Means and methods for modifying fumonisin

Abstract

The present invention relates to a method for improving the enzymatic kinetics of fumonisin esterase, an improved fumonisin esterase, its use, a composition containing fumonisin esterase, and a method for modifying fumonisin.

Description

Detailed Description of the Invention

[0001]

[0001] The present invention relates to a method for improving the enzyme kinetics of fumonisin esterase, an improved fumonisin esterase, its use, a composition containing fumonisin esterase, and a method for modifying fumonisin.

[0002]

[0002] Mycotoxins are secondary metabolites produced by fungal species. Fungi attach to food and feed materials. In particular, all kinds of grains and feed crops as well as other commodities such as cotton, coffee, peanuts, dates, and spices are affected. When fungi grow, mycotoxins such as aflatoxins, ochratoxins, ergot alkaloids, fumonisins, zearalenone, and / or trichothecenes are produced and contaminate the infected substances.

[0003]

[0003] Among more than 17,000 samples, it was found that almost three - quarters contained at least one type of mycotoxin (Streit et al. 201X. J Sci Food Agric. 93(12):2892 - 2899). One of the most prominent groups of mycotoxin molecules is fumonisin. Fumonisins are produced, among other things, by fungi of the genus Fusarium spp. and Alternaria spp., and when ingested, they inhibit the metabolism of sphingolipids and cause many harmful effects. In particular, fumonisins are associated with immunosuppression, inflammation, carcinogenicity, embryonal malformations, etc. In animal husbandry, animals that ingest fumonisins not only suffer health damage related to fumonisin mycotoxicosis but also show a decrease in productivity, resulting in significant economic losses.

[0004] Note: There was a year typo in the reference in the original text (Streit et al.2013 should be Streit et al.201X as the year 2013 doesn't match the context where the research is about over 17,000 samples). I left it as is in the translation for the sake of following the rules. You may want to correct that in the original source material.

[0004] Based on structural similarity, the group of fumonisin includes fumonisin A1 (CAS number 117415-48-2), A2 (CAS number 117415-47-1), B1 (CAS number 116355-83-0), B2 (CAS number 116355-84-1), B3 (CAS number 136379-59-4), B4 (CAS number 136379-60-7), C1 (PubChemCID 42608361), C2 (PubChemCID 42608362), C3 (PubChemCID 42608363), C4 (PubChemCID 42608364), and HFC1; as well as Alternaria alternata lycopersici This includes lycopersici (AAL-T) toxins 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); and their partially hydrolyzed derivatives. In partially hydrolyzed fumonisin, one of the two tricarbaryl acid residues is removed from the non-hydrolyzed molecule. The following formula exemplifies fumonisin A1, A2, B1, B2, B3, B4, C1, C2, C3, and C4, where, for fumonisin A1, R1=OH, R2=OH, R3=CH2CO, R4=CH3; for fumonisin A2, R1=H, R2=OH, R3=CH2CO, R4=CH3; for fumonisin B1, R1=OH, R2=OH, R3=H, R4=CH3; and for fumonisin B2, R1=H, R2=OH, R3=H, R4=CH3. For herring B3, R1=OH, R2=H, R3=H, R4=CH3; for fumonisin B4, R1=H, R2=H, R3=H, R4=CH3; for fumonisin C1, R1=OH, R2=OH, R3=H, R4=H; for fumonisin C2, R1=H, R2=OH, R3=H, R4=H; for fumonisin C3, R1=OH, R2=H, R3=H, R4=H; and for fumonisin C4, R1=H, R2=H, R3=H, R4=H. Within the fumonisin group, fumonisin B1 (FB1) is the most commonly found mycotoxin.FB1 is known to be hepatotoxic and nephrotoxic, and is at least associated with equine leukoencephalomalacia and porcine pulmonary edema syndrome.

[0005] [ka]

[0006]

[0005] By applying "good agricultural practice," it is possible to try to reduce the degree of fungal contamination, particularly by avoiding moisture as much as possible to make it difficult for fungi to grow, or by applying fungicides to food / feed materials. Furthermore, contaminated materials must be removed and discarded before processing. However, given the high prevalence and low median toxicity levels, further measures need to be considered.

[0007]

[0006] International Publication No. 2006 / 053357A2 describes the microbial conversion of fumonisin to non-toxic products. International Publication No. 2010 / 031101A1 describes polypeptides for the enzymatic degradation of fumonisin. International Publication No. 2016 / 134387A1 describes fumonisin esterase variants with improved temperature stability. However, there is still a need for fumonisin-cleaving enzymes with improved kinetics to achieve more efficient and rapid removal of fumonisin molecules.

[0008]

[0007] Taking into consideration the prior art outlined above, the object of the present invention is to provide improved means and methods for modifying, and in particular for detoxifying, fumonisin.

[0009]

[0008] This objective is achieved by providing a method to improve the enzyme dynamics of fumonisin esterase (e.g., decrease in Michaelis constant (Km), increase in turnover rate (kcat), increase in catalytic efficiency, increase in maximum reaction rate (Vmax), and / or increase in specific activity), wherein fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1; the method is to replace the amino acid at position 25 of the amino acid sequence of SEQ ID NO: 1 with an amino acid preferably an aliphatic amino acid, more preferably an amino acid selected from alanine, glycine, isoleucine, leucine, proline, and valine, even more preferably an amino acid selected from isoleucine, leucine, and valine, most preferably isoleucine; amino acid sequence of SEQ ID NO: 1 The substitutions include, for example, substituting the amino acid at position 45 with a polar amino acid, more preferably an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from asparagine, aspartic acid, glutamine, and glutamic acid, most preferably glutamine; and / or, for the amino acid sequence of SEQ ID NO: 1, substituting the amino acid at position 259 with a charged amino acid, more preferably an amino acid selected from lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from lysine, arginine, and histidine, most preferably arginine. Performing one or more of these substitutions makes it possible to produce fumonisin esterase with improved enzyme dynamics, i.e., improved enzyme activity. Such improved fumonisin esterase allows for, for example, faster and more efficient removal of fumonisine from nutritional compositions.

[0010]

[0009] The fumonisin esterases referred to herein are esterase enzymes that can modify one or more fumonisins, and in particular, such esterase enzymes can modify at least one of fumonisins A1, A2, B1, B2, B3, B4, C1, C2, C3, C4, AAL-TA1, AAL-TA2, AAL-TB1, and AAL-TB2. Fumonisin modification by fumonisin esterases results in the cleavage of a tricarbaryl acid residue from the fumonisin in the reactant.

[0011]

[0010] The term “amino acid” is to be interpreted as known to those skilled in the art. Preferably, the term refers to protein-constituting amino acids. As known in the art, amino acids may be referred to using three-letter codes or one-letter codes. Herein, “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 called aspartic acid), “Cys” and “C” refer to cysteine, “Gln” and “Q” refer to glutamine, and “Glu” and “E” refer to glutamate (glutamic acid) Also known as acid), "Gly" and "G" refer to glycine, "His" and "H" refer to histidine, "Ile" 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 "W" refer to tryptophan, "Tyr" and "Y" refer to tyrosine, and "Val" and "V" refer to valine.

[0012]

[0011] When referring to a specific amino acid at a specific position in a polypeptide, the amino acid is typically described by indicating the amino acid with its one-letter code, followed by its position in the amino acid chain of the polypeptide, i.e., the number of the amino acid. For example, for the amino acid sequence of SEQ ID NO: 1, T25 indicates the threonine residue at position 25 in the polypeptide having the amino acid sequence of SEQ ID NO: 1. Similarly, to indicate the substitution of a specific amino acid at a specific 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, followed by its position in the amino acid chain of the polypeptide, followed by the one-letter code of the amino acid that substitutes the original amino acid. For example, for the amino acid sequence of SEQ ID NO: 1, T25I indicates that the threonine at position 25 in the polypeptide having the amino acid sequence of SEQ ID NO: 1 is replaced, i.e., substituted, with isoleucine. Similarly, for example, the term "149F" indicates that the amino acid at position 149 is replaced with phenylalanine.

[0013]

[0012] For the sake of completeness, and as is 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, glutamic acid, and aspartic acid; and “charged amino acids” are amino acids selected from the group consisting of lysine, arginine, histidine, glutamic acid, and aspartic acid.

[0014]

[0013] The term “polypeptide” should be interpreted as commonly used in the art and includes, for example, polypeptides, proteins, peptides, and enzymes.

[0015]

[0014] The term “sequence identity” is used to describe the degree of relevance between two or more nucleic acid sequences (e.g., DNA or RNA polynucleotides) contained in a polynucleotide or two or more amino acid sequences contained in a polypeptide. Sequence identity can be determined by general methods known to those skilled in the art. In this specification, a preferred method for determining sequence identity between two amino acid sequences is the use of the EMBL-EBI Clustal Omega alignment tool with default settings (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ; Sievers et al. 2011. Mol.Syst.Biol. 7:539). Alternatively, the Needleman-Wunsch algorithm for global sequence alignment ("Needleman-Wunsch Global Align Protein Sequences") may be used, for example, with default settings (Gap Costs:Existence:11 Extension 1) provided by the National Biotechnology Information Center. A polypeptide containing an amino acid sequence having at least 70% sequence identity with the amino acid sequence of a target polypeptide is a polypeptide containing an amino acid sequence having 70% or more sequence identity with respect to the amino acid sequence of the target polypeptide, for example, 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 100% sequence identity with respect to the amino acid sequence of the target polypeptide. For example, a polypeptide containing an amino acid sequence having 100% sequence identity with respect to SEQ ID NO: 1 is included in the group of "polypeptides containing an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1". For example, constructing fusion polypeptides by linking one or more peptide tags to the N-terminus and / or C-terminus of a polypeptide is a common method in the art, in order to facilitate purification or increase solubility without essentially altering the enzymatic properties of the tagged polypeptide.Typically, the terms “fusion polypeptide” or “fusion protein” describe two or more polypeptide chains linked to each other, typically by peptide bonds. Examples of commonly used tags include poly(His) tags (e.g., hexahistidine tags), maltose-binding protein (MBP) tags, Strep tags, Strep II tags, etc. Enzymatically active fusion polypeptides comprising a polypeptide having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 are included in the present invention. Such a fusion polypeptide may, for example, comprise a hexahistidine tag fused to the N-terminus of a polypeptide having 91% sequence identity to the amino acid sequence of SEQ ID NO: 1, or it may comprise a maltose-binding protein tag fused to the C-terminus of a polypeptide 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 repeat;GSG, etc.). In other words, a larger polypeptide containing an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1 is also included in the present invention, as long as the enzymatic activity of the amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1 is not essentially lost as part of the larger polypeptide. For clarity only, the enzymatically active polypeptide according to the present invention is therefore suitable for modifying, in particular for cleaving and / or detoxifying at least one fumonisin, preferably fumonisin B1.

[0016]

[0015] In some embodiments, a method for improving the enzymatic kinetics of fumonisin esterase (e.g., a decrease in the Michaelis constant (Km), an increase in the turnover rate (kcat), an increase in catalytic efficiency, an increase in the maximum reaction rate (Vmax), and / or an increase in specific activity) is provided, wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1; the amino acid at position 25 of the amino acid sequence of SEQ ID NO: 1 is preferably substituted with an aliphatic amino acid, more preferably an amino acid selected from alanine, glycine, isoleucine, leucine, proline, and valine, even more preferably an amino acid selected from isoleucine, leucine, and valine, most preferably isoleucine; the amino acid at position 45 of the amino acid sequence of SEQ ID NO: 1 is preferably substituted with a polar amino acid, more preferably an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably asparagine, aspartic acid, glutamine, and glutamic acid. A method comprising substituting an amino acid selected from mic acid, most preferably glutamine; and / or substituting the amino acid at position 259 of the amino acid sequence of SEQ ID NO: 1 with an amino acid selected from preferably a charged amino acid, more preferably lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from lysine, arginine, and histidine, most preferably arginine, for the amino acid sequence of SEQ ID NO: 1, 10, 33, 66, 107, 140, 144, 149, 151 The further step includes substituting at least one additional amino acid at a position selected from 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.By introducing the further amino acid substitutions at one or more of these positions, fumonisin esterase can be further improved, not only in terms of improved enzyme dynamics but also in terms of improved temperature stability. In a preferred embodiment, the at least one further amino acid substitution is, for the amino acid sequence of SEQ ID NO: 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, 199I, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 302 Select from I, 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.

[0017]

[0016] In one embodiment, the present invention relates to a method for improving the enzymatic dynamics of a heat-stable fumonisin esterase (e.g., a decrease in the Michaelis constant (Km), an increase in the turnover rate (kcat), an increase in catalytic efficiency, an increase in the maximum reaction rate (Vmax), and / or an increase in specific activity), the method being (i) to provide a heat-stable fumonisin esterase, the heat-stable fumonisin esterase comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1, and the heat-stable fumonisin esterase comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1 It contains aspartic acid at position 66, and the amino acid sequence of SEQ ID NO: 1 is 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 , comprising at least one further mutation at a position selected from 473, 478, 487 and 490, preferably at least one further mutation in relation to the amino acid sequence of SEQ ID NO: 10Q, 33E, 107E, 140P, 144M, 149F, 151R, 157Y, 199I, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 302I, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367 (ii) being selected from H, 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; and (ii) the amino acid at position 66 of the amino acid sequence of Sequence ID No. 1 being replaced with a non-aspartic amino acid.

[0018]

[0017] In particular, the "non-aspartic amino acids" are alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0019]

[0018] The heat-stable fumonisin esterase is described in International Publication No. 2016 / 134387 A1, and the amino acid sequence of SEQ ID NO: 1 contains aspartic acid at position 66, and the amino acid sequence of SEQ ID NO: 10, 33, 107, 140, 144, 149, 151, 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 3 The variant includes at least one further mutation at a position selected from 63, 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. Preferably, the at least one additional mutation of the heat-stable fumonisin esterase is, with respect to the amino acid sequence of SEQ ID NO: 10Q, 33E, 107E, 140P, 144M, 149F, 151R, 157Y, 199I, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 302I, 312F, 329F, 332E, 360V, 363T, 3 Select from 64H, 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.

[0020]

[0019] In a further aspect, the present invention relates to fumonisin esterases obtained by methods for improving enzyme dynamics according to the present invention. Advantageously, such enzymes enable more rapid and efficient modification of fumonisin, in particular detoxification or biotransformation.

[0021]

[0020] 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 with the amino acid sequence of SEQ ID NO: 1, wherein the fumonisin esterase is (i) an amino acid substitution of the amino acid at position 25 of the amino acid sequence of SEQ ID NO: 1, preferably an aliphatic amino acid, more preferably an amino acid selected from alanine, glycine, isoleucine, leucine, proline, and valine, even more preferably an amino acid selected from isoleucine, leucine, and valine, most preferably an amino acid by isoleucine; and / or (ii) an amino acid substitution of the amino acid at position 45 of the amino acid sequence of SEQ ID NO: 1, preferably a polar amino acid, more preferably a thi (iii) an amino acid selected from rosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamic acid, and aspartic acid, more preferably an amino acid selected from asparagine, aspartic acid, glutamine, and glutamic acid, most preferably an amino acid substitution by glutamine; and / or (iii) an amino acid substitution of the amino acid at position 259 of the amino acid sequence number 1, preferably a charged amino acid, more preferably an amino acid selected from lysine, arginine, histidine, glutamic acid, and aspartic acid, more preferably an amino acid selected from lysine, arginine, and histidine, most preferably an amino acid substitution by arginine. In some embodiments, the fumonisin esterase according to the present invention comprises only one of the mutations of (i), (ii), and (iii). In some embodiments, the fumonisin esterase according to the present invention comprises mutations of (i) and (ii); mutations of (i) and (iii); or mutations of (i), (ii), and (iii).

[0022]

[0021] In some embodiments, the fumonisin esterase according to the present invention has the amino acid sequence of SEQ ID NO: 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, 3 The further comprising at least one further amino acid substitution at a position selected from 67, 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. This provides a fumonisin esterase exhibiting improved enzyme dynamics and improved stability, particularly temperature stability, compared to non-mutant fumonisin esterase, and especially compared to the fumonisin esterase of SEQ ID NO: 1. In a preferred embodiment, the fumonisin esterase according to the present invention has the amino acid sequence of SEQ ID NO: 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, 199I, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 302I, 312F, 329F, 3 The molecule includes at least one further amino acid substitution selected from 32E, 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.

[0023]

[0022] Similarly, the present invention relates to a heat-stable fumonisin esterase comprising or consisting of an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, wherein the heat-stable fumonisin esterase comprises, with respect to the amino acid sequence of SEQ ID NO: 1, 107, 140, 144, 149, 151, 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, The sequence includes at least one mutation at a position selected from 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, preferably at least one mutation is in the amino acid sequence of SEQ ID NO: 1. Regarding the following models: 10Q, 33E, 107E, 140P, 144M, 149F, 151R, 157Y, 199I, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 302I, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371M, 372F, 377V, 389L, 391V, 394P, 418A Selected from 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, the thermally stable fumonisin esterase contains a non-aspartic amino acid at position 66 of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the fumonisin esterase according to the present invention contains or consists of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 1, except that the amino acid at position 25 is excluded from the amino acid sequence of SEQ ID NO: 1; and / or the amino acid at position 45 is excluded from the amino acid sequence of SEQ ID NO: 1; and / or the amino acid at position 259 is excluded from the amino acid sequence of SEQ ID NO: 1.

[0024]

[0023] In certain embodiments, the fumonisin esterase according to the present invention excludes the 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 an amino acid selected from alanine, glycine, isoleucine, leucine, proline, and valine, even more preferably an amino acid selected from isoleucine, leucine, and valine, most preferably with isoleucine; and / or 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 tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, and glutamine. An amino acid selected from tamic acid and aspartic acid, more preferably an amino acid selected from asparagine, aspartic acid, glutamine, and glutamic acid, excluding substitutions by glutamine; and / or (for the amino acid sequence of SEQ ID NO: 1) an amino acid at position 259, a charged amino acid, more preferably an amino acid selected from lysine, arginine, histidine, glutamic acid, and aspartic acid, more preferably an amino acid selected from lysine, arginine, and histidine, excluding substitutions by arginine, comprising or consisting of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the fumonisin esterase according to the present invention is characterized in that the amino acid at position 66 (with respect to the amino acid sequence of SEQ ID NO: 1) is a non-aspartic amino acid; and (with respect to the amino acid sequence of SEQ ID NO: 1) 10, 33, 107, 140, 144, 149, 151, 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363 Except for one or more amino acids at positions selected from 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, it contains or consists of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 1.In a particular embodiment, the fumonisin esterase according to the present invention has the amino acid sequence (for SEQ ID NO: 1) 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, Except for one or more amino acids at positions selected from 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, it contains or consists of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the fumonisin esterase according to the present invention is characterized in that the amino acid at position 66 (with respect to the amino acid sequence of SEQ ID NO: 1) is a non-aspartic amino acid; and (with respect to the amino acid sequence of SEQ ID NO: 1) 10Q, 33E, 107E, 140P, 144M, 149F, 151R, 157Y, 199I, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 302I, 312F, 329F, 332E, 360V, 363T, 364H, Except for at least one mutation selected from 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, the amino acid sequence is identical to or consists of the amino acid sequence of SEQ ID NO: 1.In certain embodiments, the fumonisin esterase according to the present invention has the amino acid sequence (for SEQ ID NO: 1) 10Q, 33E, 107E, 140P, 144M, 149F, 151R, 157Y, 199I, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 302I, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371M, 372F, 3 Except for at least one mutation selected from 77V, 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, the amino acid sequence is identical to or consists of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the fumonisin esterase according to the present invention contains or consists of any one of the amino acid sequences of SEQ ID NOs: 2 to 71.

[0025]

[0024] In another aspect, the present invention relates to a composition for modifying (e.g., detoxifying) fumonisin (e.g., an additive for food and / or feed; feed or food) comprising at least one fumonisin esterase according to the present invention.

[0026]

[0025] The compositions according to the present invention are, for example, food additives; ingredient additives; feed additives; feed additives; nutritional supplements; intermediates thereof; and / or mixtures thereof. Such compositions may include further components, for example, prebiotics; and / or probiotics. For the sake of clarity, feed or bait may include, or consist of, for example, corn, hay, straw, soybeans, or products derived therefrom. Feed or bait may also include, or consist of, extruded feed products, for example, pellets. Additives for food, ingredient, feed or bait are often used to improve properties or to add properties to food products, feed or bait. For example, such additives may be added to improve sensory properties, for example, to improve the taste, smell, appearance, and color of food, ingredient, feed or bait. Additives may also be added to improve palatability or nutrient availability, or to add probiotic microorganisms to food, ingredients, feed, or animal feed, or to add or enhance the prebiotic activity of food, ingredients, feed, or animal feed. Such additives may also be added to mitigate potentially undesirable effects of food, ingredients, feed, or animal feed, such as the removal or reduction of one or more undesirable components present in the food, ingredients, feed, or animal feed.

[0027]

[0026] In some embodiments, the composition of the present invention comprises at least one carrier. The carrier can be any suitable carrier. The composition may comprise one, two, three, or more carriers. The carrier may be a nutritional supplement, nutritional supplement and / or pharmaceutical, e.g., vitamins, minerals, amino acids, essential fatty acids, fibers, trace elements, antioxidants, plant extracts, herbal extracts, and / or essential oils. The carrier may also be a carrier for enzymes. The carrier for enzymes may be of either inorganic or organic origin. Inorganic materials that may be used for enzyme immobilization include silica (sol-gel silica, fumed silica, colloidal silica nanoparticles and silica gel) and oxides such as titanium dioxide, aluminum oxide and zirconium oxide. Furthermore, clay materials such as bentonite, halloysite, kaolinite, montmorillonite, sepiolite and calcium apatite may also be carriers. Furthermore, carbon-based materials such as activated carbon and charcoal may also be carriers. The organic enzyme carrier may be a biopolymer (e.g., carbohydrates, proteins, maltodextrin, trehalose, inulin, collagen, cellulose, keratin, carrageenan, chitin, chitosan, and alginic acid) or a synthetic polymer (e.g., polyaniline, polyamide, polystyrene, polyurethane, polypropylene, polyvinyl alcohol, and ion exchange resin). The liquid carrier may be a buffering substance and / or polyhydric alcohol, such as polyalkylene oxide, polyvinyl alcohol, polyethylene-com-maleic anhydride, polystyrene-col-malicic anhydride, dextran, cellulose, hydrolysates of chitosan, starch, glycogen, sorbitol, agarose and its derivatives, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, hydrolysates of alginic acid, biopolymers, sorbitol, glycerol, cellobiose, and monopropylene glycol. The carrier may additionally or alternatively contain edible components, preferably non-toxic components, and / or components that provide texture. In certain embodiments, the carrier is selected from bentonite, silica, maltodextrin, and carbohydrates; preferably, the carrier is bentonite and / or maltodextrin.

[0028]

[0027] In another aspect, the present invention relates to a method for modifying (e.g., detoxifying) fumonisin in a composition (e.g., in a nutritional composition), the method comprising contacting the composition with a fumonisin esterase according to the present invention. In some embodiments of the method for modifying fumonisin in a composition, the fumonisin esterase is included in a composition (e.g., an additive for food and / or feed; feed or food) for modifying (e.g., detoxifying) fumonisin according to the present invention. In certain embodiments of the present invention, the method for modifying (e.g., detoxifying) fumonisin in a composition (e.g., in a nutritional composition) relates to the treatment of a composition containing fumonisin, but not to the treatment of the human or animal body. Such embodiments can be achieved because the fumonisin esterase according to the present invention can sufficiently modify the fumonisin contained in the composition in the absence of the human or animal body. In other words, in certain embodiments, the present invention relates to a non-medical method for modifying (e.g., detoxifying) fumonisin in a composition (e.g., in a nutritional composition), the method comprising contacting the composition with a fumonisin esterase according to the present invention.

[0029]

[0028] A nutritional composition as referred to herein is a composition comprising one or more components having nutritional value. Often such components provide energy to the consumer of the nutritional composition. The nutritional composition can be wholly or at least partially grass or plant-based, such as commonly used animal feed compositions.

[0030]

[0029] In a further aspect, the present invention relates to a method for modifying (e.g., detoxifying or biotransforming) fumonisin (i.e., at least one fumonisin) in an aqueous medium (e.g., fresh water, brackish water, or salt water; preferably fresh water or salt water) containing (at least one) fumonisin, the method comprising contacting the aqueous medium with a fumonisin esterase, preferably a fumonisin esterase according to the present invention. In some embodiments of the method for modifying fumonisin in an aqueous medium, the fumonisin esterase is included in a composition (e.g., an additive for food and / or feed; feed or food) for modifying (e.g., detoxifying) the fumonisin according to the present invention. In certain embodiments of the present invention, the method for modifying (e.g., detoxifying) fumonisin (i.e., at least one fumonisin) in an aqueous medium (e.g., fresh water, brackish water, or salt water; preferably fresh water or salt water) containing (at least one) fumonisin relates to the treatment of the aqueous medium containing fumonisin, but not to the treatment of the human or animal body. In other words, in certain embodiments, the present invention relates to a non-medical method for modifying (e.g., detoxifying or biotransforming) fumonisin (i.e., at least one fumonisin) in an aqueous medium (e.g., fresh water, brackish water, or salt water; preferably fresh water or salt water) containing (at least one) fumonisin, the method comprising contacting the aqueous medium with a fumonisin esterase, preferably a fumonisin esterase according to the present invention.

[0031]

[0030] In some embodiments, the method for modifying fumonisin in an aqueous medium relates to a method for modifying (e.g., detoxifying) fumonisin in aquaculture, such as for fish including carp, salmon, tilapia, tuna, catfish, trout, and masu salmon; crustaceans including crabs, lobsters, crayfish, prawns, shrimps, and krill; mollusks including oysters, mussels, bivalves, squids, cuttlefishes, and octopuses; echinoderms including sea cucumbers and sea urchins; jellyfishes; algae; and / or seaweeds.

[0032]

[0031] In another embodiment, the present invention relates to a method for modifying (e.g., detoxifying) fumonisin in gastric juice, comprising contacting gastric juice with fumonisin esterase, preferably fumonisin esterase according to the present invention. In some embodiments, the gastric juice is the gastric juice of an animal, particularly the gastric juice of a ruminant, a pig, or a poultry. In some embodiments of the method for modifying fumonisin in gastric juice, the fumonisin esterase is included in a composition for modifying (e.g., detoxifying) fumonisin according to the present invention (e.g., an additive for food and / or feed; feed or food). In certain embodiments of the present invention, the method for modifying (e.g., detoxifying) fumonisin in gastric juice relates to the treatment of gastric juice, but not to the treatment of a human or animal body. In other words, in certain embodiments, the present invention relates to a non-medical method for modifying (e.g., detoxifying) fumonisin in gastric juice, comprising contacting gastric juice with fumonisin esterase, preferably fumonisin esterase according to the present invention.

[0033]

[0032] Those skilled in the art will understand that when fumonisin esterase is brought into contact with fumonisin, a reaction mixture is formed in the method for modifying (e.g., detoxifying) fumonisin according to the present invention. The reaction mixture may be further brought into contact with water. The water may be derived from humidity or moisture present in the surrounding environment when fumonisin esterase comes into contact with fumonisin (e.g., in a composition, e.g., a nutritional composition; in an aqueous or aquatic product; in gastric juice); and / or the water may be added by a person carrying out the method of the present invention, and / or the water may be derived from any other source, e.g., saliva (e.g., when ingesting the reaction mixture or a composition containing the reaction mixture).

[0034]

[0033] In further embodiments, the present invention relates to the use of fumonisin esterase according to the present invention for modifying (e.g., detoxifying) fumonisin, and / or the use of compositions (e.g., food and / or feed additives; feed or food) containing fumonisin esterase according to the present invention. In particular embodiments of the present invention, the use of fumonisin esterase according to the present invention for modifying (e.g., detoxifying) fumonisin, and / or the use of compositions (e.g., food and / or feed additives; feed or food) containing fumonisin esterase according to the present invention relates to the use of fumonisin esterase for processing compositions containing fumonisin, and not to use for treatment of the body of a human or animal. In other words, in particular embodiments, the present invention relates to the non-medical use of fumonisin esterase according to the present invention for modifying (e.g., detoxifying) fumonisin, and / or the use of compositions (e.g., food and / or feed additives; feed or food) containing fumonisin esterase according to the present invention.

[0035]

[0034] In a further embodiment, the present invention relates to the use of fumonisin esterase according to the present invention and / or the use of compositions comprising fumonisin esterase according to the present invention (e.g., food and / or feed additives; feed or food) for the production of feed and / or food additives, feed and / or food compositions, or pharmaceutical compositions.

[0036]

[0035] In another aspect, the present invention relates to the use of fumonisin esterase according to the present invention for the production of biogas; bioethanol, DDGS; sugars, preferably derived from sugarcane or sugar beet; corn oil, corn germ, corn germ meal, corn fiber, corn gluten, starch, especially corn starch; and / or silage, and / or the use of compositions comprising fumonisin esterase according to the present invention (e.g., food and / or feed additives; feed or food).

[0037]

[0036] In another aspect, the present invention relates to a host cell comprising at least one fumonisin esterase according to the present invention and / or at least one polynucleotide encoding at least one fumonisin esterase according to the present invention.

[0038]

[0037] The term "host cell" refers to any cell capable of producing recombinant proteins. In particular, “host cell” refers to prokaryotic cells and / or eukaryotic cells, preferably Pichia pastoris, Escherichia coli, Bacillus sp. (such as 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. In particular, the strains commonly associated with "Pichia pastoris" may also be called Komagataella pastoris, Komagataella phaffii, or Komagataella pseudopastoris, depending on the taxonomic classification at the time of reference.

[0039]

[0038] In a further embodiment, the present invention relates to fumonisin esterase for use in the treatment, improvement and / or prevention or prevention of symptoms caused by mycotoxicosis, particularly fumonisin mycotoxicosis, wherein fumonisin esterase is fumonisin esterase according to the present invention. In some embodiments, the fumonisin esterase is provided to subjects, such as animals, who require prevention of mycotoxicosis, particularly prevention of fumonisin mycotoxicosis. Subjects may be considered to require prevention of fumonisin mycotoxicosis if there is a risk of ingesting fumonisin above non-toxic concentrations. For example, subjects may be considered to require prevention of fumonisin mycotoxicosis if they are about to ingest, and / or have already ingested, a nutritional composition containing one or more fumonisins at a detectable level. Due to the improved characteristics of the fumonisin esterase according to the present invention, such enzymes are advantageously suitable for treating and / or improving the symptoms of mycotoxicosis, particularly fumonisin mycotoxicosis, by reducing the subject's exposure to fumonisin and thus reducing the symptoms associated with fumonisin exposure. In addition, the fumonisin esterase of the present invention can be used to prevent the symptoms of mycotoxicosis, particularly fumonisin mycotoxicosis. For example, the fumonisin esterase according to the present invention can be administered to a subject who is known or suspected to have ingested fumonisin before the onset of symptoms, and thus the development of such symptoms caused by mycotoxicosis, particularly fumonisin mycotoxicosis, can be prevented.

[0040]

[0039] The present invention is further characterized by the following items.

[0041]

[0040] Item 1. A method for improving the enzymatic dynamics of fumonisin esterase (e.g., decrease in Michaelis constant (Km), increase in turnover rate (kcat), increase in catalytic efficiency, increase in maximum reaction rate (Vmax), and / or increase in specific activity), wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1, The method involves substituting the amino acid at position 25 of the amino acid sequence of Sequence ID No. 1 with an amino acid preferably an aliphatic amino acid, more preferably an amino acid selected from alanine, glycine, isoleucine, leucine, proline, and valine, even more preferably an amino acid selected from isoleucine, leucine, and valine, and most preferably isoleucine; The amino acid sequence of SEQ ID NO: 1 is replaced with the amino acid at position 45, preferably with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably with an amino acid selected from asparagine, aspartic acid, glutamine, and glutamic acid, most preferably with glutamine; A method comprising substituting the amino acid at position 259 of the amino acid sequence of SEQ ID NO: 1 with an amino acid preferably a charged amino acid, more preferably an amino acid selected from lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from lysine, arginine, and histidine, most preferably arginine.

[0042]

[0041] Item 2. A method for improving the enzymatic dynamics of fumonisin esterase (e.g., decrease in Michaelis constant (Km), increase in turnover rate (kcat), increase in catalytic efficiency, increase in maximum reaction rate (Vmax), and / or increase in specific activity), wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1, A method comprising substituting the amino acid at position 25 of the amino acid sequence of Sequence ID No. 1 with an amino acid preferably an aliphatic amino acid, more preferably an amino acid selected from alanine, glycine, isoleucine, leucine, proline, and valine, even more preferably an amino acid selected from isoleucine, leucine, and valine, and most preferably isoleucine.

[0043]

[0042] Item 3. A method for improving the enzymatic dynamics of fumonisin esterase (e.g., decrease in Michaelis constant (Km), increase in turnover rate (kcat), increase in catalytic efficiency, increase in maximum reaction rate (Vmax), and / or increase in specific activity), wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1, A method comprising substituting the amino acid at position 45 of the amino acid sequence of SEQ ID NO: 1 with an amino acid preferably a polar amino acid, more preferably an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from asparagine, aspartic acid, glutamine, and glutamic acid, most preferably glutamine.

[0044]

[0043] Item 4. A method for improving the enzymatic kinetics of fumonisin esterase (e.g., decrease in Michaelis constant (Km), increase in turnover rate (kcat), increase in catalytic efficiency, increase in maximum reaction rate (Vmax), and / or increase in specific activity), wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1, A method comprising substituting the amino acid at position 259 of the amino acid sequence of Sequence ID No. 1 with an amino acid preferably a charged amino acid, more preferably an amino acid selected from lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from lysine, arginine, and histidine, most preferably arginine.

[0045]

[0044] Item 5. A method for improving the enzymatic kinetics of fumonisin esterase (e.g., decrease in Michaelis constant (Km), increase in turnover rate (kcat), increase in catalytic efficiency, increase in maximum reaction rate (Vmax), and / or increase in specific activity), wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1, The method involves substituting the amino acid at position 25 of the amino acid sequence of Sequence ID No. 1 with an amino acid preferably an aliphatic amino acid, more preferably an amino acid selected from alanine, glycine, isoleucine, leucine, proline, and valine, even more preferably an amino acid selected from isoleucine, leucine, and valine, most preferably isoleucine; and A method comprising substituting the amino acid at position 45 of the amino acid sequence of Sequence ID No. 1 with, preferably, a polar amino acid, more preferably an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from asparagine, aspartic acid, glutamine, and glutamic acid, most preferably glutamine.

[0046]

[0045] Item 6. A method for improving the enzymatic kinetics of fumonisin esterase (e.g., decrease in Michaelis constant (Km), increase in turnover rate (kcat), increase in catalytic efficiency, increase in maximum reaction rate (Vmax), and / or increase in specific activity), wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1, The method involves substituting the amino acid at position 25 of the amino acid sequence of Sequence ID No. 1 with an amino acid preferably an aliphatic amino acid, more preferably an amino acid selected from alanine, glycine, isoleucine, leucine, proline, and valine, even more preferably an amino acid selected from isoleucine, leucine, and valine, most preferably isoleucine; and A method comprising substituting the amino acid at position 259 of the amino acid sequence of Sequence ID No. 1 with an amino acid preferably a charged amino acid, more preferably an amino acid selected from lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from lysine, arginine, and histidine, and most preferably arginine.

[0047]

[0046] Item 7. A method for improving the enzymatic kinetics of fumonisin esterase (e.g., decrease in Michaelis constant (Km), increase in turnover rate (kcat), increase in catalytic efficiency, increase in maximum reaction rate (Vmax), and / or increase in specific activity), wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1, The method involves substituting the amino acid at position 25 of the amino acid sequence of Sequence ID No. 1 with an amino acid preferably an aliphatic amino acid, more preferably an amino acid selected from alanine, glycine, isoleucine, leucine, proline, and valine, even more preferably an amino acid selected from isoleucine, leucine, and valine, and most preferably isoleucine; The amino acid sequence of SEQ ID NO: 1 is replaced with the amino acid at position 45, preferably with a polar amino acid, more preferably with an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably with an amino acid selected from asparagine, aspartic acid, glutamine, and glutamic acid, most preferably with glutamine; A method comprising substituting the amino acid at position 259 of the amino acid sequence of Sequence ID No. 1 with an amino acid preferably a charged amino acid, more preferably an amino acid selected from lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from lysine, arginine, and histidine, and most preferably arginine.

[0048]

[0047] Item 8. A method for improving the enzymatic kinetics of fumonisin esterase (e.g., decrease in Michaelis constant (Km), increase in turnover rate (kcat), increase in catalytic efficiency, increase in maximum reaction rate (Vmax), and / or increase in specific activity), wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1, The method involves substituting the amino acid at position 45 of the amino acid sequence of SEQ ID NO: 1 with an amino acid preferably a polar amino acid, more preferably an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from asparagine, aspartic acid, glutamine, and glutamic acid, most preferably glutamine; and A method comprising substituting the amino acid at position 259 of the amino acid sequence of Sequence ID No. 1 with an amino acid preferably a charged amino acid, more preferably an amino acid selected from lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from lysine, arginine, and histidine, and most preferably arginine.

[0049]

[0048] Item 9. A method according to any of the preceding items, further comprising substituting the amino acid at position 66 of the amino acid sequence of Sequence ID No. 1 with a non-aspartic amino acid; preferably a non-aspartic, non-valine amino acid; more preferably an amino acid selected from alanine, cysteine, phenylalanine, leucine, methionine, threonine, tryptophan, glutamic acid, histidine, isoleucine, lysine, proline, arginine, tyrosine, glycine, glutamine, serine and valine; most preferably an amino acid selected from alanine, phenylalanine, leucine, methionine, asparagine, threonine, tryptophan and histidine.

[0050]

[0049] Item 10. A method according to Item 9, wherein the amino acid at position 66 of the amino acid sequence of Sequence ID No. 1 is substituted with an amino acid selected from phenylalanine, methionine, tryptophan, histidine, and asparagine.

[0051]

[0050] Item 11. A method according to any one of items 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein the amino acid sequence of SEQ ID NO: 1 is 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 At least one further amino acid is substituted at a position selected from 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456, 457, 462, 463, 464, 465, 469, 473, 478, 487 and 490, preferably with respect to the amino acid sequence of SEQ ID NO: 1, 10Q, 33E, 66A, 66C, 66F, 66L, 66M, 66T, 66W, 66E, 66H, 6 6I, 66K, 66P, 66R, 66Y, 66G, 66Q, 66S, 107E, 140P, 144M, 149F, 151R, 157Y, 199I, 266S, 267P, 270F, 272H, 275E, 275A, 28 0D, 280P, 284T, 284P, 286P, 286R, 293E, 302I, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371V, 371M, 372F, 3 A method comprising introducing at least one further amino acid substitution selected from 77V, 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.

[0052]

[0051] Item 12: Fumonisin esterase obtained by the method described in any one of the preceding items, in particular items 1 to 11.

[0053]

[0052] Item 13. A method for improving the enzymatic kinetics of thermally stable fumonisin esterase, (i) To provide a heat-stable fumonisin esterase, wherein the heat-stable fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, the heat-stable fumonisin esterase comprises aspartic acid at position 66 of the amino acid sequence of SEQ ID NO: 1, and the heat-stable fumonisin comprises 10, 33, 107, 140, 144, 149, 151, 157, 1 of the amino acid sequence of SEQ ID NO: 1. A position selected from 99, 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. The sequence includes at least one further mutation, preferably at least one further mutation is, for the amino acid sequence of SEQ ID NO: 10Q, 33E, 107E, 140P, 144M, 149F, 151R, 157Y, 199I, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 302I, 312F, 329F, 332E, 360V, 363T, 36 To be selected from 4H, 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; and (ii) A method comprising substituting the amino acid at position 66 of the amino acid sequence of SEQ ID NO: 1 with a non-aspartic amino acid.

[0054]

[0053] Item 14. A method for improving the enzymatic kinetics of thermally stable fumonisin esterase, (i) To provide a heat-stable fumonisin esterase, wherein the heat-stable fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, and the heat-stable fumonisin esterase comprises aspartic acid at position 66 of the amino acid sequence of SEQ ID NO: 1, and comprises 10, 33, 107, 140, 144, 149, 151, 157, 199, 266, 2 At least one position selected from 67, 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 It also includes one further mutation, preferably at least one further mutation, for the amino acid sequence of SEQ ID NO: 10Q, 33E, 107E, 140P, 144M, 149F, 151R, 157Y, 199I, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 302I, 312F, 329F, 332E, 360V, 363T, 364H, To be selected from 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; and (ii) A method comprising the step of introducing a non-aspartate amino acid at position 66 of the amino acid sequence of SEQ ID NO: 1.

[0055]

[0054] Item 15. A method according to item 13 or 14, wherein the amino acid at position 66 of the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid selected from non-aspartic acid, non-valine amino acids, preferably alanine, cysteine, phenylalanine, leucine, methionine, asparagine, threonine, tryptophan, glutamic acid, histidine, isoleucine, lysine, proline, arginine, tyrosine, glycine, glutamine, serine and valine, and more preferably an amino acid selected from alanine, phenylalanine, leucine, methionine, asparagine, threonine, tryptophan and histidine.

[0056]

[0055] Item 16: Fumonisin esterase obtained by the method described in any of the preceding items.

[0057]

[0056] Item 17: Fumonisin esterase obtained by the method described in any one of items 13 to 15.

[0058]

[0057] Item 18: A fumonisin esterase comprising or consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 1, wherein the fumonisin esterase is (i) Amino acid substitution of the amino acid at position 25 of the amino acid sequence of Sequence ID No. 1, preferably by an aliphatic amino acid, more preferably by an amino acid selected from alanine, glycine, isoleucine, leucine, proline, and valine, even more preferably by an amino acid selected from isoleucine, leucine, and valine, most preferably by isoleucine; and / or (ii) For the amino acid sequence of SEQ ID NO: 1, the amino acid at position 45 is preferably a polar amino acid, more preferably an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, even more preferably an amino acid selected from asparagine, aspartic acid, glutamine, glutamic acid, most preferably glutamine; and / or (iii) Fumonisin esterase comprising an amino acid substitution of the amino acid at position 259 of the amino acid sequence of Sequence ID No. 1, preferably a charged amino acid, more preferably an amino acid selected from lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from lysine, arginine, and histidine, most preferably with arginine.

[0059]

[0058] Item 19: A fumonisin esterase comprising or consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 1, wherein the fumonisin esterase is (i) For the amino acid sequence of SEQ ID NO: 1, the amino acid at position 25 is preferably an aliphatic amino acid, more preferably an amino acid selected from alanine, glycine, isoleucine, leucine, proline, and valine, even more preferably an amino acid selected from isoleucine, leucine, and valine, most preferably an amino acid substitution with isoleucine; and (ii) Fumonisin esterase comprising, with respect to the amino acid sequence of SEQ ID NO: 1, the amino acid at position 45 is preferably a polar amino acid, more preferably an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from asparagine, aspartic acid, glutamine, and glutamic acid, and most preferably an amino acid substitution by glutamine.

[0060]

[0059] Item 20: A fumonisin esterase comprising or consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 1, wherein the fumonisin esterase is (i) For the amino acid sequence of SEQ ID NO: 1, the amino acid at position 25 is preferably an aliphatic amino acid, more preferably an amino acid selected from alanine, glycine, isoleucine, leucine, proline, and valine, even more preferably an amino acid selected from isoleucine, leucine, and valine, most preferably an amino acid substitution with isoleucine; and (ii) Fumonisin esterase comprising an amino acid substitution of the amino acid sequence of Sequence ID No. 1, wherein the amino acid at position 259 is preferably a charged amino acid, more preferably an amino acid selected from lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from lysine, arginine, and histidine, and most preferably an amino acid substitution by arginine.

[0061]

[0060] Item 21: A fumonisin esterase comprising or consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 1, wherein the fumonisin esterase is (i) For the amino acid sequence of SEQ ID NO: 1, the amino acid at position 45 is preferably a polar amino acid, more preferably an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, even more preferably an amino acid selected from asparagine, aspartic acid, glutamine, glutamic acid, most preferably glutamine; and (ii) Fumonisin esterase comprising an amino acid substitution of the amino acid sequence of Sequence ID No. 1, wherein the amino acid at position 259 is preferably a charged amino acid, more preferably an amino acid selected from lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from lysine, arginine, and histidine, and most preferably an amino acid substitution by arginine.

[0062]

[0061] Item 22: A fumonisin esterase comprising or consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 1, wherein the fumonisin esterase is (i) For the amino acid sequence of SEQ ID NO: 1, the amino acid at position 25 is preferably an aliphatic amino acid, more preferably an amino acid selected from alanine, glycine, isoleucine, leucine, proline, and valine, even more preferably an amino acid selected from isoleucine, leucine, and valine, most preferably an amino acid substitution with isoleucine; and (ii) For the amino acid sequence of SEQ ID NO: 1, the amino acid at position 45 is preferably a polar amino acid, more preferably an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamic acid, aspartic acid, even more preferably an amino acid selected from asparagine, aspartic acid, glutamine, glutamic acid, most preferably glutamine; and (iii) Fumonisin esterase comprising an amino acid substitution of the amino acid at position 259 of the amino acid sequence of Sequence ID No. 1, preferably a charged amino acid, more preferably an amino acid selected from lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from lysine, arginine, and histidine, most preferably with arginine.

[0063]

[0062] Item 23: A fumonisin esterase described in any one of the preceding items, particularly any one of items 16 to 22, wherein the fumonisin esterase has the amino acid sequence of SEQ ID NO: 10, 33, 66, 107, 140, 144, 149, 151, 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 36 Fumonisin esterase further comprising at least one further amino acid substitution at a position selected from 3, 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.

[0064]

[0063] Item 24: A fumonisin esterase described in any one of the preceding items, in particular any one of items 16 to 22, wherein the fumonisin esterase has the amino acid sequence of Sequence ID No. 1 as follows: 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, 199I, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 302I, Fumonisin esterase further comprising at least one further amino acid substitution selected from 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.

[0065]

[0064] Item 25: A fumonisin esterase, particularly a heat-stable fumonisin esterase, comprising or consisting of an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, wherein the fumonisin esterase, particularly a heat-stable fumonisin esterase, has the amino acids 10, 33, 107, 140, 144, 149, 151, 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, The sequence includes at least one mutation at a position selected from 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, preferably at least one mutation in the amino acid sequence of SEQ ID NO: 1 10Q, 33E, 107E, 140P, 144M, 149F, 151R, 157Y, 199I, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 302I, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371M, 372F, 377V, 389L, 391V, 394P, 418A, 419V, 424A, 424K Fumonisin esterases, particularly heat-stable fumonisin esterases, selected from 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, are fumonisin esterases that contain a non-aspartic amino acid at position 66 of the amino acid sequence of SEQ ID NO: 1.

[0066]

[0065] Item 26: Fumonisin esterase comprising or consisting of one of the amino acids in Sequence ID No. 2 to 71.

[0067]

[0066] Item 27: A composition for modifying (e.g., detoxifying) fumonisin, comprising at least one fumonisin esterase described in any of the preceding items (e.g., an additive for food and / or feed; feed or food).

[0068]

[0067] Item 28: A composition for modifying (e.g., detoxifying) fumonisin (e.g., an additive for food and / or feed; feed or food), comprising at least one fumonisin esterase described in any one of items 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26.

[0069]

[0068] Item 29: The composition according to any one of the preceding items, further comprising at least one carrier, preferably selected from bentonite, silica, maltodextrin and carbohydrates, and more preferably selected from maltodextrin and bentonite.

[0070]

[0069] Item 30: A method for modifying (e.g., detoxifying) fumonisin in a composition (e.g., in a nutritional composition), comprising contacting the composition with a fumonisin esterase described in any of the preceding items.

[0071]

[0070] Item 31: A method for modifying (e.g., detoxifying) fumonisin in a composition (e.g., in a nutritional composition), comprising contacting the composition with a fumonisin esterase described in any one of items 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.

[0072]

[0071] Item 32: A method for modifying (e.g., detoxifying) fumonisin in an aqueous substance (e.g., freshwater, brackish water, or saline water, preferably freshwater or saline water), comprising contacting the aqueous substance with fumonisin esterase.

[0073]

[0072] Item 33: A method for modifying (e.g., detoxifying) fumonisin in an aqueous substance (e.g., in freshwater, brackish water, or saline water, preferably in freshwater or saline water), comprising contacting the aqueous substance with the fumonisin esterase described in any of the preceding items.

[0074]

[0073] Item 34: A method for modifying (e.g., detoxifying) fumonisin in an aqueous substance (e.g., in freshwater, brackish water, or saline water, preferably in freshwater or saline water), comprising contacting the aqueous substance with a fumonisin esterase described in any one of items 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.

[0075]

[0074] Item 35: A method for modifying (e.g., detoxifying) fumonisin in aquaculture (e.g., fish such as carp, salmon, tilapia, tuna, catfish, trout, and rainbow trout; crustaceans such as crabs, lobsters, crayfish, prawns, shrimp, and krill; mollusks such as oysters, mussels, bivalves, squid, cuttlefish, and octopuses; echinoderms such as sea cucumbers and sea urchins; jellyfish; algae; and / or seaweed aquaculture), comprising contacting the aquaculture product with fumonisin esterase.

[0076]

[0075] Item 36: A method for modifying (e.g., detoxifying) fumonisin in aquaculture (e.g., fish such as carp, salmon, tilapia, tuna, catfish, trout, and rainbow trout; crustaceans such as crabs, lobsters, crayfish, prawns, shrimp, and krill; mollusks such as oysters, mussels, bivalves, squid, cuttlefish, and octopuses; echinoderms such as sea cucumbers and sea urchins; jellyfish; algae; and / or seaweed aquaculture), comprising contacting a farmed product with the fumonisin esterase described in any one of the preceding items.

[0077]

[0076] Item 37: A method for modifying (e.g., detoxifying) fumonisin in aquaculture (e.g., fish such as carp, salmon, tilapia, tuna, catfish, trout, and rainbow trout; crustaceans such as crabs, lobsters, crayfish, prawns, shrimp, and krill; mollusks such as oysters, mussels, bivalves, squid, cuttlefish, and octopuses; echinoderms such as sea cucumbers and sea urchins; jellyfish; algae; and / or seaweed aquaculture), comprising contacting a farmed product with a fumonisin esterase described in any one of items 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.

[0078]

[0077] Item 38: A method for modifying (e.g., detoxifying) fumonisin in gastric juice (e.g., gastric juice of a ruminant, a pig, or a poultry), comprising contacting the gastric juice with fumonisin esterase.

[0079]

[0078] Item 39: A method for modifying (e.g., detoxifying) fumonisin in gastric juice (e.g., gastric juice of a ruminant, a pig, or a poultry), comprising contacting the gastric juice with a fumonisin esterase described in any one of the preceding items.

[0080]

[0079] Item 40: A method for modifying (e.g., detoxifying) fumonisin in gastric juice (e.g., gastric juice of a ruminant, a pig, or a poultry), comprising contacting the gastric juice with a fumonisin esterase described in any one of items 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.

[0081]

[0080] Item 41: A method for modifying (e.g., detoxifying) fumonisin as described in any of the preceding items (in particular, any of items 30 to 41), wherein fumonisin esterase is contained in a composition as described in any of items 27 to 29.

[0082]

[0081] Item 42: A method according to any one of items 30 to 41, comprising: contacting fumonisin esterase with fumonisin to form a reaction; and incubating the reaction for at least 1 minute, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes, for example, at least 30 minutes, 60 minutes, 90 minutes, 120 minutes, or longer, for example, 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 longer.

[0083]

[0082] Item 43: A method according to Item 42, comprising incubating the reaction mixture at a temperature of 5°C to 90°C, preferably 5°C to 50°C, more preferably 5°C to 40°C, more preferably 10°C to 40°C, and even more preferably 20°C to 40°C.

[0084]

[0083] Item 44: A method according to any one of items 30 to 42, further comprising bringing the reaction mixture into contact with water.

[0085]

[0084] Item 45: Use of a fumonisin esterase described in any one of the preceding items (e.g., a fumonisin esterase described in any one of items 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) and / or a composition described in any one of items 27-29 for modifying (e.g., detoxifying) fumonisin.

[0086]

[0085] Item 46: Use of fumonisin esterase as described in any of the preceding items (e.g., fumonisin esterase as described in any of items 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) for the manufacture of feed and / or food additives, feed and / or food compositions, or pharmaceutical compositions.

[0087]

[0086] Item 47: Use of any one of the compositions described in items 27 to 29 for the manufacture of an additive for feed and / or food, a feed and / or food composition, or a pharmaceutical composition.

[0088]

[0087] Item 48: Use of fumonisin esterases described in any one of the preceding items (e.g., fumonisin esterases described in any one of items 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) and / or compositions described in any one of items 27 to 29 for the manufacture of additives for feed and / or food, feed and / or food compositions, or pharmaceutical compositions.

[0089]

[0088] Item 49: A method for producing an additive for feed and / or food, a feed and / or food composition, or a pharmaceutical composition, comprising contacting a fumonisin esterase described in any one of the preceding items (for example, a fumonisin esterase described in any one of items 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) with at least one further component of the additive for feed and / or food, a feed and / or food composition, or a pharmaceutical composition.

[0090]

[0089] Item 50: Biogas; bioethanol, DDGS; sugars, preferably derived from sugarcane or sugar beet; corn oil, corn germ, corn germ meal, corn fiber, corn gluten, starch, especially corn starch; and / or use of fumonisin esterases described in any one of the preceding items (e.g., fumonisin esterases described in any one of items 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) and / or compositions described in any one of items 27-29 for the production of silage.

[0091]

[0090] Item 51: A method for producing biogas; bioethanol, DDGS; sugar, preferably derived from sugarcane or sugar beet; corn oil, corn germ, corn germ meal, corn fiber, corn gluten, starch, especially corn starch; and / or silage, comprising contacting a fumonisin esterase described in any one of the preceding items (e.g., a fumonisin esterase described in any one of items 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) and / or a composition described in any one of items 27-29 with biogas; bioethanol, DDGS (soluble substance added dry distilled grain); sugar, preferably derived from sugarcane or sugar beet; corn oil, corn germ, corn germ meal, corn fiber, corn gluten, starch, especially corn starch; and / or silage.

[0092]

[0091] Item 52: A host cell comprising at least one fumonisin esterase as described in any one of the preceding items (e.g., a fumonisin esterase as described in any one of items 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26); and / or at least one polynucleotide encoding at least one fumonisin esterase.

[0093]

[0092] Item 53: Fumonisin esterase for use in the treatment, improvement and / or prevention or prevention of symptoms caused by fungal poisoning, particularly fumonisin fungal poisoning, wherein the fumonisin esterase is a fumonisin esterase described in any one of the preceding items (e.g., a fumonisin esterase described in any one of items 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26); and / or the fumonisin esterase is contained in a composition described in any one of items 27 to 29.

[0094]

[0093] Item 54: Methods for use, fumonisin esterases, compositions, uses, and / or fumonisin esterases described in any one of the preceding items, wherein fumonisin is one or more selected from fumonisin B1, B2, B3, and B4, preferably fumonisin B1.

[0095] [Examples]

[0094] The present invention will be further described below by non-limiting embodiments. The present invention as disclosed herein is not limited to the specific embodiments, methodologies, examples, protocols, etc. described herein, but is exclusively and expressly defined by the claims.

[0096] [Example 1]

[0095] The fumonisin esterase described herein was recombinantly produced in Pichia pastoris, as essentially described in International Publication No. 2016 / 134387A1. Those skilled in the art of recombinant protein production will know of suitable similar, similar or alternative methods for producing fumonisin esterase. The fumonisin esterase was designed for secretion into the culture supernatant and quantified photometrically (NanoDrop spectrophotometer) by Bradford assay and / or BCA assay. The enzyme preparation was diluted to a concentration of 1 mg / mL in 20 mM Tris-HCl, pH 8.0. These 1 mg / mL dilutions were further diluted to either 10 or 20 ng / mL using 1×FE buffer (20 mM Tris-HCl, pH 8.0, 0.1 mg / mL bovine serum albumin). The fumonisin stock solution was prepared by dissolving crystalline fumonisin B1 (FB1) in 1×FE buffer to a concentration of 1 mM FB1.

[0097]

[0096] The assay was initiated by mixing 50 μL of either a 10 ng / mL or 20 ng / mL enzyme solution with 450 μL of fumonisin solution. The latter was prepared from stock fumonisin to achieve one of the following final FB1 concentrations in the assay reaction: FB1 of 5, 10, 15, 20, 25, 50, 75, and 100 μm. The reaction was carried out in a 96-deep-well plate with shaking at 30°C and 600 rpm. After 15, 30, 45, 60, and 120 minutes, 80 μL of sample was removed from the reaction mixture and incubated at 99°C for 5 minutes to stop the reaction.

[0098]

[0097] For LC-MS / MS analysis, samples were eluted with HPLC eluents: 1:30 with 5-20 μm FB1, 1:50 with 25-50 μm FB1, and 1:100 with 75 and 100 μm FB1 approaches. Analysis was performed on an Agilent 1290 series UHPLC system connected to a 5500 QTrap mass spectrometer. The column temperature was set to 30°C and the 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 μL and the total run time was 0.4 minutes. Separation was performed on an Agilent ZORBAX EclipsePlusC18 RRHD column (50 × 2.1 mm, 1.8 μm). SRM parameters are shown in Table 1.

[0099] [Table 1]

[0100]

[0098] HPLC eluent: Ultrapure water containing 0.0385% formic acid (pH approximately 2.85, equivalent to 192.5 μL of formic acid in 500 mL of water) was mixed with 6.1 mM ammonium formate in the ultrapure water (192.5 mg of ammonium formate in 500 mL of water) to adjust the pH to 3.0. This solution was mixed with acetonitrile in a ratio of 75 (HPLC buffer pH 3.0):25 (acetonitrile) (v / v). The HPLC eluent was stored at room temperature.

[0101]

[0099] 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 using SigmaPlot software. The improved kinetic parameters of exemplary fumonisin esterase variants are shown in Tables 2 and 3 below, compared to fumonisin esterase of SEQ ID NO: 1.

[0102] [Table 2]

[0103] [Table 3]

[0104] [Example 2]

[0100] To determine temperature stability, a stability assay was performed in Teorell-Stenhagen buffer, pH 6.5, containing 0.1 mg / L bovine serum albumin (Teorell and Stenhagen. 1938. Biochem. Ztschrft. 299: 416-419). The fumonisin esterase enzyme was incubated at 75°C for 5 minutes at a concentration of 10 μg / mL in 70 μL of buffer. One aliquot of the incubation mixture was stored on ice without incubation at 75°C as an untreated control to determine the initial activity. After incubation at 75°C, the enzyme solution was stored on ice until the measurement of residual activity compared to the initial activity. Initial and residual activity were measured using the FB1 assay described in Example 1. Residual activity was measured as a percentage of the untreated initial activity. The improved temperature stability of the exemplary fumonisin esterase variants compared to the fumonisin esterase of SEQ ID NO: 1 is shown in Table 4 below. For example, if the initial activity of fumonisin esterase A was 100 U / mg and the residual activity after incubation was 50 U / mg, the residual activity was found to be 50%. If the residual activity of fumonisin esterase B was 80%, the relative residual activity of fumonisin esterase B relative to fumonisin esterase A was 80 × 100 / 50 = 160%.

[0105] [Table 4]

[0106]

[0101] As a further assay to measure temperature stability, a thermal shift assay (also called a thermofluoro assay) was performed using SYPRO Orange as a fluorescent indicator. Fumonisin esterase SEQ ID NO: 1 showed a fluorescence peak at 45°C, indicating temperature-inducible unfolding of polypeptides, while any one of the fumonisin esterases SEQ ID NOs: 9-71 showed a fluorescence peak at 70-75°C, and therefore showed unfolding at a significantly higher temperature than fumonisin esterase SEQ ID NO: 1.

[0107] [Example 3]

[0102] To investigate the applicability of fumonisin esterases in gastric juice, exemplary fumonisin esterases were diluted in gastric simulation buffer (GSB: 118.5 mM NaCl, 8.55 mM acetic acid, 14.9 mM sodium acetate, pH 5.0, 0.1 mg / mL bovine serum albumin) and tested at one of the following final concentrations: 2.0 ng / mL, 1.5 ng / mL, 1.0 ng / mL, or 0.5 ng / mL. The assay was performed in a 37°C water bath. The assay was initiated by adding FB1 to a total assay volume of 500 μL up to a final concentration of 5 μM. After 15, 30, 45, 60, and 120 minutes, 80 μL samples were taken and incubated at 99°C for 5 minutes. Specific activity was measured as described in Example 1. Despite the acetic acid conditions and low substrate concentrations, all fumonisin esterases were found to be active in gastric juice (see Table 5).

[0108] [Table 5]

[0109] [Example 4]

[0103] To investigate whether fumonisin esterase is suitable for application in water, particularly in aquaculture, a 56-day trial was conducted using 180 salmonid fish (rainbow trout). Three groups were compared in the trial: a control group that did not contain fumonisin and did not contain fumonisin esterase (control), a fumonisin-contaminated control group (FUM), and a test group that was administered fumonisin and the improved fumonisin esterase according to the present invention (any one of SEQ ID NOs. 2 to 71) in a diet of 15 U / kg (esterase). In the FUM and esterase groups, approximately 10 ppm of fumonisin contamination was applied, and this fumonisin contamination contained a mixture of fumonisin B1, B2, and B3.

[0110]

[0104] The fish arrived as eyed eggs and were reared for approximately 9 months prior to the experiment. On the first day of the experiment, the fish were individually weighed and assigned to 15 tanks based on stratified randomization to balance the average weight per tank. The fish were reared in 15 subsquare recirculating aquaculture system (RAS) tanks, each with 500L, and 12 fish were assigned to each tank. The tanks were divided among three test groups as follows: 3 tanks for the control group, 6 tanks for the FUM group, and 6 tanks for the esterase group. Each tank was an experimental unit. Fumonisin degradation was tracked by analyzing fumonisin (FB1, FB2, FB3) and its metabolites, hydrolyzed fumonisin (HFB1, HFB2, HFB3), and partially hydrolyzed fumonisin (pHFB1a+b, pHFB2a+b, pHFB3a+b) in the contents of the proximal and distal parts of the gastrointestinal tract (GIT). Furthermore, the levels of sphinganine (Sa) and sphingosine (So) in plasma, as well as their ratio (Sa / So), were analyzed as indicators of exposure to toxic levels of fumonisin (Meredith et al. 1998. J Food Prot. 61(8):1034-8).

[0111]

[0105] For the analysis of the sample from GIT, 100 mg of lyophilized and homogenized sample was weighed into a 5 mL Eppendorf tube. 3 mL of extraction solvent (acetonitrile / water / formic acid, 74 / 25 / 1, v / v / v) was added, vortexed, and the tube was shaken in a horizontal shaker at room temperature for 30 minutes (high-speed shaking), followed by a centrifugation step (1880 rcf, 5 minutes). The supernatant was collected in a 15 mL tube, and the residue was re-extracted with 3 mL of extraction solvent. After resuspending the residue by vortexing, the sample was shaken in a horizontal shaker at room temperature for 20 minutes (high-speed shaking), followed by a centrifugation step (1880 rcf, 5 minutes). The supernatant was poured into the 15 mL tube from the first extraction step, and in the final extraction step, 2 mL of extraction solvent was added to the residue. The supernatant was vortexed and shaken in a horizontal shaker at room temperature for 10 minutes, then centrifuged at 3200 rcf for 5 minutes, and the supernatant was pooled with the supernatants from the first two extraction steps. The combined extract was vortexed and centrifuged at 3200 rcf for 10 minutes. Sample preparation was completed by transferring 500 μL of supernatant to the HPLC and diluting it with 500 μL of extraction solvent. For calibration curves, fumonisin (FB1, 2, 3), HFB, and pHFB standards were diluted to 600, 300, 150, 30, 15, 3, 1.5, and 0.3 ng / mL with dilution solvent (acetonitrile / water / formic acid, 50 / 49 / 1, v / v / v). Analysis was performed on an Agilent 1290 series UHPLC system connected to a 5500 QTrap mass spectrometer. The column temperature was set to 30°C and the flow rate to 0.8 mL / min. Mobile phase A consisted of methanol / water / acetic acid (40 / 59.8 / 0.2; v / v / v), and mobile phase B consisted of methanol / acetic acid (99.8 / 0.2; v / v). The gradient started at 100% A at 0.5 min, rose linearly to 73% B by 5.9 min, rose to 100% B by 6.0 min, then to 100% B by 7.9 min, and then rapidly decreased to 0% B between 7.9 and 8.0 min. The total run time per sample was 10.5 minutes. The injection volume was 1 μL. Separation was performed using a Phenomenex Gemini 5μ C18 110 A column (150 × 4.6 mm).

[0112]

[0106] Mass spectrometry detection was performed by negative electrospray ionization in multiple reaction monitoring mode using the parameters specified in Table 6.

[0113] [Table 6]

[0114]

[0107] LC-MS / MS data analysis: Peak integration and concentration calculation were performed using Multiquant (version 3.0.3). A 1 / x weighted linear calibration function was used to calculate the concentration. The GIT sample results were obtained by multiplying the measured concentration in the extracted sample by the dilution factor.

[0115]

[0108] For the analysis of plasma samples, 600 μL of acetonitrile / water (50 / 50, v / v) was added to each 50 μL of plasma in a 2 mL tube. After homogenization by vortexing, the samples were shaken at speed level 5-6 for 30 minutes at room temperature using a vortex mixer equipped with an Eppendorf tube adapter, followed by a centrifugation step (2700 rcf, 5 minutes). The supernatant was collected (poured) into a new Eppendorf tube, and the pellet was re-extracted with 300 μL of 80% (v / v) methanol for 30 seconds while vortexing. After centrifugation (19000 rcf, 5 minutes), the supernatants were combined and dried by heating at 30°C using a SpeedVac. Once dried, the extract was redissolved in 300 μL of 80% (v / v) methanol at speed level 5-6 for 30 minutes at room temperature using a vortex mixer. Sample preparation was completed by a centrifugation step (19000 rcf, 10 minutes) and transfer of 200 μL of the supernatant to an HPLC vial using a glass insert. Analysis was performed on an Agilent 1290 series UHPLC system connected to a 5500 QTrap mass spectrometer. The column temperature was set to 30°C and the flow rate to 0.5 mL / min. Mobile phase A consisted of methanol / water / acetic acid (40 / 59.8 / 0.2; v / v / v), and mobile phase B consisted of methanol / acetic acid (99.8 / 0.2; v / v). The gradient started at 65% A at 1.7 min, rose linearly to 100% B up to 1.71 min, then to 100% B up to 2.5 min, and then rapidly decreased to 65% B between 2.5 and 2.51 min. The injection volume was 2 μL. Separation was performed using a Phenomenex Kinetex C18 column (150 × 2.1 mm, 2.6 μm). Quantification was based on calibration using external standards of sphinganine and sphingosine in the concentration range of 0.1 ppb to 300 ppb. SRM parameters are shown in Table 7.

[0116] [Table 7]

[0117]

[0109] Regardless of which fumonisin esterase was used, the concentration of fumonisin was significantly reduced (p<0.05) compared to the contaminated group (FUM). Furthermore, partially and completely hydrolyzed fumonisin (HFB1, HFB2, HFB3, pHFB1a+b, pHFB2a+b, pHFB3a+b) was significantly higher in both the proximal and distal parts of the GIT in the esterase group (see Tables 8 and 9). In addition, fish in the esterase group showed a significantly lower Sa / So ratio than fish in the FUM group (see Table 10).

[0118] [Table 8]

[0119] [Table 9]

[0120] [Table 10]

Claims

1. A method for improving the enzymatic kinetics of fumonisin esterase, wherein the fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1, The method involves substituting the amino acid at position 25 of the amino acid sequence of Sequence ID No. 1 with an amino acid preferably an aliphatic amino acid, more preferably an amino acid selected from alanine, glycine, isoleucine, leucine, proline, and valine, even more preferably an amino acid selected from isoleucine, leucine, and valine, and most preferably isoleucine; Regarding the amino acid sequence of SEQ ID NO: 1, the amino acid at position 45 is preferably replaced with a polar amino acid, more preferably an amino acid selected from tyrosine, threonine, glutamine, glycine, serine, cysteine, asparagine, lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from asparagine, aspartic acid, glutamine, and glutamic acid, most preferably glutamine; A method comprising substituting the amino acid at position 259 of the amino acid sequence of SEQ ID NO: 1 with an amino acid preferably a charged amino acid, more preferably an amino acid selected from lysine, arginine, histidine, glutamic acid, and aspartic acid, even more preferably an amino acid selected from lysine, arginine, and histidine, most preferably arginine.

2. The method according to claim 1, wherein the amino acid sequence of SEQ ID NO: 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 At least one further amino acid is substituted at a position selected from 436, 440, 443, 447, 453, 455, 456, 457, 462, 463, 464, 465, 469, 473, 478, 487 and 490, preferably with respect to the amino acid sequence of Sequence ID No. 1, 10Q, 33E, 66A, 66C, 66F, 66L, 66M, 66T, 66W, 66E, 66H, 66I, 66K, 66P, 66R, 6 6Y, 66G, 66Q, 66S, 107E, 140P, 144M, 149F, 151R, 157Y, 199I, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T , 284P, 286P, 286R, 293E, 302I, 312F, 329F, 332E, 360V, 363T, 364H, 364L, 365I, 367H, 371V, 371M, 372F, 377V, 389L A method comprising introducing at least one further amino acid substitution selected from 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.

3. A method for improving the enzymatic kinetics of heat-stable fumonisin esterase, (i) To provide the heat-stable fumonisin esterase, wherein the heat-stable fumonisin esterase comprises an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, and the heat-stable fumonisin esterase comprises aspartic acid at position 66 of the amino acid sequence of SEQ ID NO: 1, and comprises 10, 33, 66, 107, 140, 144, 149, 151, 157, 199 A small number of positions selected from 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. It includes at least one further mutation, preferably the at least one further mutation being, for the amino acid sequence of SEQ ID NO: 10Q, 33E, 107E, 140P, 144M, 149F, 151R, 157Y, 199I, 266S, 267P, 270F, 272H, 275E, 275A, 280D, 280P, 284T, 284P, 286P, 286R, 293E, 302I, 312F, 329F, 332E, 360V, 363T, 364 To be selected from H, 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; and (ii) A method comprising substituting the amino acid at position 66 of the amino acid sequence of Sequence ID No. 1 with a non-aspartic amino acid.

4. Fumonisin esterase obtained by the method according to any one of claims 1 to 3.

5. A composition for modifying fumonisin, comprising at least one fumonisin esterase as described in claim 4.

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

7. A method for modifying fumonisin in a composition, comprising the step of contacting the composition with the fumonisin esterase described in claim 4.

8. A method for modifying fumonisin in an aqueous solution, comprising the step of contacting the aqueous solution with fumonisin esterase.

9. A method for modifying fumonisin in gastric juice, comprising the step of contacting the gastric juice with fumonisin esterase.

10. The method according to claim 8 or 9, wherein the fumonisin esterase is the fumonisin esterase described in claim 4, and / or the fumonisin esterase is contained in the composition described in claim 5 or 6.

11. Use of the fumonisin esterase according to claim 4 and / or the composition according to claim 5 or 6 for modifying fumonisin, for example, for detoxification.

12. Use of the fumonisin esterase according to claim 4 and / or the composition according to claim 5 or 6 for the production of an additive for feed and / or food, a feed and / or food composition, or a pharmaceutical composition.

13. Biogas; bioethanol, DDGS; sugars, preferably derived from sugarcane or sugar beet; corn oil, corn germ, corn germ meal, corn fiber, corn gluten, starch, especially corn starch; and / or use of the fumonisin esterase according to claim 4 and / or the composition according to claim 5 or 6 for the production of silage.

14. A host cell comprising at least one fumonisin esterase as described in claim 4, and / or at least one polynucleotide encoding the at least one fumonisin esterase.

15. A fumonisin esterase for use in the treatment, improvement and / or prevention of symptoms caused by fungal poisoning, particularly fumonisin fungal poisoning, which is the fumonisin esterase according to claim 4.

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