EP4673536A1

EP4673536A1 - Means and methods for modifying fumonisins

Info

Publication number: EP4673536A1
Application number: EP24707531.0A
Authority: EP
Inventors: David Bednar; Jiri Damborsky; Corinna KERN; Veronika Laskova; Wulf-Dieter Moll; Veronika Stepankova; Michaela THAMHESL; Gudrun Vogtentanz
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2023-02-28
Filing date: 2024-02-27
Publication date: 2026-01-07
Also published as: JP2026509151A; AR131996A1; TW202436327A; MX2025010058A; WO2024180045A1; CN120787259A; AU2024228703A1

Abstract

The present invention relates to a method for improving enzyme kinetics of a fumonisin esterase, an improved fumonisin esterase, use thereof, a composition comprising the fumonisin esterase, and a method for modifying a fumonisin.

Description

MEANS AND METHODS FOR MODIFYING FUMONISINS

[001] The present invention relates to a method for improving enzyme kinetics of a fumonisin esterase, an improved fumonisin esterase, use thereof, a composition comprising the fumonisin esterase, and a method for modifying a fumonisin.

[002] Mycotoxins are secondary metabolites produced by fungal species. Fungi infest food as well as feed material. In particular all types of grains and feed crops as well as other commodities such as cotton, coffee, peanuts, dates, spices etc. are affected. Upon fungal growth, mycotoxins such as aflatoxins, ochratoxin, ergot alkaloids, fumonisins, zearalenone and/or trichothecenes are produced, contaminating the infested material.

[003] Among more than 17,000 samples, almost three quarters were found to contain at least one mycotoxin (Streit et al. 2013. J Sci Food Agric. 93(12): 2892-2899). One of the most prominent groups of mycotoxin molecules are the fumonisins. Fumonisins are produced inter alia by Fusarium spp. and Alternaria spp. fungi and impair the sphingolipid metabolism upon ingestion, causing numerous adverse effects. In particular, fumonisins are associated with immunosuppression, inflammation, cancerogenity, embryonic malformation etc. In livestock farming, animals ingesting fumonisins are not only subjected to the health hazards associated with fumonisin mycotoxicosis but also show decreased performance, resulting in considerable financial losses.

[004] Based on structural similarity, the group of fumonisins comprises fumonisin A1 (CAS No. 117415-48-2), A2 (CAS No. 117415-47-1), B1 (CAS No. 116355-83-0), B2 (CAS No. 116355-84- 1), B3 (CAS No. 136379-59-4), B4 (CAS no. 136379-60-7), C1 (PubChemCID 42608361), C2 (PubChemCID 42608362), C3 (PubChemCID 42608363), C4 (PubChemCID 42608364), and HFC1 ; as well as the Alternaria alternata lycopersici toxins (AAL-T) A1 (CAS No. 79367-52-5), A2 (CAS No. 79367-51-4), B1 (CAS No. 149849-90-1), and B2 (CAS No. 149849-91-2); as well as partially hydrolyzed derivatives thereof. In partially hydrolyzed fumonisins, one of two tricarballylic acid residues has been removed from the non-hydrolyzed molecule. The formula below exemplarily shows fumonisins A1 , A2, B1 , B2, B3, B4, C1 , C2, C3, and C4, wherein R1=OH, R2=OH, R3=CH2CO, R4=CH3 for fumonisin A1 , R1=H, R2=OH, R3=CH2CO, R4=CH3 for fumonisin A2, R1=OH, R2=OH, R3=H, R4=CH3 for fumonisin B1 , R1=H, R2=OH, R3=H, R4=CH3 for fumonisin B2, R1=OH, R2=H, R3=H, R4=CH3 for fumonisin B3, R1=H, R2=H, R3=H, R4=CH3 for fumonisin B4, R1=OH, R2=OH, R3=H, R4=H for fumonisin C1 , R1=H, R2=OH, R3=H, R4=H for fumonisin C2, R1=OH, R2=H, R3=H, R4=H for fumonisin C3, and R1=H, R2=H, R3=H, R4=H for fumonisin C4. Within the group of fumonisins, fumonisin B1 (FB1) is the most prevalent mycotoxin. FB1 has been described to be hepatoxic and nephrotoxic and is associated at least with equine leukoencephalomalacia and porcine pulmonary oedema syndrome.

[005] By applying "good agricultural practice", it can be attempted to contain the degree of fungal contamination, in particular by avoiding moisture as much as possible to disfavor fungal growth, or by applying fungicides to the food/feed material. Additionally, contaminated materials should be removed and discarded prior to processing. However, due to the high prevalence and to the low median toxic dose, further measures have to be considered.

[006] In WO 2006/053357 A2, microbial conversion of fumonisins to non-toxic products is described. In WO 2010/031101 A1 , polypeptides for the enzymatic degradation of fumonisins are described. In WO 2016/134387 A1 , fumonisin esterase variants with improved temperature stability are described. However, in order to achieve an even more efficient and faster removal of fumonisin molecules, there is still a need for fumonisin cleaving enzymes with improved kinetics.

[007] In view of the prior art as outlined above, it is an objective of the present invention to provide improved means and methods for modifying, in particular for detoxifying fumonisins.

[008] This objective is achieved by providing a method for improving enzyme kinetics (e.g. decreased Michaelis constant (Km), increased turnover number (kcat), increased catalytic efficiency, increased maximum reaction rate (Vmax), and/or increased specific activity) of a fumonisin esterase, wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 ; the method comprising substituting the amino acid at position 25 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with an aliphatic amino acid, more preferably with an amino acid selected from alanine, glycine, isoleucine, leucine, proline, valine, even more preferably with an amino acid selected from isoleucine, leucine, valine, most preferably with isoleucine; substituting the amino acid at position 45 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid select from asparagine, aspartate, glutamine, glutamate, most preferably with glutamine; and/or substituting the amino acid at position 259 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a charged amino acid, more preferably with an amino acid selected from lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid selected from lysine, arginine, histidine, most preferably with arginine. When performing one or more of these substitutions, it is possible to produce a fumonisin esterase with improved enzyme kinetics, i.e. improved enzyme activity. Such an improved fumonisin esterase allows faster and more efficient removal of fumonisins e.g. from nutritional compositions.

[009] A fumonisin esterase as referred to herein is an esterase enzyme capable of modifying one or more fumonisin(s), in particular such an esterase enzyme is capable of modifying at least one of fumonisins A1 , A2, B1 , B2, B3, B4, C1 , C2, C3, C4, AAL-TA1 , AAL-TA2, AAL-TB1 , AAL- TB2. Upon fumonisin modification by a fumonisin esterase, tricarballylic acid residue(s) are cleaved from the reactant fumonisin.

[010] The term "amino acids" is to be interpreted as known to a person skilled in the art. Preferably, the term refers to proteinogenic amino acids. As known in the art, amino acids may be referred to by using a three-letter-code or a one-letter-code. Therein, "Ala" and "A" refer to alanine, "Arg" and "R" refer to arginine, "Asn" and "N" refer to asparagine, "Asp" and "D" refer to aspartate (also referred to as aspartic acid), "Cys" and "C" refer to cysteine, "Gin" and "Q" refer to glutamine, "Glu" and "E" refer to glutamate (also referred to as glutamic acid), "Gly" and "G" refer to glycine, "His" and "H" refer to histidine, "lie" and "I" refer to isoleucine, "Leu" and "L" refer to leucine, "Lys" and "K" refer to lysine, "Met" and "M" refer to methionine, "Phe" and "F" refer to phenylalanine, "Pro" and "P" refer to proline, "Ser" and "S" refer to serine, "Thr" and "T" refer to threonine, "Trp" and "W¹ refer to tryptophan, "Tyr" and "Y" refer to tyrosine, "Vai" and "V" refer to valine.

[011] When referring to a certain amino acid at a certain position in a polypeptide, said amino acid is typically described by indicating said amino acid by its one-letter-code letter, followed by indicating the position, i.e. the number of said amino acid in the amino acid chain of the polypeptide. For instance, T25 with respect to the amino acid sequence of SEQ ID NO: 1 indicates the threonine residue at position 25 in the polypeptide having the amino acid sequence of SEQ ID NO: 1 . Similarly, in order to indicate a substitution of a certain amino acid at a certain position with another amino acid, the amino acid to be substituted (i.e. the "original" amino acid) is first indicated with its one-letter-code letter, followed by its position in the amino acid chain of the polypeptide, followed by the one-letter-code letter of the amino acid which shall replace the original amino acid. For instance, T25I with respect to the amino acid sequence of SEQ ID NO: 1 indicates that the threonine at position 25 in the polypeptide having the amino acid sequence of SEQ ID NO: 1 will be replaced, i.e. substituted, by an isoleucine. Analogously, for instance the term "149F" indicates that the amino acid at position 149 shall be replaced with a phenylalanine.

[012] Merely for the sake of completeness and as known in the art, "aliphatic amino acids" are amino acids selected from the group consisting of alanine, glycine, isoleucine, leucine, methionine, proline, and valine; "polar amino acids" are amino acids selected from the group consisting of tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamate, and aspartate; "charged amino acids" are amino acids selected from the group consisting of lysine, arginine, histidine, glutamate, and aspartate.

[013] The term "polypeptide" is to be construed as commonly used in the art and encompasses e.g. polypeptides, proteins, peptides, enzymes.

[014] The term "sequence identity" is used to describe the degree of relatedness of two or more nucleic acid sequences (e.g. DNA or RNA polynucleotides) comprised in polynucleotides or of two or more amino acid sequences comprised in polypeptides. The sequence identity can be determined by common methods known to a skilled person. Herein, the preferred method for determination of sequence identity among two amino acid sequences is the use of the Clustal Omega alignment tool of EMBL-EBI (https://www.ebi.ac.uk/Tools/msa/clustalo/; Sievers et al. 2011. Mol. Syst. Biol. 7: 539) with default settings. Alternatively, the Needleman-Wunsch algorithm for global sequence alignment may be used, e.g. as provided by the National Center for Biotechnology Information (“Needleman-Wunsch Global Align Protein Sequences”) using default settings (Gap Costs: Existence: 11 Extension 1). Polypeptides comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence of a target polypeptide may be polypeptides comprising an amino acid sequence having 70% or more % sequence identity, e.g. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity to the amino acid sequence of the target polypeptide. For instance, a polypeptide comprising an amino acid sequence having 100% sequence identity to SEQ ID NO: 1 , is encompassed by the group of "polypeptides comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1". It is common practice in the art to construct fusion polypeptides linking one or more peptide tag(s) to the N- and/or C- terminus of a polypeptide, e.g. in order to facilitate easier purification or to increase solubility, without essentially altering the enzymatic properties of the tagged polypeptide. Typically, the term "fusion polypeptide" or "fusion protein" describes two or more polypeptide chains linked to one another, typically by peptide bonds. Examples for commonly used tags are poly(His)-tags (e.g. hexahistidine tag), maltose-binding-protein (MBP)-tag, Strep-tag, Strep ll-tag etc. Thus, enzymatically active fusion polypeptides comprising a polypeptide comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO. 1 , are encompassed by the present invention. Such fusion polypeptides may e.g. comprise a hexahistidine tag fused to the N-terminus of a polypeptide comprising an amino acid sequence having 91 % sequence identity to the amino acid sequence of SEQ ID NO: 1 , or e.g. comprise a maltose-binding-protein tag fused to the C-terminus of a polypeptide comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 1 via a linker peptide (e.g. GG; GPG; EA or EA repeats; GSG; etc.). In other words, larger polypeptides comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 as a part of said larger polypeptide are also encompassed by the present invention, as long as the enzymatic activity of said amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 , is not essentially obliterated. Merely for the sake of clarification, the enzymatically active polypeptide(s) according to the invention are capable of and thus suitable for modifying, in particular cleaving and/or detoxifying, at least one fumonisin, preferably fumonisin B1.

[015] In some embodiments, the method for improving enzyme kinetics (e.g. decreased Michaelis constant (Km), increased turnover number (kcat), increased catalytic efficiency, increased maximum reaction rate (Vmax), and/or increased specific activity) of a fumonisin esterase, wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 ; the method comprising substituting the amino acid at position 25 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with an aliphatic amino acid, more preferably with an amino acid selected from alanine, glycine, isoleucine, leucine, proline, valine, even more preferably with an amino acid selected from isoleucine, leucine, valine, most preferably with isoleucine; substituting the amino acid at position 45 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid select from asparagine, aspartate, glutamine, glutamate, most preferably with glutamine; and/or substituting the amino acid at position 259 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a charged amino acid, more preferably with an amino acid selected from lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid selected from lysine, arginine, histidine, most preferably with arginine; further comprises the step of substituting at least one further amino acid at a position selected from 10, 33, 66, 107, 140, 144, 149, 151 , 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363, 364, 365, 367, 371 , 372, 377, 389, 391 , 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456, 457, 462, 463, 464, 465, 469, 473, 478, 487 and 490 with respect to the amino acid sequence of SEQ I D NO: 1 . By introducing said further amino acid substitution(s) at one or more of these position(s), the fumonisin esterase can be improved even further, in particular not only with respect to improved enzyme kinetics characteristics but also with respect to improved temperature stability. In a preferred embodiment, said at least one further amino acid substitution is selected from 10Q, 33E, 66A, 66C, 66F, 66L, 66M, 66T, 66W, 66E, 66H, 661, 66K, 66P, 66R, 66Y, 66G, 66Q, 66S, 107E, 140P, 144M, 149F, 151 R, 157Y, 1991, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 3021, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371V, 371M, 372F, 377V, 389L, 391V, 394P, 418A, 419V, 424A, 424K, 427V, 429P, 430A, 436A, 436S, 440G, 440S, 443T, 447A, 453R, 455S, 456Q, 457T, 462Y, 463D, 464I, 465H, 465S, 465G, 469K, 473A, 478D, 487N and 490P with respect to the amino acid sequence of SEQ ID NO: 1 .

[016] In an aspect, the invention relates to a method for improving enzyme kinetics (e.g. decreased Michaelis constant (Km), increased turnover number (kcat), increased catalytic efficiency, increased maximum reaction rate (Vmax), and/or increased specific activity) of a thermostable fumonisin esterase, the method comprising (i) providing the thermostable fumonisin esterase, wherein the thermostable fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 , wherein the thermostable fumonisin esterase comprises an aspartic acid at position 66 with respect to the amino acid sequence of SEQ ID NO: 1 , and at least one additional mutation at a position selected from 10, 33, 107, 140, 144, 149, 151 , 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363, 364, 365, 367, 371 , 372, 377, 389, 391 , 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456, 457, 462, 463, 464, 465, 469, 473, 478, 487 and 490 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably wherein the at least one additional mutation is selected from 10Q, 33E, 107E, 140P, 144M, 149F, 151 R, 157Y, 1991, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 3021, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371 M, 372F, 377V, 389L, 391V, 394P, 418A, 419V, 424A, 424K, 427V, 429P, 430A, 436A, 436S, 440G, 440S, 443T, 447A, 453R, 455S, 456Q, 457T, 462Y, 463D, 464I, 465H, 465S, 465G, 469K, 473A, 478D, 487N and 490P with respect to the amino acid sequence of SEQ ID NO: 1 ; and (ii) substituting the amino acid at position 66 with respect to the amino acid sequence of SEQ ID NO: 1 with a non-aspartic acid amino acid.

[017] In particular, "non aspartic acid amino acids" are alanine, arginine, asparagine, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[018] Thermostable fumonisin esterases are described in WO 2016/134387 A1 and comprise an aspartic acid at position 66 with respect to the amino acid sequence of SEQ ID NO: 1 , and at least one additional mutation at a position selected from 10, 33, 107, 140, 144, 149, 151 , 157,

199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363, 364, 365, 367,

371 , 372, 377, 389, 391 , 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456,

457, 462, 463, 464, 465, 469, 473, 478, 487 and 490 with respect to the amino acid sequence of

SEQ ID NO: 1. Preferably said at least one additional mutation of said thermostable fumonisin esterase(s) is selected from 10Q, 33E, 107E, 140P, 144M, 149F, 151 R, 157Y, 1991, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 3021, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 3651, 367H, 371 M, 372F, 377V, 389L, 391V, 394P, 418A, 419V, 424A, 424K, 427V, 429P, 430A, 436A, 436S, 440G, 440S, 443T, 447A, 453R, 455S, 456Q, 457T, 462Y, 463D, 464I, 465H, 465S, 465G, 469K, 473A, 478D, 487N and 490P with respect to the amino acid sequence of SEQ ID NO: 1.

[019] In a further aspect, the present invention relates to a fumonisin esterase obtainable by a method for improving enzyme kinetics according to the invention. Advantageously, such an enzyme allows for faster and more efficient modification, in particular detoxification or biotransformation of fumonisins.

[020] In some embodiments, the present invention relates to a fumonisin esterase comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 , wherein the fumonisin esterase comprises (i) an amino acid substitution of the amino acid at position 25 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with an aliphatic amino acid, more preferably with an amino acid selected from alanine, glycine, isoleucine, leucine, proline, valine, even more preferably with an amino acid selected from isoleucine, leucine, valine, most preferably with isoleucine; and/or (ii) an amino acid substitution of the amino acid at position 45 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid select from asparagine, aspartate, glutamine, glutamate, most preferably with glutamine; and/or (iii) an amino acid substitution of the amino acid at position 259 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a charged amino acid, more preferably with an amino acid selected from lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid selected from lysine, arginine, histidine, most preferably with arginine. In some embodiments, a fumonisin esterase according to the invention comprises only one of the mutations of (i), (ii) and (iii). In some embodiments, a fumonisin esterase according to the invention comprises the mutations of (i) and (ii); of (i) and (iii); of (ii) and (iii); or of (i) and (ii) and (iii).

[021] In some embodiments, a fumonisin esterase according to the invention further comprises at least one additional amino acid substitution at a position selected from 10, 33, 66, 107, 140, 144, 149, 151 , 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363, 364, 365, 367, 371 , 372, 377, 389, 391 , 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456, 457, 462, 463, 464, 465, 469, 473, 478, 487 and 490 with respect to the amino acid sequence of SEQ ID NO: 1 . Hereby, a fumonisin esterase is provided, which fumonisin esterase shows improved enzyme kinetics as well as improved stability, in particular temperature stability, compared to a non-mutated fumonisin esterase, in particular compared to the fumonisin esterase of SEQ ID NO: 1. In preferred embodiments, fumonisin esterase according to the invention comprises at least one additional amino acid substitution selected from 10Q, 33E, 66A, 66C, 66F, 66L, 66M, 66T, 66W, 66E, 66H, 661, 66K, 66P, 66R, 66Y, 66G, 66Q, 66S, 107E, 140P, 144M, 149F, 151 R, 157Y, 1991, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 3021, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371V, 371 M, 372F, 377V, 389L, 391V, 394P, 418A, 419V, 424A, 424K, 427V, 429P, 430A, 436A, 436S, 440G, 440S, 443T, 447A, 453R, 455S, 456Q, 457T, 462Y, 463D, 464I, 465H, 465S, 465G, 469K, 473A, 478D, 487N and 490P with respect to the amino acid sequence of SEQ ID NO: 1.

[022] Similarly, the present invention relates to a thermostable fumonisin esterase comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 , wherein the thermostable fumonisin esterase comprises at least one mutation at a position selected from 10, 33, 107, 140, 144, 149, 151 , 157, 199, 266, 267, 270,

272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363, 364, 365, 367, 371 , 372, 377, 389,

391 , 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456, 457, 462, 463, 464,

465, 469, 473, 478, 487 and 490 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably wherein the at least one mutation is selected from 10Q, 33E, 107E, 140P, 144M, 149F, 151 R, 157Y, 1991, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 3021, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371 M, 372F, 377V, 389L, 391V, 394P, 418A, 419V, 424A, 424K, 427V, 429P, 430A, 436A, 436S, 440G, 440S, 443T, 447A, 453R, 455S, 456Q, 457T, 462Y, 463D, 464I, 465H, 465S, 465G, 469K, 473A, 478D, 487N and 490P with respect to the amino acid sequence of SEQ ID NO: 1 , and wherein the thermostable fumonisin esterase comprises a non-aspartic acid amino acid at position 66 with respect to the amino acid sequence of SEQ I D NO: 1.

[023] In certain embodiments, a fumonisin esterase according to the present invention comprises or consists of an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1 , except for the amino acid at position 25 with respect to the amino acid sequence of SEQ ID NO: 1 ; and/or except for the amino acid at position 45 with respect to the amino acid sequence of SEQ ID NO: 1 ; and/or except for the amino acid at position 259 with respect to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, a fumonisin esterase according to the present invention comprises or consists of an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1 , except for a substitution of the amino acid at position 25 (with respect to the amino acid sequence of SEQ ID NO: 1) with an aliphatic amino acid, more preferably with an amino acid selected from alanine, glycine, isoleucine, leucine, proline, valine, even more preferably with an amino acid selected from isoleucine, leucine, valine, most preferably with isoleucine; and/or except for a substitution of the amino acid at position 45 (with respect to the amino acid sequence of SEQ ID NO: 1) with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid select from asparagine, aspartate, glutamine, glutamate, most preferably with glutamine; and/or except for a substitution of the amino acid at position 259 (with respect to the amino acid sequence of SEQ ID NO: 1) with a charged amino acid, more preferably with an amino acid selected from lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid selected from lysine, arginine, histidine, most preferably with arginine. In certain embodiments, a fumonisin esterase according to the present invention comprises or consists of an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1 , except for the amino acid at position 66 (with respect to the amino acid sequence of SEQ ID NO: 1) being a non-aspartic acid amino acid; and except for any one or more amino acid(s) at position(s) selected from 10, 33, 107, 140, 144, 149, 151 , 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363,

364, 365, 367, 371 , 372, 377, 389, 391 , 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447,

453, 455, 456, 457, 462, 463, 464, 465, 469, 473, 478, 487 and 490 (with respect to the amino acid sequence of SEQ ID NO: 1). In certain embodiments, a fumonisin esterase according to the present invention comprises or consists of an amino acid sequence identical to the amino acid sequence of SEQ I D NO: 1 , except for any one or more amino acid(s) at position(s) selected from 10, 33, 107, 140, 144, 149, 151 , 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363, 364, 365, 367, 371 , 372, 377, 389, 391 , 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456, 457, 462, 463, 464, 465, 469, 473, 478, 487 and 490 (with respect to the amino acid sequence of SEQ ID NO: 1). In certain embodiments, a fumonisin esterase according to the present invention comprises or consists of an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1 , except for the amino acid at position 66 (with respect to the amino acid sequence of SEQ ID NO: 1) being a non-aspartic acid amino acid; and except for at least one mutation selected from 10Q, 33E, 107E, 140P, 144M, 149F, 151 R, 157Y, 1991, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 3021, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371 M, 372F, 377V, 389L, 391V, 394P, 418A, 419V, 424A, 424K, 427V, 429P, 430A, 436A, 436S, 440G, 440S, 443T, 447A, 453R, 455S, 456Q, 457T, 462Y, 463D, 464I, 465H, 465S, 465G, 469K, 473A, 478D, 487N and 490P (with respect to the amino acid sequence of SEQ ID NO: 1). In certain embodiments, a fumonisin esterase according to the present invention comprises or consists of an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1 , except for at least one mutation selected from 10Q, 33E, 107E, 140P, 144M, 149F, 151 R, 157Y, 1991, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 3021, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371M, 372F, 377V, 389L, 391V, 394P, 418A, 419V, 424A, 424K, 427V, 429P, 430A, 436A, 436S, 440G, 440S, 443T, 447A, 453R, 455S, 456Q, 457T, 462Y, 463D, 464I, 465H, 465S, 465G, 469K, 473A, 478D, 487N and 490P (with respect to the amino acid sequence of SEQ ID NO: 1). In certain embodiments, a fumonisin esterase according to the present invention comprises or consists of an amino acid sequence of any one of SEQ ID NOs. 2-71 . [024] In another aspect, the invention relates to a composition (e.g. an additive for food and/or feed; feed or food) for modifying (e.g. detoxifying) a fumonisin, comprising at least one fumonisin esterase according to the invention.

[025] A composition according to the invention can be e.g. food additive(s); foodstuff additive(s); fodder additive(s); feed additive(s); nutritional supplement(s); intermediate(s) thereof; and/or mixture(s) thereof. Such compositions may comprise further components, e.g. prebiotic(s); and/or probiotic(s). Merely for clarification, fodder or feed may e.g. comprise or consist of corn, hay, straw litter, soy, or products obtained therefrom. Also, fodder or feed may comprise or consist of extruded feed products, e.g. pellets. Additives for food, foodstuff, fodder or feed are often used to improve or add properties to the foodstuff, fodder or feed. For instance, such additives may be added to improve organoleptic properties, e.g. to improve taste, smell, appearance, color of the food, foodstuff, fodder or feed. Also, additives may be added to improve palatability, nutrient availability, or to add probiotic microorganisms to the food, foodstuff, fodder or feed, or to add or enhance prebiotic activity of the food, foodstuff, fodder or feed. Also, such additives may be added to counteract potentially undesirable effects of the food, foodstuff, fodder or feed, such as removal or reduction of one or more undesirable components comprised in the food, foodstuff, fodder or feed.

[026] In some embodiments, the composition of the invention comprises at least one carrier. The carrier can be any suitable carrier. The composition may comprise one, two, three or even more carriers. The carrier may be dietary supplement(s), nutraceutical(s) and/or pharmaceutical(s), e.g. vitamin(s), mineral(s), amino acid(s), essential fatty acid(s), fiber(s), trace element(s), antioxidant(s), plant extract(s), herbal extract(s), and/or essential oil(s). The carrier can also be a carrier for enzyme(s). Carriers for enzymes can be both of inorganic and organic origin. Potential inorganic materials used for the immobilization of enzymes are silica (sol-gel silica, fumed silica, colloidal silica nanoparticles and silica gels) and oxides such as titanium oxide, aluminum oxide and zirconium oxide. Furthermore, clay materials such as bentonite, halloysite, kaolinite, montmorillonite, sepiolite and calcium apatite may be carriers. Additionally, carbonbased materials such as activated carbons and charcoal can be carriers. Organic enzyme carriers may be biopolymers (e.g. carbohydrates, proteins, maltodextrin, trehalose, inulin, collagen, cellulose, keratins, carrageenan, chitin, chitosan and alginate) or synthetic polymers (e.g. polyaniline, polyamides, polystyrene, polyurethane, polypropylene, polyvinyl alcohol and ion exchange resins). Liquid carriers could be e.g. buffer substances and/or polyalcohols such as polyalkylene oxides, poly-vinyl alcohols, polyethylene-co-maleic acid anhydrides, polystyrene-co- malic acid anhydrides, dextrans, celluloses, hydrolyzates of chitosan, starches, glycogen, sorbitol, agarose and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolyzates, bio-polymers, sorbitol, glycerol, cellobiose, and mono propylene glycol. The carrier may additionally or alternatively be an eatable component, preferably a non-toxic component and/or a component providing for a texture. In a particular embodiment, the carrier is selected from bentonite, silica, maltodextrin and carbohydrates; preferably the carrier is bentonite and/or maltodextrin.

[027] In another aspect, the invention relates to a method for modifying (e.g. detoxifying) a fumonisin in a composition (e.g. in a nutritional composition), comprising contacting the composition with a fumonisin esterase according to the invention. In some embodiments of the method for modifying a fumonisin in a composition, the fumonisin esterase is comprised in a composition (e.g. an additive for food and/or feed; feed or food) for modifying (e.g. detoxifying) a fumonisin according to the invention. In particular embodiments of the invention, the method for modifying (e.g. detoxifying) a fumonisin in a composition (e.g. in a nutritional composition) relates to a treatment of the composition comprising the fumonisin, but not to a treatment of a human or animal body. Such embodiments can be achieved since a fumonisin esterase according to the invention is well capable of modifying a fumonisin comprised in a composition in the absence of a human or animal body. In other words, in particular embodiments, the invention relates to a non-medical method for modifying (e.g. detoxifying) a fumonisin in a composition (e.g. in a nutritional composition), comprising contacting the composition with a fumonisin esterase according to the invention.

[028] A nutritional composition as referred to herein is a composition comprising one or more component(s) having nutritional value. Often such components provide energy to the consumer of the nutritional composition. A nutritional composition may be entirely or at least partly herbal or plant-based, such as commonly used animal feed compositions.

[029] In a further aspect, the invention relates to a method for modifying (e.g. detoxifying or biotransforming) a fumonisin (i.e. at least one fumonisin) in an aquatic body (e.g. in fresh water, in brackish water, or in salt water; preferably in fresh water or in salt water) comprising the (at least one) fumonisin, comprising contacting the aquatic body with a fumonisin esterase, preferably with a fumonisin esterase according to the invention. In some embodiments of the method for modifying a fumonisin in an aquatic body, the fumonisin esterase is comprised in a composition (e.g. an additive for food and/or feed; feed or food) for modifying (e.g. detoxifying) a fumonisin according to the invention. In particular embodiments of the invention, the method for modifying (e.g. detoxifying) a fumonisin (i.e. at least one fumonisin) in an aquatic body (e.g. in fresh water, in brackish water, or in saltwater; preferably in fresh water or in saltwater) comprising the (at least one) fumonisin relates to a treatment of the aquatic body comprising the fumonisin, but not to a treatment of a human or animal body. In other words, in particular embodiments, the invention relates to a non-medical method for modifying (e.g. detoxifying or biotransforming) a fumonisin (i.e. at least one fumonisin) in an aquatic body (e.g. in fresh water, in brackish water, or in salt water; preferably in fresh water or in salt water) comprising the (at least one) fumonisin, comprising contacting the aquatic body with a fumonisin esterase, preferably with a fumonisin esterase according to the invention.

[030] In some embodiments, said method for modifying a fumonisin in an aquatic body relates to a method for modifying (e.g. detoxifying) a fumonisin in aquaculture, e.g. for the cultivation of fish such as carp, salmon, tilapia, tuna, catfish, trout, rainbow trout; of crustaceans such as crabs, lobsters, crayfish, prawn, shrimp, krill; of mollusks such as oyster, mussels, clams, squid, cuttlefish, and octopuses; of echinoderms such as sea cucumbers, sea urchins; of jellyfish; of algae; and/or of seaweed.

[031] In another aspect, the invention relates to a method for modifying (e.g. detoxifying) a fumonisin in gastric juice, comprising contacting the gastric juice with a fumonisin esterase, preferably with a fumonisin esterase according to the invention. In some embodiments, said gastric juice is animal gastric juice, in particular gastric juice of a ruminant, pig, or poultry. In some embodiments of the method for modifying a fumonisin in gastric juice, the fumonisin esterase is comprised in a composition (e.g. an additive for food and/or feed; feed or food) for modifying (e.g. detoxifying) a fumonisin according to the invention. In particular embodiments of the invention, the method for modifying (e.g. detoxifying) a fumonisin in gastric juice relates to a treatment of the gastric juice, but not to a treatment of a human or animal body. In other words, in particular embodiments, the invention relates to a non-medical method for modifying (e.g. detoxifying) a fumonisin in gastric juice, comprising contacting the gastric juice with a fumonisin esterase, preferably with a fumonisin esterase according to the invention.

[032] A person skilled in the art will understand that upon contacting a fumonisin esterase with a fumonisin, a reaction mixture is formed in a method for modifying (e.g. detoxifying) a fumonisin according to the invention. Said reaction mixture may be further contacted with water. Said water may come from humidity or moisture present in the surrounding or environment in which the fumonisin esterase is contacted with the fumonisin (e.g. in the composition, e.g. nutritional composition; in the aquatic body or aquaculture, in the gastric juice), and/or said water may be added by a person performing a method of the invention, and/or said water may stem from any other source, e.g. from saliva (e.g. upon ingestion of the reaction mixture, or a composition comprising the reaction mixture).

[033] In a further aspect, the invention relates to a use of a fumonisin esterase according to the invention and/or to a use of a composition (e.g. an additive for food and/or feed; feed or food) comprising a fumonisin esterase according to the invention, for modifying (e.g. detoxifying) a fumonisin. In particular embodiments of the invention, the use of a fumonisin esterase according to the invention and/or the use of a composition (e.g. an additive for food and/or feed; feed or food) comprising a fumonisin esterase according to the invention, for modifying (e.g. detoxifying) a fumonisin relates to a use of the fumonisin esterase for treatment of a composition comprising the fumonisin, but not to a use for a treatment of a human or animal body. In other words, in particular embodiments, the invention relates to a non-medical use of a fumonisin esterase according to the invention and/or to a use of a composition (e.g. an additive for food and/or feed; feed or food) comprising a fumonisin esterase according to the invention, for modifying (e.g. detoxifying) a fumonisin.

[034] In a further aspect, the invention relates to a use of a fumonisin esterase according to the invention and/or to a use of a composition (e.g. an additive for food and/or feed; feed or food) comprising a fumonisin esterase according to the invention, for manufacturing an additive for feed and/or food, a feed and/or food composition, or a pharmaceutical composition.

[035] In another aspect, the invention relates to a use of a fumonisin esterase according to the invention and/or to a use of a composition (e.g. an additive for food and/or feed; feed or food) comprising a fumonisin esterase according to the invention, for the manufacture of biogas; bioethanol, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and/or silage.

[036] In another aspect, the invention relates to a host cell comprising at least one fumonisin esterase according to the invention and/or comprising at least one polynucleotide encoding the at least one fumonisin esterase according to the invention.

[037] The term "host cell" refers to any cell capable of recombinant protein production. In particular, "host cell" refers to prokaryotic and/or eukaryotic cells, preferably Pichia pastoris, Escherichia coli, Bacillus sp. such as e.g. Bacillus subtilis or Bacillus amyloliquefaciens, Streptomyces sp., Hansenula sp., Trichoderma sp., Lactobacillus sp., Aspergillus sp., plant cells and/or spores of Bacillus, Trichoderma or Aspergillus. Notably, strains commonly associated with "Pichia pastoris" are sometimes referred to as Komagataella pastoris, Komagataella phaffii or Komagataella pseudopastoris, depending on the respective taxonomic classification at the time of reference.

[038] In a further aspect, the invention relates to a fumonisin esterase for use in treatment, amelioration and/or prevention or prophylaxis of symptoms caused by mycotoxicosis, in particular by fumonisin mycotoxicosis, wherein the fumonisin esterase is a fumonisin esterase according to the invention. In some embodiments, said fumonisin esterase is provided to a subject, e.g. an animal, in need of prevention of mycotoxicosis, in particular in need of prevention of fumonisin mycotoxicosis. A subject may be considered to be in need of prevention of fumonisin mycotoxicosis in case of a risk of fumonisin ingestion beyond a non-toxic concentration. For instance, a subject may be considered to be in need of prevention of fumonisin mycotoxicosis when said subject is about to ingest, and/or already has ingested a nutritional composition comprising one or more fumonisin(s) at detectable levels. Owing to the improved features of a fumonisin esterase according to the invention, such an enzyme is advantageously suitable for treating and/or ameliorating symptoms of mycotoxicosis, in particular symptoms of fumonisin mycotoxicosis, by reducing the exposure of a subject to fumonisins and thus reducing the symptoms associated with fumonisin exposure. In addition, a fumonisin esterase according to the invention can be used to prevent symptoms of mycotoxicosis, in particular symptoms of fumonisin mycotoxicosis. For instance, a fumonisin esterase according to the invention can be administered to a subject known or suspected to have ingested fumonisin(s) prior to the onset of symptoms, thus preventing an occurrence of such symptoms caused by mycotoxicosis, in particular symptoms of fumonisin mycotoxicosis.

[039] The invention is further characterized by the following items:

[040] Item 1. Method for improving enzyme kinetics (e.g. decreased Michaelis constant (Km), increased turnover number (kcat), increased catalytic efficiency, increased maximum reaction rate (Vmax), and/or increased specific activity) of a fumonisin esterase, wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ I D NO: 1 , the method comprising substituting the amino acid at position 25 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with an aliphatic amino acid, more preferably with an amino acid selected from alanine, glycine, isoleucine, leucine, proline, valine, even more preferably with an amino acid selected from isoleucine, leucine, valine, most preferably with isoleucine; substituting the amino acid at position 45 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid select from asparagine, aspartate, glutamine, glutamate, most preferably with glutamine; and/or substituting the amino acid at position 259 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a charged amino acid, more preferably with an amino acid selected from lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid selected from lysine, arginine, histidine, most preferably with arginine.

[041] Item 2. Method for improving enzyme kinetics (e.g. decreased Michaelis constant (Km), increased turnover number (kcat), increased catalytic efficiency, increased maximum reaction rate (Vmax), and/or increased specific activity) of a fumonisin esterase, wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ I D NO: 1 , the method comprising substituting the amino acid at position 25 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with an aliphatic amino acid, more preferably with an amino acid selected from alanine, glycine, isoleucine, leucine, proline, valine, even more preferably with an amino acid selected from isoleucine, leucine, valine, most preferably with isoleucine. [042] Item 3. Method for improving enzyme kinetics (e.g. decreased Michaelis constant (Km), increased turnover number (kcat), increased catalytic efficiency, increased maximum reaction rate (Vmax), and/or increased specific activity) of a fumonisin esterase, wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ I D NO: 1 , the method comprising substituting the amino acid at position 45 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid select from asparagine, aspartate, glutamine, glutamate, most preferably with glutamine.

[043] Item 4. Method for improving enzyme kinetics (e.g. decreased Michaelis constant (Km), increased turnover number (kcat), increased catalytic efficiency, increased maximum reaction rate (Vmax), and/or increased specific activity) of a fumonisin esterase, wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ I D NO: 1 , the method comprising substituting the amino acid at position 259 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a charged amino acid, more preferably with an amino acid selected from lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid selected from lysine, arginine, histidine, most preferably with arginine.

[044] Item 5. Method for improving enzyme kinetics (e.g. decreased Michaelis constant (Km), increased turnover number (kcat), increased catalytic efficiency, increased maximum reaction rate (Vmax), and/or increased specific activity) of a fumonisin esterase, wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ I D NO: 1 , the method comprising substituting the amino acid at position 25 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with an aliphatic amino acid, more preferably with an amino acid selected from alanine, glycine, isoleucine, leucine, proline, valine, even more preferably with an amino acid selected from isoleucine, leucine, valine, most preferably with isoleucine; and substituting the amino acid at position 45 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid select from asparagine, aspartate, glutamine, glutamate, most preferably with glutamine.

[045] Item 6. Method for improving enzyme kinetics (e.g. decreased Michaelis constant (Km), increased turnover number (kcat), increased catalytic efficiency, increased maximum reaction rate (Vmax), and/or increased specific activity) of a fumonisin esterase, wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ I D NO: 1 , the method comprising substituting the amino acid at position 25 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with an aliphatic amino acid, more preferably with an amino acid selected from alanine, glycine, isoleucine, leucine, proline, valine, even more preferably with an amino acid selected from isoleucine, leucine, valine, most preferably with isoleucine; and substituting the amino acid at position 259 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a charged amino acid, more preferably with an amino acid selected from lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid selected from lysine, arginine, histidine, most preferably with arginine.

[046] Item 7. Method for improving enzyme kinetics (e.g. decreased Michaelis constant (Km), increased turnover number (kcat), increased catalytic efficiency, increased maximum reaction rate (Vmax), and/or increased specific activity) of a fumonisin esterase, wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ I D NO: 1 , the method comprising substituting the amino acid at position 25 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with an aliphatic amino acid, more preferably with an amino acid selected from alanine, glycine, isoleucine, leucine, proline, valine, even more preferably with an amino acid selected from isoleucine, leucine, valine, most preferably with isoleucine; substituting the amino acid at position 45 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid select from asparagine, aspartate, glutamine, glutamate, most preferably with glutamine; and substituting the amino acid at position 259 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a charged amino acid, more preferably with an amino acid selected from lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid selected from lysine, arginine, histidine, most preferably with arginine.

[047] Item 8. Method for improving enzyme kinetics (e.g. decreased Michaelis constant (Km), increased turnover number (kcat), increased catalytic efficiency, increased maximum reaction rate (Vmax), and/or increased specific activity) of a fumonisin esterase, wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ I D NO: 1 , the method comprising substituting the amino acid at position 45 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid select from asparagine, aspartate, glutamine, glutamate, most preferably with glutamine; and substituting the amino acid at position 259 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a charged amino acid, more preferably with an amino acid selected from lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid selected from lysine, arginine, histidine, most preferably with arginine.

[048] Item 9. The method of any of the preceding items, further comprising substituting the amino acid at position 66 with respect to the amino acid sequence of SEQ ID NO: 1 with a nonaspartate amino acid; preferably with a non-aspartate, non-valine amino acid; more preferably with an amino acid selected from alanine, cysteine, phenylalanine, leucine, methionine, threonine, tryptophan, glutamate, histidine, isoleucine, lysine, proline, arginine, tyrosine, glycine, glutamine, serine and valine; most preferably with an amino acid selected from alanine, phenylalanine, leucine, methionine, asparagine, threonine, tryptophan and histidine.

[049] Item 10. The method of item 9, wherein the amino acid at position 66 with respect to the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid selected from phenylalanine, methionine, tryptophan, histidine, and asparagine.

[050] Item 11. The method of any one of items 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein at least one further amino acid at a position selected from 10, 33, 66, 107, 140, 144, 149, 151 , 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363, 364, 365, 367, 371 , 372, 377, 389, 391 , 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456, 457, 462, 463, 464, 465, 469, 473, 478, 487 and 490 with respect to the amino acid sequence of SEQ ID NO: 1 is substituted, preferably wherein at least one further amino acid substitution selected from 10Q, 33E, 66A, 66C, 66F, 66L, 66M, 66T, 66W, 66E, 66H, 66I, 66K, 66P, 66R, 66Y, 66G, 66Q, 66S, 107E, 140P, 144M, 149F, 151R, 157Y, 1991, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 3021, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371V, 371M, 372F, 377V, 389L, 391V, 394P, 418A, 419V, 424A, 424K, 427V, 429P, 430A, 436A, 436S, 440G, 440S, 443T, 447A, 453R, 455S, 456Q, 457T, 462Y, 463D, 464I, 465H, 465S, 465G, 469K, 473A, 478D, 487N and 490P with respect to the amino acid sequence of SEQ ID NO: 1 is introduced.

[051] Item 12: Fumonisin esterase obtainable by the method of any one of the preceding items, in particular items 1-11.

[052] Item 13. Method for improving enzyme kinetics of a thermostable fumonisin esterase, the method comprising (i) providing the thermostable fumonisin esterase, wherein the thermostable fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 , wherein the thermostable fumonisin esterase comprises an aspartic acid at position 66 with respect to the amino acid sequence of SEQ ID NO: 1 , and wherein the thermostable fumonisin comprises at least one additional mutation at a position selected from 10, 33, 107, 140, 144, 149, 151 , 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363, 364, 365, 367, 371 , 372, 377, 389, 391 , 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456, 457, 462, 463, 464, 465, 469, 473, 478, 487 and 490 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably wherein the at least one additional mutation is selected from 10Q, 33E, 107E, 140P, 144M, 149F, 151 R, 157Y, 1991, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 3021, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371 M, 372F, 377V, 389L, 391V, 394P, 418A, 419V, 424A, 424K, 427V, 429P, 430A, 436A, 436S, 440G, 440S, 443T, 447A, 453R, 455S, 456Q, 457T, 462Y, 463D, 464I, 465H, 465S, 465G, 469K, 473A, 478D, 487N and 490P with respect to the amino acid sequence of SEQ ID NO: 1 ; and

(ii) substituting the amino acid at position 66 with respect to the amino acid sequence of SEQ ID NO: 1 with a non-aspartic acid amino acid.

[053] Item 14. Method for improving enzyme kinetics of a thermostable fumonisin esterase, the method comprising

(i) providing the thermostable fumonisin esterase, wherein the thermostable fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 , wherein the thermostable fumonisin esterase comprises an aspartic acid at position 66 with respect to the amino acid sequence of SEQ ID NO: 1 , and at least one additional mutation at a position selected from 10, 33, 107, 140, 144, 149, 151 , 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363, 364, 365, 367, 371 , 372, 377, 389, 391 , 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456, 457, 462, 463, 464, 465, 469, 473, 478, 487 and 490 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably wherein the at least one additional mutation is selected from 10Q, 33E, 107E, 140P, 144M, 149F, 151 R, 157Y, 1991, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 3021, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371M, 372F, 377V, 389L, 391V, 394P, 418A, 419V, 424A, 424K, 427V, 429P, 430A, 436A, 436S, 440G, 440S, 443T, 447A, 453R, 455S, 456Q, 457T, 462Y, 463D, 464I, 465H, 465S, 465G, 469K, 473A, 478D, 487N and 490P with respect to the amino acid sequence of SEQ ID NO: 1 ; and

(ii) comprising the step of introducing a non-aspartic acid amino acid at position 66 with respect to the amino acid sequence of SEQ ID NO: 1. [054] Item 15. The method of item 13 or 14, wherein the amino acid at position 66 with respect to the amino acid sequence of SEQ ID NO: 1 is substituted with a non-aspartic acid, non-Valin amino acid, preferably with an amino acid selected from alanine, cysteine, phenylalanine, leucin, methionine, asparagine, threonine, tryptophan, glutamate, histidine, isoleucine, lysine, proline, arginine, tyrosine, glycine, glutamine, serine and valine; more preferably selected from alanine, phenylalanine, leucine, methionine, asparagine, threonine, tryptophan and histidine.

[055] Item 16: Fumonisin esterase obtainable by the method of any of the preceding items.

[056] Item 17: Fumonisin esterase obtainable by the method of any one of items 13-15.

[057] Item 18: Fumonisin esterase comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 , wherein the fumonisin esterase comprises

(i) an amino acid substitution of the amino acid at position 25 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with an aliphatic amino acid, more preferably with an amino acid selected from alanine, glycine, isoleucine, leucine, proline, valine, even more preferably with an amino acid selected from isoleucine, leucine, valine, most preferably with isoleucine; and/or

(ii) an amino acid substitution of the amino acid at position 45 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid select from asparagine, aspartate, glutamine, glutamate, most preferably with glutamine; and/or

(iii) an amino acid substitution of the amino acid at position 259 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a charged amino acid, more preferably with an amino acid selected from lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid selected from lysine, arginine, histidine, most preferably with arginine.

[058] Item 19: Fumonisin esterase comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 , wherein the fumonisin esterase comprises

(i) an amino acid substitution of the amino acid at position 25 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with an aliphatic amino acid, more preferably with an amino acid selected from alanine, glycine, isoleucine, leucine, proline, valine, even more preferably with an amino acid selected from isoleucine, leucine, valine, most preferably with isoleucine; and

(ii) an amino acid substitution of the amino acid at position 45 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid select from asparagine, aspartate, glutamine, glutamate, most preferably with glutamine.

[059] Item 20: Fumonisin esterase comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 , wherein the fumonisin esterase comprises

(ii) an amino acid substitution of the amino acid at position 259 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a charged amino acid, more preferably with an amino acid selected from lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid selected from lysine, arginine, histidine, most preferably with arginine.

[060] Item 21 : Fumonisin esterase comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 , wherein the fumonisin esterase comprises

(i) an amino acid substitution of the amino acid at position 45 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid select from asparagine, aspartate, glutamine, glutamate, most preferably with glutamine; and

[061] Item 22: Fumonisin esterase comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 , wherein the fumonisin esterase comprises

(ii) an amino acid substitution of the amino acid at position 45 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid select from asparagine, aspartate, glutamine, glutamate, most preferably with glutamine; and

[062] Item 23: The fumonisin esterase of any one of the preceding items, in particular of any one of items 16-22, wherein the fumonisin esterase further comprises at least one additional amino acid substitution at a position selected from 10, 33, 66, 107, 140, 144, 149, 151 , 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363, 364, 365, 367, 371 , 372, 377, 389, 391 , 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456, 457, 462, 463, 464, 465, 469, 473, 478, 487 and 490 with respect to the amino acid sequence of SEQ ID NO: 1.

[063] Item 24: The fumonisin esterase of any one of the preceding items, in particular of any one of items 16-22, wherein the fumonisin esterase further comprises at least one additional amino acid substitution selected from 10Q, 33E, 66A, 66C, 66F, 66L, 66M, 66T, 66W, 66E, 66H, 66I, 66K, 66P, 66R, 66Y, 66G, 66Q, 66S, 107E, 140P, 144M, 149F, 151 R, 157Y, 1991, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 3021, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371V, 371M, 372F, 377V, 389L, 391V, 394P, 418A, 419V, 424A, 424K, 427V, 429P, 430A, 436A, 436S, 440G, 440S, 443T, 447A, 453R, 455S, 456Q, 457T, 462Y, 463D, 464I, 465H, 465S, 465G, 469K, 473A, 478D, 487N and 490P with respect to the amino acid sequence of SEQ ID NO: 1 .

[064] Item 25: Fumonisin esterase, in particular thermostable fumonisin esterase, comprising or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 , wherein the fumonisin esterase, in particular the thermostable fumonisin esterase, comprises at least one mutation at a position selected from 10, 33, 107, 140, 144, 149, 151 , 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363, 364, 365, 367, 371 , 372, 377, 389, 391 , 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456, 457, 462, 463, 464, 465, 469, 473, 478, 487 and 490 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably wherein the at least one mutation is selected from 10Q, 33E, 107E, 140P, 144M, 149F, 151 R, 157Y, 1991, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 3021, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371M, 372F, 377V, 389L, 391V, 394P, 418A, 419V, 424A, 424K, 427V, 429P, 430A, 436A, 436S, 440G, 440S, 443T, 447A, 453R, 455S, 456Q, 457T, 462Y, 463D, 464I, 465H, 465S, 465G, 469K, 473A, 478D, 487N and 490P with respect to the amino acid sequence of SEQ ID NO: 1 , and wherein the fumonisin esterase, in particular the thermostable fumonisin esterase, comprises a non-aspartic acid amino acid at position 66 with respect to the amino acid sequence of SEQ ID NO: 1.

[065] Item 26: Fumonisin esterase comprising or consisting of an amino acid sequence of any one of SEQ ID NOs. 2-71.

[066] Item 27: Composition (e.g. additive for food and/or feed; feed or food) for modifying (e.g. detoxifying) a fumonisin, comprising at least one fumonisin esterase of any one of the preceding items.

[067] Item 28: Composition (e.g. additive for food and/or feed; feed or food) for modifying (e.g. detoxifying) a fumonisin, comprising at least one fumonisin esterase of any one of items 12, 16,

17, 18, 19, 20, 21 , 22, 23, 24, 25, 26.

[068] Item 29: The composition of any one of the preceding items, wherein the composition further comprises at least one carrier, preferably selected from bentonite, silica, maltodextrin and carbohydrates, more preferably from maltodextrin and bentonite.

[069] Item 30: Method for modifying (e.g. detoxifying) a fumonisin in a composition (e.g. in a nutritional composition), comprising contacting the composition with a fumonisin esterase of any one of the preceding items.

[070] Item 31 : Method for modifying (e.g. detoxifying) a fumonisin in a composition (e.g. in a nutritional composition), comprising contacting the composition with a fumonisin esterase of any one of items 12, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26.

[071] Item 32: Method for modifying (e.g. detoxifying) a fumonisin in an aquatic body (e.g. in fresh water, in brackish water, or in salt water; preferably in fresh water or in salt water), comprising contacting the aquatic body with a fumonisin esterase.

[072] Item 33: Method for modifying (e.g. detoxifying) a fumonisin in an aquatic body (e.g. in fresh water, in brackish water, or in salt water; preferably in fresh water or in salt water), comprising contacting the aquatic body with a fumonisin esterase of any one of the preceding items.

[073] Item 34: Method for modifying (e.g. detoxifying) a fumonisin in an aquatic body (e.g. in fresh water, in brackish water, or in salt water; preferably in fresh water or in salt water), comprising contacting the aquatic body with a fumonisin esterase of any one of items 12, 16, 17,

18, 19, 20, 21 , 22, 23, 24, 25, 26.

[074] Item 35: Method for modifying (e.g. detoxifying) a fumonisin in aquaculture (e.g. in aquaculture for the cultivation of fish such as carp, salmon, tilapia, tuna, catfish, trout, rainbow trout; of crustaceans such as crabs, lobsters, crayfish, prawn, shrimp, krill; of mollusks such as oyster, mussels, clams, squid, cuttlefish, and octopuses; of echinoderms such as sea cucumbers, sea urchins; of jellyfish; of algae; and/or of seaweed), comprising contacting the aquaculture with a fumonisin esterase.

[075] Item 36: Method for modifying (e.g. detoxifying) a fumonisin in aquaculture (e.g. in aquaculture for the cultivation of fish such as carp, salmon, tilapia, tuna, catfish, trout, rainbow trout; of crustaceans such as crabs, lobsters, crayfish, prawn, shrimp, krill; of mollusks such as oyster, mussels, clams, squid, cuttlefish, and octopuses; of echinoderms such as sea cucumbers, sea urchins; of jellyfish; of algae; and/or of seaweed), comprising contacting the aquaculture with a fumonisin esterase of any one of the preceding items.

[076] Item 37: Method for modifying (e.g. detoxifying) a fumonisin in aquaculture (e.g. in aquaculture for the cultivation of fish such as carp, salmon, tilapia, tuna, catfish, trout, rainbow trout; of crustaceans such as crabs, lobsters, crayfish, prawn, shrimp, krill; of mollusks such as oyster, mussels, clams, squid, cuttlefish, and octopuses; of echinoderms such as sea cucumbers, sea urchins; of jellyfish; of algae; and/or of seaweed), comprising contacting the aquaculture with a fumonisin esterase of any one of items 12, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26.

[077] Item 38: Method for modifying (e.g. detoxifying) a fumonisin in gastric juice (e.g. gastric juice of a ruminant, pig, or poultry), comprising contacting the gastric juice with a fumonisin esterase.

[078] Item 39: Method for modifying (e.g. detoxifying) a fumonisin in gastric juice (e.g. gastric juice of a ruminant, pig, or poultry), comprising contacting the gastric juice with a fumonisin esterase of any one of the preceding items.

[079] Item 40: Method for modifying (e.g. detoxifying) a fumonisin in gastric juice (e.g. gastric juice of a ruminant, pig, or poultry), comprising contacting the gastric juice with a fumonisin esterase of any one of items 12, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26.

[080] Item 41 : The method for modifying (e.g. detoxifying) a fumonisin of any of the preceding items (in particular of any one of items 30-41), wherein the fumonisin esterase is comprised in a composition of any one of items 27-29.

[081] Item 42: The method of any one of items 30-41 , wherein the method comprises forming a reaction mixture by contacting the fumonisin esterase with the fumonisin, and incubating the reaction mixture for at least 1 minute, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 minutes, etc., e.g. at least 30, 60, 90, or 120 minutes, or even longer, e.g. at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 hours or even longer.

[082] Item 43: The method of item 42, wherein the method comprises incubating the reaction mixture at a temperature of at least 5 °C and at most 90 °C, preferably at a temperature of at least 5 °C and at most 50 °C, more preferably at a temperature of at least 5 °C and at most 40 °C, more preferably at a temperature of at least 10 °C and at most 40 °C, even more preferably at a temperature of at least 20 °C and at most 40 °C.

[083] Item 44: The method of any one of items 30-42, further comprising contacting the reaction mixture with water.

[084] Item 45: Use of a fumonisin esterase of any one of the preceding items (e.g. a fumonisin esterase of any one of items 12, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26) and/or a composition of any one of items 27-29 for modifying (e.g. detoxifying) a fumonisin.

[085] Item 46: Use of a fumonisin esterase of any one of the preceding items (e.g. a fumonisin esterase of any one of items 12, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26) for manufacturing an additive for feed and/or food, a feed and/or food composition, or a pharmaceutical composition.

[086] Item 47: Use of a composition of any one of items 27-29 for manufacturing an additive for feed and/or food, a feed and/or food composition, or a pharmaceutical composition.

[087] Item 48: Use of a fumonisin esterase of any one of the preceding items (e.g. a fumonisin esterase of any one of items 12, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26) and/or a composition of any one of items 27-29 for manufacturing an additive for feed and/or food, a feed and/or food composition, or a pharmaceutical composition.

[088] Item 49: Method for manufacturing an additive for feed and/or food, a feed and/or food composition, or a pharmaceutical composition, comprising contacting a fumonisin esterase of any one of the preceding items (e.g. a fumonisin esterase of any one of items 12, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26) with at least one further component of an additive for feed and/or food, a feed and/or food composition, or a pharmaceutical composition.

[089] Item 50: Use of a fumonisin esterase of any one of the preceding items (e.g. a fumonisin esterase of any one of items 12, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26) and/or a composition of any one of items 27-29 for the manufacture of biogas; bioethanol, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and/or silage.

[090] Item 51 : Method for manufacture of biogas; bioethanol, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and/or silage, comprising contacting a fumonisin esterase of any one of the preceding items (e.g. a fumonisin esterase of any one of items 12, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26) and/or a composition of any one of items 27-29 with at least one further component of biogas; bioethanol, DDGS (dried distillers grains with solubles); sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and/or silage. [091] Item 52: Host cell comprising at least one fumonisin esterase of any one of the preceding items (e.g. a fumonisin esterase of any one of items 12, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26); and/or at least one polynucleotide encoding the at least one fumonisin esterase.

[092] Item 53: Fumonisin esterase for use in treatment, amelioration and/or prevention or prophylaxis of symptoms caused by mycotoxicosis, in particular by fumonisin mycotoxicosis, wherein the fumonisin esterase is the fumonisin esterase of any one of the preceding items (e.g. a fumonisin esterase of any one of items 12, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26); and/or wherein the fumonisin esterase is comprised in a composition of any one of items 27-29.

[093] Item 54: The method, fumonisin esterase, composition, use, and/or fumonisin esterase for use of any one of the preceding items, wherein the fumonisin is one or more selected from fumonisin B1 , B2, B3, and B4, preferably wherein the fumonisin is fumonisin B1.

Examples

[094] In the following, the present invention is further described by non-limiting examples. The present invention as disclosed herein is not limited to specific embodiments, methodology, examples, protocols etc. described herein but solely defined by the claims.

Example 1

[095] Fumonisin esterases described herein were recombinantly produced in Pichia pastoris, essentially as described in WO 2016/134387 A1. A person skilled in the art of recombinant protein production will be aware of suitable analogous, similar or alternative methods for producing the fumonisin esterases. Fumonisin esterases were designed for secretion to the cultivation supernatant and quantified photometrically (NanoDrop spectrophotometer), by Bradford assay and/or by BCA assay. Enzyme preparations were diluted with 20 mM Tris-HCI, pH 8.0, to a concentration of 1 mg/mL. These 1 mg/mL dilutions were further diluted to either 10 or 20 ng/mL using 1x FE buffer (20 mM Tris-HCI, pH 8.0, 0.1 mg/mL bovine serum albumin). A fumonisin stock solution was prepared by dissolving crystalline fumonisin B1 (FB1) in 1x FE buffer to a concentration of 1 mM FB1 .

[096] The assay was started by mixing 50 pL of either a 10 ng/mL or a 20 ng/mL enzyme solution with 450 pL of a fumonisin solution. The latter was prepared from the fumonisin stock solution to achieve either of the following final FB1 concentrations in the assay reaction: 5, 10, 15, 20, 25, 50, 75, 100 pM of FB1. The reaction was performed in a 96-deep well plate, 30 °C, 600 rpm shaking. After 15, 30, 45, 60 and 120 minutes, 80 pL samples were drawn from the reaction mixture and incubated at 99 °C for 5 minutes to stop the reaction. [097] For LC-MS/MS analysis the samples were diluted with HPLC-eluent: 1 :30 for 5-20 pM FB1 , 1 :50 for 25 and 50 pM FB1 and 1 : 100 for 75 and 100 pM FB1 approaches. Analyses were performed on an Agilent 1290 series LIHPLC system coupled to a 5500 QTrap mass spectrometer. Column temperature was set to 30 °C and flow rate to 1.0 mL/min. The mobile phase consisted of methanol/water/acetic acid (5/94/1 , v/v/v). The injection volume was 1 pL and the total runtime 0.4 minutes. Separation was performed on an Agilent ZORBAX EclipsePlusC18 RRHD column (50 x 2.1 mm, 1.8 pm). SRM parameters are shown in table 1.

Table 1. Selected Reaction Monitoring transitions in negative ion mode. Declustering potential (DP), collision energy (CE), collision cell exit potential (CXP). Entrance potential (EP) was -10.0 V for all analytes.

[098] HPLC-eluent: Ultrapure water containing 0.0385% formic acid (pH~2.85, corresponding to 192.5 pL formic acid in 500 mL of water) was adjusted to pH3.0 by addition of 6.1 mM ammonium formate in ultrapure water (192.5 mg of ammonium formate in 500 mL of water). This solution was mixed with acetonitrile at a ratio of 75 (HPLC buffer pH 3.0): 25 (acetonitrile) (v/v). The HPLC eluent is stored at room temperature.

[099] After LC-MS/MS measurement, data analysis was performed using MultiQuant 3.0.3 software (Sciex). The results were exported to Microsoft Excel for statistical analysis. Michaelis- Menten curves were generated by using SigmaPlot software. Improved kinetic parameters of exemplary fumonisin esterase variants are shown in tables 2 and 3 below relative to the fumonisin esterase of SEQ ID NO: 1.

Table 2: Relative kinetic parameters of exemplary fumonisin esterases. Michaelis constants (Km) were determined in pM; turnover numbers (kcat) in sec¹; catalytic efficiencies in pM^-1 sec¹.

Table 3: Relative kinetic parameters of exemplary fumonisin esterases. Specific activities were determined in ll/rng, wherein 1 unit (II) was defined as the threefold amount of enzyme that releases 1 pmol tricarballylic acid (TCA) per minute from 100 pM fumonisin B1 (FB1) in 20 mM Tris-CI buffer pH 8.0 with 0.1 mg/mL bovine serum albumin (BSA) at 30 °C.

Example 2

[100] To determine temperature stability, a stability assay was performed in Teorell-Stenhagen buffer, pH 6.5, (Teorell and Stenhagen. 1938. Biochem. Ztschrft. 299: 416-419) containing 0.1 mg/L bovine serum albumin. Fumonisin esterase enzymes were incubated for 5 minutes at

75 °C at a concentration of 10 pg/mL in 70 pL buffer. One aliquot of the incubation mixture was stored on ice without incubation at 75 °C as untreated control to determine initial activity. After incubation at 75 °C, the enzyme solutions were stored on ice until determination of residual activity compared to initial activity. Initial and residual activities were determined using the FB1 assay described in Example 1 . Residual activity was determined in % of the non-treated initial activity. The improvements in temperature stabilities of exemplary fumonisin esterase variants are shown in table 4 below relative to the fumonisin esterase of SEQ ID NO: 1. For instance, in case the initial activity of a fumonisin esterase A was 100 ll/rng and the residual activity after incubation was 50 ll/rng, the residual activity was found to be 50%. In case a residual activity of a fumonisin esterase B was 80%, the relative residual activity of fumonisin esterase B relative to the fumonisin esterase A was 80*100/50 = 160%.

Table 4: Relative residual activity of exemplary fumonisin esterases relative to the fumonisin esterase of SEQ ID NO: 1. Residual activities were determined in % of untreated initial activities.

[101] As a further assay to determine temperature stability, thermal shift assays (also referred to as thermofluor assays) were performed using SYPRO Orange as fluorescent indicator. While the fumonisin esterase of SEQ ID NO: 1 showed a fluorescence peak at 45 °C, indicating temperature-induced unfolding of the polypeptide, fumonisin esterases of any one of SEQ ID NOs: 9-71 showed a fluorescence peak at 70-75 °C, thus indicating unfolding at a markedly higher temperature than the fumonisin esterase of SEQ ID NO: 1.

Example 3

[102] To study the applicability of fumonisin esterases in gastric juices, exemplary fumonisin esterases were tested at final concentrations of either 2.0 ng/mL, 1.5 ng/mL, 1.0 ng/mL or 0.5 ng/mL, diluted in gastric simulation buffer (GSB: 118.5 mM NaCI, 8.55 mM acetic acid, 14.9 mM sodium acetate, pH 5.0, 0.1 mg/mL bovine serum albumin). The assay was performed in a water bath at 37 °C. The assay was started by adding FB1 to a final concentration of as little as 5 pM in a total assay volume of 500 pL. After 15, 30, 45, 60 and 120 minutes, 80 pL samples were drawn and incubated at 99 °C for 5 minutes. Specific activities were determined as described in Example 1. Despite the acetic conditions and the low substrate concentration, all fumonisin esterases were found active in gastric juice, see table 5.

Table 5: Specific activities of exemplary fumonisin esterases in gastric juice.

Example 4

[103] In order to study whether fumonisin esterases are suitable for application in water, in particular in aquaculture, a 56-day trial with 180 salmonid fish (rainbow trouts) was carried out. Three groups were compared in the trial: a control without fumonisins and without fumonisin esterase (control), a fumonisin-contaminated control group (FUM), and a trial group (esterase) receiving fumonisin and an improved fumonisin esterase according to the invention (any one of SEQ ID NOs. 2-71) at 15 ll/kg feed. In the FUM and the esterase group, a fumonisin contamination of approximately 10 ppm was applied, which fumonisin contamination comprised a mixture of fumonisins B1 , B2 and B3.

[104] The fish arrived as eyed eggs and were reared for a period of approximately 9 months prior to the trial. On the first day of the trial, fish were individually weighed and allocated to 15 tanks based on stratified randomization with the aim to balance the mean body weight per tank. Fish were kept in 15 sub-square recirculating aquaculture system (RAS) tanks of 500 L each, twelve fish were allocated to each tank. The tanks were divided between the three trial groups as follows: Three tanks for the control group, six for the FUM group, and six for the esterase group. The tank was the experimental unit. Fumonisin degradation was followed by analyzing fumonisins (FB1 , FB2, FB3), and their metabolites, hydrolyzed fumonisins (HFB1 , HFB2, HFB3) and partially hydrolyzed fumonisins (pHFB1a+b, pHFB2a+b, pHFB3a+b) in contents of the proximal and distal part of the gastrointestinal tract (GIT). Furthermore, the levels of sphinganine (Sa) and sphingosine (So) and their ratio in the blood plasma (Sa/So) were analyzed as indicator for exposure to toxic levels of fumonisins (Meredith et al. 1998. J Food Prot. 61 (8): 1034-8).

[105] For analysis of the samples from the GIT, 100 mg lyophilized and homogenized sample was weighted into 5 mL Eppendorf tubes. Three mL extraction solvent (acetonitrile/water/formic acid, 74/25/1 , v/v/v) were added, vortexed and the tubes were shaked on a horizontal shaker at room temperature for 30 minutes (fast shaking) followed by a centrifugation step (1880 ref for 5 minutes). Supernatants were collected into 15 mL tubes and the residues was re-extracted with 3 mL extraction solvent. After resuspension of the residuals by vortexing, samples were shaked again on a horizontal shaker at room temperature for 20 minutes (fast shaking) followed by a centrifugation step (1880 ref for 5 minutes). Supernatants were transferred to the 15 mL tubes from the first extraction step by pouring and for the last extraction step, 2 mL extraction solvent were added to the residues. After vortexing and shaking for 10 minutes on a horizontal shaker at room temperature, centrifugation was carried out at 3200 ref for 5 minutes and the supernatants pooled with the supernatants from the first two extraction steps. Combined extracts were vortexed and centrifuged at 3200 ref for 10 minutes. Sample preparation was finalized with the transfer of 500 pL supernatant into an HPLC and diluted with 500 pL extraction solvent. For the calibration curve, standards for fumonisins (FB1 , 2, 3), HFBs and pHFBs were diluted in dilution solvent (acetonitrile/water/formic acid, 50/49/1 , v/v/v) to 600, 300, 150, 30, 15, 3, 1.5 and 0.3 ng/mL. Analyses were performed on an Agilent 1290 series LIHPLC system coupled to a 5500 QTrap mass spectrometer. Column temperature was set to 30 °C and flow rate to 0.8 mL/min. Mobile phases A consisted of methanol/water/acetic acid (40/59.8/0.2; v/v/v) and mobile phase B of methanol/acetic acid (99.8/0.2; v/v). The gradient started with 100 % A for 0.5 minutes and continued with a linear increase to 73 % B until 5.9 minutes and to 100 % B until 6.0 minutes followed by 100 % B until 7.9 minutes and a steep decrease to 0 % B between 7.9 and 8.0 minutes. Total runtime per sample was 10.5 minutes. The injection volume was 1 pL. Separation was performed on a Phenomenex Gemini 5p C18 110 A column (150 x 4.6 mm).

[106] Mass spectrometric detection was performed with negative electrospray ionization in multiple reaction monitoring mode with parameters according to table 6.

Table 6. Parameters mass transitions. DP = Declustering Potential; EP = Entrance Potential; CE = Collision Energy; CXP = Cell Exit Potential.

[107] Data analysis for LC-MS/MS: Peak integration and calculation of the concentration was performed using Multiquant (Version 3.0.3). To calculate the concentration a 1/x weighted linear calibration function was used. Measured concentrations in the extracted samples were multiplied by the dilution factor to receive the results for the GIT samples.

[108] For analysis of the plasma samples, 50 pL plasma in a 2 mL tube were added with 600 pL of acetonitrile/water (50/50, v/v) each. After homogenization by vortexing, samples were shaked at room temperature using a vortexer with Eppendorf tube adaptor at speed level 5-6 for 30 minutes followed by a centrifugation step (2700 ref for 5 minutes). Supernatants were collected in fresh Eppendorf tubes (pouring) and a re-extract of the pellet with 300 pL of 80% (v/v) methanol for 30 seconds while vortexing was carried out. After centrifugation (19 000 ref for 5 minutes), the supernatants were combined and dried using a SpeedVac at 30 °C heating. When dry, the extracts were re-dissolved in 300 pL of 80% (v/v) methanol using vortexer at speed level 5-6 at room temperature for 30 minutes. Sample preparation was finalized with a centrifugation step (19 000 ref for 10 minutes) and the transfer of 200 pL supernatant into an HPLC vial with glass insert. Analyses were performed on an Agilent 1290 series LIHPLC system coupled to a 5500 QTrap mass spectrometer. Column temperature was set to 30 °C and flow rate to 0.5 mL/min. Mobile phases A consisted of methanol/water/acetic acid (40/59.8/0.2; v/v/v) and mobile phase B of methanol/acetic acid (99.8/0.2; v/v). The gradient started with 65 % B for 1.7 minutes and continued with a linear increase to 100 % B until 1.71 minutes followed by 100 % B until 2.5 minutes and a steep decrease to 65 % B between 2.5 and 2.51 minutes. The injection volume was 2 pL. Separation was performed on a Phenomenex Kinetex C18 column (150 x 2.1 mm, 2.6 pm). Quantification was based on calibration with external standards of sphinganine and sphingosine in a concentration range from 0.1 ppb to 300 ppb. SRM parameters are shown in table 7.

Table 7. Selected Reaction Monitoring transitions in positive ion mode. DP = Declustering Potential; EP = Entrance Potential; CE = Collision Energy; CXP = Cell Exit Potential.

[109] Using any fumonisin esterase, the concentrations of the fumonisins were significantly (p<0.05) decreased compared to the contaminated group (FUM). Furthermore, levels of partially and fully hydrolyzed fumonisins (HFB1 , HFB2, HFB3, pHFB1a+b, pHFB2a+b, pHFB3a+b) were significantly higher in both the proximal and the distal part of the GIT of the esterase group, see tables 8 and 9. Also, fish in the esterase group exhibited a significantly lower Sa/So ratio than the fish in the FUM group, see table 10. Table 8: Fumonisins and its metabolites in the proximal Gl tract of rainbow trout (group means in pg/g lyophilized GIT content) (n=6 for FUM and esterase, and n=3 for control group), p-value refers to comparison between FUM and esterase group. Table 9: Fumonisins and its metabolites in the distal Gl tract of rainbow trout (group means in pg/g lyophilized GIT content) (n=6 for FUM and esterase, and n=3 for control group), p-value refers to comparison between FUM and esterase group.

Table 10: Plasma sphinganine to sphingosine ratio (Sa/So) in rainbow trout (n=6 for FUM and esterase, n=3 for control group.

Claims

1. Method for improving enzyme kinetics of a fumonisin esterase, wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 , the method comprising substituting the amino acid at position 25 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with an aliphatic amino acid, more preferably with an amino acid selected from alanine, glycine, isoleucine, leucine, proline, valine, even more preferably with an amino acid selected from isoleucine, leucine, valine, most preferably with isoleucine; substituting the amino acid at position 45 with respect to the amino acid sequence of SEQ ID NO:

1 , preferably with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid select from asparagine, aspartate, glutamine, glutamate, most preferably with glutamine; and/or substituting the amino acid at position 259 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably with a charged amino acid, more preferably with an amino acid selected from lysine, arginine, histidine, glutamate, aspartate, even more preferably with an amino acid selected from lysine, arginine, histidine, most preferably with arginine.

2. The method of claim 1 , wherein at least one further amino acid at a position selected from 10, 33, 66, 107, 140, 144, 149, 151 , 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363, 364, 365, 367, 371 , 372, 377, 389, 391 , 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456, 457, 462, 463, 464, 465, 469, 473, 478, 487 and 490 with respect to the amino acid sequence of SEQ ID NO: 1 is substituted, preferably wherein at least one further amino acid substitution selected from 10Q, 33E, 66A, 66C, 66F, 66L, 66M, 66T, 66W, 66E, 66H, 66I, 66K, 66P, 66R, 66Y, 66G, 66Q, 66S, 107E, 140P, 144M, 149F, 151 R, 157Y, 1991, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 3021, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371V, 371M, 372F, 377V, 389L, 391V, 394P, 418A, 419V, 424A, 424K, 427V, 429P, 430A, 436A, 436S, 440G, 440S, 443T, 447A, 453R, 455S, 456Q, 457T, 462Y, 463D, 464I, 465H, 465S, 465G, 469K, 473A, 478D, 487N and 490P with respect to the amino acid sequence of SEQ ID NO: 1 is introduced.

3. Method for improving enzyme kinetics of a thermostable fumonisin esterase, the method comprising (i) providing the thermostable fumonisin esterase, wherein the thermostable fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 , wherein the thermostable fumonisin esterase comprises an aspartic acid at position 66 with respect to the amino acid sequence of SEQ ID NO: 1 , and at least one additional mutation at a position selected from 10, 33, 107, 140, 144, 149, 151 , 157, 199, 266,

267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363, 364, 365, 367, 371 , 372,

377, 389, 391 , 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456, 457, 462,

463, 464, 465, 469, 473, 478, 487 and 490 with respect to the amino acid sequence of SEQ ID NO: 1 , preferably wherein the at least one additional mutation is selected from 10Q, 33E, 107E, 140P, 144M, 149F, 151 R, 157Y, 1991, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 3021, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371M, 372F, 377V, 389L, 391V, 394P, 418A, 419V, 424A, 424K, 427V, 429P, 430A, 436A, 436S, 440G, 440S, 443T, 447A, 453R, 455S, 456Q, 457T, 462Y, 463D, 464I, 465H, 465S, 465G, 469K, 473A, 478D, 487N and 490P with respect to the amino acid sequence of SEQ ID NO: 1 ; and

4. Fumonisin esterase obtainable by the method of any one of the preceding claims.

5. Composition for modifying a fumonisin, comprising at least one fumonisin esterase of claim 4.

6. The composition of claim 5, further comprising at least one carrier, preferably selected from bentonite, silica, maltodextrin and carbohydrates.

7. Method for modifying a fumonisin in a composition comprising contacting the composition with a fumonisin esterase of claim 4.

8. Method for modifying a fumonisin in an aquatic body, comprising contacting the aquatic body with a fumonisin esterase.

9. Method for modifying a fumonisin in gastric juice, comprising contacting the gastric juice with a fumonisin esterase.

10. The method of claim 8 or 9, wherein the fumonisin esterase is the fumonisin esterase of claim 4 and/or wherein the fumonisin esterase is comprised in a composition of claim 5 or 6.

11. Use of a fumonisin esterase of claim 4 and/or a composition of claim 5 or 6 for modifying, e.g. detoxifying a fumonisin.

12. Use of a fumonisin esterase of claim 4 and/or a composition of claim 5 or 6 for manufacturing an additive for feed and/or food, a feed and/or food composition, ora pharmaceutical composition.

13. Use of a fumonisin esterase of claim 4 and/or a composition of claim 5 or 6 for the manufacture of biogas; bioethanol, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and/or silage.

14. Host cell comprising at least one fumonisin esterase of claim 4 and/or at least one polynucleotide encoding the at least one fumonisin esterase.

15. Fumonisin esterase for use in treatment, amelioration and/or prevention of symptoms caused by mycotoxicosis, in particular by fumonisin mycotoxicosis, wherein the fumonisin esterase is the fumonisin esterase of claim 4.