Trichothecene-transforming alcohol dehydrogenase, method for transforming trichothecenes and trichothecene-transforming additive
11001812 · 2021-05-11
Assignee
Inventors
Cpc classification
A23K50/80
HUMAN NECESSITIES
A61K38/443
HUMAN NECESSITIES
International classification
Abstract
An alcohol dehydrogenase of sequence ID numbers 2, 3 or 4 containing metal ions and a quinone cofactor, or in addition, a functional variant exhibiting a sequence identity of at least 80%, preferably at least 86%, especially preferred at least 89% and at least one redox cofactor for the transformation of at least one trichothecene exhibiting a hydroxyl group on the C-3 atom, as well as a method for the enzymatic transformation of trichothecenes and a trichothecene-transforming additive.
Claims
1. A method for the enzymatic transformation of trichothecenes, wherein at least one trichothecene exhibiting a hydroxyl group on the C-3 atom is brought into contact with an alcohol dehydrogenase of sequence ID numbers 2, 3 or 4 containing metal ions and a quinone cofactor, or with a functional variant thereof having a sequence identity of at least 99% thereto, with at least one redox cofactor and water, and if necessary at least one excipient.
2. The method according to claim 1, wherein the trichothecene exhibiting a hydroxyl group on the C-3 atom is transformed at a temperature between 5° C. and 55° C.
3. The method according to claim 1, wherein at least one trichothecene exhibiting a hydroxyl group on the C-3 atom is brought into contact with the alcohol dehydrogenase containing metal ions and a quinone cofactor, or at least a functional variant thereof, with the redox factor, with water, and if necessary, with the excipient, for at least one minute.
4. The method according to claim 1, wherein the quinone cofactor is selected from the group pyrroloquinoline quinone (PCC), tryptophan tryptophylquinone (TTC), topaquinone (TPC), lysine tyrosylquinone (LTC), and cysteine tryptophylquinone (CTC).
5. The method according to claim 1, wherein the quinone cofactor is bound to the alcohol dehydrogenase by at least one metal ion selected from the group Li.sup.+, Na.sup.+, K.sup.+, Mg.sup.2+, Ca.sup.2+, Zn.sup.2+, Zn.sup.3+, Mn.sup.2+, Mn.sup.3+, Fe.sup.2+, Fe.sup.3+, Cu.sup.2+, Cu.sup.3+, Co.sup.2+ and Co.sup.3+.
6. The method according to claim 1, wherein the at least one redox cofactor is selected from the group phenazine methosulphate (PMS), PMS derivatives, potassium hexacyanoferrate (III), sodium hexacyanoferrate (III), cytochrome C, coenzyme Q1, coenzyme Q10, methylene blue, and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The invention is explained below based on embodiments and a drawing. Herein:
(2)
(3)
DETAILED DESCRIPTION OF THE INVENTION
Example 1: Cloning of the Genes and Purification of the Alcohol Dehydrogenase
(4) The codon-optimised nucleotide sequences of the alcohol dehydrogenase of SEQ ID numbers 1 to 4 for the respective host cell were taken from DNA2.0 and contained restriction sites at the nucleic acid level on the 5′ end and on the 3′ end of the sequence, and at the amino acid level, additionally a C- or N-terminal 6×His tag. These nucleotide sequences were integrated by means of standard methods in expression vectors for the expression in Escherichia coli or Komagataella pastoris, and transformed to E. coli or K. pastoris, and expressed in E. coli or K. pastoris (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).
(5) The alcohol dehydrogenases with SEQ ID numbers 1 to 4 were selectively fortified chromatographically from cell lysates in the case of the expression in E. coli and from the intercellular expression in K. pastoris or from the culture supernatant in the case of the extracellular expression in K. pastoris by means of standard methods via nickel sepharose columns. The selectively fortified eluates were incubated and activated in the presence of metal ions and quinone cofactors, in which case “activated” means that the alcohol dehydrogenases exhibit both the metal ion and the quinone cofactor as bound. These activated alcohol dehydrogenases were used to determine the enzymatic properties of the alcohol dehydrogenases with SEQ ID numbers 1 to 4 in examples 3 to 7 below. The total protein concentration was determined photometrically with the Bradford reagent (Sigma #B6916), in which case the absorptions were measured in a microplate photometer (plate reader, Biotek, Synergy HT) at a wavelength of 595 nm. The protein concentration was ascertained based on a calibration curve that was determined using the Bradford assay by measuring the bovine serum albumin (BSA, Sigma #A4919) solutions with concentrations up to a maximum of 1500 μg/ml.
Example 2: Determination of the Sequence Identity
(6) The percent sequence identity over the entire length of the amino acid sequence of the alcohol dehydrogenases with SEQ ID numbers 1-4 relative to each other was determined using the BLAST program (Basic Local Alignment Search Tool), especially BLASTP, which is available for use on the homepage of the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/), with which it is possible to compare two or more sequences with each other according to the algorithm by Altschul et al., 1997 (Nucleic Acids Res. (1997) 25:3389-3402). The basic settings were used as program settings, especially: “max target sequence”=100; “expected threshold”=10; “word size”=3; “matrix”=BLOSOM62; “gap costs”=“existence: 11; extension: 1”; “computational adjustment”=“conditional compositional score matrix adjustment”. The percentage identities of the amino acid sequences to one another are shown in Table 1:
(7) TABLE-US-00001 TABLE 1 SEQ ID SEQ ID SEQ ID SEQ ID no. 1 no. 2 no. 3 no. 4 SEQ ID no. 1 100% 87% 89% 86% SEQ ID no. 2 87% 100% 99% 90% SEQ ID no. 3 89% 99% 100% 91% SEQ ID no. 4 86% 90% 91% 100%
Example 3: Transformation of Trichothecene Exhibiting a Hydroxyl Group on the C-3 Atom
(8) To determine their suitability to transform trichothecenes that exhibit a hydroxyl group on the C-3 atom, especially DON, nivalenol and T-2 toxin, the alcohol dehydrogenases with SEQ ID numbers 1-4 were produced with a C-terminal 6×His tag in E. coli, as described in Example 1.
(9) A transformation is then present when the quantity of the trichothecene exhibiting a hydroxyl group on the C-3 atom is reduced by bringing it into contact with an activated alcohol dehydrogenase, i.e., an alcohol dehydrogenase that contains metal ions and a quinone cofactor.
(10) In each case, 100 ml of an E. coli culture with an optical density (OD600 nm) of 2.0-2.5 were harvested by centrifugation at 4° C. and resuspended in 20 ml potassium phosphate buffer. The cell suspensions were lysed by French press treatment 3 times at 20,000 psi. The cell lysates were separated into soluble and insoluble parts by centrifugation. The supernatant was filtered sterilely and the alcohol dehydrogenase was fortified by means of standard methods via nickel sepharose columns. Following this, a buffer exchange was performed by dialysis with specific tubes with a cut-off of ten kilodaltons. The resulting total protein concentration was measured by Bradford assay.
(11) The quinone cofactors and the metal ions were bound to the alcohol dehydrogenase by incubation in an aqueous solution. Here, the quinone cofactor, such as pyrroloquinoline quinone (PCC, CAS no. 72909-34-3), tryptophan tryptophylquinone (TTC, CAS no. 134645-25-3), topaquinone (TPC, CAS no. 64192-68-3), lysine tyrosylquinone (LTC, CAS no. 178989-72-5) and cysteine tryptophylquinone (CTC, CAS no. 400616-72-0), is added to the existing total protein concentration as an aqueous solution in an approximately twentyfold molar excess. The metal ions selected from Li.sup.+, Na.sup.+, K.sup.+, Mg.sup.2+, Ca.sup.2+, Zn.sup.2+, Zn.sup.3+, Mn.sup.2+, Mn.sup.3+, Fe.sup.2+, Fe.sup.3+, Cu.sup.2, Cu.sup.3+, Co.sup.2+ and Co.sup.3+ are used as an aqueous solution of a salt thereof. Unless otherwise indicated, the alcohol dehydrogenases were normally used with PCC (Sigma Aldrich #D7783) and Ca.sup.2+, activated as a 5 mM CaCl.sub.2 solution. The enzymes purified and activated in this manner were used for in vitro transformation assays of a trichothecene exhibiting a hydroxyl group on the C-3 atom. Unless otherwise indicated, the terms “enzyme or “alcohol dehydrogenase” are always understood to refer to the appropriately activated alcohol dehydrogenases containing metal ions and a quinone cofactor.
(12) The transformation assays were carried out in an aqueous solution with the following components: 100 mM Tris-HCl pH 7.5 or 10% Teorell Stenhagen pH 7.5; synthetic redox cofactor selected from group 1 mM phenazine methosulphate PMS (Sigma Aldrich #P9625), 1 mM methylene blue (Sigma #M9140), 1 mM coenzyme Q10 (Sigma #C9538), 1 mM coenzyme Q1 (Sigma #C9538) and 20 mM potassium hexacyanoferrate (111) PFC (111) (Fluke #60300); 10 ppm up to a maximum of 100 ppm of a trichothecene exhibiting a hydroxyl group on the C-3 atom by adding the desired quantity of a toxin substrate stock solution; and 10 nM to 100 nM, maximum 300 nM of an activated alcohol dehydrogenases of SEQ ID no. 1, 2, 3 or 4 containing metal ions and a quinone cofactor. Unless otherwise indicated, the Tris-HCl buffer, the redox cofactor PMS, DON and the alcohol dehydrogenase of SEQ ID no. 1 are normally used. Each transformation assay was carried out in a 1.5 ml brown Eppendorf reaction vessel. The reaction mixtures were incubated at 30° C. in a thermoblock for up to 120 minutes, at least 40 minutes. After 0, 10, 20, 30, and 40 minutes, a sample of 0.1 ml was taken in each case and mixed with 0.1 ml methanol and stored at −20° C., or alternatively analysed immediately by LC-MS/MS or HPLC.
(13) A sterilely filtered, aqueous 2000 ppm DON solution was used as the DON substrate stock solution. To produce this solution, DON in crystalline form (Biopure Standard from Romer Labs, art. no. 001050, pureness at least 98%) was weighed and dissolved. To quantify the trichothecenes exhibiting a hydroxyl group on the C-3 atom and their transformation metabolites, HPLC analyses were performed, wherein the substances were separated chromatographically by means of a Phenomenex C18 Gemini NX column with dimensions of 150 mm×4.6 mm and a particulate size of 5 μm. A methanol/water mixture with an ammonium acetate concentration of 5 mM was used as the eluant. The UV signal was recorded and evaluated at 220 nm. For the quantification by means of LC-MS/MS analyses, the substances were separated chromatographically by means of a Zorbax eclipse C8 column with dimensions of 150 mm×4.6 mm and a particulate size of 5 μm. A methanol/water mixture with an ammonium acetate concentration of 5 mM was used as the eluant. The UV signal at 220 nm was recorded. Electrospray ionisation (ESI) was used as the ionisation source. The trichothecenes exhibiting a hydroxyl group on the C-3 atom were quantified by means of a QTrap/LC/MS/MS (triple quadrupole, applied biosystems) in “enhanced mode”.
(14) The negative slope of the transformation lines (=reduction in the toxin concentration over time) in the linear range were used as a standard for the activity of the alcohol dehydrogenases. To determine the residual activities, the measured activities for different parameters relative to the basic activity, measured under standard conditions, especially 30° C. and pH 7.5, were applied and usually represented as percentages.
(15)
(16) To compare the efficiency of the quinone cofactors, in the transformation assays, 10 nM of the alcohol dehydrogenase of SEQ ID no. 1 activated with quinone cofactors PCC, TTC, TPC, LTC, and CTC, 10 ppm DON, and 1 mM synthetic redox factor PMS each were mixed in 100 mM Tris-HCl pH 7.5 and incubated at 30° C. The DON concentrations were determined by means of LC-MS/MS after 30 minutes. The results are shown in Table 2.
(17) To compare the efficiency of the redox cofactors, in the transformation assays, 10 nM activated enzyme (alcohol dehydrogenase of SEQ ID no. 1), 10 ppm DON, and 1 mM or 20 mM of the synthetic redox cofactors to be tested respectively were mixed in 100 mM Tris-HCl pH 7.5 and incubated at 30° C. The DON concentrations were determined by LC-MS/MS after 30 minutes. The results are shown in Table 2.
(18) TABLE-US-00002 TABLE 2 Quinone cofactor DON [ppm] Redox cofactor DON [ppm] PCC 1.94 1 mM PMS 1.95 TTQ 2.32 20 mM PFC (III) 2.11 TPQ 2.41 1 mM coenzyme Q1 8.58 LTQ 2.04 1 mM methylene blue 6.88
(19) To test the influence of the metal ions in the activated enzyme on the transformation, the alcohol dehydrogenase of SEQ ID no. 1 and PCC were activated, but with different metal ions in each case, namely, Mg.sup.2+, Ca.sup.2+, Zn.sup.2+, Mn.sup.2+, Fe.sup.2+ and Cu.sup.2+. The transformation assays contained 10 nM activated alcohol dehydrogenase, 10 ppm DON, and 1 mM PMS in 100 mM Tris-HCl pH 7.5 respectively, and were incubated at 30° C. The DON concentrations were determined by means of LC-MS/MS after 30 minutes. The results are shown in Table 3.
(20) TABLE-US-00003 TABLE 3 Metal ion DON [ppm] Metal ion DON [ppm] Mg.sup.2+ 1.90 Mn.sup.2+ 2.57 Ca.sup.2+ 1.98 Fe.sup.2+ 2.17 Zn.sup.2+ 2.46 Cu.sup.2+ 2.61
(21) Analogously to the above-mentioned DON transformation assays, transformation assays were carried out with other trichothecenes exhibiting a hydroxyl group on the C-3 atom. In these assays, instead of 50 ppm DON, 50 ppm T-2 toxin or 50 ppm nivalenol were used. All four alcohol dehydrogenases of SEQ ID numbers 1 to 4 containing metal ions and a quinone cofactor were also able to transform T-2 toxin and nivalenol, in which case, more than half the originally used toxin was transformed within 30 minutes.
Example 4: Measurement of the Activity Areas
(22) To determine the capacity of alcohol dehydrogenases of SEQ ID numbers 1-4 to transform DON under different conditions, alcohol dehydrogenase of SEQ ID no. 1 was used as an example.
(23) The alcohol dehydrogenase of SEQ ID no. 1 was produced and activated with Ca.sup.2+ and PCC as described in Example 3. To determine the activity of the enzyme over a temperature range from 10° C. to 50° C. and over a pH range from 3.0 to 9.0, a 10% Teorell Stenhagen buffer was used instead of the 100 mM Tris-HCl pH 7.5 buffer.
(24) The transformation assays to determine the activities at different temperatures were carried out in an aqueous solution with the following components: 10% Teorell Stenhagen pH 7.5, 1 mM synthetic redox cofactor PMS, 50 ppm DON, and 10 nM activated alcohol dehydrogenases of SEQ ID no. 1. The transformation assays were incubated up to 60 min in a thermocycler (Eppendorf) with a temperature gradient from 10° C. to 50° C. After 0, 10, 20, 30, 40, and 60 minutes, a sample of 0.05 ml was taken in each case and mixed with 0.05 ml methanol to stop the reaction, and stored at −20° C. The samples were prepared for the LC-MS/MS, as described in Example 3, and analysed by means of LC-MS/MS. The course of the DON reduction was determined for each temperature and the activity was calculated, as described in Example 3. The slope of the linear range of the transformation line at 30° C. was used as a reference value to calculate the residual activity at the other temperatures. Table 4 shows the temperatures in ° C. and the associated residual activities in percent. Surprisingly, it has been shown that the alcohol dehydrogenase of SEQ ID no. 1 is active over a broad temperature range. At 10° C., a residual activity of 48% was measured, and at approximately 50° C., a residual activity of 67%.
(25) TABLE-US-00004 TABLE 4 Temperature Residual activity Temperature Residual activity [° C.] [%] [° C.] [%] 10.0 48 32.8 105 12.7 60 33 108 15 69 35.3 105 17.6 73 38.4 120 20.5 86 40.7 116 23.3 89 43.2 108 26.2 82 45.9 96 28.3 100 48.2 89 30.2 100 49.8 67
(26) The transformation assays to determine the activity in a pH range from 4.0 to 9.0 were carried out in an aqueous solution with the following components: 10% Teorell Stenhagen pH 4.0 to pH 10.0, 20 mM synthetic redox cofactor PFC, 100 ppm DON, and 20 nM activated alcohol dehydrogenases of SEQ ID no. 1. The transformation assays were incubated up to 60 min in a thermocycler at 30° C. After 0, 10, 20, 30, 40 and 60 minutes, a sample of 0.05 ml was taken in each case and mixed with 0.05 ml methanol to stop the reaction, and stored at −20° C. As described in Example 3, the samples were diluted and analysed by means of LC-MS/MS. The course of the DON reduction was determined at each pH value and the activity was calculated, as described in Example 3. The slope of the linear range of the transformation line at pH 7.5 was used as a reference value to calculate the residual activity at the other temperatures. Table 5 shows the pH values and the associated residual activities (DON reduction based on the reference pH value of 7.5) in percent.
(27) TABLE-US-00005 TABLE 5 Residual pH activity 4.0 10% 5.0 18% 6.0 20% 6.5 52% 7.0 105% 7.5 100% 8.0 69% 9.0 105%
Example 5: Determining the Temperature Stability
(28) The temperature stability of the alcohol dehydrogenase of SEQ ID no. 1 was determined over a range from 30° C. to 55° C. To do this, the activated alcohol dehydrogenase was incubated in a 100 mM Tris-HCl buffer, pH 7.5 for up to 60 min at a specific temperature in a thermocycler (Eppendorf). After 0, 5, 10, 15, 20, 30, 40, and 60 minutes, an aliquot of the alcohol dehydrogenase was taken and the activity was determined in a DON transformation assay, as described in Example 3. The transformation assays contained the following components: 100 mM Tris-HCl, pH 7.5, 1 mM PMS, 50 ppm DON, 10 nM activated alcohol dehydrogenases of SEQ ID no. 1. As described in Example 3, the reactions were incubated and the sampling to determine the activity was done after 0, 10, 20, 30, 40 and 60 min. The course of the DON reduction was determined for each temperature for each incubation time. The slope of the linear range of the DON transformation line was calculated to determine the temperature stability. The slope of the linear range of the DON transformation line of the respective temperature at the time of t=0 min was used as the reference value for the calculation of the residual activities. Table 6 shows the temperatures in ° C., the incubation time in minutes, and the associated residual activities in percent. The alcohol dehydrogenase of SEQ ID no. 1 was the steadiest when stored for an hour at temperatures of 30° C. and 37° C. In comparison to this, the alcohol dehydrogenase still had 73% residual activity at 40° C. after being stored an hour. A 50% residual activity was measured after being stored at 45° C. for 30 min. Surprisingly, a residual activity of 84% was detected after being stored 5 min at 50° C.
(29) TABLE-US-00006 TABLE 6 Incubation time 5 10 15 20 30 60 0 min min min min min min 40 min min 30° C. 100% 99% 98% 94% 88% 99% 100% 95% 37° C. 100% 92% 94% 92% 90% 91% 47% 79% 40° C. 100% 90% 83% 77% 75% 83% 82% 73% 45° C. 100% 85% 78% 77% 60% 57% 47% 19% 50° C. 100% 84% 30% 36% 13% 12% 10% 0%
Example 6: Determining the pH Stability
(30) The pH stability of the activated alcohol dehydrogenase of SEQ ID no. 1 was determined over a range from pH 4.0 to pH 10.0. To do this, a tenfold concentration of the activated alcohol dehydrogenase (100 nM) was stored in 10% Teorell Stenhagen buffer pH 4.0 to pH 10.0 for up to 120 minutes at a temperature of 30° C. After 0, 60, and 120 minutes, an aliquot of the alcohol dehydrogenase was taken and the activity in a transformation assay was determined, and as described in Example 3, carried out at 30° C. with the following components: 100 mM Tris-HCl, pH 7.5, 1 mM PMS, 50 ppm DON, 10 nM activated alcohol dehydrogenases of SEQ ID no. 1. The sampling to determine the activity was taken after 0, 10, 20, 30, and 40 min. The course of the DON reduction was determined for each pH value for each time. To determine the stability, the slope of the linear range of the DON transformation line was calculated for each pH value at the respective time. The slope of the linear range of the DON transformation line of the respective pH value at the time of t=0 min was used as the reference value for the calculation of the activities of the following incubation times. Table 7 shows the pH value, the time of the pH incubation in minutes, and the associated residual activities in percent. The alcohol dehydrogenase of SEQ ID no. 1 was stable at pH 5.0 to pH 9.0 after a 60-minute incubation. Surprisingly, the alcohol dehydrogenase exhibited particularly good stability in an acidic environment (no activity loss at pH 5.0) and in a heavily alkaline environment (no activity loss in an incubation after 120 min at pH 9.0).
(31) TABLE-US-00007 TABLE 7 Incubation time 60 min 120 min pH 4.0 72% 51% pH 5.0 111% 109% pH 6.0 92% 88% pH 7.0 87% 85% pH 8.0 83% 73% pH 9.0 93% 60% pH 10.0 69% 55%
Example 7: Transformation of DON in Complex Matrices
(32) To determine the capability of the activated alcohol dehydrogenases to transform trichothecenes in complex matrices also without an external addition of synthetic redox cofactors, the activated alcohol dehydrogenase of SEQ ID no. 1 was produced as described in Example 3, and DON transformation assays were carried out in complex matrices. Here, complex matrices are defined as the rumen fluid of cattle, intestinal contents from the jejunum of swine, gastric juice of swine, saliva of humans and swine, granulated piglet feed, and granulated piglet feed mixed with saliva, rumen fluid, or intestine contents, among other things. In order to have a comparison with the buffer system, inspections were carried out with Tris-HCl, as described in Example 3. For the piglet feed, a standard feed based on maize, soya, and barley was used.
(33) To determine the alcohol dehydrogenase activity in rumen fluid (pH 5.9), 1 ml of sterile rumen fluid filtrate was added to 100, 200, and 300 nM of activated alcohol dehydrogenase of SEQ ID no. 1 and 50 ppm DON in each case. The control batches were tested in aqueous solution, as described in Example 3. The transformation assays were incubated at 30° C. in a thermoblock for up to 24 hours. Samples were taken after 0, 0.5, 1.0, 5.0, and 24.0 hours, in which case a 0.1 ml sample was taken at each time, and the reaction was stopped with 0.1 ml methanol. The samples were stored at −20° C., defrosted, and centrifuged for 10 min at 13,000 rpm with an Eppendorf tabletop centrifuge, and filtered sterilely with a 0.2 μM Spartan filter. For the LC-MS/MS, the samples were diluted as described in Example 3 and analysed by means of LC-MS/MS. The concentration of DON at the time of t=0 h was used as the reference value (100%) for the following values. Table 8 shows the percentage of DON concentration that was measured at a certain time relative to the time of t=0 h. For the activity in the Tris-HCl buffer, the presence of an externally added synthetic redox cofactor is necessary, because the transformation of DON occurs slowly, and was detectable only 24 hours later with an alcohol dehydrogenase concentration of 300 nM. Surprisingly, it has been demonstrated that DON is transformed without the addition of an external synthetic redox cofactor in a sterile rumen fluid filtrate at a pH value of 5.9. This shows clearly that there are substances in the rumen fluid that serve as natural redox cofactors. With a concentration of 300 nM, only 42% of the initial DON quantity is contained in the preparation after 5 hours incubation. After 24 hours incubation, DON is detectable only in low quantities with an alcohol dehydrogenase concentration greater than 200 nM.
(34) TABLE-US-00008 TABLE 8 Rumen fluid Rumen fluid Tris-HCl pH 7.5 with synthetic without synthetic without synthetic redox cofactor redox cofactor redox cofactor 100 100 200 300 100 200 300 nM nM nM nM nM nM nM 0 h 100% 100% 100% 100% 100% 100% 100% 0.5 h 0% 100% 100% 87% 100% 99% 95% 1.0 h 0% 100% 100% 83% 100% 99% 89% 5.0 h 0% 94% 75% 42% 99% 88% 86% 24.0 h 0% 53% 3% 0% 97% 84% 67%
(35) To determine the alcohol dehydrogenase activity in swine gastric juice without mash with a pH value of about 3, in swine intestinal contents with a pH value of about 6, and in swine and human saliva, 300 nM activated alcohol dehydrogenase SEQ ID no. 1, about 20 ppm DON, was mixed with 1 ml gastric juice (sterilely filtered), 1 ml mushy intestinal contents, or 1 ml saliva in each case. As a negative check, assays containing only digestion fluids with 20 ppm DON were included, and as a positive check, transformation assays containing all the components, including 20 mM of the synthetic redox cofactor PFC (III), were used. Samples were taken after 0, 3.0, 5.0, and 24.0 hours, in which case a 0.1 ml sample was taken at each time, and the reaction was stopped with 0.1 ml methanol. The samples were stored at 20° C., defrosted, and centrifuged for 10 min at 13,000 rpm with an Eppendorf tabletop centrifuge, and filtered sterilely (0.2 μM Spartan filter). For the LC-MS/MS, the samples were diluted 1:10 in the eluant (see Example 3) and analysed by means of LC-MS/MS as in Example 3. Table 9 shows the respective DON concentrations that were measured at the time of the sampling. Surprisingly, a reduction of DON in the saliva occurred without an externally added synthetic redox cofactor (regardless of the species). This shows clearly that there are substances in the saliva secretions of humans and swine that are suitable as natural redox cofactors for the transformation of DON with the alcohol dehydrogenase SEQ ID no. 1. No substantial reduction of the DON concentration was measured in the pure gastric juice without mush. A reduction of the DON concentration occurred in the intestinal contents only by adding the synthetic redox cofactor.
(36) TABLE-US-00009 TABLE 9 DON [ppm] Sample 0 h 3 h 5 h 24 h Saliva Negative check 20 19 18 18 (human) 0 mM PFC (III) 20 13 12 8 Positive check 20 18 0 0 0 mM PFC (III) Saliva Negative check 20 20 19 18 (swine) 0 mM PFC (III) 21 10 8 5 Positive check 20 20 0 0 0 mM PFC (III) Gastric Negative check 22 22 21 21 juice 0 mM PFC (III) 22 21 21 20 Positive check 20 24 21 19 18 mM PFC (III) Intestinal Negative check 21 20 20 20 contents 0 mM PFC (III) 24 23 22 22 Positive check 20 23 9 8 4 mM PFC (III)
(37) To determine the activity of the alcohol dehydrogenases in piglet feed, 100 mg of piglet feed was mixed with 400 μl 100 mM Tris-HCl buffer, pH 7.5, 400 μl swine saliva, 400 μl sterile swine gastric juice or 400 μl swine intestinal contents respectively. These piglet feed suspensions were stored overnight at 4° C. Following this, about 20 ppm DON, and/or 300 nM activated alcohol dehydrogenases of SEQ ID no. 1, and/or 20 mM of the synthetic redox cofactor PFC (III) were added to all the samples. The preparations without alcohol dehydrogenase and without the external synthetic redox cofactor were used as the negative check. The preparations with the added alcohol dehydrogenase and synthetic redox cofactor were used as the positive check. Samples were taken after 0, 3.0, 5.0, and 24.0 hours. One entire sample was used each time. For the sample, 500 μl methanol was added, followed by 30 min homogenization on a shaker with 300 rpm. Following this, the samples were centrifuged for 15 min (Eppendorf tabletop centrifuge, 13,000 rpm) and the supernatant was filtered with a syringe through a 0.2 μM Spartan filter. The supernatants were stored at −20° C., defrosted, and for the LC-MS/MS diluted 1:10 in the eluant, and analysed by means of LC-MS/MS as described in Example 3.
(38) Table 10 shows the DON concentration that was present in the samples at the respective times. In the piglet feed buffer mixture there were substances that can assume the role of the externally added synthetic redox cofactors, because the DON concentration decreases continuously in the absence of the external synthetic redox cofactor. These substances come from the piglet feed, because as shown before, no DON transformation could be measured in the buffer without an external synthetic redox cofactor. In the presence of the external synthetic redox cofactor, the transformation of DON in the piglet feed buffer mixture occurs faster in comparison.
(39) In the mixture of piglet feed and saliva, the alcohol dehydrogenase also exhibited activity independently of the presence of the external synthetic redox cofactor; whereas a faster reduction of DON occurred in the transformation assays that contained the external synthetic redox cofactor.
(40) Surprisingly, the alcohol dehydrogenase of SEQ ID no. 1 in the piglet feed mixture is also active without adding the external synthetic redox cofactor. By adding piglet feed to the gastric juice, on the one hand, the pH of the gastric juice was increased, and on the other hand, naturally occurring redox cofactors that can replace the external synthetic redox cofactor were released from the piglet feed. Activity of the alcohol dehydrogenase was ascertained in the intestinal contents only when an external synthetic redox cofactor was added to the transformation assay.
(41) TABLE-US-00010 TABLE 10 DON [ppm] Sample 0 h 3 h 5 h 24 h Piglet feed Negative check 21 20 20 20 in buffer 0 mM PFC (III) 20 10 9 5 Positive check 21 0 0 0 20 mM PFC (III) Piglet feed Negative check 20 20 20 20 in saliva 0 mM PFC (III) 20 12 9 8 Positive check 21 1 0.8 0.5 20 mM PFC (III) Piglet feed Negative check 21 21 20 20 in gastric 0 mM PFC (III) 20 7 5 2 juice Positive check 20 5 0.7 0 20 mM PFC (III) Piglet feed Negative check 21 20 20 20 in intestinal 0 mM PFC (III) 21 20 18 16 contents Positive check 20 5 3 0.7 20 mM PFC (III)