Fumonisins enzymatic degradation

Abstract

The present invention relates to the use of an enzyme comprising an amino acid sequence having at least 80% sequence identity with sequence SEQ ID NO: 1 for degrading fumonisins in a sample. Furthermore, the present invention relates to a method of degrading fumonisins based on incubating a contaminated sample with an enzyme comprising an amino acid sequence having at least 80% sequence identity to the sequence SEQ ID NO: 1.

Description

Technical Field

[0001] The present invention belongs to the fields of agricultural food and biotechnology. Specifically, the present invention describes the use of enzymes for fumonisin degradation and methods thereof. Background Art

[0002] Many types of fungi can grow in grains and animal feeds, producing secondary metabolites called mycotoxins. Mycotoxins are the biggest challenge faced by animal feed producers, and therefore, regular monitoring and remediation are necessary because these toxins are very harmful to both humans and animals.

[0003] Fumonisins are a group of mycotoxins that are widespread in nature and pose harmful health risks to humans and animals due to their contamination of a variety of food and feed matrices. In fact, the economic losses in the agricultural food sector are estimated to be as high as billions of dollars, and thus the economic potential of protective or fumonisin removal measures is correspondingly high.

[0004] Currently, various different measures are available, such as mechanical, thermal, or chemical methods. However, such methods also have a related drawback, that is, they may produce unknown or unexpected by-products, the danger of which may be no less than that of the original toxin.

[0005] In this context, enzymatic detoxification is a very promising research and development topic at present because enzymes can remove mycotoxins in a defined manner. Some documents in the prior art disclose the enzymatic degradation of fumonisins:

[0006] WO2010031101A1 discloses the enzyme FumD for degrading fumonisin B1 toxin. This document describes an additive for the enzymatic degradation of fumonisins in plant raw materials and mixtures containing plant raw materials, which is characterized in that it contains an enzyme having an amino acid sequence corresponding to the FumD hydrolase for degrading FB1.

[0007] In Heinl S, et al., J Biotechnol. 2010 Jan 15; 145(2):120-9, two genes, fumD and fumI, of Coleophora sp. MTA144 were reported, and their gene products degrade the mycotoxin FB1 through consecutive activities. In particular, their isolation, small-scale expression, and enzymatic activities were described.

[0008] However, considering the significant importance of fumonisin degradation in the agricultural food industry and the few known enzymatic fumonisin detoxification methods in the prior art, alternative enzymatic fumonisin degradation methods are needed. Summary of the Invention

[0009] The present invention describes the use of an enzyme in the enzymatic degradation of a group of mycotoxins, namely fumonisins, said enzyme comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, and a method for fumonisin degradation based on the incubation of a target sample with said enzyme.

[0010] The inventors have demonstrated that the enzyme (also referred to herein as "FB1 hydr") is able to degrade different concentrations of fumonisin B1 (FB1) (Tables 4 and 5) in a rapid manner (Table 7), and, as observed in the thermal stability test (Table 8) and the pH stability test (Table 9) respectively, retains its ability to degrade fumonisins after incubation at different temperatures and different pH values. Moreover, the degradation of fumonisins has been achieved in different types of samples, such as in naturally contaminated maize samples (Table 10) and in gastric and intestinal fluid samples (Table 13). In addition, the FB1 hydr enzyme is able to degrade not only FB1, but also fumonisin B2 (FB2) and fumonisin B3 (FB3) (Table 14).

[0011] Based on these results, since mycotoxins pose a challenge in the agro-food field, the present invention shows clear industrial applicability. In fact, the use of FB1 hydr for fumonisin degradation presents several advantages. Compared to other methods, the enzymatic degradation of fumonisins avoids or reduces the possibility of generating unknown or unexpected by-products, which may be as dangerous as the original mycotoxins, since the enzyme targets specific toxins and removes them in a defined manner. Moreover, the degradation of fumonisins is accomplished in a rapid manner with a low dose of enzyme. Additionally, FB1 hydr has relevant properties of thermal and pH stability, retaining its ability to degrade fumonisins under different conditions.

[0012] Accordingly, one aspect of the present invention relates to the use of an enzyme in the degradation of fumonisins in a sample, said enzyme comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1; hereinafter referred to as "the use of the present invention".

[0013] As used herein, the term "fumonisin degradation" refers to the enzymatic modification of one or more fumonisin molecules, resulting in a decrease or loss of the concentration and / or activity of said fumonisin molecule. Methods and techniques for assessing the degradation of a compound / metabolite (specifically fumonisin molecules in the present invention) are known in the art and include, but are not limited to, mass spectrometry (MS) techniques.

[0014] The term "fumonisin" (abbreviated as "FB") refers to a group of mycotoxins produced by pathogenic fungi. Chemically, fumonisins are characterized by having two methyl groups (–CH3), one amino group (-NH2), one to four hydroxyl groups (-OH-), and two tricarboxylate ester groups, which are located at different positions on the linear polyketide backbone.

[0015] More than 15 fumonisin homologs have been discovered and identified to date, including fumonisin A, B, C, and P types. Among the fumonisin B types, FB1, FB2, and FB3 are the most abundant, and FB1, as the most toxic form, can coexist with other forms of fumonisins such as FB2 and FB3. These forms (FB1, FB2, and FB3) are the main food contaminants.

[0016] In the present invention, the enzyme used for fumonisin degradation is referred to in this document as "FB1 hydr" (or simply "FB1 enzyme"), and may also be referred to hereinafter as "the enzyme of the present invention", which comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:1.

[0017] SEQ ID NO:1

[0018] MSTARLTPTSASTLKLLSVAVAALTLSACLSGGGGGGADGPNPLAIETSEGKVVGISNDGIRVFRGIPYAAPPVGDLRLAPPQPPASRSETLRLSEEFGNSCPQSDLTTGQQVGNEDCLYLNVYAPAEAEDLPVMVWIHGGAFVFGNGGGEYDPTRLVEQDVIVVTLNYRLGNLGFLAHPALESDAGNFALMDQQLALAWVKENITAFGGDPANVTIFGESAGGHSVMSHIVSPRAEEADLFQRAIVQSGSYAPFQMPKATAQFLGTSVANGLGCTDPETAASCLRSLPVSAFLAAQGSQSIPVVDPDDDLLPKSIQQALADGDFNSSLDIMIGSNQNEGTLFVALDEVGGDPIDGEAEYRERVAEFFQPYQASIPFDDDQIATDYLDFVDGEAKPFEAALSGIWTDFMFACNAYSQASTFAGASMNTFQYWFRDEDAPWTLVPPFAV

[0019] Preferred embodiments provide for the use of the present invention, wherein the enzyme comprises an amino acid sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO:1. More preferably, the enzyme comprises or consists of the amino acid sequence SEQ ID NO:1.

[0020] Yet another more preferred embodiment provides for the use of the present invention, wherein the enzyme comprises an amino acid sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO:2. Even more preferably, the enzyme comprises or consists of the amino acid sequence SEQ ID NO:2.

[0021] SEQ ID NO:2

[0022] MAHHHHHHVGTGSNDDDDKSPDPMSTARLTPTSASTLKLLSVAVAALTLSACLSGGGGGGADGPNPLAIETSEGKVVGISNDGIRVFRGIPYAAPPVGDLRLAPPQPPASRSETLRLSEEFGNSCPQSDLTTGQQVGNEDCLYLNVYAPAEAEDLPVMVWIHGGAFVFGNGGGEYDPTRLVEQDVIVVTLNYRLGNLGFLAHPALESDAGNFALMDQQLALAWVKENITAFGGDPANVTIFGESAGGHSVMSHIVSPRAEEADLFQRAIVQSGSYAPFQMPKATAQFLGTSVANGLGCTDPETAASCLRSLPVSAFLAAQGSQSIPVVDPDDDLLPKSIQQALADGDFNSSLDIMIGSNQNEGTLFVALDEVGGDPIDGEAEYRERVAEFFQPYQASIPFDDDQIATDYLDFVDGEAKPFEAALSGIWTDFMFACNAYSQASTFAGASMNTFQYWFRDEDAPWTLVPPFAVSFPLGATHAGEIPYVLYPQAIMEQRYTGDPDDLNSLAGEMVDYWTQFAKTGDPNTTDGVAAAWQQAATGNLMTLDVPNASNANTLGFLGYHHCSYWADPPLVLR

[0023] It can be seen that SEQ ID NO:2 contains the amino acid sequence of SEQ ID NO:1.

[0024] In the present invention, "sequence identity" refers to the degree of similarity obtained by aligning two nucleotide or amino acid sequences. The degree of identity, expressed as a percentage, can be obtained based on the number of shared residues between the aligned sequences. The degree of identity between two nucleotide or amino acid sequences can be determined by conventional methods, such as using standard sequence alignment algorithms known in the art, such as BLAST [Altschul S.F. et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5; 215(3): 403-10]. The BLAST programs (such as BLASTN, BLASTX, TBLASTX, BLASTP, and TBLASTN) are publicly available on the website of the National Center for Biotechnology Information (NCBI).

[0025] The enzyme of the present invention can be obtained by techniques known in the prior art. Examples of techniques for obtaining a protein (such as the enzyme of the present invention) include, but are not limited to: chemical or biosynthesis, gene recombination, or expression of a polynucleotide encoding the enzyme of the present invention.

[0026] As shown hereinafter, the inventors have demonstrated that FB1 hydr can effectively degrade other toxins besides FB1, particularly FB2 and FB3. Thus, in a preferred embodiment of the use of the present invention (alone or in combination with other preferred embodiments of the present invention), the fumonisin degradation comprises the degradation of at least one fumonisin selected from the list consisting of FB1, FB2, FB3, and any combination thereof. More preferably, the fumonisin is FB1.

[0027] FB1, FB2, and FB3 are different types of fumonisins, and their chemical structures are summarized as follows:

[0028]

[0029] In another preferred embodiment of the use of the present invention (alone or in combination with other preferred embodiments), the fumonisin concentration is 1 to 25 ppm (parts per million). More preferably, the fumonisin concentration is 2 to 20 ppm. Even more preferably, the fumonisin concentration is selected from the list consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 ppm.

[0030] Furthermore, any sample contaminated with, capable of producing, or susceptible to fumonisins is suitable for the use of the present invention. However, in a preferred embodiment of the use of the present invention (alone or in combination with other preferred embodiments), the sample is a raw material sample or a mixture containing raw materials.

[0031] The term "raw material" refers to any resource existing in nature and materials that are unprocessed or minimally processed, including plant-based raw materials.

[0032] In a more preferred embodiment of the present invention, the raw material is a plant-based raw material.

[0033] Plant-based raw materials refer to raw materials of plant origin. Examples of plant-based raw materials include, but are not limited to, grains, herbs, fruits, vegetables, and intermediate products containing these substances for the production of food and feed, such as corn, wheat, and distillers grains.

[0034] In a further more preferred embodiment of the use of the present invention, the plant-based raw material is selected from the list consisting of corn, wheat, and distillers grains.

[0035] In another preferred embodiment of the use of the present invention (alone or in combination with other preferred embodiments), the sample is a food or feed product.

[0036] As used herein, the terms "food" and "feed product" have the same meaning and are interchangeable in this specification. The food product can be in liquid, powder, or solid form. The food is for human or non-human animal consumption, including feed and animal food. The food includes functional foods well known in the art. Examples of food include, but are not limited to, feed, dairy products, plant products, meat products, snacks, confectionery products, forage, beverages, baby food, grains, fried foods, industrial bakery products, and biscuits. Examples of dairy products include, but are not limited to, fermented dairy products (such as, but not limited to, yogurt or cheese) or non-fermented dairy products (such as, but not limited to, ice cream, butter, margarine, or whey). Plant products such as, but not limited to, grains in any form. In this context, "food and feed products" also refer to parts or small amounts separated therefrom.

[0037] In another preferred embodiment of the use of the present invention (alone or in combination with other preferred embodiments), the sample is a separated biological sample. Preferably, the separated biological sample is intestinal fluid and / or gastric fluid. More preferably, the separated biological sample is porcine intestinal fluid and / or porcine gastric fluid.

[0038] The term "separated biological sample" refers to any sample separated from a subject, including but not limited to biological fluids obtained from an individual by any method known to those skilled in the art for this purpose.

[0039] The inventors have achieved effective fumonisin degradation using different concentrations of the enzyme of the present invention. Preferred embodiments provide for the use of the present invention wherein the concentration of the enzyme of the present invention is from 2.5 μg / mL to 10,000 μg / mL (including the end point values of this range). More preferred embodiments provide for the use of the present invention wherein the concentration of the enzyme of the present invention is from 5 μg / mL to 1,000 μg / mL (including the end point values of this range). Even more preferably, the concentration of the enzyme of the present invention is selected from the list consisting of the following concentration values: 5, 10, 20, 25, 30, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 950 and 1,000 μg / mL.

[0040] In addition, the inventors have conducted fumonisin degradation tests under different pH conditions. Preferred embodiments (either alone or in combination with other preferred embodiments) provide for the use of the present invention wherein the pH value is from 5 to 8 (including the end point values of this range); more preferably, wherein the pH value is 6.0 - 8.0.

[0041] As described previously herein, applying the enzyme of the present invention to a sample has proven to achieve effective fumonisin degradation. The inventors of the present invention have also developed a method for fumonisin degradation.

[0042] Accordingly, a second aspect of the present invention relates to a method for degrading fumonisin in a sample (hereinafter referred to as "the method of the present invention"), comprising the following steps:

[0043] a) incubating the sample with an enzyme comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO:1.

[0044] Preferred embodiments provide for the method of the present invention wherein the enzyme comprises an amino acid sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO:1. More preferably, the enzyme of the present invention comprises or consists of the amino acid sequence SEQ ID NO:1.

[0045] Still more preferred embodiments provide for the method of the present invention wherein the enzyme comprises an amino acid sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO:2. Even more preferably, the enzyme comprises or consists of the amino acid sequence SEQ ID NO:2.

[0046] As used herein, the phrase "incubating a sample with an enzyme" means bringing the enzyme of the present invention into contact in a reaction mixture. As is known to those skilled in the art, the reaction mixture may contain other reagents and / or buffers known in the prior art. In a preferred embodiment of the method of the present invention (alone or in combination with other preferred embodiments), step (a) comprises incubating the sample with the enzyme of the present invention in a phosphate buffer, preferably at a concentration of 10 - 100 mM (including the end points of this range).

[0047] As described previously herein, the inventors have achieved effective fumonisin degradation using different concentrations of the enzyme of the present invention. Accordingly, a preferred embodiment provides a method of the present invention wherein the concentration of the enzyme of the present invention is from 2.5 μg / mL to 10,000 μg / mL (including the end points of this range). A more preferred embodiment provides a method of the present invention wherein the concentration of the enzyme of the present invention is from 5 μg / mL to 1,000 μg / mL (including the end points of this range). Even more preferably, the concentration of the enzyme of the present invention is selected from the list consisting of the following concentration values: 5, 10, 20, 25, 30, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 950 and 1,000 μg / mL.

[0048] In addition, step (a) may be carried out under specific incubation conditions. In a preferred embodiment of the method of the present invention, step (a) comprises incubating the sample with the enzyme of the present invention for 2.5 minutes to 24 hours (including the end points of this range). Preferably, step (a) comprises incubating the sample with the enzyme of the present invention for 2.5 minutes to 90 minutes (including the end points of this range). More preferably, step (a) comprises incubating the sample with the enzyme of the present invention for 5 to 30 minutes (including the end points of this range). Even more preferably, step (a) comprises incubating the sample with the enzyme of the present invention for a time selected from the list consisting of 5, 10, 15, 20, 25 and 30 minutes.

[0049] In another preferred embodiment of the method of the present invention (alone or in combination with other preferred embodiments), step (a) comprises incubating the sample with the enzyme of the present invention at a temperature of 30°C to 90°C (including the end point values of this range). More preferably, step (a) comprises incubating the sample with the enzyme of the present invention at a temperature selected from the list consisting of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90°C.

[0050] In another preferred embodiment of the method of the present invention (alone or in combination with other preferred embodiments), step (a) comprises incubating the sample with the enzyme of the present invention at a pH value of 5 to 8 (including the end point values of this range). Preferably, step (a) comprises incubating the sample with the enzyme of the present invention at a pH value selected from the list consisting of pH 5, pH 6, pH 7 and pH 8.

[0051] In an even more preferred embodiment of the method of the present invention, step (a) comprises incubating the sample with the enzyme of the present invention at a temperature of 37°C to 90°C (including the end point values of this range) and at a pH of 5 to pH 8 (including the end point values of this range) for 5 minutes to 90 minutes (including the end point values of this range).

[0052] As described in the foregoing aspect of the present invention, the inventors have demonstrated that FB1 hydr can effectively degrade other toxins in addition to FB1, especially FB2 and FB3. Therefore, in another preferred embodiment of the method of the present invention (alone or in combination with other preferred embodiments of the present invention), fumonisin degradation comprises the degradation of at least one fumonisin selected from the list consisting of FB1, FB2, FB3 and any combination thereof. More preferably, the fumonisin is FB1.

[0053] In another preferred embodiment of the method of the present invention (alone or in combination with other preferred embodiments), the fumonisin concentration is 1 to 25 ppm (parts per million). More preferably, the fumonisin concentration is 2 to 20 ppm. Even more preferably, the fumonisin concentration is selected from the list consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 ppm.

[0054] In addition, as described in the foregoing aspect of the present invention, the enzyme of the present invention can effectively degrade fumonisins in various samples.

[0055] In a preferred embodiment of the method of the present invention (alone or in combination with other preferred embodiments of the method of the present invention), the sample is a raw material sample or a mixture containing raw materials. In a more preferred embodiment of the method of the present invention, the raw material is a plant-based raw material.

[0056] In a further more preferred embodiment of the method of the present invention, the plant-based raw material is selected from the list consisting of corn, wheat, and distillers grains.

[0057] In another preferred embodiment of the method of the present invention, the sample is a food or feed product.

[0058] In another preferred embodiment of the method of the present invention (alone or in combination with other preferred embodiments), the sample is a separated biological sample. Preferably, the separated biological sample is intestinal fluid and / or gastric fluid. More preferably, the separated biological sample is porcine intestinal fluid and / or porcine gastric fluid.

[0059] The terms used to define the method of the present invention have been described in the foregoing aspects of the invention, and these terms and their preferred embodiments are equally applicable to the method of the present invention. Detailed Description of the Invention

[0060] Examples

[0061] 1. Materials and Methods

[0062] 1.1. Enzyme source, production and purification

[0063] Using the DIAMOND search tool, with the FumD sequence (SEQ ID NO:3) as the search target and using (internal and public) sequence databases, the sequence encoding the FB1hydr enzyme (SEQ ID NO:4) was selected. The DIAMOND search used default parameters (sequence identity > 60%; alignment length > 70; e-value < 1e -5 )). The identified sequence encoding FB1hydr (SEQ ID NO:4) was used as a template for gene synthesis, and the gene was codon-optimized to maximize its expression in Escherichia coli (E. coli).

[0064] SEQ ID NO:3

[0065] MKEHQCRGGRASPAAPATWLARISVSRGASAIAWTFMLGATAIPVAAQTDDPKLVRHTQSGAVEGVEGDVETFLGIPFAAPPVGDLRWRPPAPPRAWAGTRDGRRFAPDCIGNERLREGSRAAGTSEDCLYLNIWSPKQVGKGGLPVMIWVYGGGFSGGSGAVPYYDGSALAQKGVVVVTFNYRAGILGFLAHPALSKESPNGVSGNYGLLDMLAAFKWVQNNIREFGGDPNRVTVFGESAGASALGLLLTSPLSESAFNQAILQSPGLARPLATLSESEANGLELGADISALRRADAGLTKIAQSRIPMSRQFTKPRPMGPILDGYVLRTLDVDAFAKGAFRKIPVLVGGNADEGRAFTDRLPVKTVLEYRAYLTEQFGDEADAWERCYPANSDADVPAAVARLFGDSQFNNGIELLSAAFAKWRTPLWRYRFTGIPGAGRRPATHGDEIPYVFANLGPSSVSMFGSLEGGAGASDIKLATEMSAAWVSFAVHGVPDQGTKSHWPRFERRGEIMTFGSQVGSGEGLGVSPSKACQPSK

[0066] SEQ ID NO:4

[0067]

[0068] The two sides of this gene are restricted enzyme digestion sites of BamHI and HindIII (stop codon), and it is delivered into the pET-45b(+) expression vector (GenScript, US) with an ampicillin resistance screening marker. The above plasmid introduced into Escherichia coli BL21(DE3) can support the expression of N-terminal histidine (His) fusion protein. Soluble His-tagged protein is produced and purified at 4 °C after binding to Ni-NTA His-Bind resin (Merck Life Science S.L.U., Madrid, Spain). SDS-PAGE analysis is carried out using a Mini PROTEAN electrophoresis system (Bio-Rad, Madrid, Spain), and the purity is evaluated to be >98%. The purified protein is stored at a concentration of 10 mg·mL -1 in 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.0) and stored at -86 °C for later use. In addition, the vector pPIC9 also synthesizes FB1hydr (optimizing codon usage for yeast), and this vector supports the expression of N-terminal histidine (his) fusion protein using methanol as an inducer, and FB1hydr is also synthesized in Pichia pastoris (P. pastoris) using methanol as an inducer. The pPIC9 vector inserted with this enzyme gene carries a functional HIS4 gene, while the used Pichia pastoris strain GS115 (phenotype Mut+) has a defect (his4) in this gene. The expression yields in Escherichia coli and Pichia pastoris are shown in Table 1.

[0069] Table 1. Expression yields of FB1hydr

[0070] Vector Host Yield (g / L) pET-45b(+) E.coli BL21(DE3) 1 g / L culture pPIC9 P.pastoris 0.05 g / L culture

[0071] The following details the detailed protocol for expressing His6-tagged enzyme that degrades FB1 in the pET-45b(+) vector and Escherichia coli BL21(DE3) host:

[0072] - Pick a single colony and use it to inoculate a 50 mL Luria Bertani (LB) broth medium (0.1 - 0.25 L flask) containing an antibiotic (ampicillin [Amp] 50 μg / mL). Terrific Broth medium can be used to replace the LB medium.

[0073] - Subsequently, incubate the culture at 37 °C and 150 rpm until the OD value reaches 0.8 - 1 (OD at 600 nm).

[0074] - The next day, add the above pre-inoculum to a 2.5 L flask containing 1 L of LB + Amp 50 μg / mL and grow it at 37 °C and 150 rpm until the optical density (OD) value reaches between 0.8 - 1 (OD measured at 600 nm).

[0075] - The method of inducing the culture depends on the plasmid. For the FB1-degrading enzyme, the plasmid is pET45b(+), and 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) is used. Induce the culture overnight at 16 °C and 200 rpm (EH730 can also be obtained in the plasmid pET45b(+)). The standard expression temperature is 16 °C, but the enzyme can also be efficiently produced at 30 - 37 °C

[0076] as well.

[0077] - Centrifuge the culture at 4 °C and 8000 rpm for 5 minutes.

[0078] - Resuspend the pellet in 20 mL of HEPES (40 mM, pH 7) to wash the pellet, and centrifuge at 8000 rpm for 5 minutes at 4 °C.

[0079] - Resuspend the pellet in 20 mL of 40 mM HEPES, and then lyophilize. Approximately 1 g of lyophilized product is obtained per liter of culture. The protein can also be purified using HIS-Select nickel affinity gel.

[0080] The detailed protocol for expressing the His6-tagged enzyme that degrades FB1 in pPIC9 and the host Pichia pastoris GS115 is described in detail below:

[0081] - Inoculate a single colony into 25 mL of BMG (buffered minimal glycerol) in a 250 mL flask (optional: baffled flask). Grow it at 28 - 30 °C and 200 - 250 rpm until OD600 = 4 - 8.

[0082] - Collect the cells by centrifugation at 1500 g.

[0083] - Resuspend the cells in BMM medium (100 - 200 mL of BMM (buffered minimal methanol) medium) to OD600 = 1, and place them in a 1 L flask, preferably with baffles.

[0084] - When transferring to BMM medium and every 24 hours thereafter, add methanol to a final concentration of 0.5%.

[0085] - To detect the maximum protein expression level, collect aliquots at 0, 6, 12, 24 hours, and then every 12 hours (ideally, but collecting 2 aliquots per day is sufficient, e.g., once in the morning and once in the afternoon).

[0086] - After determining the optimal expression conditions, the cells were separated from the supernatant by centrifugation at 1500 g, and then the supernatant containing the enzyme was obtained. This supernatant can be directly freeze-dried or precipitated, or purified using HIS-Select nickel affinity gel because the C-terminus of this protein contains a 6-His tag.

[0087] 1.2. Preparation of FB1hydr samples for degradation tests

[0088] Three different types of samples were prepared to test their ability to degrade FB1 toxin.

[0089] - The first sample is a sample consisting of purified FB1hydr, hereinafter referred to as FB1hydr (pure product). To prepare this sample, soluble His-tagged protein was first prepared and purified at room temperature by binding to nickel-nitrilotriacetic acid (Ni-NTA) His-Bind resin (purchased from Merck Life Science S.L.U., Madrid, Spain). Briefly, monoclonal Escherichia coli (E. coli) (previously grown at 37 °C on solid Luria Bertani (LB) agar medium containing 100 μg / mL ampicillin (Amp)) was picked and used to inoculate 50 mL of LB-Amp medium (in a 0.25 L conical flask), and then cultured overnight at 37 °C and 150 rpm. Then, 50 mL of the above culture was inoculated into 1 L of LB-Amp medium (in a 2.5 L conical flask) and cultured at 37 °C until the OD600nm reached 0.7 - 0.9. Protein expression was induced by adding IPTG to a final concentration of approximately 1 mM, followed by incubation at 16 °C for 16 hours. Cells were collected by centrifugation at 8000 g for 10 minutes, yielding a pellet of 2 - 3 grams (wet weight) per liter. The wet cell pellet was resuspended in 15 mL of 50 mM sodium phosphate (pH 8.0), 10 mM imidazole, and 300 mM NaCl. 15 μL of lysozyme bioprocessing reagent (Novagen, Darmstadt, Germany) was added, and the mixture was incubated with rotation and stirring on ice for 60 minutes. The cell suspension was sonicated for a total of 5 minutes and centrifuged at 15000 × g for 15 minutes at 4 °C, and the supernatant was retained. After binding to HIS-Select nickel affinity gel (Merck Life Science S.L.U., Madrid, Spain), the soluble His-tagged protein was purified at 4 °C and eluted according to the corresponding protocol. Briefly, once the supernatant had flowed through the column, the resin was washed three times with 50 mM sodium phosphate buffer (pH 8) containing 0.3 M NaCl and 10 mM imidazole to remove proteins not bound to the Ni-resin. Finally, the His-tagged protein was eluted with 50 mM sodium phosphate buffer (pH 8) containing 0.3 M NaCl and 250 mM imidazole. The purified protein solution was ultrafiltered and concentrated through a low-binding hydrophilic 10000 nominal molecular weight limit cut-off membrane (regenerated cellulose, Amicon). Then, the protein solution in 40 mM HEPES buffer (pH 7.0) was dialyzed extensively using a Pur-A-LyzerTM Maxi 1200 dialysis kit (Sigma-Aldrich, Missouri, USA) as follows. At room temperature, 0.5 - 3 mL of the concentrated protein solution was dialyzed against 2 L of buffer for 1 hour, after which the buffer was changed to another 2 L of buffer and maintained for more than 1 hour. Then, the buffer was changed again, and dialysis was carried out overnight at 4 °C. The dialyzed protein solution was recovered and then lyophilized.

[0090] - The second sample is a sample consisting of a protein extract containing FB1 hydr, herein referred to as FB1 hydr (semi-pure). To prepare this sample, a soluble His-tagged protein was first prepared and purified at room temperature by binding to nickel-nitrilotriacetic acid (Ni-NTA) His-Bind resin (purchased from Merck Life Science S.L.U., Madrid, Spain). Briefly, monoclonal Escherichia coli (E. coli) (previously grown at 37 °C on solid LB agar medium containing 100 μg / mL ampicillin (Amp)) was picked and inoculated into 50 mL of LB-Amp medium (0.25 L conical flask), and then cultured overnight at 37 °C and 150 rpm. Then, 50 mL of the above culture was inoculated into 1 L of LB-Amp medium (2.5 L conical flask) and cultured at 37 °C until the OD600nm reached 0.7–0.9. Protein expression was induced by adding IPTG to a final concentration of approximately 1 mM, followed by incubation at 16 °C for 16 hours. Cells were collected by centrifugation at 8000g for 10 minutes, yielding a pellet of 2-3 grams (wet weight) per liter. The wet cell pellet was resuspended in 15 mL of 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.0). 15 μl of lysozyme bioprocessing reagent (Novagen, Darmstadt, Germany) was added, and the mixture was incubated with rotation and stirring on ice for 60 minutes. The cell suspension was sonicated for a total of 5 minutes and centrifuged at 15000×g for 15 minutes at 4 °C. The supernatant was retained and then freeze-dried.

[0091] - The third sample is a sample composed of FB1 hydr immobilized on Lifetech ECR8806M Purolite resin, referred to herein as FB1 hydr (Purolite). To prepare this sample, a soluble His-tagged protein was first prepared and purified at room temperature by binding to nickel-nitrilotriacetic acid (Ni-NTA) His-Bind resin (purchased from Merck Life Science S.L.U., Madrid, Spain). Briefly, a monoclonal Escherichia coli (E. coli) (previously grown at 37 °C on solid LB agar medium containing 100 μg / mL ampicillin (Amp)) was picked and inoculated into 50 mL of LB-Amp medium (in a 0.25 L conical flask), and then cultured overnight at 37 °C and 150 rpm. Then, 50 mL of the above culture was inoculated into 1 L of LB-Amp medium (in a 2.5 L conical flask) and cultured at 37 °C until the OD600nm reached 0.7–0.9. Protein expression was induced by adding IPTG to a final concentration of approximately 1 mM, followed by incubation at 16 °C for 16 hours. Cells were collected by centrifugation at 8000 g for 10 minutes, yielding a pellet of 2-3 grams (wet weight) per liter. The wet cell pellet was resuspended in 15 mL of 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.0). 15 μL of lysozyme bioprocessing reagent (Novagen, Darmstadt, Germany) was added, and the mixture was incubated with rotation and stirring on ice for 60 minutes. The cell suspension was sonicated for a total of 5 minutes and centrifuged at 15000×g for 15 minutes at 4 °C, and the supernatant was retained. Then, 1 gram of Lifetech ECR8806M Purolite resin was added, and the suspension was kept at 150 rpm at room temperature overnight. Subsequently, the suspension was filtered, and the resin was retained and dried under vacuum.

[0092] 1.3. FB1 degradation test

[0093] The ability of different batches of the prepared samples - FB1 hydr (pure), FB1 hydr (semi-pure), and FB1 hydro (Purolite) - to degrade FB1 toxin was tested separately. The experimental procedure is as follows:

[0094] - The lyophilized enzyme material was redissolved in 0.1 M phosphate buffer (pH 7.0).

[0095] - A certain amount of the enzyme solution was taken and added to a solution containing 2 ppm of FB1.

[0096] - The solution was incubated at 37 °C for 5 minutes to 24 hours.

[0097] - The degradation of FB1 was evaluated by MS / MS.

[0098] 1.4. Ability of FB1 hydr mutants to degrade FB1

[0099] To demonstrate the ability of the FB1 hydr enzyme with the sequence SEQ ID NO:1 (hereinafter referred to as FB1h) to degrade fumonisins (especially FB1), the inventors selected two regions for mutant design, namely the active site pocket (hereinafter referred to as the "pocket", or FB1h_p) and the overall protein structure (hereinafter referred to as the "non-pocket", or FB1h_np). The inventors designed two mutants in each region (a total of 4 mutants, see Table 2). The first type is called the soft mutant: FB1h_p_s or FB1h_np_s, which have 1 or 8 mutations respectively; the second type is called the hard mutant: FB1h_p_h or FB1h_np_h, which have 9 or 81 mutations respectively. The inventors observed that sequences with different identity levels would retain or not retain the ability to degrade FB1.

[0100] Table 2. Number of mutations introduced in each region and each recognition sequence

[0101]

[0102] To design the mutants, the following protocol was applied. First, the inventors used three-dimensional modeling to obtain a three-dimensional model of the FB1h enzyme. Second, the pocket region and the non-pocket region were determined by fpocket computational analysis. Finally, evolutionary trace analysis was used to select positions that are less important from an evolutionary perspective and less harmful amino acid substitutions; this final analysis was necessary to avoid mutating residues that are crucial for maintaining the enzyme structure and function. Briefly, the pocket region consists of 43 amino acids and the non-pocket region consists of 405 amino acids.

[0103] In the following SEQ ID NO:1 sequence, the amino acids that conform to the active site pocket are shown in bold, and all other amino acids (those not shown in bold) constitute the pocketless region:

[0104] MSTARLTPTSASTLKLLSVAVAALTLSACLSGGGGGGADGPNPLAIETSEGKVVGISNDGIRVFRGIPYAAPPVGDLRLAPPQPPASRSETLRLSEEFGNSCPQSDLTTGQQVGNEDCLYLNVYAPAEAEDLPVMVWIHGGAFVFGNGGGEYDPTRLVEQDVIVVTLNYRLGNLGFLAHPALESDAGNFALMDQQLALAWVKENITAFGGDPANVTIFGESAGGHSVMSHIVSPRAEEADLFQRAIVQSGSYAPFQMPKATAQFLGTSVANGLGCTDPETAASCLRSLPVSAFLAAQGSQSIPVVDPDDDLLPKSIQQALADGDFNSSLDIMIGSNQNEGTLFVALDEVGGDPIDGEAEYRERVAEFFQPYQASIPFDDDQIATDYLDFVDGEAKPFEAALSGIWTDFMFACNAYSQASTFAGASMNTFQYWFRDEDAPWTLVPPFAV

[0105] Four mutants (SEQ ID NO:5 to SEQ ID NO:8) were synthesized, produced, and purified as in the wild type. Briefly, the mutants were produced in Escherichia coli. After expression, cell-free sonicated protein extracts were produced and freeze-dried. Approximately 1 g of freeze-dried material was produced for shipment. Along with the 4 mutants, the inventors also shipped the wild-type enzyme. All samples were shipped in the same form (freeze-dried material).

[0106] FB1h_p_s

[0107] SEQ ID NO:5

[0108] MSTARLTPTSASTLKLLSVAVAALTLSACLSGGGGGGADGPNPLAIETSEGKVVGISNDGIRVFRGIPYAAPPVGDLRLAPPQPPASRSETLRLSEEFGNSCPQSDLTTGQQVGNEDCLYLNVYAPAEAEDLPVMVWIHGGAFVFGNGGGEYDPTRLVEQDVIVVTLNYRLGNLGFLAHPALESDAGNFALMDQQLALAWVKENITAFGGDPANVTIFGESAGGHSVMSHIVSPRAEEADLFQRAIVQSGSYAPFQMPKATAQFLGTSVANGLGCTDPETAASCLRSLPVSAFLAAQGSQSIPVVDPDDDLLPKSIQQALADGDFNSSLDIMIGSNQNEGTLFVALDEVGGDPIDGEAEYRERVAEFFQPYQASIPFDDDQIATDYLDFVDGEAKPFEAALSGIYTDFMFACNAYSQASTFAGASMNTFQYWFRDEDAPWTLVPPFAV

[0109] FB1h_p_h

[0110] SEQ ID NO:6

[0111] MSTARLTPTSASTLKLLSVAVAALTLSACLSGGGGGGADGPNPLAIETSEGKVVGISNDGIRVFRGIPYAAPPVGDLRLAPPQPPASRSETLRLSEEFGNSCPQSDLTTGQQVGNEDCLYLNVYAPAEAEDLPVMVWIHGGAFVFGNGGGEYDPTRLVEQDVIVVTLNYRLGNLGFLAHPALESDAGNFALMDQQLALAWVKENITAFGGDPANVTIFGESAGGHSVMSHIVSPRAEEADLFQRAIVQSGSYAPFQMPKATAQFLGTSVANGLGCTDPETAASCLRSLPVSAFLAAQGSQSIPVVDPDDDLLPKSINQALADGDFNSSLDIMIGSNQNEGTLFVALDEVGGDPIDGEAEYRERVADYFQPYQGTIPFDDDQIATEYLDFVDGEAKPFEAALSGLYTDYMFACNAYSQASTFAGASMNTFQYWFRDEDAPWTLVPPFAV

[0112] FB1h_np_s

[0113] SEQ ID NO:7

[0114] MSTARLTPTSASTLKLLSVAVAALTLSACLSGGGGGGADGPNPLAIETSEGKVVGISNDGIRVFRGIPYAAPPVGDLRLAPPQPPASRSETLRLSEEFGNSCPQSDLTTGQQVGNEDCLYLNVYAPAEAEDLPVMVWIHGGAFVFGNGGGEYDPTRLVEQDVIVVTLNYRLGNLGFLAHPALESDAGNFALMDQQLALAWVKENITAFGGDPANVTIFGESAGGHSVMSHIVSPRAEEADLFQRAIVQSGSYAPFQMPKATAQFLGTSVANGLGCTDPETAASCLRSLPVSAFLAAQGSQSIPVVDPDDDLLPKSIQQALAEGEFNSSIDIMIGSNQNEGTLFVALDEVGGDPIDGEAEYRERVAEFFQPYQASIPFDDDQIATDYLDFVDGEAKPFEAALTAIWSDFMFACNAFSQASTFAGASLNTFQYWFRDEDAPWTLVPPFAV

[0115] FB1h_np_h

[0116] SEQ ID NO:8

[0117] MSTARLTPTSASTLKLLSVAVAALTLSACLSGGGGGGADGPNHLLIDTTEGRVVGLTNEAIRVFRGIPYAAPPVGELRLAPPQHPATKSETLRLSEDFGNTCPQTELSTGQNVGNEDCLYLNVYAPGDGDDLPVMVWIHGGAFLYAQGAAEYDPTRLVEQDVIVVTLNYRLGNLGFLAHPALDSDGGNFALMDQQLALAWVKENITAFGGDPGNVTIFGESAGGHSVMSHIVSPRAEDADLFQRAIVQSGSYAPFQMPKATAQFLGTSVANGLGCTDPETAASCLRTLPVTGFIGGQGSQSIPVVDPEEELLPRTIQNALGEGEYNTSIDIMIGSNQNEGTLFVALDEVGGEPIDADGEYRDKVGEFFQPYQASIPFDDDNIATDYLDFVEGDAKPFEAGLTAIWSDFMFACNAFSQASSFGAASLNTFQYWFRDEDAPWTLVPPFAV

[0118] 2. Results

[0119] 2.1. General characteristics of FB1 hydr

[0120] The identified sequence, SEQ ID NO:1, showed significant differences from FumD, with an identity of 30.1% at the amino acid level. To verify the activity of this enzyme, the gene encoding FB1hydr was synthesized, and the protein was produced and purified.

[0121] Degradation assays using different batches of protein samples (purified, semi-purified, and immobilized) confirmed its ability to degrade FB1. The results showed that FB1hydr was active against FB1: 2 ppm of FB1 was 100% degraded after 5 minutes of reaction (Table 3).

[0122] Table 3. FB1 degradation efficiency (tested at 37 °C)

[0123]

[0124]

[0125] When experiments were conducted with 10 ppm FB1, 100% degradation was found after 5 minutes. A full MS / MS scan was performed on the reaction mixture (in which 10 ppm FB1 was 100% degraded) to screen for possible metabolites. The data obtained were compared with the literature data, and possible metabolites were identified: confirmed as HFB1 (hydrolyzed fumonisin B1), a non-toxic metabolite of FB1.

[0126] The thermal stability within 3 minutes at 90 °C was further tested. For this purpose, the enzyme materials in powder form (FB1-hydr (purolite) and FB1-hydr (semi-purified)) were incubated at 90 °C for 3 minutes, then buffer and FB1 toxin were added, and the degradation percentage was evaluated. The results summarized in Table 4 showed that both “FB1hydr” enzyme materials (FB1-hydr (semi-purified) and purolite) could degrade 2 ppm FB1 within 5 minutes and maintained thermal stability at 90 °C.

[0127] Table 4. Thermal stability test (90 °C, 3 minutes)

[0128]

[0129] 2.2. In-depth characterization of semi-purified FB1 hydr

[0130] 2.2.1. Enzyme dose-dependent degradation

[0131] The degradation efficiency of different doses of FB1 hydr enzyme was further evaluated. As shown in Table 5, when the enzyme concentration was tested from 5 to 0.1 mg / mL, we found that the enzyme activity was the highest at 0.1 mg / mL (100 μg / mL). When experiments were conducted with a low dose of FB1 enzyme, we found that 2 ppm FB1 could be 100% degraded after 30 minutes of incubation under the condition that the enzyme concentration was as low as 10 μg / mL (Table 6).

[0132] FB1 enzyme dose, conditions:

[0133] - 2 ppm FB1

[0134] - 30 minutes

[0135] - pH 7.0

[0136] Table 5. Degradation efficiency of enzyme dose

[0137]

[0138]

[0139] 1 Use semi-purified FB1 enzyme.

[0140] Table 6. Degradation Efficiency of Enzyme Dosage

[0141]

[0142] 1 Use semi-pure FB1 enzyme.

[0143] 2.2.2. Toxin dose-dependent degradation

[0144] The degradation efficiency of different dosages of FB1 toxin was further evaluated. As shown in Table 7, at an enzyme concentration of 25 μg / mL, 100% degradation was achieved when using 2 - 20 ppm of FB1 toxin. It can be considered that for low contamination cases of 2 ppm or lower, an enzyme concentration of 10 μg / mL is appropriate.

[0145] Toxin dosage, conditions:

[0146] - 25 μg / mL, 10 μg / mL.

[0147] - 2 ppm FB1, 5 ppm FB1, 10 ppm FB1, 20 ppm FB1

[0148] - 30 minutes

[0149] - pH 7.0

[0150] Table 7. Degradation Efficiency of Toxin Dosage at Two Enzyme Dosages

[0151]

[0152]

[0153] 1 Use semi-pure FB1 enzyme.

[0154] 2 The content of acetonitrile (ACN) in the experimental solution was 8.3%, which had a negative impact on the performance of the enzyme. Therefore, the optimal FB1 concentration was 10 ppm because it had an ACN content of no more than 4% and the enzyme remained stable.

[0155] 2.2.3. Time course of FB1 degradation

[0156] Take 25 μg / mL FB1 enzyme, 2 ppm and 10 ppm FB1 toxin, and sample at 5, 10, 20, and 30 minutes respectively under the condition of pH 7.0 to evaluate the degradation efficiency. As shown in Table 8, the degradation was quite rapid, and complete degradation of 2 ppm and 10 ppm was achieved after 5 minutes of incubation.

[0157] Table 8. Time Course of FB1 Degradation

[0158]

[0159] 1 Use semi-purified FB1 enzyme.

[0160] 2.2.4. Thermal stability of FB1 enzyme

[0161] The thermal stability of FB1 enzyme was tested by degrading FB1 toxin as described below:

[0162] - Take 25 μg / mL of FB1 enzyme and incubate at 90 °C for 3 minutes.

[0163] - Then add 2 ppm and 10 ppm of FB1 toxin and take samples at 30 minutes.

[0164] - The buffer pH is 7.0.

[0165] - As shown in Table 8, the degradation was quite rapid, and complete degradation to 2 ppm and 10 ppm was achieved after 5 minutes of incubation.

[0166] As shown in Table 9, the enzyme was quite stable after incubation at 90 °C and retained the ability to degrade 100% of the toxin (2 - 10 ppm) under the condition of low-dose enzyme (25 μg / mL).

[0167] Table 9. Thermal stability of FB1 enzyme

[0168] <![CDATA[Enzyme concentration (μg / mL) 1 > FB1 (ppm) Reaction time (min) DEG, % 25 2 30 100 25 10 30 100

[0169] 1 Use semi-purified FB1 enzyme.

[0170] 2.2.5. pH stability of FB1 enzyme

[0171] When degradation tests were carried out at different pH values (3, 4, 5, 6, 7, 8), almost complete degradation was found at pH values from 5 to 8 (Table 10).

[0172] Table 10. pH stability of FB1 enzyme

[0173]

[0174] 1 Use semi-purified FB1 enzyme.

[0175] 2.3. Detection of FB1 in corn matrix

[0176] The ability of FB1 enzyme to degrade FB1 toxin in naturally contaminated corn samples was further evaluated as follows:

[0177] - The total amount of corn ground was 1802 μg / kg (FB1 and FB2).

[0178] - Weigh 1 g of the sample, add 3 mL of 0.1 M phosphate buffer (pH 7.0), according to Wang et al.,

[0179] Liquid Chromatography-Tandem Mass Spectrometry for Determination of Aflatoxin B1, Deoxynivalenol and Zearalenone in Artificial Porcine Gastrointestinal Digestive Juice, Chin J Anal Chem, 2015, 43(1), 1-6. Set the ratio to 1:3 and set three replicates.

[0180] As shown in Table 11, almost complete degradation was observed after 30 - 90 minutes of incubation when the enzyme was supplied at a rate of 10 - 25 mg per kg of naturally contaminated corn. This was observed in both cases when two different concentrations of FB1 were used in the corn (Table 11).

[0181] Table 11. Degradation efficiency in naturally contaminated corn

[0182]

[0183] 1 Use semi-pure FB1 enzyme. 10 mg / Kg: For 1 kg / ton of feed, it is equivalent to 10 × 1000 mg / kg of the product, or 10 g / kg = MycoRaid addition amount of 1%.

[0184] 2.4. Addition cost of FB1 enzyme

[0185] The efficiency of pure and semi-pure FB1 enzymes was further evaluated as follows:

[0186] - Take each enzyme (pure or semi-pure) at 5 μg / mL and FB1 at 2 ppm and 10 ppm for testing, and sample at 5, 10, and 30 minutes.

[0187] - The thermal stability of each enzyme (pure or semi-pure), with a dosage of 10 μg / mL, 2 ppm FB1, and the test lasts for 30 minutes.

[0188] - Use pure or semi-pure FB1 enzyme to degrade FB1 in corn matrix, with dosages of 10 μg / mL and 25 μg / mL, and the test lasts for 30 minutes.

[0189] As shown in Table 12, we found that both enzyme preparations (pure or semi-pure) were very efficient and could completely degrade 2 ppm toxin at a dose of 5 μg enzyme / mL after 5 - 30 minutes of incubation.

[0190] Table 12. Degradation tests at 5, 10, and 30 minutes using purified or semi-purified enzyme (5 μg / mL) and 2 ppm and 10 ppm of FB1

[0191]

[0192] The efficiency of pure and semi-purified enzymes in degrading FB1 was also tested in naturally contaminated maize (Table 13). For this purpose, 10 μg / mL and 25 μg / mL of each enzyme preparation were used and incubated for 30 minutes. As shown in Table 13, almost complete or complete degradation was obtained in both cases.

[0193] Table 13. Degradation tests at 30 minutes when using purified or semi-purified enzymes to treat naturally contaminated maize

[0194]

[0195]

[0196] Based on the above results, it may be necessary to add the FB1 enzyme (pure or semi-purified enzyme) at a ratio of 10 - 25 mg per 1 kg of product. Using this ratio, it was finally found that the FB1 toxin was completely degraded in the gastric and intestinal fluids of pigs (Table 14).

[0197] Table 14. Degradation tests in porcine intestinal fluid

[0198]

[0199] 1 Use semi-purified FB1 enzyme.

[0200] 2.5. Toxin degradation test

[0201] The efficiency of the semi-purified FB1 enzyme in degrading toxins other than FB1, specifically AFB1, FB2, FB3, T-2, ZEN, and OTA, was further evaluated. The test method was the same as that for FB1, and the results are summarized in Table 15.

[0202] Table 15. Degradation tests of various toxins by FB1 hydr (semi-purified); DON = deoxynivalenol; AFB1 = aflatoxin B1; FB1 = fumonisin B1; FB2 = fumonisin B2; FB3 = fumonisin B3; T-2 = T-2 toxin; Zen = zearalenone; OTA = ochratoxin A.

[0203]

[0204] As shown in Table 15, this enzyme can not only effectively degrade FB1, but also effectively degrade FB2 and FB3, and its chemical structure has been given in the previous text of this document.

[0205] 2.6. Ability of FB1 hydr mutants to degrade FB1

[0206] As shown in Table 16, the results of the degradation experiment using an enzyme material with a concentration of 10 μg / ml showed that the wild-type enzyme (FB1h_WT) and the mutant FB1h_p_s had 98% identity at the active site pocket (99% identity when referring to the amino acid sequence with a total length of 448) and only one mutation, and both could completely degrade the FB1 toxin. Variants with a higher number of mutations (tested 8 to 81 mutations) in the active site (catalytic "pocket" region) or the overall protein structure ("non-pocket" region) retained the degradation activity, but the efficiency was significantly reduced. In fact, the mutants FB1h_p_h (incorporated 9 mutations in the active site pocket), FB1h_np_s (incorporated 8 mutations in the "non-pocket"), and FB1h_np_h (incorporated 81 mutations in the "non-pocket") could degrade the FB1 toxin under the same conditions, but the degradation percentage was 0.4 - 17.5%, depending on the enzyme concentration. This indicates that sequences with 99% identity to SEQ ID NO:1 retained the ability to degrade the FB1 toxin, while sequences with 98 - 82% identity could not degrade FB1 as effectively as the wild type.

[0207] Table 16. FB1 degradation experiments using wild-type and variants designed for each region

[0208]

[0209]

[0210] This confirms that enzymes containing amino acid sequences with at least 82% sequence identity to the full-length sequence of SEQ ID NO:1 do show the ability to degrade fumonisin B, and the degradation level is significantly lower than that of the enzyme of SEQ ID NO:1; this depends on the number and nature of the mutations or different amino acids.

[0211] In addition to the design and testing of the above 4 mutants, two other sequences were selected for testing, FB1-4a (from the MarDB database; MMP09287984; Victivallales bacterium) and FB1-1a (from the MarDB database; MMP06457154; Phycisphaerales bacterium), which have approximately 26% and 25% sequence identity with FB1hydr (SEQ ID NO:1), respectively. The inventors found that both enzymes that are more distantly related to FB1hydr degrade the FB1 toxin, but at a lower level (Table 17).

[0212] Table 17. Degradation of FB1 Distant Homologous Proteins

[0213]

[0214] FB1-1a (from the MarDB database; MMP06457154; Phycisphaerales bacterium).

[0215] SEQ ID NO:9

[0216] MGFVLLVFMCNAMVKAQDTVTIKTNAGPVVGEVSDGIRVFKGIPYAAPPVGDLRWRAPQAVAPWQSPRQCQNFSSRCPQPDVSDRPWLGKAGPMDEDCLYLNVWAPSDVKKPLPVMVWIHGGGFTIGAGSLDFYEGKAFAKEQVVLVTINYRLGPFGFFGHEALAKESPNHVSGNYGLLDQIAALQWVQRNIQSFGGDPNCVTIFGESAGSVAVTCLMVSPLAKGLFHRAIAQSGTGTSIYQQLNEPHKRSKSLHATGNVLADKLGIDASDTDALAKLRNVSAQKLLEISNPQLISQGLQGTKFGPVIDGYVLPANPGKMFANGQQHPIPLMIGSNQQDGILHASALPIRRVQGYKWILNRLFGKDGPAVFDLFPAKTNDDIKQAKLDLMTVSAFAAPSRRLAAAMQNVATPAYLYEFTRISPAAKRKGLGAVHGAEIVYLFDTFGHPTGYDQTDHQLANQMRTYWINFATTGNPNGSNLPHWPTYDVTDQHMQFGDTSQAGKHLYKQACDIFDRVVIQEQ

[0217] FB1-4a (from MarDB database; MMP09287984; Victivallales bacterium).

[0218] SEQ ID NO:10

[0219] MRYPFLAFALFFAVAAPAAEPVVPCVAGPVRGERTAEGLCVFRGIPYAAPPVGELRWRAPQPVAPWTEVRDATAFSPACPQPPRPIPAVQSVGETNEDCLYLNVWTPELAPAKRSVMVWIHGGGMTIGAGSSEVYDGSALARQDVVVVTINYRLGVLGFLAHPELSAESPDKVSGNYGLLDQIAALRWVRDNIAAFGGDPGNVTIFGESAGSVSVGCLLVAPAAEGLFHRAIMESGAPSGVAEPLRDGKNSAEEQGLDIARRLKAKTLADLRAIPAADLVAGCPAAIGPTAKRKDNYKFGPCVDGVVLPDQPLRLLAEGKFHHVPILLGTNRDEMTLFLNREEKGKPKRKMGLRFVVRSLYKDQAEAVLAAFPCDRDEDAAFAFRDLMTVACFTSPCRYVANAAAAGGAPVYLYEFSRVASTVGRAGLGATHGIEIPYVFGTLKDTFGDGTDRTLSKAMGAYWVAFAHTGVPSATGLPAWPRYAAPADSYLEFGDTVTVGQGLRRAQCDAVEPALRERLSLPER

Claims

1. Use of an enzyme comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 1 in degrading fumonisins in a sample.

2. The use according to claim 1, wherein the enzyme comprises the amino acid sequence SEQ ID NO:

1.

3. The use according to claim 1 or 2, wherein the degradation of the fumonisins comprises the degradation of at least one fumonisin selected from the list consisting of fumonisin B1 (FB1), fumonisin B2 (FB2), fumonisin B3 (FB3), and any combination thereof.

4. The use according to any one of claims 1 to 3, wherein the fumonisin is FB1.

5. The use according to any one of claims 1 to 4, wherein the sample is a raw material sample or a mixture containing raw materials.

6. The use according to claim 5, wherein the raw material is a plant-based raw material.

7. The use according to claim 6, wherein the plant-based raw material is selected from the list consisting of corn, wheat, and distillers grains.

8. The use according to any one of claims 1 to 4, wherein the sample is a food or feed product.

9. The use according to any one of claims 1 to 4, wherein the sample is a separated biological sample, preferably wherein the separated biological sample is intestinal fluid and / or gastric fluid.

10. The use according to any one of claims 1 to 9, wherein the concentration of the enzyme is 5 μg / mL to 1000 μg / mL.

11. A method for degrading fumonisins in a sample, comprising the following steps: a) incubating the sample with an enzyme, the enzyme comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO:

1.

12. The method according to claim 11, wherein the degradation of the fumonisins comprises the degradation of at least one fumonisin selected from the list consisting of FB1, FB2, FB3, and any combination thereof.

13. The method according to claim 11 or 12, wherein the sample is a raw material sample or a mixture containing raw materials, preferably wherein the raw material is a plant-based raw material.

14. The method according to claim 11 or 12, wherein the sample is a food or feed product.

15. The method according to claim 11 or 12, wherein the sample is a separated biological sample.

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