Fusarium toxin-cleaving polypeptide variants, additive containing same, use of same, and method for splitting fusarium toxins

Abstract

The invention relates to fusarium toxin-cleaving polypeptide variants of a fusarium toxin carboxyl esterase with the SEQ ID NO:46. Each of the polypeptide variants has an amino acid sequence shortened by 47 amino acids at the N terminus, and the amino acid sequences have at least 70%, preferably 80%, in particular 100%, sequence identity, namely SEQ ID NO:1, to the amino acid sequence portion 48-540 of the SEQ ID NO:46. The invention also relates to isolated polynucleotides which code for the polypeptide variants, to a fusarium toxin-cleaving additive containing at least one polypeptide variant and optionally at least one auxiliary agent, to the use of the polypeptide variants or the additive, and to a method for hydrolytically cleaving at least one fusarium toxin.

Claims

1. A fusarium toxin-cleaving polypeptide variant of a fusarium toxin carboxylesterase of SEQ ID NO: 46, wherein the polypeptide variant possesses an amino acid sequence truncated by 47 amino acids at the N-terminus, the amino acid sequence sharing at least 90% sequence identity with the amino acid sequence section 48-540 of SEQ ID NO: 46, wherein the temperature stability (T(50%)) of the polypeptide of SEQ ID No. 46 is determined to be 42 C., the temperature stability (T(50%)) of the polypeptide of SEQ ID NO: 1 is determined to be 45 C. and, the polypeptide variant has a relative increase of temperature stability (T(50%)) compared to the polypeptide of SEQ ID NO: 1; wherein the amino acid sequence of the polypeptide variant comprises at least one amino acid substitution at a position according to the numbering of SEQ ID NO: 1 selected from the group consisting of position 10, 33, 66, 107, 140, 144, 149, 151, 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363, 364, 365, 367, 371, 372, 377, 389, 391, 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456, 457, 462, 463, 464, 465, 469, 473, 478, 487 and 490, and that the amino acid substitution at positions 10 and 456 are selected from Q, E, N, H, K and R, at positions 33, 107, 293 and 332 from E, Q, D, K, R and N, at positions 66, 463 and 478 from D, E, K, N, Q and R, at positions 140 and 490 from P, A, S and N, at positions 144 and 367 from I, L, M and V, at positions 149, 270, 312, 329 and 372 from F, Y, W and H, at positions 151 and 453 from D, E, K and R, at positions 157 and 462 from F, H, W and Y, at positions 199, 302, 365 and 464 from I, L, M and V, at positions 266 and 455 and from A, S and T, at positions 267, 394 and 429 from A, N, P and S, at position 272 from H, N, Q and S, at position 275 from A, D, E, G, K, N, Q, R and S, at position 280 from A, D, E, K, N, P, Q, R and S, at position 284 from A, N, P, S, T and V, at position 286 from A, D, E, K, N, P, R and S, at positions 360, 377, 391, 419 and 427 from A, 1, L S, T and V, at positions 363, 443 and 457 from A, S, T and V, at position 364 from H, 1, L, M, N, Q, S and V, at position 371 from A, 1, L, M, S, T and V, at position 389 from I, L, M and V, at positions 418, 430, 447 and 473 from A, G and S, at position 424 from A, D, E, G, K, R and S, at position 436 from A, G, S and T, at position 440 from A, G, S and T, at position 465 from A, G, H, N, Q, S and T, at position 469 from D, E, K and R and/or at position 487 from N, D, Q, H and S; wherein the amino acid sequence of the polypeptide variant comprises at least two amino acid substitutions at positions according to the numbering of SEQ ID NO: 1, selected from the group consisting of 10, 33, 66, 107, 140, 144, 149, 151, 157, 199, 266, 267, 270, 272, 275, 280, 284, 286, 293, 302, 312, 329, 332, 360, 363, 364, 365, 367, 371, 372, 377, 389, 391, 394, 418, 419, 424, 427, 429, 430, 436, 440, 443, 447, 453, 455, 456, 457, 462, 463, 464, 465, 469, 473, 478, 487 and 490, wherein the at least two amino acid substitutions are selected from the group consisting of 10Q, 66D, 144M, 151R, 199I, 266S, 267P, 272H, 275E, 275A, 280D, 284T, 286P, 286R, 293E, 302I, 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; and wherein the amino acid sequence does not include SEQ ID NO: 1.

2. The polypeptide variant according to claim 1, wherein the amino acid substitutions are selected from the group consisting of 10Q, 33E, 66D, 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, 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. The polypeptide variant according to claim 1, wherein the polypeptide variant comprises an amino acid substitution on at least one position selected from the group consisting of 66, 199, 302, 377, 394, 424, 430 and 463.

4. The polypeptide variant according to claim 1, wherein at least one of the amino acid substitutions is selected from the group consisting of 66D, 199I, 302I, 377V, 394P, 424A, 430A and 463D.

5. The polypeptide variant according to claim 1, wherein the amino acid sequence of the polypeptide variant comprises combinations of several amino acid substitutions, wherein the combinations of the positions being selected from the group consisting of 66/199/302/394/424/430, 66/199/302/377/394/424/430, 66/199/302/377/394/424/430/463, 66/144/199/302/360/372/377/394/424/430/443/463, 199/302/377/394/424/430/463, 66/199/302/377/394, 66/199/302/364/377/394/424/430/463, 66/199/302/377/394/424/430/463/465, 66/199/302/377/394/424/430/440/463, 66/199/302/377/394/424/430/447/463, 66/199/302/377/394/418/424/430/463, 66/199/302/377/394/424/436/430/463, 66/199/302/364/377/394/424/430/463, 66/199/302/377/394/424/430/463/490, 66/199/302/377/394/424/430/463/469, 66/199/302/377/389/394/424/430/463, 66/199/302/377/394/424/430/463/465, 66/199/302/377/394/424/430/463/464, 66/199/302/377/394/424/430/463/465, 66/199/302/377/394/424/430/440/463, 66/199/302/377/394/424/430/457/463, 66/199/302/377/394/424/430/436/463, 66/199/302/363/3711/377/394/424/430/463, 66/199/302/377/394/424/430/447/453/463, 66/199/302/377/394/424/430/456/462/463, 66/199/302/377/394/419/424/427/430/463, 66/199/302/365/377/394/424/430/463/487 and 66/199/302/371/377/394/424/430/463/487.

6. The polypeptide variant according to claim 5, wherein the amino acid sequence of the polypeptide variant is selected from the group consisting of SEQ ID Nos. 2 to 29.

7. The polypeptide variant according to claim 1, wherein the amino acid sequence of the polypeptide variant comprises combinations of several amino acid substitutions, the combinations of the positions being selected from the group consisting of 66/99/302/364/377/389/394/419/424/427/430/447/463/465/469, 66/199/302/377/389/394/419/424/427/430/447/463/465/469, 66/199/302/363/364/371/377/389/394/419/424/427/430/447/463/464/465/469, 66/199/302/363/371/377/389/394/419/424/427/430/447/463/464/465/469, 66/199/302/364/367/371/377/389/394/418/419/424/427/430/436/440/447/463/464/465/469/490, 66/199/302/367/371/377/389/394/418/419/424/427/430/436/440/447/463/464/465/469/490, 66/199/302/363/367/371/377/394/424/430/463/490, 66/199/302/377/394/418/419/424/427/430/436/440/447/463, 66/199/302/377/389/394/424/430/457/463/464/465/469, 66/199/302/363/371/377/389/394/419/424/427/430/440/447/457/463/464/469/490, 66/199/302/377/394/424/430/463/447/490/469/465, 66/199/302/377/394/424/430/463/490/46/490/469/465/419/427/371/487, 66/199/302/371/377/394/419/424/427/430/447/453/463/465/469/487/490, 66/199/302/367/371/377/389/394/418/419/424/427/429/430/436/440/447/457/463/464/465/469/490, 66/199/302/371/377/389/394/419/424/427/430/436/447/453/456/462/463/465/469/490/487 and 66/199/302/367/371/377/389/394/418/419/424/427/429/430/436/440/447/453/456/457/462/463/464/465/469/487/490.

8. The polypeptide variant according to claim 7, wherein the amino acid sequence of the polypeptide variant is selected from the group consisting of SEQ ID Nos. 30 to 45.

9. An isolated polynucleotide, wherein the polynucleotide comprises a nucleotide sequence encoding a polypeptide variant according to claim 1.

10. A preparation for prophylaxis and/or treatment of fusarium toxin mycotoxicoses, the preparation comprising a polypeptide variant according to claim 1.

11. A method for hydrolytically cleaving at least one fusarium toxin, wherein at least one fusarium toxin is contacted with at least one polypeptide variant according to claim 1 and that the mixture of the polypeptide variant and the fusarium toxin is subjected to a temperature treatment at at least 50 C. and that the mixture is contacted with an amount of moisture sufficient for hydrolytic cleavage either during or after the temperature treatment.

12. The method according to claim 11, wherein the polypeptide variant is mixed with a feed or food product contaminated with at least one fusarium toxin, and the temperature treatment is optionally performed during a pelletizing process.

13. The method according to claim 12, wherein the polypeptide variant is added at a concentration range from 5 U to 500 U per kilogram of feed or food product.

14. A fusarium toxin-cleaving additive, wherein said additive comprises at least one polypeptide variant according to claim 1 and optionally at least one supplement material.

15. The additive according to claim 14, wherein the supplement material is selected from the group consisting of inert carriers, vitamins, minerals, phytogenic substances, enzymes and other components for detoxifying mycotoxins, the other components optionally selected from mycotoxin-degrading enzymes, in particular aflatoxin oxidases, ergotamine hydrolases, ergotamine amidases, zearalenone esterases, zearalenone lactonases, zearalenone hydrolases, ochratoxin amidases, fumonisin am inotransferases, aminopolyol am inoxidases, deoxynivalenol epoxide hydrolases, deoxynivalenol dehydrogenases, deoxynivalenol oxidases, trichothecene dehydrogenases, trichothecene oxidases; and mycotoxin-degrading microorganisms; and mycotoxin-binding substances, for instance microbial cell walls or inorganic materials such as bentonite.

16. A method for cleaving at least one fusarium toxin comprising the step of contacting the additive according to claim 14 with the at least one fusarium toxin in pelletized, food or feed products, for pigs, poultry, cattle, horses, fishes or aquaculture.

17. A method for cleaving at least one fusarium toxin comprising the step of contacting the additive according to claim 14 with at least one fusarium toxin in food or feed products in a process at temperatures of at least 50 C.

18. A method for hydrolytically cleaving at least one fusarium toxin, wherein at least one fusarium toxin is contacted with at least one additive according to claim 14 and the mixture of the at least one additive and the at least one fusarium toxin is subjected to a temperature treatment at at least 50 C. and that the mixture is contacted with an amount of moisture sufficient for hydrolytic cleavage either during or after the temperature treatment.

19. The method according to claim 18, wherein the at least one additive is mixed with a feed or food product contaminated with at least one fusarium toxin, and the temperature treatment is optionally performed during a pelletizing process.

20. The method according to claim 19, wherein the polypeptide variant is added at a concentration range from 5 U to 500 U per kilogram of feed or food product.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) In the following, the invention will be explained in more detail by way of examples.

(2) Polypeptide Variants

Example 1: Modification, Cloning and Expression of Polynucleotides Encoding Fusarium Toxin-Cleaving Polypeptides

(3) Amino acid substitutions, insertions or deletions were performed by mutations of the nucleotide sequences by means of PCR using the QuikChange site-directed mutagenesis kit (Stratagene) according to instructions. Alternatively, also complete nucleotide sequences were synthesized (GeneArt). The nucleotide sequences generated by PCR mutagenesis and those obtained from GeneArt were integrated by standard methods in expression vectors for the expression in E. coli or P. pastoris, were transformed in E. coli or P. pastoris, and were expressed in E. coli or P. pastoris, respectively (J. M. Cregg, Pichia Protocols, second Edition, ISBN-10: 1588294293, 2007; J. Sambrook et al. 2012, Molecular Cloning, A Laboratory Manual 4th Edition, Cold Spring Harbor), wherein any other suitable host cell may also be used for this task.

(4) The term expression vector refers to a DNA construct capable of expressing a gene in vivo or in vitro. In particular, it encompasses DNA constructs suitable for transferring the polypeptide-encoding nucleotide sequence into the host cell so as to be integrated in the genome or freely located in the extrachromosomal space, and to intracellularly express the polypeptide-encoding nucleotide sequence and, optionally, transport the polypeptide out of the cell. The term host cell refers to any cell that contains either a nucleotide sequence to be expressed or an expression vector and is able to produce an enzyme or polypeptide according to the invention. In particular, this refers to prokaryotic and/or eukaryotic cells, preferably P. pastoris, E. coli, Bacillus subtilis, Streptomyces, Hansenula, Trichoderma, Lactobacillus, Aspergillus, plant cells and/or spores of Bacillus, Trichoderma oder Aspergillus. The soluble cell lysate in the case of E. coli and the culture supernatant in the case of P. pastoris, respectively, were used to determine the catalytic properties of the polypeptide variants.

Example 2: Determination of the Catalytic Activity and Specific Activity of Fusarium Toxin-Degrading Polypeptides

(5) The corresponding genes encoding fusarium toxin-degrading polypeptides were cloned in Escherichia coli using standard methods, intracellularly expressed, and subsequently lyzed by ultrasonic treatment and centrifuged. The clear supernatant was diluted with 20 mM Tris-HCl buffer (pH 8.0) containing 0.1 mg/l bovine serum albumin (about 10.sup.3 to 10.sup.5) and used in the FB1-degradation mixture so as to degrade 10% to 90% of the amount of FB1 contained in the degradation mixture by the polypeptide.

(6) In order to determine the catalytic enzyme activity, tests on the hydrolytic cleavage of fumonisin B1 (FB1) were carried out, the tests having been performed in a 20 mM Tris-HCl buffer (pH 8.0) containing 0.1 mg/l bovine serum albumin at a temperature of 30 C. for 30 minutes. In addition, the mixture contained a substrate concentration of 100 M FB1 (Biopure Referenzsubstanzen GmbH Tulin, Austria, BRM 001007) and one of the polypeptides to be tested. After an incubation of 30 minutes, the mixture was heat-inactivated at 99 C. for 5 min to stop the reaction.

(7) In order to determine the enzymatic activity of feed samples, the fusarium toxin-transforming polypeptide variants have to be extracted from the feed samples prior to testing. To this end, 10 grams of feed were dissolved in 100 ml 20 mM Tris-HCl buffer (pH 8.0) containing 0.1 mg/ml bovine serum albumin and shaken at 150 rpm for 1 hour at 20 C. After this, the samples were centrifuged at 4000 g for 15 min, and the clear supernatant was diluted as required (10.sup.2 to 10.sup.3) and used in the FB1 solution.

(8) The quantification of FB1 was performed by LC-MS (liquid chromatography-mass spectroscopy) according to the method of Heinl et al. (J. of Biotechnology, 2010, 145, pp. 120-129, 2.6.3. Liquid chromatography-mass spectrometry). To this end, a calibration with FB1 standards additionally containing a complete .sup.13C-labeled, internal FB1 standard (Biopure Referenzsubstanzen GmbH Tulin, Austria) was done. As opposed to Heinl et al. (2010), only the degradation of FB1 was measured to determine the catalytic enzyme activity of the polypeptide solutions used. The catalytic enzyme activity of the used polypeptide solutions is indicated in units per ml, one unit being defined as reduction of 1 mol FB1 per minute under the above-identified reaction conditions in the test.

(9) For determining the specific activities, the enzyme concentrations were determined by quantitative Western blot or ELISA. The specific enzyme activities were calculated by the activities (units) having been based on the used amounts of enzyme and are indicated in units per mg.

Example 3: Temperature Stability of Fusarium Toxin-Degrading Polypeptides

(10) The expression and quantification of the fusarium toxin-degrading polypeptides were performed as described in Examples 1 and 2. Prior to the determination of the activity, the amount of cell lysate was divided into several portions (of 60 l each). Two to 10 portions were subjected to a heat treatment for 5 min in a commercially available PCR cycler (e.g. Eppendorf Matercycler Gradient), each portion having been incubated at different temperatures. Meanwhile, another portion of the cell lysate, the 100% control, was incubated on ice. Following the heat treatment, all of the samples/test mixtures were incubated at 10 C. for 1 minute to equalize the temperatures. The enzymatic activity of both the heat-treated samples and the 100% control were determined as described in Example 2. The activity remaining after the heat treatment is referred to as residual activity. The temperature at which the residual activity is 50% as compared to the non-heat-treated 100% control, is abbreviated by T(50%), constituting the measure for the temperature stability of the polypeptide.

(11) The increases of T(50%), indicated in degree Celsius, of polypeptide variants relative to the polypeptide of SEQ ID No. 46 or SEQ ID No. 1, respectively, is a measure for the increased temperature stability. The increase in the T(50%) value can be indicated in C., yet also in percent relative to the T(50%) value of the parental polypeptide. The following example serves for illustration: If the parental enzyme had a catalytic activity of 50 U/ml after a 5-minute incubation on ice and a catalytic activity of 25 U/ml after a 5-minute incubation at 48 C., the T(50%) value would be 48 C. If a polypeptide variant had a T(50%) value of 51 C., the relative increase in the temperature stability (T(50%)) would be 6.25. This results from the difference between the two T(50%) values of 3 C., divided by the T(50%) value of the parental starting enzyme of 48 C., multiplied by 100.

(12) Instead of the catalytic activity, the specific activity may also be used for determining the temperature stability.

(13) The determination of the temperature stability may also be performed by alternative enzymatic assays and even without determining the catalytic activity of the FB1 reaction. What is important in this respect, is that equal amounts of thermally treated polypeptide and of the 100% control are used, which is, for instance, ensured by the use of equal volumes of cell lysate.

(14) Instead of the catalytic activity, also the measurement signals of enzymatic degradation assays (e.g. MS signal, extinction, etc.) may be used for determining the temperature stability. If the measurement signal is directly proportional to the enzymatic activity (e.g. peak surface of reacted FB1), the T(50%) value of a polypeptide is the temperature at which the value of the measurement signal of the heat-treated polypeptide comprises 50% of the value of the measurement signal of the 100% control of the polypeptide.

(15) The temperature stability (T(50%)) of the polypeptide of SEQ ID No. 46 was determined to be 42 C., that of the polypeptide of SEQ ID No. 1 to be 45 C. Thus, the relative increase in the temperature stability that could be achieved by truncating the N-terminal sequence was about 7%. Moreover, an increase in the enzymatic activity of the polypeptide of SEQ ID No. 1 relative to the parental polypeptide of SEQ ID No. 46 could also be determined.

(16) The temperature stability of the polypeptide of SEQ ID No. 1 could be further increased by the selective substitution of individual amino acids. The relative increases in the temperature stability of these polypeptide variants relative to the parental polypeptide of SEQ ID No. 1 are illustrated in Table 1.

(17) TABLE-US-00002 TABLE 1 Modifications of the polypeptide variants and their relative increases in the temperature stability in percent as compared to the parental enzyme of SEQ ID No. 1 SEQ ID No. of Relative the polypeptide increase in containing the Modification(s) of SEQ ID No. 1 T (50%) modifications H10Q 4.4% A33E 4.4% N66D 6.7% G107E 4.4% I140P 4.4% L144M 4.4% L149F 4.4% K151R 4.4% V157Y 4.4% L199I 6.7% R266S 4.4% Q267P 4.4% K270F 4.4% R272H 4.4% G275E 4.4% G275A 4.4% G280D 4.4% G280P 4.4% R284T 4.4% R284P 4.4% L286P 4.4% L286R 4.4% K293E 4.4% L302I 6.7% A312F 4.4% L329F 4.4% Q332E 4.4% F360V 4.4% S363T 4.4% Q364H 4.4% Q364L 4.4% F365I 4.4% N367H 4.4% L371V 4.4% L371M 4.4% L372F 4.4% A377V 6.7% T389L 4.4% I391V 4.4% A394P 6.7% S418A 4.4% M419V 4.4% E424A 6.7% E424K 4.4% A427V 4.4% A429P 4.4% S430A 6.7% T436A 4.4% T436S 4.4% A440G 4.4% A440S 4.4% V443T 4.4% V447A 4.4% Q453R 4.4% T455S 4.4% K456Q 4.4% S457T 4.4% F462Y 4.4% E463D 6.7% R464I 4.4% R465H 4.4% R465S 4.4% R465G 4.4% M469K 4.4% S473A 4.4% G478D 4.4% K487N 4.4% Q490P 4.4% L199I/A394P 13.3% N66D/L199I 11.1% L199I/L302I 13.3% L199I/A377V 15.6% L199I/E424A 11.1% L199I/S430A 11.1% L199I/E463D 13.3% L199I/L302I/A394P 20.0% L199I/L302I/A377V 17.8% L199I/L302I/E424A 17.8% L199I/A377V/A394P 22.2% L199I/A394P/A429P 17.8% L372F/A394P/V443T 17.8% L199I/L302I/L372F 15.6% L144M/L199I/L302I 20.0% F360V/A394P/V443T 17.8% H10Q/K151R/A302I 15.6% R266S/A377V/E424K 20.0% Q267/A394P/T436S 17.8% R272H/G280D/E463D 20.0% G275A/L302I/F360V 17.8% N66D/L286P/N367H 15.6% R284T/L286R/S430A 15.6% K293E/E424A/M469K 20.0% S363T/A377V/K456Q 20.0% Q364H/L371V/S430A 17.8% L199I/Q364L/Q490P 15.6% F365I/A394P/R464I 15.6% L371M/A377V/A429P 17.8% L302I/L372F/Q453R 15.6% T389L/M419V/E463D 17.8% I391V/A394P/A440G 20.0% S418A/S430A/F462Y 20.0% N66D/A427V/V443T 17.8% A440S/S457T/E463D 20.0% L199I/V447A/T455S 20.0% A377V/R465H/K487N 17.8% L302I/R465S/G478D 15.6% A377V/R465G/S473A 20.0% N66D/L199I/L302I/A394P/E424A/S430A 26.7% SEQ ID No. 2 N66D/L199I/L302I/A377V/A394P/E424A/S430A 31.1% SEQ ID No. 3 N66D/L199I/L302I/A377V/A394P/E424A/S430A/E463D 37.8% SEQ ID No. 4 N66D/L144M/L199I/L302I/F360V/L372F/A377V/ 33.3% SEQ ID No. 5 A394P/E424A/S430A/V443T/E463D L199I/L302I/A377V/A394P/E424A/S430A/E463D 31.1% SEQ ID No. 6 N66D/L199I/L302I/A377V/A394P 26.7% SEQ ID No. 7 N66D/L199I/L302I/Q364H/A377V/A394P/ 35.5% SEQ ID No. 8 E424A/S430A/E463D N66D/L199I/L302I/A377V/A394P/E424A/ 40.0% SEQ ID No. 9 S430A/E463D/R465H N66D/L199I/L302I/A377V/A394P/E424A/ 37.8% SEQ ID No. 10 S430A/A440G/E463D N66D/L199I/L302I/A377V/A394P/E424A/ 35.5% SEQ ID No. 11 S430A/V447A/E463D N66D/L199I/L302I/A377V/A394P/S418A/ 35.5% SEQ ID No. 12 E424A/S430A/E463D N66D/L199I/L302I/A377V/A394P/E424A/ 35.5% SEQ ID No. 13 T436A/S430A/E463D N66D/L199I/L302I/Q364L/A377V/A394P/ 35.5% SEQ ID No. 14 E424A/S430A/E463D N66D/L199I/L302I/A377V/A394P/E424A/ 37.8% SEQ ID No. 15 S430A/E463D/Q490P N66D/L199I/L302I/A377V/A394P/E424A/ 37.8% SEQ ID No. 16 S430A/E463D/M469K N66D/L199I/L302I/A377V/T389L/A394P/ 40.0% SEQ ID No. 17 E424A/S430A/E463D N66D/L199I/L302I/A377V/A394P/E424A/ 35.5% SEQ ID No. 18 S430A/E463D/R465S N66D/L199I/L302I/A377V/A394P/E424A/ 40.0% SEQ ID No. 19 S430A/E463D/R464I N66D/L199I/L302I/A377V/A394P/E424A/ 35.5% SEQ ID No. 20 S430A/E463D/R465G N66D/L199I/L302I/A377V/A394P/E424A/ 33.3% SEQ ID No. 21 S430A/A440S/E463D N66D/L199I/L302I/A377V/A394P/E424A/ 37.8% SEQ ID No. 22 S430A/S457T/E463D N66D/L199I/L302I/A377V/A394P/E424A/ 35.5% SEQ ID No. 23 S430A/T436S/E463D N66D/L199I/L302I/S363T/L371V/A377V/ 40.0% SEQ ID No. 24 A394P/E424A/S430A/E463D N66D/L199I/L302I/A377V/A394P/E424A/ 35.5% SEQ ID No. 25 S430A/V447A/Q453R/E463D N66D/L199I/L302I/A377V/A394P/E424A/ 40.0% SEQ ID No. 26 S430A/K456Q/F462Y/E463D N66D/L199I/L302I/A377V/A394P/M419V/ 35.5% SEQ ID No. 27 E424A/A427V/S430A/E463D N66D/L199I/L302I/F365I/A377V/A394P/ 33.3% SEQ ID No. 28 E424A/S430A/E463D/K487N N66D/L199I/L302I/L371M/A377V/A394P/ 33.3% SEQ ID No. 29 E424A/S430A/E463D/K487N N66D/L199I/L302I/Q364L/A377V/T389L/ 51.1% SEQ ID No. 30 A394P/M419V/E424A/A427V/S430A/V447A/E463D/ R465S/M469K N66D/L199I/L302I/A377V/T389L/A394P/M419V/ 46.7% SEQ ID No. 31 E424A/A427V/S430A/V447A/E463D/R465S/ M469K N66D/L199I/L302I/S363T/Q364L/L371V/ 60.0% SEQ ID No. 32 A377V/T389L/A394P/M419V/E424A/A427V/S430A/ V447A/E463D/R464I/R465S/M469K N66D/L199I/L302I/S363T/L371V/A377V/T389L/ 57.8% SEQ ID No. 33 A394P/M419V/E424A/A427V/S430A/V447A/ E463D/R464I/R465S/M469K N66D/L199I/L302I/Q364L/N367H/L371V/A377V/ 62.2% SEQ ID No. 34 T389L/A394P/S418A/M419V/E424A/A427V/ S430A/T436A/A440S/V447A/E463D/R464I/ R465S/M469K/Q490P N66D/L199I/L302I/N367H/L371V/A377V/T389 62.2% SEQ ID No. 35 L/A394P/S418A/M419V/E424A/A427V/S430A/ T436A/A440S/V447A/E463D/R464I/R465S/ M469K/Q490P N66D/L199I/L302I/S363T/N367H/L371V/ 42.2% SEQ ID No. 36 A377V/A394P/E424A/S430A/E463D/Q490P N66D/L199I/L302I/A377V/A394P/S418A/ 46.7% SEQ ID No. 37 M419V/E424A/A427V/S430A/T436A/A440S/V447A/ E463D N66D/L199I/L302I/A377V/T389L/A394P/E424A/ 48.9% SEQ ID No. 38 S430A/S457T/E463D/R464I/R465S/M469K N66D/L199I/L302I/S363T/L371V/A377V/ 55.5% SEQ ID No. 39 T389L/A394P/M419V/E424A/A427V/S430A/A440S/ V447A/S457T/E463D/R464I/M469K/Q490P N66D/L199I/L302I/A377V/A394P/E424A/S430A/ 46.7% SEQ ID No. 40 V447A/E463D/R465S/M469K/Q490P N66D/L199I/L302I/L371M/A377V/A394P/ 51.1% SEQ ID No. 41 M419V/E424A/A427V/S430A/E463D/R465S/ M469K/K487N/Q490P N66D/L199I/L302I/L371M/A377V/A394P/ 55.5% SEQ ID No. 42 M419V/E424A/A427V/S430AA/447A/Q453R/ E463D/R465S/M469K/K487N/Q490P N66D/L199I/L302I/N367H/L371V/A377V/T389L/ 64.4% SEQ ID No. 43 A394P/S418A/M419V/E424A/A427V/A429P/ S430A/T436A/A440S/V447A/S457T/E463D/ R464I/R465S/M469K/Q490P N66D/L199I/L302I/L371M/A377V/T389L/A394P/ 62.2% SEQ ID No. 44 M419V/E424A/A427V/S430A/T436A/V447A/ Q453R/K456Q/F462Y/E463D/R465S/M469K/ K487N/Q490P N66D/L199I/L302I/N367H/L371V/A377V/T389L/ 65.2% SEQ ID No. 45 A394P/S418A/M419V/E424A/A427V/A429P/ S430A/T436A/A440S/V447A/Q453R/K456Q/ S457T/F462Y/E463D/R464I/R465S/M469K/ K487N/Q490P

Example 4: Temperature-Dependent Activity (Temperature Activity) of Fusarium Toxin-Degrading Polypeptides

(18) The fusarium toxin-degrading polypeptide variants to be tested for their temperature-dependent activities were purified prior to carrying out the tests. To this end, the polypeptide variants were purified from fermentation supernatants in a two-step process using anion exchange chromatography and subsequently size exclusion chromatography. The polypeptide variants were adjusted to concentrations of 1 mg/ml and used in the reaction mixture at dilutions of 10.sup.5 to 10.sup.6 in reaction volumes of 1 ml. Activity determinations were performed by tests as described in Example 3, by FB1 hydrolysis and subsequent quantification of FB1 by LC-MS, the tests have been carried out at different temperatures. Incubation was performed using two heating blocks (Eppendorf, ThermoMixer) at temperatures of 10 C., 20 C., 30 C., 35 C., 40 C., 45 C., 50 C., 55 C., 60 C., 65 C. and 70 C. Thirty minutes after the beginning of the heat exposure, 100 l of the reaction mixture were each taken and heat-inactivated at 99 C. for 5 min. The test performed at 30 C. in the heating block served as a 100% control. Exemplary results are indicated in Table 2.

(19) TABLE-US-00003 TABLE 2 Temperature-dependent activities of fusarium toxin-degrading polypeptides. <LOQ values are below detection limits (level of quantification: <0.15 U/l in the test preparation) Sequence ID incl. amino Relative activity, based on acid substitutions Temperature the activity at 30 C. [%] SEQ ID No. 1 10 C. 35 20 C. 59 30 C. 100 35 C. 123 40 C. 100 45 C. 105 50 C. 90 55 C. 26 60 C. <LOQ 65 C. <LOQ 70 C. <LOQ SEQ ID No. 1 with 30 C. 100 L199I/L302I/A394P 40 C. 100 50 C. 95 60 C. 62 70 C. 16 SEQ ID No. 1 with 30 C. 100 L144M/L199I/L302I 40 C. 104 50 C. 91 60 C. 60 70 C. 18 SEQ ID No. 1 with 30 C. 100 R266S/A377V/E424K 40 C. 110 50 C. 97 60 C. 63 70 C. 29 SEQ ID No. 1 with 30 C. 100 K293E/E424A/M469K 40 C. 98 50 C. 81 60 C. 50 70 C. <LOQ SEQ ID No. 1 with 30 C. 100 F365I/A394P/R464I 40 C. 105 50 C. 84 60 C. 57 70 C. 14 SEQ ID No. 1 with 30 C. 100 S418A/S430A/F462Y 40 C. 103 50 C. 89 60 C. 53 70 C. <LOD SEQ ID No. 4 10 C. 33 20 C. 58 30 C. 100 35 C. 109 40 C. 110 45 C. 114 50 C. 148 55 C. 113 60 C. 94 65 C. 83 70 C. 46 SEQ ID No. 43 10 C. 31 20 C. 56 30 C. 100 35 C. 113 40 C. 120 45 C. 126 50 C. 157 55 C. 168 60 C. 134 65 C. 102 70 C. 98 SEQ ID No. 44 10 C. 33 20 C. 59 30 C. 100 35 C. 110 40 C. 124 45 C. 131 50 C. 162 55 C. 174 60 C. 126 65 C. 99 70 C. 95 SEQ ID No. 45 10 C. 41 20 C. 68 30 C. 100 35 C. 115 40 C. 136 45 C. 141 50 C. 164 55 C. 177 60 C. 137 65 C. 121 70 C. 100 Enzyme Palletizing temperature Residual activity % SEQ ID No. 1 75 C. 15 80 C. <LOQ 85 C. <LOQ 90 C. <LOQ SEQ ID No. 4 75 C. 57 80 C. 46 85 C. 37 90 C. 14 SEQ ID No. 43 75 C. 78 80 C. 73 85 C. 58 90 C. 31 SEQ ID No. 44 75 C. 70 80 C. 59 85 C. 48 90 C. 25 SEQ ID No. 45 75 C. 72 80 C. 68 85 C. 48 90 C. 30

Example 5: Determination of the Pelletizing Stability of Fusarium Toxin-Degrading Polypeptides

(20) Selected polypeptide variants were cloned in Pichia pastoris in a bioreactor using standard methods under controlled aerobic conditions and extracellularly secreted. The clear supernatant was separated from the biomass, supplemented with a carrier substance (maltodextrin) and processed to a pelletizable powder using a spray-dryer. The fusarium toxin-degrading polypeptide variants present in power form were admixed to piglet rearing feed, each at the same concentration of 100 U/kg, and processed to feed pellets in a controlled process. During the pelletizing process, the feed was moistened by hot steaming and heated in individual batches at precisely defined temperatures (75 to 95 C. in 5 C. steps). This preparation phase was followed by the pelletizing process proper. The residual activities of the fusarium toxin-degrading polypeptide variants contained in the pellets were determined as described in Example 2, non-pelletized feed containing the respective fusarium toxin-degrading polypeptide variants serving as 100% controls. The enzyme activity remaining after the pelletizing process is therefore defined as residual activity. The values are indicated in Table 3.

(21) TABLE-US-00004 TABLE 3 Pelletizing temperatures and residual activities of fusarium toxin- degrading polypeptides. <LOQ values are below detection limits (level of quantification: <0.15 U/l in the test mixture) Enzyme Pelletizing temperature Residual activity % SEQ ID No. 1 75 C. 15 80 C. <LOQ 85 C. <LOQ 90 C. <LOQ SEQ ID No. 4 75 C. 57 80 C. 46 85 C. 37 90 C. 14 SEQ ID No. 43 75 C. 78 80 C. 73 85 C. 58 90 C. 31 SEQ ID No. 44 75 C. 70 80 C. 59 85 C. 48 90 C. 25 SEQ ID No. 45 75 C. 72 80 C. 68 85 C. 48 90 C. 30