped with a diode array detector (DAD) operating at 274 nm. When separated on a Zorbax SB-Aq, 4.6 x 150 mm, 5 μm column (Agilent Technologies) at 35°C using solvent A: 20% methanol in water + 5 mM ammonium acetate and solvent B: 90% methanol in water + 5 mM ammonium acetate and the following gradient: 0-0.1 min 0% B phase, 0.1-3 min linear increase to 90% B phase, 3-5 min linear increase to 100% B phase for 1.9 min, then drop to 0% B phase in 0.1 min, the retention time of the analytes was 7.03 min for ZEN, 5.17 min for HZEN and 5.95 min for DHZEN. The column was conditioned for 2.0 min before starting the next run. The flow rate was set to 0.8 ml / min and the injection volume was set to 15 μl.

[0211] Quantification is based on calibration using ZEN, HZEN and DHZEN external standards. The enzyme activity in units per liter (U / l) is calculated from the slope of the linear range of ZEN degradation, which is determined by a plot of the ZEN concentration in the sample versus the sampling time point. In order to determine the amount of enzyme activity in a sample in U / l, the slope of the linear range in the plot as described above can be calculated in μM ZEN / hour and divided by 60 to determine μM / min. By taking into account possible dilutions and by including these appropriate dilution factors in the calculation, the enzyme activity in the sample can be determined in U / l. The following example is used for illustration: If the slope of the linear range is 10 μM / h, the enzyme activity of the undiluted sample is 0.17 U / l; calculated by 10 / 60=0.17.

[0212] In the context of this article, it should be noted that the term "unit" or "U" refers to a measure of the catalytic activity of an enzyme and is defined as the amount of micromoles (μmol) of substrate (here zearalenone) reacted or cleaved per minute under defined conditions. The enzyme concentration of an enzyme or polypeptide solution is defined by the "activity" of the enzyme or polypeptide solution, expressed in units per milliliter (U / ml) or units per liter (U / l) of solution.

[0213] The present invention also relates to a composition comprising the α / β-hydrolase described herein. The composition may be a food or feed additive or a food or feed product.

[0214] Methods for preparing such food and / or feed compositions are known to the person skilled in the art and are described inter alia in WO 99 / 35240.

[0215] The present invention also relates to an α / β-hydrolase or composition as described herein for use in the treatment or prevention of a disease. Such a disease may be a disease affecting hormone balance, such as estrogen balance, in particular mycotoxinosis caused by ZEN.

[0216] The present invention also relates to a kit comprising the α / β-hydrolase or composition described herein.

[0217] The present invention is also characterized by the following items:

[0218] 1. A method for increasing the stability of an α / β-hydrolase, the α / β-hydrolase comprising a sequence corresponding to positions 145 to 218 of SEQ ID NO: 1, 3, 4, 5, and / or a sequence corresponding to positions 144 to 217 of SEQ ID NO: 2, and / or a sequence corresponding to positions 161 to 235 of SEQ ID NO: 6, and / or a sequence having 58% or more sequence identity with the sequence corresponding to positions 145 to 218 of SEQ ID NO: 1, 3, 4, 5, and / or a sequence having 58% or more sequence identity with the sequence corresponding to positions 144 to 217 of SEQ ID NO: 2, and / or a sequence having 58% or more sequence identity with the sequence corresponding to positions 161 to 235 of SEQ ID NO: 6 (CAP-domain; 58% identity with the CAP-domain of SEQ ID NO: 1, 2, 3, 4, 5, 6), the method comprising:

[0219] Substitute at least one amino acid at the following positions:

[0220] - a position corresponding to positions 160 to 205 of SEQ ID NO: 1, or

[0221] - a position corresponding to positions 159 to 204 of SEQ ID NO: 2, or

[0222] - a position corresponding to position 160 to 205 of SEQ ID NO: 3, 4 or 5, or

[0223] - a position corresponding to positions 176 to 222 of SEQ ID NO: 6,

[0224] wherein the amino acid is replaced with an amino acid having a more negative hydropathic index than the replaced amino acid,

[0225] The hydropathic index is determined by the Kyte and Doolittle hydropathic index.

[0226] Thereby, α / β-hydrolases with increased stability are obtained.

[0227] 2. The method according to item 1, wherein the hydropathic index of a certain amino acid is:

[0228]

[0229]

[0230] 3. The method according to item 1 or 2, wherein the method comprises replacing at least one amino acid at the following position:

[0231] - positions corresponding to positions 185 to 191 of SEQ ID NO: 1, and / or

[0232] - a position corresponding to positions 184 to 190 of SEQ ID NO: 2, and / or

[0233] - a position corresponding to position 185 to 191 of SEQ ID NO: 3, 4 or 5, and / or

[0234] - positions corresponding to positions 201 to 208 of SEQ ID NO: 6.

[0235] 4. The method according to any of the preceding items, wherein the amino acid is replaced by an amino acid selected from R, K, D, Q, D, N, E, P, G, T, S or H.

[0236] 5. A method according to any of the preceding items, wherein the amino acid is replaced by an amino acid selected from R, K, N, Q, D, E, H, P, Y, W, S, T, G, A, M, C, F, L or V.

[0237] 6. A method according to any of the preceding items, wherein the amino acid is replaced by an amino acid selected from R, K, N, Q, D, E, H, P, Y, W, S, T or G.

[0238] 7. The method according to any of the preceding items, wherein the amino acid is replaced by an amino acid selected from R, K, N, Q, D, E, H or P.

[0239] 8. The method according to any of the preceding items, wherein the amino acid is replaced by an amino acid selected from S, P, R, D, H, G or N.

[0240] 9. The method according to any one of the preceding items, wherein the amino acid is replaced by an amino acid selected from R, D, H, G, N or P.

[0241] 10. The method according to any one of the preceding items, wherein the amino acid is replaced by an amino acid selected from R, D, H, G or N.

[0242] 11. The method according to any of the preceding items, wherein the amino acid is replaced by an amino acid selected from P, S, R or H.

[0243] 12. The method according to any one of the preceding items, wherein the amino acid is replaced by an amino acid selected from R or N.

[0244] 13. A method according to any of the preceding items, wherein the amino acid substitution is selected from one or more of the following: V→A, G→R, G→S, A→P, A→R, A→D, A→H, A→N, A→G, S→D, P→H, M→D, G→E, I→A, I→V, H→N and / or Q→K.

[0245] 14. A method according to any of the preceding items, wherein the amino acid substitution is selected from one or more of the following: V160A, G185R, G185S, A186P, A186R, A188D, A188H, A188N, A188G, A188R, S189D, P190H, M191D, G199E, I200A, I200V, H203N and / or Q205K.

[0246] 15. A method according to any of the preceding items, wherein the amino acid substitution is selected from one or more of the following: V→A, G→R, G→S, A→P, A→R, A→D, A→H, A→N, A→G, S→D, P→H, M→D, G→E, I→A, I→V, H→N and / or Q→K.

[0247] 16. A method according to any of the preceding items, wherein the amino acid substitution is selected from G185R, A186R, A188R, A188D, A188H, A188N and / or M191D.

[0248] 17. The method according to any one of the preceding items, wherein the increased stability is a decrease in GRAVY value, an increase in pH stability and / or an increase in temperature stability.

[0249] 18. The method according to any of the preceding items, wherein the GRAVY value is calculated by dividing the sum of the hydropathic values ​​(index) of all amino acids by the number of amino acid residues in the sequence (calculated according to Kyte and Doolittle).

[0250] 19. The method according to any one of the preceding items, wherein the GRAVY value is calculated by dividing the sum of the hydropathic values ​​(indexes) of all amino acids in the sequence by the total number of amino acids in the sequence.

[0251] 20. The method according to any one of the preceding items, wherein the amino acid is replaced by an amino acid selected from R, D, H, G, N or P.

[0252] 21. The method according to any one of the preceding items, wherein the method comprises replacing at least one amino acid at the following position:

[0253] - a position corresponding to positions 185 to 191 of SEQ ID NO: 1, or

[0254] - a position corresponding to positions 184 to 190 of SEQ ID NO: 2, or

[0255] - a position corresponding to position 185 to 191 of SEQ ID NO: 3, 4 or 5, or

[0256] - a position corresponding to positions 201 to 208 of SEQ ID NO: 6,

[0257] and wherein the amino acid is replaced with an amino acid selected from R, D, H, G, N or P.

[0258] 22. An α / β-hydrolase obtainable by the method according to any one of the preceding items.

[0259] 23. An α / β-hydrolase comprising a sequence corresponding to positions 145 to 218 of SEQ ID NO: 1, 3, 4, 5, and / or a sequence corresponding to positions 144 to 217 of SEQ ID NO: 2, and / or a sequence corresponding to positions 161 to 235 of SEQ ID NO: 6, and / or a sequence having 58% or more sequence identity with the sequence corresponding to positions 145 to 218 of SEQ ID NO: 1, 3, 4, 5, and / or a sequence having 58% or more sequence identity with the sequence corresponding to positions 144 to 217 of SEQ ID NO: 2, and / or a sequence having 58% or more sequence identity with the sequence corresponding to positions 161 to 235 of SEQ ID NO: 6 (CAP-domain; 58% identity with the CAP-domain of SEQ ID NO: 1, 2, 3, 4, 5, 6),

[0260] wherein the polypeptide sequence comprises at least one amino acid substitution at the following position:

[0261] - a position corresponding to positions 160 to 205 of SEQ ID NO: 1, or

[0262] - a position corresponding to positions 159 to 204 of SEQ ID NO: 2, or

[0263] - a position corresponding to position 160 to 205 of SEQ ID NO: 3, 4 or 5, or

[0264] - a position corresponding to positions 176 to 222 of SEQ ID NO: 6,

[0265] The α / β-hydrolase has a GRAVY value that is at least 0.6% more negative than the GRAVY value of the α / β-hydrolase having the polypeptide sequence of SEQ ID NO:1.

[0266] 24. An α / β-hydrolase having a polypeptide sequence comprising a sequence corresponding to positions 161 to 235 of SEQ ID NO: 6, or comprising a sequence having a sequence identity of greater than 58% to a sequence corresponding to positions 161 to 235 of SEQ ID NO: 6,

[0267] Wherein, the polypeptide sequence comprises at least one amino acid substitution at the following position:

[0268] - a position corresponding to positions 160 to 205 of SEQ ID NO: 1, or

[0269] - a position corresponding to positions 159 to 204 of SEQ ID NO: 2, or

[0270] - a position corresponding to position 160 to 205 of SEQ ID NO: 3, 4 or 5, or

[0271] - a position corresponding to positions 176 to 222 of SEQ ID NO: 6,

[0272] The α / β-hydrolase has a GRAVY value that is at least 0.6% more negative than the GRAVY value of the α / β-hydrolase having the polypeptide sequence of SEQ ID NO:6.

[0273] 25. An α / β-hydrolase having a polypeptide sequence, the polypeptide sequence comprising a sequence corresponding to positions 145 to 218 of SEQ ID NO: 1, 3, 4, 5, and / or a sequence corresponding to positions 144 to 217 of SEQ ID NO: 2, and / or a sequence corresponding to positions 161 to 235 of SEQ ID NO: 6, and / or a sequence having 58% or more sequence identity with a sequence corresponding to positions 145 to 218 of SEQ ID NO: 1, 3, 4, 5, and / or a sequence having 58% or more sequence identity with a sequence corresponding to positions 144 to 217 of SEQ ID NO: 2, and / or a sequence having 58% or more sequence identity with a sequence corresponding to positions 161 to 235 of SEQ ID NO: 6 (CAP-domain; 58% identity with the CAP-domain of SEQ ID NO: 1, 2, 3, 4, 5, 6), comprising

[0274] wherein the polypeptide sequence comprises at least one amino acid substitution at the following position:

[0275] - a position corresponding to positions 185 to 191 of SEQ ID NO: 1, or

[0276] - a position corresponding to positions 184 to 190 of SEQ ID NO: 2, or

[0277] - a position corresponding to position 185 to 191 of SEQ ID NO: 3, 4 or 5, or

[0278] - a position corresponding to positions 201 to 208 of SEQ ID NO: 6,

[0279] Wherein, the amino acid substitution is selected from V→A, G→R, G→S, A→P, A→R, A→D, A→H, A→N, A→G, S→D, P→H, M→D, G→E, I→A, I→V, H→N, Q→K, F→Y and / or V→C, and / or

[0280] The amino acid is replaced by an amino acid selected from P, R, D, H, G or N. Preferably, the amino acid used is selected from R, D, H, G or N. More preferably, the amino acid used is selected from R or N.

[0281] 26. The α / β-hydrolase according to any one of the preceding items, wherein the α / β-hydrolase comprises the following amino acid substitutions:

[0282] -G→R and A→N, preferably G185R and A188N;

[0283] -G→S and A→R, preferably G185S and A188R;

[0284] - G→R, A→R, A→H, S→D, P→H and M→D, preferably G185R, A186R, A188H, S189D, P190H and M191D;

[0285] - V→A, G→R, A→N, G→E, I→A, H→N and Q→K, preferably V160A, G185R, A188N, G199E, I200A, H203N and Q205K;

[0286] - V→A, G→S, A→R, G→E, I→A, H→N and Q→K, preferably V160A, G185S, A188R, G199E, I200A, H203N and Q205K;

[0287] - V→A, G→E, I→A, H→N and Q→K, preferably V160A, G199E, I200A, H203N and Q205K;

[0288] - V→A, G→R, A→R, A→H, G→E, I→A, H→N and Q→K, preferably V160A, G185R, A186R, A188H, G199E, I200A, H203N and Q205K, and / or

[0289] - V→A, G→R, A→R, A→H, G→E, I→V, H→N and Q→K, preferably V160A, G185R, A186R, A188H, G199E, I200V, H203N and Q205K;

[0290] - V→A, G→R, A→R, A→H, S→D, P→H, M→D, G→E, I→V, H→N and Q→K, preferably V160A, G185R, A186R, A188H, S189D, P190H, M191D, G199E, I200V, H203N and Q205K; and / or

[0291] -F→Y and V→C, preferably F183Y and V197C.

[0292] 27. Use of the α / β-hydrolase described in any one of the preceding items for degrading zearalenone (ZEN).

[0293] 28. A composition comprising the α / β-hydrolase according to any one of the preceding items, preferably, the composition is a food or feed additive or a food or feed product.

[0294] 29. The α / β-hydrolase or composition according to any one of the preceding items, for use in treating or preventing a disease.

[0295] 30. A kit comprising the α / β-hydrolase or the composition according to any one of the preceding items.

[0296] 31. A method for increasing the stability of an α / β-hydrolase comprising a sequence corresponding to positions 160 to 205 of SEQ ID NO: 1, 3, 4, 5, and / or a sequence corresponding to positions 159 to 204 of SEQ ID NO: 2, and / or a sequence corresponding to positions 176 to 222 of SEQ ID NO: 6, and / or a sequence having 58% or more sequence identity with the sequence corresponding to positions 160 to 205 of SEQ ID NO: 1, 3, 4, 5, and / or a sequence having 58% or more sequence identity with the sequence corresponding to positions 159 to 204 of SEQ ID NO: 2, and / or a sequence having 58% or more sequence identity with the sequence corresponding to positions 176 to 222 of SEQ ID NO: 6 (VI-domain; 58% identity with the VI-domain of SEQ ID NO: 1, 2, 3, 4, 5, 6), the method comprising:

[0297] Substitute at least one amino acid at the following positions:

[0298] - a position corresponding to positions 160 to 205 of SEQ ID NO: 1, or

[0299] - a position corresponding to positions 159 to 204 of SEQ ID NO: 2, or

[0300] - a position corresponding to position 160 to 205 of SEQ ID NO: 3, 4 or 5, or

[0301] - a position corresponding to positions 176 to 222 of SEQ ID NO: 6,

[0302] wherein the amino acid is replaced with an amino acid having a more negative hydropathic index than the replaced amino acid,

[0303] The hydropathic index is determined by the Kyte and Doolittle hydropathic index.

[0304] Thereby, α / β-hydrolases with increased stability are obtained.

[0305] 32. A method for increasing the stability of an α / β-hydrolase, the α / β-hydrolase comprising the sequence of SEQ ID NO: 1, 2, 3, 4, 5 or 6, the method comprising

[0306] Substitute at least one amino acid at the following positions:

[0307] - a position corresponding to positions 160 to 205 of SEQ ID NO: 1, or

[0308] - a position corresponding to positions 159 to 204 of SEQ ID NO: 2, or

[0309] - a position corresponding to position 160 to 205 of SEQ ID NO: 3, 4 or 5, or

[0310] - a position corresponding to positions 176 to 222 of SEQ ID NO: 6,

[0311] wherein the amino acid is replaced with an amino acid having a more negative hydropathic index than the replaced amino acid,

[0312] The hydropathic index is determined by the Kyte and Doolittle hydropathic index.

[0313] Thereby, α / β-hydrolases with increased stability are obtained.

[0314] 33. An α / β-hydrolase comprising a sequence corresponding to positions 160 to 205 of SEQ ID NO: 1, 3, 4, 5, and / or a sequence corresponding to positions 159 to 204 of SEQ ID NO: 2, and / or a sequence corresponding to positions 176 to 222 of SEQ ID NO: 6, and / or a sequence having 58% or more sequence identity to a sequence corresponding to positions 160 to 205 of SEQ ID NO: 1, 3, 4, 5, and / or a sequence having 58% or more sequence identity to a sequence corresponding to positions 159 to 204 of SEQ ID NO: 2, and / or a sequence having 58% or more sequence identity to a sequence corresponding to positions 176 to 222 of SEQ ID NO: 6 (VI-domain; 58% identity to the VI-domain of SEQ ID NO: 1, 2, 3, 4, 5, 6), wherein the polypeptide sequence comprises at least one amino acid substitution at the following positions:

[0315] - a position corresponding to positions 160 to 205 of SEQ ID NO: 1, or

[0316] - a position corresponding to positions 159 to 204 of SEQ ID NO: 2, or

[0317] - a position corresponding to position 160 to 205 of SEQ ID NO: 3, 4 or 5, or

[0318] - a position corresponding to positions 176 to 222 of SEQ ID NO: 6,

[0319] The α / β-hydrolase has a GRAVY value that is at least 0.6% more negative than the GRAVY value of the α / β-hydrolase having the polypeptide sequence of SEQ ID NO:1.

[0320] 34. An α / β-hydrolase having a polypeptide sequence, the polypeptide sequence comprising a sequence corresponding to positions 145 to 218 of SEQ ID NO: 1, 3, 4, 5, and / or a sequence corresponding to positions 144 to 217 of SEQ ID NO: 2, and / or a sequence corresponding to positions 161 to 235 of SEQ ID NO: 6, and / or a sequence having 58% or more sequence identity with a sequence corresponding to positions 145 to 218 of SEQ ID NO: 1, 3, 4, 5, and / or a sequence having 58% or more sequence identity with a sequence corresponding to positions 144 to 217 of SEQ ID NO: 2, and / or a sequence having 58% or more sequence identity with a sequence corresponding to positions 161 to 235 of SEQ ID NO: 6 (CAP-domain; 58% identity with the CAP-domain of SEQ ID NO: 1, 2, 3, 4, 5, 6), comprising

[0321] wherein the polypeptide sequence comprises at least one amino acid substitution at the following position:

[0322] - a position corresponding to positions 185 to 191 of SEQ ID NO: 1, or

[0323] - a position corresponding to positions 184 to 190 of SEQ ID NO: 2, or

[0324] - a position corresponding to position 185 to 191 of SEQ ID NO: 3, 4 or 5, or

[0325] - a position corresponding to positions 201 to 208 of SEQ ID NO: 6,

[0326] wherein the amino acid substitution is selected from the group consisting of V→A, G→R, G→S, A→P, A→R, A→D, A→H, A→N, A→G, S→D, P→H, M→D, G→E, I→A, I→V, H→N and Q→K, and / or

[0327] The amino acid is replaced by an amino acid selected from P, R, D, H, G or N. Preferably, the amino acid used is selected from R, D, H, G or N. More preferably, the amino acid used is selected from R or N.

[0328] ****

[0329] It should be noted that, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" as used herein include the plural reference. Thus, for example, reference to "an agent" includes one or more such different agents, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that may modify or substitute the methods described herein.

[0330] Unless otherwise indicated, the term "at least" before a series of elements will be understood to refer to each element in the series. Those skilled in the art will recognize or use routine experimentation to determine many equivalents of the specific embodiments of the invention described herein. The present invention is intended to cover such equivalents.

[0331] The term "and / or" used herein includes the meanings of "and", "or" and "all or any other combinations of the elements connected by the term".

[0332] The term "less than" or the antonym "greater than" does not include specific numbers.

[0333] For example, less than 20 means less than the number shown. Similarly, greater than or higher means greater than or higher than the number shown, for example greater than 80% means greater than or higher than 80% of the number shown.

[0334] In this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of the stated integers or steps or sets of integers or steps, but not the exclusion of any other integers or steps or sets of integers or steps. When used herein, the term "comprising" can be replaced with the term "containing" or, as is sometimes used herein, with the term "having". When used herein, "consisting of excludes any element, step or ingredient not specified.

[0335] The term "including" means "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0336] It should be understood that the present invention is not limited to the specific methods, operating procedures, materials, reagents and substances described herein and can be varied accordingly. The terminology used herein is only for the purpose of describing specific embodiments and is not intended to limit the scope of the present invention, which is limited only by the claims.

[0337] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, specifications, etc.), whether preceding or following, are incorporated herein in their entirety. Nothing herein will be construed as an admission that the present invention is not entitled to antedate such disclosures by virtue of prior invention. To the extent that material incorporated by reference is inconsistent or inconsistent with this specification, this specification will take precedence over any such material.

[0338] The contents of all documents and patent documents cited herein are incorporated herein in their entirety.

[0339] The following sequences were used in this application.

[0340]

[0341]

[0342]

[0343] Table 2: Sequences used in this application. Motifs that may be located in the flanking regions of the VI-domain described herein are shown in SEQ ID NOs: 7 and 8. Motifs that may be located in the flanking regions of the CAP-loop described herein are shown in SEQ ID NOs: 9, 10, 11. The "X" in the motif may be any amino acid. The second amino acid in the motif of SEQ ID NO: 9 may be F or Y, represented as "(F / Y)". Both the VI-domain and the CAP-loop are contained in the CAP-domain, and the CAP-domain may be located in an excursion between the β-fold and the α-helix, such as between b6 and aD of the α / β-hydrolase, as described, for example, in Ollis et al., (1992) 'The alpha / betahydrolase fold' Protein Eng. 5(3): 197-211. The VI-domain and the CAP-loop are shown as underlined in SEQ ID NOs: 1-6.

[0344] The present invention and its advantages can be better understood through the following examples which are given for illustrative purposes only. These examples are not intended to limit the scope of the present invention in any way.

[0345] Embodiments of the present invention

[0346] Example 1: Modification, cloning and expression of polynucleotides encoding polypeptides that cleave zearalenone

[0347] Using QuikChange site-directed mutagenesis kit (Stratagene), according to the manufacturer's instructions, by means of PCR means, amino acid substitution, insertion or deletion is implemented by mutation of nucleotide sequence. Optionally, complete nucleotide sequences are also synthesized (e.g., by GeneArt GeneSynthesis of Thermo Fisher Scientific). The nucleotide sequences generated by PCR mutagenesis and those obtained from GeneArt are integrated into expression vectors for expression in Escherichia coli (E. coli.) by standard methods. Escherichia coli BL21 (DE3) is transformed with the expression vector and the nucleotide sequence is expressed in the strain (JM Cregg, Pichia Protocols, second Edition, ISBN-10: 1588294293, 2007; J. Sambrook et al., 2012, Any other suitable host cell may also be used for this work. Soluble cell lysates of E. coli are used to determine the catalytic properties of the polypeptide variants.

[0348] Example 2: Determination of ZEN-degrading α / β-hydrolases, their CAP-domains, their VI-domains and CAP-loops

[0349] In order to determine whether the amino acid sequence is an α / β-hydrolase that degrades ZEN, the target sequence is aligned with the sequence of SEQ ID NO: 1 to determine sequence identity. In addition, topology prediction and homology modeling are performed for the target sequence. Sequence alignment is performed using CLCsequence viewer 7.8.1 with the following parameters: gap opening penalty: 10.0, gap extension penalty: 1.0, gap termination penalty: any other, alignment: very accurate.

[0350] By adopting the homology modeling of the following parameters, YASARA Structure 16.7.22 (1993-2016 by Elmar Krieger, Bioinformatics 30, 2981-2982) is used to implement topology prediction and homology modeling: PSI-BLAST iteration: 3, PSI-BLAST E-value: 0.5, oligomeric state: 4, template: 5, with the same sequence: 1, each template alignment: 5, modeling speed: fast, terminal extension: 10, loop sample: 50, using PSSP: yes. The topology prediction by PSI-Pred secondary structure prediction algorithm (Jones (1999) 'Protein secondary structure prediction based on position-specific scoring matrices' J. Mol. Biol. 292: 195-202) is recorded in the YASARA homology modeling report. The Z-score of all homology models obtained is recorded. The resulting model with the best Z-score was used for structural analysis and determination of structural features of alpha / beta-hydrolase and shifts as described by Ollis et al., (1992) 'The alpha / betahydrolase fold' Protein Eng. 5(3): 197-211.

[0351] To identify the CAP-domain, VI-domain and CAP-loop in a new target sequence, the secondary structure of the α / β-hydrolase core domain was labeled according to Figure 2a in Ollis et al. (1992) 'The alpha / beta hydrolase fold' Protein Eng. 5(3): 197-211. The CAP-domain, VI-domain and CAP-loop are located within the offset between b6 and aD of the α / β-hydrolase that degrades ZEN. The CAP-domain may start shortly after the C-terminus of the b6 β-strand of the α / β-hydrolase core domain and may continue to the N-terminal start of the aD α-helix of the α / β-hydrolase core domain. The VI-domain is part of the CAP-domain and starts at the first amino acid after the QXAGP motif present in the CAP-domain and continues to the last amino acid before the EYDPE motif, wherein the EYDPE motif is not part of the VI-domain. The CAP-loop is part of the VI-domain and starts at the first amino acid after the G(F / Y)XXAA motif present in the VI-domain and continues to the last amino acid before the ARXF motif (or the QLFP motif for SEQ ID NO: 6), wherein the ARXF motif (or the QLFP motif for SEQ ID NO: 6) is not part of the CAP-loop. For example, the positions of the CAP-domain, the VI-domain and the CAP-loop are determined by the positions of the polypeptides as described herein for SEQ ID NOs: 1-6 and are shown in Figure 1 .

[0352] Example 3: Determination of the overall average hydrophilicity (GRAVY) value

[0353] The total average hydrophilicity (GRAVY) value of a polypeptide amino acid sequence is defined as the sum of the hydrophilicity values ​​(Kyte and Doolittle, 1982, cited herein) of all amino acids divided by the length of the polypeptide, which corresponds to the total number of amino acids in the polypeptide. The GRAVY values ​​of polypeptides and defined portions of polypeptides are calculated using the ProtParam program (Gasteiger E. et al., Protein Identification and Analysis Tools on the ExPASy Server, in John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press, 2005, pp. 571-607) on https: / / web.expasy.org / protparam. The CAP-domain of SEQ ID NO: 1 is defined as the portion from amino acid position 145 to 218 (inclusive), the VI-domain of SEQ ID NO: 1 is defined as the amino acid sequence from amino acid position 160 to 205 (inclusive), and the CAP-loop of SEQ ID NO: 1 is defined as the portion of the amino acid sequence from amino acid position 185 to 191 (inclusive). For the entire polypeptide of SEQ ID NO: 1, the calculated GRAVY value is -0.167; for the CAP-domain of SEQ ID NO: 1, the GRAVY value is -0.284; for the VI-domain of SEQ ID NO: 1, the GRAVY value is -0.043; and for the CAP-loop in the CAP-domain of SEQ ID NO: 1, the GRAVY value is +0.271. The CAP-domain, VI-domain and CAP-loop of SEQ ID NO: 6 are defined as the portion from amino acid positions 161-235, 176-222, 201-208, inclusive, respectively. For the entire polypeptide of SEQ ID NO: 6, the calculated GRAVY value is -0.192; for the CAP-domain of SEQ ID NO: 6, the GRAVY value is -0.388; for the VI-domain of SEQ ID NO: 6, the GRAVY value is -0.468; and for the CAP-loop of SEQ ID NO: 6, the GRAVY value is -0.362.

[0354] The percentage reduction in the GRAVY value of the entire amino acid sequence of the polypeptide resulting from at least one mutation relative to the entire amino acid sequence of the unmutated / unsubstituted polypeptide of SEQ ID NO: 1 or 6 is calculated by dividing the difference between the two GRAVY values ​​by the GRAVY value of the unmutated polypeptide and multiplying by 100. For illustration, the calculation for SEQ ID NO: 1 variant V160A is shown here: ((-0.174)-(-0.167)) / (-0.167)x100=4.2%. The results of other examples are listed in Figure 2A and 2E middle.

[0355] The percentage reduction in the GRAVY value of the CAP-domain caused by a mutation in the CAP-domain relative to the sequence of the non-mutated CAP-domain of SEQ ID NO: 1 or 6 is calculated by dividing the difference between the two GRAVY values ​​by the GRAVY value of the non-mutated CAP-domain and multiplying by 100. For illustration, the calculation for the SEQ ID NO: 1 variant V160A is shown here: ((-0.316)-(-0.284)) / (-0.284)x100=11.3%. The results of other examples are listed in Figure 2B and 2F middle.

[0356] The percentage reduction in the GRAVY value of the VI-domain relative to the sequence of the unmutated VI-domain of SEQ ID NO: 1 or 6 resulting from a mutation in the VI-domain is calculated by dividing the difference between the two GRAVY values ​​by the GRAVY value of the unmutated VI-domain and multiplying by 100. For illustration, the calculation for the SEQ ID NO: 1 variant G185R is shown here: ((-0.133)-(0.043)) / (-0.043)x100=209.3%. The results of other examples are listed in Figure 2C and 2G middle.

[0357] The percentage reduction in the GRAVY value of the CAP-loop caused by a mutation in the CAP-loop relative to the sequence of the non-mutated CAP-loop of SEQ ID NO: 1 or 6 is calculated by dividing the difference between the two GRAVY values ​​by the GRAVY value of the non-mutated CAP-loop and multiplying by 100. For illustration, the calculation for the SEQ ID NO: 1 variant G185R is shown here: ((-0.314)-(+0.271)) / (+0.271)x100=-215.9%. Since the GRAVY value of the parental CAP-loop is positive, this value is negative. To simplify the data presentation of other examples of mutations, the percentage values ​​are shown in Figure 2D Positive values ​​are shown in the figure.

[0358] Example 4: Determination of the activity of polypeptides that degrade ZEN

[0359] The corresponding gene encoding the polypeptide for degrading ZEN is cloned by standard methods, expressed intracellularly in E. coli, and the polypeptide produced is isolated from E. coli by methods known to those skilled in the art, for example, by lysis using French Press cells. The protein concentration is determined by standard methods, for example, the BCA method (Pierce BCA Protein Assay Kit Prod # 23225).

[0360] The enzyme activity was determined in a sample buffer (Teorell Stenhagen buffer (Stenhagen & Teorell. (1938) Nature 141, 415) containing 0.1 mg / ml bovine serum albumin at pH 7.5 at 37°C for up to 3 hours. The polypeptide was diluted with sample buffer and placed on ice until use. A 1500 ppm (w / v) ZEN stock solution in acetonitrile was diluted 1:10 with sample buffer and stored at 25°C until further dilution for degradation reactions. Degradation groups and a negative control were prepared in reaction tubes. As a negative control, a sample containing 5 μg / ml Sample buffer of ZEN. For the degradation reaction, the sample buffer was mixed with the polypeptide solution to reach a final ZEN concentration of 5 μg / ml and a final enzyme concentration, which achieved 90% to 100% ZEN degradation within 3 hours. The reaction was started by adding the polypeptide to the sample buffer containing ZEN. No enzyme was added to the negative control. Vortex for about 2 seconds immediately after the start of each reaction, and the sample (100 μl) of 0h was taken out and transferred to a new reaction tube. The reaction was incubated in a preheated water bath at 37°C, and the sample was heat-inactivated by incubation at 99°C for 10 minutes, centrifuged (2 minutes, 25°C, 14674xg) and 90 μl of the supernatant was transferred to an HPLC vial with an insert. The sample was stored at 4°C until HPLC-DAD measurement. Sampling was repeated after 0.5, 1.0, 2.0 and 3.0 hours.

[0361] The concentrations of ZEN, HZEN and DHZEN can be analyzed by HPLC-DAD as described in Vekiru et al. (Vekiru et al., (2016) 'Isolation and characterization of enzymatic zearalenone hydrolysis reaction products' World Mycotoxin Journal 9:353-363). The analysis was performed on an Agilent 1100 series HPLC equipped with a DAD detector operating at 274 nm. When separated on a Zorbax SB-Aq, 4.6×150 mm, 5 μm column (Agilent Technologies) at 35° C. using solvent A: 20% methanol in water + 5 mM ammonium acetate and solvent B: 90% methanol in water + 5 mM ammonium acetate and the following gradient: 0-0.1 min 0% B phase, 0.1-3 min linear increase to 90% B phase, 3-5 min linear increase to 100% B phase for 1.9 min, then drop to 0% B phase in 0.1 min, the retention time of the analytes was 7.03 min for ZEN, 5.17 min for HZEN and 5.95 min for DHZEN. The column was conditioned for 2.0 min before starting the next run. The flow rate was set to 0.8 ml / min and the injection volume was set to 15 μl. Quantification was based on calibration using external standards for ZEN, HZEN and DHZEN. The enzyme activity in units per liter (U / l) is calculated from the slope of the linear range of ZEN degradation, which is determined by a plot of the ZEN concentration in the sample versus the sampling time point. To determine the amount of enzyme activity in a sample in U / l, the slope of the linear range in the plot as described above can be calculated in μM ZEN / hour and divided by 60 to determine μM / min. By taking into account possible dilutions and by including these appropriate dilution factors in the calculation, the enzyme activity in the sample can be determined in U / l. The following example is used for illustration: If the slope of the linear range is 10 μM / h, the enzyme activity of the undiluted sample is 0.17 U / l; calculated by 10 / 60=0.17.

[0362] Example 5: Temperature stability of ZEN-degrading polypeptides

[0363] The production and quantification of polypeptides that degrade ZEN were carried out as described in the above examples. In order to evaluate the temperature stability, the enzymes that degrade ZEN were incubated in buffer solutions at different temperatures and then tested for their ability to degrade ZEN under optimal conditions. Samples were taken during the entire thermal incubation period and residual activity was calculated relative to a control that was not heat treated.

[0364] For the temperature stability test, the polypeptide was diluted to a concentration of 0.001526923U / ml using sample buffer (Teorell Stenhagen buffer containing 0.1mg / ml bovine serum albumin at pH 7.5) and placed on ice for standby use. 40 aliquots of 50μl diluted polypeptide solutions were transferred to 4 groups of 12-tubes (e.g., purchased from starlab), wherein the first tube of each group of tubes and the last tube of each group of tubes were not used but left vacant. The tubes were sealed with 12-caps (e.g., purchased from starlab). As a positive control, 4 aliquots of 50μl diluted enzyme solutions were transferred to 4 PCR tubes. All PCR tubes and tubes were placed on ice until the temperature incubation step was started. As a negative control, 4 aliquots of 50μl sample buffers were transferred to 4 PCR tubes. These tubes were stored at 25°C.

[0365] Incubate 4 sets of 12-tube strips in a preheated PCR cycler with a gradient function (e.g., Eppendorf Mastercycler gradient) at a selected temperature of + / -10°C. The PCR cycler automatically calculates the temperature gradient (+ / -10°C of the selected temperature) along the PCR cycler heating block. Incubate the PCR tubes containing the positive control on ice and the PCR tubes containing the negative control at 25°C. After 0, 5, 10, and 20 minutes, transfer 1 set of PCR strips and 1 negative control tube to be placed on ice until the end of the incubation, which is 20 minutes after the start of the incubation.

[0366] After all incubation steps were completed and all tubes and links were placed on ice, the ZEN degradation assay was started.

[0367] ZEN degradation assay buffer (Teorell Stenhagen buffer, pH 7.5, containing 0.1 mg / ml bovine serum albumin and 5.3 ppm ZEN) was prepared and aliquots of 660 μl of the assay buffer were transferred to 48 reaction tubes. The tubes were sealed and stored at 25°C until the start of the ZEN degradation assay. For the degradation assay, 40 μl of each of the 40 temperature treated samples from the PCR strip tubes, 40 μl of each of the 4 negative controls and 40 μl of each of the 4 positive controls were added to the tube containing 660 μl of the assay buffer, thereby achieving a final ZEN concentration of 5 ppm in the assay reaction. In addition, a final concentration of the polypeptide was thereby achieved to effectively degrade ZEN within three hours (i.e., 90%-100% ZEN degradation).

[0368] By adding the temperature treated sample, positive or negative control to the analysis buffer, the degradation analysis is started. The ZEN degradation reaction is incubated in a preheated water bath at 37°C. Immediately after the degradation reaction begins, vortex mix for about 2 seconds, and 100 μl of the 0h sample is transferred to a new reaction tube. After 0.5, 1.0, 2.0 and 3.0 hours, other samples are extracted from the ZEN degradation analysis reaction. Once the sample is taken out from the degradation reaction, the enzyme heat inactivation in the sample is made by incubation at 99°C for 10 minutes. Subsequently, the tube is centrifuged (2 minutes, 25°C, 14674xg) and 90 μl of supernatant is transferred to the HPLC bottle with an insert. These HPLC bottles are stored at 4°C until HPLC-DAD measurements are performed as described in Example 4.

[0369] The enzyme activity is calculated, for example, in units / liter (U / l) or units / milliliter (U / ml) using the linear decrease in ZEN concentration determined by HPLC-DAD analysis of ZEN degradation samples. One unit is defined as the amount of enzyme activity that degrades 1 μmol of ZEN in 1 minute under the conditions described. The residual activity after 0, 5, 10 and 20 minutes of incubation at different temperatures is calculated as follows: the enzyme activity in the temperature treated samples divided by the average of the enzyme activity of the 0-minute samples, multiplied by 100.

[0370] The temperature stability (T(50%)) is defined as the temperature at which the polypeptide has 50% residual activity compared to the positive control after incubation for 10 minutes at this temperature. The following example is used for illustration: the parent enzyme has an enzyme activity of 50 U / ml after incubation on ice for 10 minutes and an activity of 25 U / ml after incubation at 59.3°C for 10 minutes, then the T(50%) value is 59.3°C. If the enzyme variant has a T(50%) value of 61.0°C, the relative increase in temperature stability (T(50%)) compared to the parent enzyme is 2.9%. This is obtained by dividing the difference between the two T(50%) values, 1.7°C, by the T(50%) value of the parent enzyme, 59.3°C, and multiplying by 100.

[0371] like Figure 3A and 3B As shown, single mutations as well as combinations of mutations show an increase in temperature stability.

[0372] Example 6: pH Stability of ZEN Toxin-Degrading Polypeptides

[0373] The peptides that degrade ZEN were incubated in buffer solutions of different pH values ​​for 1 hour, and then the ability of these peptides to degrade ZEN under optimal conditions was tested. Samples were taken regularly and the concentrations of ZEN, HZEN and DHZEN were measured and analyzed using HPLC-DAD.

[0374] The pH value used for the experiment is pH 3.5, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0 and 6.0. The tested polypeptide is transferred to 8 sample tubes containing 8 different pH incubation buffers. The incubation buffer is a semi-concentrated FedState Simulated Gastric Fluid intermediate buffer (Jantratid et al., (2008) 'Dissolution mediasimulating conditions in the proximal human gastrointestinal tract: an update. 'Pharm Res. 2008 Jul; 25 (7): 1663-76) that is free of milk and is set to pH 3.5, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0 and 6.0. An aliquot of the polypeptide variant is also transferred to a tube containing sample buffer (Teorell Stenhagen buffer, pH 7.5, containing 0.1 mg / ml bovine serum albumin) as a positive control. The concentration of the test polypeptide in the incubation solution was 0.001526923U / ml and the volume was 100μl. The tube was vortexed for about 2 seconds and incubated for 1 hour at 37°C in a preheated water bath. As a negative control, 100μl of sample buffer was incubated for 1 hour at 37°C in a preheated water bath. After incubation for 1 hour, the ZEN degradation analysis was performed using the test polypeptide with a final concentration of 8.72527E-05U / ml. To start the ZEN degradation reaction, 40μl of the incubated sample was transferred to 660μl of assay buffer (Teorell Stenhagen buffer, pH 7.5, containing 0.1mg / ml bovine serum albumin and 5.3ppm ZEN). The addition of assay buffer ensured that the pH value was constant at pH 7.5 in all samples. Vortexed for about 2 seconds immediately after the start of each reaction and the 0h sample (100μl) was removed and transferred to a new reaction tube. The reaction was incubated in a preheated water bath at 37°C, the sample taken from the reaction was heat inactivated by incubation at 99°C for 10 minutes, centrifuged (2 minutes, 25°C, 14674xg) and 90 μl of supernatant was transferred to an HPLC vial with an insert. The samples were stored at 4°C until HPLC-DAD measurement. Samples were taken from each degradation analysis reaction after 0.5, 1.0, 2.0 and 3.0 hours. ZEN, HZEN and DHZEN were analyzed by HPLC-DAD as described in Example 4 and the activity was calculated as described in Example 4.

[0375] The increase in pH stability is defined as the increase in the residual activity of the polypeptide solution after incubation at pH 4.0 compared to the residual activity of the non-mutated parent enzyme solution after the same treatment. The residual activity is defined by comparing the activity of the pH-treated polypeptide solution with the activity of the same polypeptide variant solution after incubation at pH 7.5. The residual activity is calculated as follows: the enzyme activity of the pH-treated sample is divided by the enzyme activity of the control incubated at pH 7.5, and then multiplied by 100. The following example is used for illustration: if the enzyme activity of the polypeptide sample after incubation at pH 4.0 is 0.5 U / l and the enzyme activity of the polypeptide sample after incubation at pH 7.5 is 2.7 U / l, then the residual activity of the polypeptide sample will be 18.5%. Furthermore, if the residual activity of the polypeptide variant after incubation at pH 4.0 is measured to be 18.5%, and the residual activity of the parent polypeptide having SEQ ID NO: 1 after incubation at pH 4.0 is measured to be 2.5%, then the pH stability of the polypeptide variant is increased by 7.4 times compared to the parent polypeptide. The data on the increased pH stability after the introduction of mutations as described herein are shown in Figure 4 .

[0376] Example 7: Testing of polypeptide variants for detoxification of ZEN in pigs

[0377] A total of 12 weaned piglets (female; age 38-40 days) were selected and randomly assigned according to the experimental setup using 12 individual cages with 1 piglet per cage (4 groups, 3 cages per group / replicate). Three test groups received enzymes that degrade ZEN and one control group did not receive any enzyme that degrades ZEN. All piglets were of the Austrian genotype [(Landrace x Large White)×Pietrain]. All cages were equipped with slatted floors, individual cup drinkers and individual feeding troughs. Climate conditions were automatically regulated by computer and recorded daily according to standard recommendations for weaned piglets.

[0378] Once in the cages, all piglets were fed a diet containing the following ingredients in percentage (w / w): 29.70% barley, 10.00% wheat, 9.98% corn, 0.27% rapeseed oil, 15.30% full fat soybeans, 10.94% maize pressure cooked, 5.00% potato protein, 5.13% glucose, 3.75% palm kernel, coconut oil, 3.75% lactose, 1.35% lignocellulose, 1.23% monocalcium phosphate, 0.93% calcium carbonate, 0.48% sodium chloride, 0.25% magnesium phosphate, 0.42% vitamin / trace element premix, 0.70% L-lysine, 0.30% L-threonine, 0.27% DL-methionine, 0.15% L-valine, 0.07% L-tryptophan, and 0.02% sweetener.

[0379] During the experiment, the diets of all groups were supplemented with ZEN to a final concentration of 500 μg ZEN / kg food. For the test group, the parent polypeptide of SEQ ID NO:1 and its two polypeptide variants were used. The polypeptides were tested at the following concentrations: 2.5U / kg food, 5U / kg food, 10U / kg food and 20U / kg food. After a 3-day adaptation phase, the polypeptide was applied for one day at a concentration of 2.5U / kg food, followed by a wash-out day with no polypeptide and no ZEN in the diet. After the wash-out day, non-control piglets received a diet containing ZEN with the same polypeptide at a concentration of 5U / kg food, followed by a wash-out day, and so on. During the trial, urine was collected for 12 hours and fecal samples were collected once a day. The samples were stored at -20°C until LC-MS / MS measurement.

[0380] In order to standardize the volume of urine excreted, the concentration of creatinine in the urine sample was measured. In order to determine the creatinine content, the urine sample was diluted with water 1:5000. The urine sample was diluted with water to a final concentration of 2.5mM creatinine. 100μl of diluted urine sample was mixed with 20μl of 100mM PBS buffer containing 528U of β-glucuronidase and incubated overnight at 37°C. After overnight incubation, 380μl of cold methanol was added, centrifuged at 14674×g, and the supernatant was transferred to an HPLC vial and stored at -20°C until analysis. For the analysis of fecal samples, 500mg of freeze-dried feces was extracted three times (90, 30 and 30 minutes) with 5ml acetonitrile / water (50 / 50, v / v) each time. After each extraction step, the sample was clarified by centrifugation (10 minutes, 14674×g). Aliquots of the combined supernatants were centrifuged and measured by HPLC-MS / MS. Analyzed on an Agilent 1290 series UHPLC system connected to a 6500QTrap mass spectrometer. The column temperature was set at 30°C with a flow rate of 0.25 ml / min. Mobile phases A and B consisted of water / acetic acid and acetonitrile / acetic acid (both 99.9 / 0.1, v / v), respectively. The gradient started with 5% B for 0.5 min, then continued with a linear increase to 36% B until 17.0 min, and linearly increased to 100% B between 17.0 and 22.0 min, then 100% B to 24.0 min, and then rapidly decreased to 5% B between 24.0 and 24.1 min. Finally, the column was re-equilibrated to 27.0 min at 5% B. The injection volume for urine samples was 2 μl, and the injection volume for fecal samples was 3 μl. Separation was performed on a Phenomenex Kinetex C18 column (150 x 2.1 mm, 2.6 μm). Quantification was based on calibration using external standards of ZEN, α-ZEL, HZEN and DHZEN. α-ZEL is a metabolite of ZEN with high estrogenic activity, produced in pigs by hepatic biotransformation (Malekinejad et al., (2006) 'Hydroxysteroid dehydrogenases in bovine and porcine granulosa cells convert zearalenone into its hydroxylated metabolites alpha-zearalenol and beta-zearalenol. Vet Res Commun: 445-53). The selected reaction monitoring (SRM) parameters are shown in Figure 5 .

[0381] In addition to the polypeptide having SEQ ID NO: 1, two polypeptide variants of the polypeptide having SEQ ID NO: 1 have been tested: variant A and variant B.

[0382] Compared to SEQ ID NO: 1, variant A comprises the following mutations: V160A / G185R / A186R / A188H / G199E / I200V / H203N / Q205K.

[0383] Compared to SEQ ID NO: 1, variant B comprises the following mutations: V160A / G185R / A186R / A188H / S189D / P190H / M191D / G199E / I200V / H203N / Q205K.

[0384] The results obtained from the analysis of urine and feces samples are shown in Figure 6 and 7 The percent change results for the combined concentration of ZEN plus α-ZEL compared to SEQ ID NO: 1 were obtained by dividing the difference between the concentrations of the two groups by the concentration of the group with SEQ ID NO: 1 and multiplying by 100. The increase in the concentration of ZEN plus α-ZEL during the feeding trial in the control group that did not receive the peptide that degrades ZEN in the diet may be caused by the enterohepatic cycling of ZEN and ZEN derivatives in pigs and thus their accumulation (Biehl et al., (1993) 'Biliary excretion and enterohepatic cycling of zearalenone inimmature pigs.' Toxicol Appl Pharmacol. 1993 Jul; 121(1): 152-9).

[0385] Example 8: Testing of different concentrations of polypeptide variants for eliminating ZEN toxicity in chickens

[0386] For the feeding trial, 90-day-old, mixed-sex chicks (Ross 308) were used. These chicks were fed two different diets in two stages. During the adaptation period, the chicks received a phase 1 diet (during the 1st-14th day), and during the experimental period, the chicks received a phase 2 diet (during the 15th-28th day). The composition of the phase 1 diet in percentage (w / w): 55.00% corn, 29.00% soybean HP, 1.00% sunflower oil, 6.92% full-fat soybeans, 0.72% soybean protein concentrate, 1.88% palm kernel, coconut oil, 1.96% calcium carbonate, 1.89% monocalcium phosphate, 0.35% sodium bicarbonate, 0.23% sodium chloride, 0.13% magnesium phosphate, 0.24% vitamin / trace element premix, 0.34% L-lysine, 0.12% L-threonine and 0.24% DL-methionine.

[0387] Composition of the Phase 2 diet in percentage (w / w): 62.00% corn, 23.80% soy HP, 2.00% sunflower oil, 5.53% whole fat soy, 0.58% soy protein concentrate, 1.50% palm kernel, coconut oil, 1.56% calcium carbonate, 1.71% monocalcium phosphate, 0.28% sodium bicarbonate, 0.19% sodium chloride, 0.10% magnesium phosphate, 0.19% vitamin / trace element premix, 0.27% L-lysine, 0.10% L-threonine and 0.20% DL-methionine.

[0388] For the adaptation period (14 days), the chicks were randomly assigned to 3 cages. After the adaptation period, the chicks were evenly distributed into 7 cages (groups) according to the average weight, with 8 chicks in each cage. The duration of the experiment was 14 days. The climatic conditions were adjusted according to the standard recommendations of the breeding company. Manual feeding was once a day, and feed and water were available at any time. The concentration of ZEN in the diet was 400μg ZEN / kg food. The control group was fed a diet containing 400μg ZEN / kg food without adding any enzymes that degrade ZEN. Polypeptide variant A (SEQ ID NO:1 containing the following mutations: V160A / G185R / A186R / A188H / G199E / I200V / H203N / Q205K) was tested at the following concentrations: 5U / kg food, 10U / kg food, 20U / kg food, 40U / kg food, 80U / kg food and 160U / kg food. After euthanasia, crop samples were taken from chicks at the beginning and end of the experiment. The samples were frozen and lyophilized. For the analysis of ZEN, HZEN and DHZEN, 1 g of each sample was weighed into a 50 ml tube and extracted twice with 15 ml 80% acetonitrile at 25°C on a rotary shaker for 30 min each. The tube was then centrifuged at 2300 x g at 25°C for 10 min, and the supernatant was combined into a new 50 ml tube. 1 ml was taken and centrifuged again at 2300 x g at 25°C for 5 min, and the supernatant was transferred to a vial for LC-MS / MS measurement. The samples were stored at -20°C until measurement and analyzed by LC-MS / MS. The analysis was performed on an Agilent 1290 series UHPLC system connected to a 6500+QTrap mass spectrometer. The column temperature was set at 30°C and the flow rate was 0.25 ml / min. Mobile phases A and B consisted of water / acetic acid and acetonitrile / acetic acid (both 99.9 / 0.1, v / v), respectively. The gradient started with 15% B for 0.5 min, then continued with a linear increase to 60% B until 13.5 min, and increased sharply to 100% B between 13.5 and 14.0 min, then 100% B to 16.9 min, followed by a sharp decrease to 15% B between 16.9 and 17.0 min. Finally, the column was re-equilibrated at 15% B to 20.0 min. The injection volume was 2 μl. Separation was performed on a Phenomenex Kinetex C18 column (150x2.1 mm, 2.6 μm). Quantification was based on calibration using external standards of ZEN, HZEN and DHZEN. The selected reaction monitoring (SRM) parameters are shown in Figure 8 .

[0389] The results obtained from the analysis of crop samples from the end of the experiment are shown in Fig. 9The percentage reduction of ZEN concentration was calculated as follows: (ZEN concentration of the control group minus ZEN concentration of the sample group) divided by the ZEN concentration of the control group, and multiplied by 100.

[0390] References

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[0392] Altschul,J.Mol.Evol.36(1993),290-300

[0393] Altschul,J.Mol.Biol.215(1990),403-410

[0394] Biehl et al., (1993) 'Biliary excretion and enterohepatic cycling ofzearalenone in immature pigs.' Toxicol Appl Pharmacol. 1993Jul; 121(1):152-9

[0395] Brutlag Comp.App.Biosci.6(1990),237-245

[0396] Burnley and Gros (2012)'phenix.ensemble_refinement: a test study of apoand holo BACE1'Computational crystallography newsletter, volume 4, pp.51–58

[0397] Cregg,Pichia Protocols,second Edition,ISBN-10:1588294293,2007

[0398] PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES,2nd Ed.,TECreighton,WHFreeman and Company,New York(1993)

[0399] Elmar Krieger,Bioinformatics 30,2981-2982

[0400] Gasteiger E. et al., Protein Identification and Analysis Tools on the ExPASy Server, in John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press, 2005, pp. 571 - 607

[0401] Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915

[0402] Henikoff and Henikoff (1992) ‘Amino acid substitution matrices from protein blocks.’ Proc Natl Acad Sci U S A. 1992 Nov 15; 89(22):10915 - 9

[0403] Jantratid et al., (2008)’Dissolution media simulating conditions in the proximal human gastrointestinal tract: an update.’ Pharm Res. 2008 Jul; 25(7):1663 - 76

[0404] Jones (1999) ‘Protein secondary structure prediction based on position - specific scoring matrices’ J. Mol. Biol. 292:195 - 202

[0405] POST - TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B.C. Johnson, Ed., Academic Press, New York (1983), pgs. 1 - 12; Seifter, Meth. Enzymol. 182(1990), 626 - 646

[0406] Kourist et al., (2010) ‘The alpha / beta-hydrolase fold 3DM database (ABHDB) as a tool for protein engineering.’ Chembiochem. 11(12):1635-43

[0407] Kurtzman (2009) “Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis.” J Ind Microbiol Biotechnol. 36(11):1435-8

[0408] Kyte and Doolittle (1983) "A simple method for displaying the hydropathic character of a protein". J.Mol.Biol. 157(1):105–32

[0409] Lenfant et al., (2013) ‘ESTHER, the database of the α / β-hydrolase fold superfamily of proteins: tools to explore diversity of functions’ Nucleic Acids Research, Volume 41, Issue D1, D423–D429

[0410] Malekinejad et al., (2006) ‘Hydroxysteroid dehydrogenases in bovine and porcine granulosa cells convert zearalenone into its hydroxylated metabolites alpha-zearalenol and beta-zearalenol. Vet Res Commun:445-53

[0411] Mindrebo et al., (2016) ‘Unveiling the functional diversity of the Alpha-Beta hydrolase fold in plants’ Curr Opin Struct Biol. 233–246

[0412] Ollis et al., (1992) ‘The alpha / beta hydrolase fold‘ Protein Eng. 5(3):197-211

[0413] Rattan, Ann. NY Acad. Sci. 663(1992); 48-62

[0414] Schatzmayr and Streit (2013) ‘Global occurrence of mycotoxins in the food and feed chain: Facts and figures.’ World Mycotoxin Journal 6(3):213-222

[0415] Sambrook et al., 2012, Molecular Cloning, A Laboratory Manual 4th Edition, Cold Spring Harbor

[0416] Stenhagen & Teorell. (1938) Nature 141, 415

[0417] Thompson Nucl. Acids Res. 2(1994), 4673-4680

[0418] Vekiru et al., (2016) ‘Isolation and characterisation of enzymatic zearalenone hydrolysis reaction products’ World Mycotoxin Journal 9:353-363)

[0419] Yamada et al., (1995) 'The Phylogenetic Relationships of Methanol-assimilating Yeasts Based on the Partial Sequences of 18S and 26S RibosomalRNAs: The Proposal of Komagataella Gen. Nov. (Saccharomycetaceae)' Bioscience, Biotechnology and Biochemistry, Vol. 59, issue 3, pp. 439-444). Sequence Listing <110> Austrian company Elber GmbH <120> Means and methods for cracking zearalenone <130> LC19310008 <160> 11 <170> SIPOSequenceListing 1.0 <210> 1 <211> 309 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> α / β hydrolase domain <400> 1 Met Val Thr Ser Pro Ala Leu Arg Asp Val His Val Pro His Ala Tyr 1 5 10 15 Pro Glu Gln Gln Val Asp Leu Gly Glu Ile Thr Met Asn Tyr Ala Glu 20 25 30 Ala Gly Asp Pro Asp Arg Pro Ala Val Leu Leu Ile Pro Glu Gln Thr 35 40 45 Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ser Glu His 50 55 60 Phe His Val Tyr Ala Val Asp Leu Arg Gly Gln Gly Arg Ser Ser Trp 65 70 75 80 Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg 85 90 95 Phe Ile Ala Leu Val Val Lys Arg Pro Val Val Val Ala Gly Asn Ser 100 105 110 Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly 115 120 125 Gln Leu Arg Gly Val Leu Cys Glu Asp Pro Pro Phe Phe Ala Ser Glu 130 135 140 Leu Val Pro Ala His Gly His Ser Val Arg Gln Gly Ala Gly Pro Val 145 150 155 160 Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gln Trp Ser Val Gly Asp 165 170 175 Trp Glu Gly Phe Cys Arg Ala Ala Gly Ala Ser Ala Ser Pro Met Ala 180 185 190 Arg Ser Phe Val Ala Asp Gly Ile Pro Gln His Leu Gln Glu Tyr Asp 195 200 205 Pro Glu Trp Ala Arg Val Phe Tyr Glu Gly Thr Val Gly Leu Ser Cys 210 215 220 Pro His Glu Arg Met Leu Gly Gln Val Lys Thr Pro Val Leu Leu Thr 225 230 235 240 His His Met Arg Gly Ile Asp Pro Glu Thr Gly Asn Leu Leu Gly Ala 245 250 255 Leu Ser Asp Glu Gln Ala Leu Arg Ala Arg Arg Leu Met Asp Ser Ala 260 265 270 Gly Val Thr Val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met 275 280 285 His Gln Ser Ala Pro Ala Arg Tyr Val Glu Ile Phe Thr Arg Trp Ala 290 295 300 Ala Ala Leu Ala Pro 305 <210> 2 <211> 308 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> α / β hydrolase domain <400> 2 Met Ala Asp Pro Ala Gln Arg Asp Val Tyr Val Pro His Ala Tyr Pro 1 5 10 15 Glu Lys Gln Ala Asp Leu Gly Glu Ile Thr Met Asn Tyr Ala Glu Ala 20 25 30 Gly Glu Pro Asp Met Pro Ala Val Leu Leu Ile Pro Glu Gln Thr Gly 35 40 45 Ser Trp Trp Gly Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu Asn Phe 50 55 60 His Val Tyr Ala Val Asp Leu Arg Gly Gln Gly Arg Ser Ser Trp Ala 65 70 75 80 Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg Phe 85 90 95 Ile Ala Leu Val Val Lys Arg Pro Val Ile Val Ala Gly Asn Ser Ser 100 105 110 Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly Gln 115 120 125 Val Arg Gly Ala Leu Cys Glu Asp Ala Pro Phe Phe Ala Ser Glu Leu 130 135 140 Val Thr Thr Cys Gly His Ser Ile Arg Gln Ala Ala Gly Pro Met Phe 145 150 155 160 Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gln Trp Ser Val Gly Asp Trp 165 170 175 Thr Gly Tyr Cys Arg Ala Ala Asp Ala Ser Ser Ser Pro Met Ala Arg 180 185 190 Tyr Phe Val Ala Asp Glu Ile Pro Gln His Met Arg Glu Tyr Asp Pro 195 200 205 Glu Trp Ala Arg Ala Phe Trp Glu Gly Thr Val Ala Leu His Cys Pro 210 215 220 His Glu Gln Leu Leu Thr Gln Val Lys Thr Pro Val Leu Leu Thr His 225 230 235 240 His Met Arg Asp Ile Asp Pro Asp Thr Gly His Leu Val Gly Ala Leu 245 250 255 Ser Asp Glu Gln Ala Ala Arg Ala Arg Leu Leu Met Glu Ser Ala Gly 260 265 270 Val Lys Val Asp Tyr Ala Ser Val Pro Asp Ala Leu His Met Met His 275 280 285 Gln Phe Asp Pro Pro Arg Tyr Val Glu Ile Phe Thr Gln Trp Ala Ala 290 295 300 Thr Leu Ala Ala 305 <210> 3 <211> 309 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> α / β water structure <400> 3 Met Val Thr Ser Pro Ala Leu Arg Asp Val His Val Pro His Ala Tyr 1 5 10 15 Pro Glu Gln Gln Val Asp Leu Gly Glu Ile Thr Met Asn Tyr Ala Glu 20 25 30 Ala Gly Asp Pro Gly Arg Pro Ala Val Leu Leu Ile Pro Glu Gln Thr 35 40 45 Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu His 50 55 60 Phe His Val Tyr Ala Val Asp Leu Arg Gly Gln Gly Arg Ser Ser Trp 65 70 75 80 Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg 85 90 95 Phe Ile Ala Leu Val Val Arg Arg Pro Val Val Val Ala Gly Asn Ser 100 105 110 Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly 115 120 125 Gln Ile Arg Gly Val Leu Cys Glu Asp Pro Pro Phe Phe Ala Ser Glu 130 135 140 Leu Val Pro Ala His Gly His Ser Val Arg Gln Gly Ala Gly Pro Val 145 150 155 160 Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gln Trp Ser Val Gly Asp 165 170 175 Trp Glu Gly Phe Arg Ser Ala Ala Asp Ala Ser Ala Ser Pro Met Ala 180 185 190 Arg Ser Phe Val Ala Asp Thr Ile Pro Gln His Leu Lys Glu Tyr Asp 195 200 205 Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Gly Leu Asn Cys 210 215 220 Pro His Glu Arg Met Leu Asn Arg Val Asn Thr Pro Val Leu Leu Thr 225 230 235 240 His His Met Arg Gly Thr Asp Pro Glu Thr Gly Asn Leu Leu Gly Ala 245 250 255 Leu Ser Asp Glu Gln Ala Ala Gln Val Arg Arg Leu Met Glu Ser Ala 260 265 270 Gly Val Lys Val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met 275 280 285 His Gln Ser Asp Pro Ala Arg Tyr Ala Glu Ile Leu Thr Pro Trp Thr 290 295 300 Ala Ala Leo Ala Pro 305 <210> 4 <211> 309 <212> PRT <213> Artificial Sequence <220> <221> peptide <222> ()..() <223> α / β water structure <400> 4 Met Val Thr Ser Pro Ala Leu Arg Asp Val His Val Pro His Ala Tyr 1 5 10 15 Pro Glu Gln Gln Val Asp Leu Gly Glu Ile Thr Met Asn Tyr Ala Glu 20 25 30 Ala Gly Asp Pro Asp Arg Pro Ala Val Leu Leu Ile Pro Glu Gln Thr 35 40 45 Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu His 50 55 60 Phe His Val Tyr Ala Val Asp Leu Arg Gly Gln Gly Arg Ser Ser Trp 65 70 75 80 Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg 85 90 95 Phe Ile Ala Leu Val Val Lys Arg Pro Val Val Val Ala Gly Asn Ser 100 105 110 Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly 115 120 125 Gln Leu Arg Gly Val Leu Cys Glu Asp Pro Pro Phe Phe Ala Ser Glu 130 135 140 Leu Val Pro Ala His Gly His Ser Val Arg Gln Gly Ala Gly Pro Val 145 150 155 160 Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gln Trp Ser Val Ser Asp 165 170 175 Trp Glu Gly Phe Cys Arg Ala Ala Gly Ala Ser Ala Ser Pro Met Ala 180 185 190 Arg Ser Phe Val Ala Asp Gly Ile Pro Gln His Leu Lys Glu Tyr Asp 195 200 205 Pro Glu Trp Ala Arg Ala Phe His Glu Gly Thr Val Gly Leu Asn Cys 210 215 220 Pro His Glu Arg Met Leu Gly Arg Val Asn Thr Pro Val Leu Leu Thr 225 230 235 240 His His Met Arg Gly Thr Asp Pro Glu Thr Gly Asn Leu Leu Gly Ala 245 250 255 Leu Ser Asp Glu Gln Ala Ala Gln Ala Arg Leu Leu Met Glu Ser Ala 260 265 270 Gly Val Arg Val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met 275 280 285 His Gln Ser Asp Pro Ala Arg Tyr Ala Glu Ile Phe Thr Arg Trp Ala 290 295 300 Ala Ala Leu Ala Pro 305 <210> 5 <211> 309 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> α / β hydrolase domain <400> 5 Met Val Thr Ser Pro Ala Leu Arg Asp Val His Val Pro His Ala Tyr 1 5 10 15 Pro Glu Gln Gln Val Asp Leu Gly Glu Ile Thr Met Asn Tyr Ala Glu 20 25 30 Ala Gly Asp Pro Gly Arg Pro Ala Val Leu Leu Ile Pro Glu Gln Thr 35 40 45 Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu His 50 55 60 Phe His Val Tyr Ala Val Asp Leu Arg Gly Gln Gly Arg Ser Ser Trp 65 70 75 80 Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg 85 90 95 Phe Met Ala Leu Val Val Arg Arg Pro Val Val Val Ala Gly Asn Ser 100 105 110 Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly 115 120 125 Gln Ile Arg Gly Val Leu Cys Glu Asp Pro Pro Phe Phe Ala Ser Glu 130 135 140 Leu Val Pro Ala His Gly His Ser Val Arg Gln Gly Ala Gly Pro Val 145 150 155 160 Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gln Trp Ser Val Gly Asp 165 170 175 Trp Glu Gly Phe Arg Ser Ala Ala Gly Ala Ser Ala Ser Pro Met Ala 180 185 190 Arg Ser Phe Val Ala Asp Thr Ile Pro Gln His Leu Lys Glu Tyr Asp 195 200 205 Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Gly Leu Asn Cys 210 215 220 Pro His Glu Arg Met Leu Asn Arg Val Asn Thr Pro Val Leu Leu Thr 225 230 235 240 His His Met Arg Gly Thr Asp Pro Glu Thr Gly Asn Leu Leu Gly Ala 245 250 255 Leu Ser Asp Glu Gln Ala Ala Gln Ala Arg Arg Leu Met Glu Ser Ala 260 265 270 Gly Val Lys Val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met 275 280 285 His Gln Ser Asp Pro Ala Arg Tyr Ala Glu Ile Leu Thr Pro Trp Ala 290 295 300 Ala Ala Leo Ala Pro 305 <210> 6 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> peptide <222> ()..() <223> α / β water structure <400> 6 Met Ala Glu Glu Gly Thr Arg Ser Glu Ala Ala Asp Ala Ala Thr Gln 1 5 10 15 Ala Arg Gln Leu Pro Asp Ser Arg Asn Ile Phe Val Ser His Arg Phe 20 25 30 Pro Glu Arg Gln Val Asp Leu Gly Glu Val Val Met Asn Phe Ala Glu 35 40 45 Ala Gly Ser Pro Asp Asn Pro Ala Leu Leu Leu Leu Pro Glu Gln Thr 50 55 60 Gly Ser Trp Trp Ser Tyr Glu Pro Val Met Gly Leu Leu Ala Glu Asn 65 70 75 80 Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr Trp 85 90 95 Thr Pro Arg Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg 100 105 110 Phe Ile Ala Leu Val Ile Lys Arg Pro Val Val Val Ala Gly Asn Ser 115 120 125 Ser Gly Gly Leu Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro Gly 130 135 140 Gln Ile Arg Ala Ala Leu Cys Glu Asp Ala Pro Phe Phe Ala Ser Glu 145 150 155 160 Leu Val Pro Ala Tyr Gly His Ser Val Leu Gln Ala Ala Gly Pro Ala 165 170 175 Phe Glu Leu Tyr Arg Asp Phe Leu Gly Asp Gln Trp Ser Ile Gly Asp 180 185 190 Trp Lys Gly Phe Val Glu Ala Ala Lys Ala Ser Pro Ala Lys Ala Met 195 200 205 Gln Leu Phe Pro Thr Pro Asp Glu Ala Pro Gln Asn Leu Lys Glu Tyr 210 215 220 Asp Pro Glu Trp Gly Arg Ala Phe Phe Glu Gly Thr Val Ala Leu His 225 230 235 240 Cys Pro His Asp Arg Met Leu Ser Gln Val Lys Thr Pro Ile Leu Ile 245 250 255 Thr His His Ala Arg Thr Ile Asp Pro Glu Thr Gly Glu Leu Leu Gly 260 265 270 Ala Leu Ser Asp Leu Gln Ala Glu His Ala Gln Asp Ile Ile Arg Ser 275 280 285 Ala Gly Val Arg Val Asp Tyr Gln Ser His Pro Asp Ala Leu His Met 290 295 300 Met His Leu Phe Asp Pro Ala Arg Tyr Ala Glu Ile Leu Thr Ser Trp 305 310 315 320 Ser Ala Thr Leu Pro Ala Asn Asp 325 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Motif <220> <221> PEPTIDE <222> (2) <223> X is any amino acid <400> 7 Gln Xaa Ala Gly Pro 1 5 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Motif <400> 8 Glu Tyr Asp Pro Glu 1 5 <210> 9 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Motif <220> <221> PEPTIDE <222> (2) <223> X is F or Y <220> <221> PEPTIDE <222> (3)..(4) <223> X is any amino acid <400> 9 Gly Xaa Xaa Xaa Ala Ala 1 5 <210> 10 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Motif <220> <221> PEPTIDE <222> (3) <223> X is any amino acid <400> 10 Ala Arg Xaa Phe 1 <210> 11 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Motif <400> 11 Gln Leu Phe Pro 1

Claims

1. A method for increasing the stability of an α / β-hydrolase, wherein the sequence of the α / β-hydrolase is as shown in SEQ ID NO: 1 or SEQ ID NO: 6, the method comprising: The amino acids in the VI-domain within the CAP-domain of the α / β-hydrolase were substituted at the following positions: - a position corresponding to positions 160 to 205 of SEQ ID NO: 1, or - positions corresponding to positions 176 to 222 of SEQ ID NO: 6, wherein the amino acid substitutions at positions corresponding to positions 160 to 205 of SEQ ID NO: 1 are: V160A; G185R; G185S; A186P; A186R; A188D; A188H; A188N; A188G; A188R; S189D; P190H; M191D; G199E; I200A; I200V; H203N; Q205K; S173C; G185R / A188N; G185S / A188R; G185R / A186R / A188H; G185R / A186R / A188H / S189D / P190H / M191D; V160A / G185R / A188N / G199E / I200A / H203N / Q205K; V160A / G185S / A188R / G199E / I200A / H203N / Q205K; V160A / G199E / I200A / H203N / Q205K; V160A / G185R / A186R / A188H / G199E / I200V / H203N / Q205K; or V160A / G185R / A186R / A188H / S189D / P190H / M191D / G199E / I200V / H203N / Q205K; wherein the amino acid substitutions at positions corresponding to positions 176 to 222 of SEQ ID NO: 6 are: F183Y; V197C; or F183Y / V197C; Thereby an α / β-hydrolase having increased stability compared to the α / β-hydrolase before substitution of the amino acid is obtained.

2. The method according to claim 1, wherein the increased stability is a decrease in GRAVY value, an increase in pH stability and / or an increase in temperature stability.

3. An α / β-hydrolase variant, wherein the α / β-hydrolase variant has an amino acid substitution at positions corresponding to positions 160 to 205 of SEQ ID NO: 1 relative to a parent α / β-hydrolase, the sequence of the parent α / β-hydrolase being as shown in SEQ ID NO: 1, wherein the amino acid substitution is: G185R; G185S; A186P; A186R; A188D; A188H; A188N; A188G; A188R; S189D; P190H; M191D; G185R / A188N; G185S / A188R; G185R / A186R / A188H; G185R / A186R / A188H / S189D / P190H / M191D; V160A / G185R / A188N / G199E / I200A / H203N / Q205K; V160A / G185S / A188R / G199E / I200A / H203N / Q205K; V160A / G199E / I200A / H203N / Q205K; V160A / G185R / A186R / A188H / G199E / I200V / H203N / Q205K; or V160A / G185R / A186R / A188H / S189D / P190H / M191D / G199E / I200V / H203N / Q205K.

4. An α / β-hydrolase variant, wherein the α / β-hydrolase variant has amino acid substitutions at positions corresponding to positions 176 to 222 of SEQ ID NO: 6 relative to a parent α / β-hydrolase, wherein the sequence of the parent α / β-hydrolase is as shown in SEQ ID NO: 6, wherein the amino acid substitution is: F183Y; V197C; or F183Y / V197C.

5. Use of an α / β-hydrolase variant for degrading zearalenone (ZEN), wherein the α / β-hydrolase variant has amino acid substitutions at positions corresponding to positions 160 to 205 of SEQ ID NO: 1 relative to a parent α / β-hydrolase, wherein the sequence of the parent α / β-hydrolase is as shown in SEQ ID NO: 1, in, The amino acid substitutions are: V160A / G185R / A186R / A188H / G199E / I200V / H203N / Q205K; or V160A / G185R / A186R / A188H / S189D / P190H / M191D / G199E / I200V / H203N / Q205K.

6. A composition comprising the α / β-hydrolase variant of claim 3 or 4.

7. The composition according to claim 6, wherein the composition is a food or feed additive.

8. Use of an α / β-hydrolase variant or a composition comprising the α / β-hydrolase variant in the preparation of a medicament for ZEN detoxification, wherein the α / β-hydrolase variant has an amino acid substitution at positions corresponding to positions 160 to 205 of SEQ ID NO: 1 relative to a parent α / β-hydrolase, the sequence of the parent α / β-hydrolase being as shown in SEQ ID NO: 1, in, The amino acid substitutions are: V160A / G185R / A186R / A188H / G199E / I200V / H203N / Q205K; or V160A / G185R / A186R / A188H / S189D / P190H / M191D / G199E / I200V / H203N / Q205K.

9. A kit comprising the α / β-hydrolase variant of claim 3 or 4.

10. A kit comprising the composition of claim 6 or 7.

Images