Deoxynivalenol-degrading enzyme mutants independent of exogenous mediators and application thereof
By mutating the amino acid composition of sorbitol dehydrogenase, electron transfer and substrate binding are improved, solving the problem of PMS dependence in existing DON-degrading enzymes. This achieves efficient and safe DON degradation, making it suitable for detoxification in food and feed.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTH CHINA AGRICULTURAL UNIVERSITY
- Filing Date
- 2026-04-13
- Publication Date
- 2026-07-24
AI Technical Summary
Existing DON degrading enzymes have slow catalytic rates, poor thermal stability, and heavy dependence on exogenous cofactor PMS, resulting in high usage costs and insufficient safety, which limits their application in the food and feed industries.
By analyzing the electron transport pathway and electrostatic potential energy of sorbitol dehydrogenase (SDH), amino acid mutations were designed to obtain highly efficient vomitoxin-degrading enzyme mutants SDHF103A/S454E/T492E that do not depend on the exogenous mediator PMS, thus improving the substrate binding region and electron transport efficiency.
The mutant enzyme achieves a 95% degradation rate of DON in the presence of PMS and an 82% degradation rate in the absence of PMS. It has a wide range of applicability and is suitable as a feed detoxification enzyme, reducing dependence on PMS and toxicity risks.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to the molecular modification of a deoxynivalenol-degrading enzyme from Fusarium nivale and its application in feed and grain samples. Background Technology
[0002] Deoxynivalenol (DON) is a type B trichothecene toxin produced by the metabolism of Fusarium fungi. It has extremely high contamination rates in major grains such as wheat, corn, DDGS, and wheat bran, as well as their processed products. This toxin not only causes significant economic losses to agricultural production but also poses a serious threat to the health and safety of humans and animals due to its cytotoxic, immunotoxic, and potential teratogenic effects.
[0003] Currently, DON detoxification methods are mainly divided into three categories: physical, chemical, and biological. Physical and chemical methods often suffer from drawbacks such as high processing costs, high energy consumption, and the potential for secondary pollution, limiting their large-scale application in the feed industry. In contrast, enzymatic degradation methods have attracted significant attention due to their high specificity and environmental friendliness, and are considered a promising green detoxification technology. The core of DON's toxicity lies in its C3 hydroxyl group, and various enzymes (such as DepA and QDDH) can oxidize this site to generate low-toxicity 3-keto-DON. However, existing DON-degrading enzymes generally suffer from slow catalytic rates, poor thermal stability, and heavy reliance on exogenous cofactors (such as methyl phenazine sulfate, PMS). PMS itself is photosensitive and has some toxicity to mammalian cells, which not only increases usage costs and process complexity but also hinders its safe application in the food and feed industries.
[0004] Sorbitol dehydrogenase (SDH) naturally possesses the ability to convert DON, oxidizing it to 3-keto-DON. However, wild-type SDH exhibits low catalytic activity towards DON due to its narrow substrate-binding pocket and inefficient electron transport pathways, hindering its direct practical application. Previous studies have indicated that the catalytic performance of SDH is closely related to its surface charge distribution and electron transport efficiency. Based on this understanding, this invention, through electrostatic potential energy calculations of SDH, identifies serine residue 454 as a key residue affecting electron transport. Building upon this, this invention designs a "co-mutation" strategy to simultaneously improve the substrate-binding region, surface charge environment, and internal electron transport pathway, ultimately obtaining a triple-mutated SDH enzyme variant with excellent catalytic performance and significantly reduced dependence on exogenous cofactors, providing a new technical solution for the practical application of DON biodetoxification. Summary of the Invention
[0005] In order to improve the degradation efficiency of vomitoxin, the present invention provides a sorbitol dehydrogenase mutant and its application.
[0006] The purpose of this invention is to obtain a highly efficient vomitoxin-degrading enzyme mutant that does not rely on the exogenous mediator PMS by designing amino acid mutations based on the natural vomitoxin degradation potential of SDH through systematic analysis of electron transport pathways and protein surface electrostatic potential energy, thereby reducing application costs and improving safety.
[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: The amino acid sequence of the vomitoxin degrading enzyme mutant described in this invention is: SEQ ID NO.1.
[0008] In one embodiment of the present invention, the amino acid at position 103 is mutated.
[0009] In one embodiment, the amino acid at position 492 is mutated based on the mutation at position 103.
[0010] In one embodiment, in addition to mutating amino acids at positions 103 and 492, amino acid at position 454 is further mutated.
[0011] In one embodiment, the carrier is a pET28a carrier.
[0012] In one embodiment, the recombinant bacteria may be constructed using Escherichia coli or yeast as hosts.
[0013] Preferably, in the presence of PMS, the mutant SDH F103A / T492E SDH F103A / S454E / T492E They all have high DON degradation rates.
[0014] Preferably, in the absence of PMS, the mutant SDH F103A / T492E It has a DON degradation rate of 62%.
[0015] Preferably, in the absence of PMS, compared to SDH F103A / T492E SDH F103A / S454E / T492E The degradation rate of DON reached 82%.
[0016] The present invention has the following advantages and effects: The present invention provides a vomitoxin-degrading enzyme mutant SDH F103A / S454E / T492EIt contains three mutation sites: F103A, S454E, and T492E. Combining electron transport principles with the surface electrostatic potential of SDH, it was found that the negative charge on the protein surface facilitates electron transport, significantly reducing dependence on PMS, and providing a foundation for subsequent enzyme preparation research.
[0017] In the presence of PMS, the mutant enzyme SDH F103A / S454E / T492E The degradation rate of DON can reach 95% within 15 minutes. Furthermore, in a system with PMS at temperatures ranging from 20°C to 40°C and pH values ranging from 4 to 10, SDH... F103A / S454E / T492E The relative activity towards DON can be maintained at over 80%. In the absence of PMS, the wild type cannot degrade DON, and SDH... F103A / T492E Only 62% of DON can be degraded within 15 minutes, while SDH... F103A / S454E / T492E The degradation rate can reach 82% within 15 minutes. Based on its ability to degrade DON better without the presence of PMS, it not only avoids the potential toxicity of PMS to organisms, making it more suitable as a feed detoxification enzyme, but also has a wider range of applicable conditions. Therefore, this enzyme has good application prospects in the field of vomitoxin biological detoxification. Attached Figure Description
[0018] Figure 1 SDS-PAGE images of mutant expression and purification; Figure 2 The following are electrostatic potential energy diagrams before and after the S454 site mutation: a; Front view of the overall electrostatic potential energy of the protein before the S454 site mutation; b; Side view of the overall electrostatic potential energy of the protein before the S454 site mutation; c; Local electrostatic potential energy diagram of the protein before the S454 site mutation; d; Local electrostatic potential energy diagram of the protein after the S454 site mutation; e; Overall electrostatic potential energy diagram of the protein after the S454 site mutation. Figure 3 The exogenous mediator PMS for wild-type SDH and different mutant SDH F103A SDH F103A / T492E SDH F103A / S454E / T492E Comparison of the effects of degradation rate; degradation rates of DON by wild-type enzyme and mutant under PMS addition and degradation rates of DON by wild-type enzyme and mutant without PMS addition. Figure 4 To investigate the effects of wild-type enzymes and triple mutant SDH on different grain matrices (wheat, corn, DDGS, bran) with and without PMS, F103A / S454E / T492E Comparison of DON degradation rates; Detailed Implementation
[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.
[0020] Example 1 Construction of expression vector for vomitoxin degrading enzyme mutant The gene sequence of the original sorbitol dehydrogenase (SDH) was synthesized by Shanghai Sangon Biotech Co., Ltd., as pET28a-SDH. F103A / T492E Using a template, a mutant was obtained by point mutation at amino acid position 454. The nucleotide sequence of the final mutant is shown in SEQ ID NO:2, and the amino acid sequence is shown in SEQ ID NO:1.
[0021] The primers used for site-directed mutagenesis are as follows: S454E-F:5′- GAA GTAGAACGCGCAGCAGCAAATTATAGCCCGGTT-3′ S454E-R: 5′-TGCTGCGCGTTCTAC TTC CCACAGGGTACGGCCCGT-3′ PCR amplification was performed using the primers described above, as follows: <![CDATA[KOD-FLEX TM PCR Master Mix(2X)]]> 25 μL Upstream primer (N454E-F) 1.5 μL Downstream primer (N454E-R) 1.5 μL template 1 μL sterile water 21 μL total 50 μL The PCR reaction conditions were as follows: Stage 1: 94°C pre-denaturation for 2 min; Stage 2: 98°C denaturation for 10 sec, 58°C annealing for 10 s, 68°C extension for 1 min 40 sec, repeated 30 times; Stage 3: 72°C final extension for 5 min. The obtained PCR products were digested with DnPI enzyme, detected by 1% agarose gel electrophoresis, purified by gel recovery, and transformed into DH5α competent cells. Plasmids of positive clones were extracted through antibiotic screening and sent for sequencing. The correctly sequenced expression plasmid pET28a-SDH was then used. F103A / S454E / T492E Transformation into E. coli BL21(DE3) successfully constructs the mutant expression engineered bacteria.
[0022] Example 2: Mutant Expression and Purification The engineered bacteria were inoculated onto 10 mL of LB solid medium containing 50 μL / mL kanamycin and incubated overnight upside down at 37°C. Single colonies were transferred from the overnight LB solid medium and inoculated into 5 mL of TB liquid medium containing 50 μL / mL kanamycin, and incubated overnight at 37°C and 150 rpm. The overnight culture was then inoculated at a 1:100 ratio into 500 mL of TB liquid medium containing 50 μL / mL kanamycin and incubated at 37°C and 150 rpm for 2–3 h, until the bacterial OD reached the target value. 600 The concentration was increased to 0.6-0.8. IPTG was added to a final concentration of 0.4 mM, and the culture was induced for another 24 h at 25°C and 150 rpm. After 24 h of induction, the bacterial culture was placed on ice for 30 min, then centrifuged at 4500 rpm and 4°C for 20 min. The supernatant was discarded, and the bacterial cells were retained. Each g of bacterial cells was resuspended in 7 mL of pre-cooled buffer (50 mM Tris-HCl, 1 mM PMSF), and the sample was sonicated on ice for 30 min. After the bacterial culture was clear, it was centrifuged at 10000 rpm and 4°C for 25 min. The supernatant was filtered through a 0.45 μm aqueous filter membrane.
[0023] Purification was performed using a rapid and efficient protein chromatography system (ÄKTA pure). The equilibration buffer formulation was: 50 mM Tris-HCl, 50 mM NaCl, 15 mM imidazole, pH 7.4. The elution buffer formulation was: 50 mM Tris-HCl, 50 mM NaCl, 500 mM imidazole, pH 7.4. The collected proteins were analyzed by SDS-PAGE electrophoresis to determine the expression and purification results of the target protein.
[0024] The results are shown in Figure 1.
[0025] Example 3: Electrostatic Potential Energy Analysis of SDH Surface In PyMol software, the serine at position 454 was simulated to be mutated to glutamic acid, and then the surface electrostatic potential energy map of the mutant was generated by PyMol.
[0026] The results are as follows Figure 2 As shown, a and b are the overall electrostatic potential energy diagrams before the S454 site mutation, viewed from the front and side, respectively; c and d are the locally magnified electrostatic potential energy diagrams before and after the S454 site mutation, respectively; and e is the overall electrostatic potential energy diagram after the S454 site mutation. As can be seen from d, the electrostatic potential energy of the S454 site before mutation is higher than that of nearby amino acids. Mutating the S454 site results in a lower negative charge in the region where this site is located, which is beneficial for the presentation of electron transport pathways, thereby increasing the activity of the SDH mutant.
[0027] Example 4: Determination of the degradation rate of standard DON by SDH mutant mutant SDH WT SDH F103A SDH F103A / T492E SDH F103A / S454E / T492E Three replicate experiments were performed with DON under the same conditions, using Tris-HCl buffer for 15 min.
[0028] Add PMS incubation system: final concentration of 100 mM Tris-HCl buffer (pH 7.4), 10 μM PQQ, 40 mM CaCl2, 1 mM PMS, 50 μg / mL protein, 15 μg / mL DON.
[0029] Incubation system without PMS: final concentration of 100 mM Tris-HCl buffer (pH 7.4), 10 μM PQQ, 40 mM CaCl2, 1 mM PMS, 50 μg / mL protein, 15 μg / mL DON.
[0030] After the reaction, 600 μL of ethyl acetate was added and thoroughly shaken for extraction. The mixture was centrifuged at 12000 rpm for 1 min, and the supernatant ethyl acetate was dried under nitrogen. The mixture was resuspended in 200 μL of 50% methanol and filtered through a 0.22 μm filter membrane. The residual DON content in the system was detected by high performance liquid chromatography.
[0031] The reduction in DON was detected using an Agilent 1260 HPLC system. The mobile phase was water and acetonitrile. The elution program was as follows: 0-15 min, acetonitrile concentration increased from 12% to 33%; 15-16 min, acetonitrile concentration increased from 33% to 90%; 16-18 min, acetonitrile concentration remained unchanged; 18-19 min, acetonitrile concentration decreased from 90% back to 12%; reequilibration for 4 min.
[0032] Degradation rate of DON (%) = (1 - amount of DON remaining in the treatment group / amount of DON in the control group) × 100% The results are as follows Figure 3 As shown, SDH F103A / S454E / T492E The degradation rate of DON reaches over 95%, and the degradation rate reaches 82% without the addition of PMS.
[0033] Example 5: Determination of the activity of SDH mutant in degrading DON in complex matrices mutant SDH F103A SDH F103A / S454E / T492E For moldy rice, DDGS, wheat bran, and corn steep liquor, three replicates were performed for each condition, and the mixture was incubated with Tris-HCl buffer for 2 h.
[0034] The degradation system is as follows: Add the PMS incubation system: final concentration of 50 mM Tris-HCl buffer (pH 7.4), 20 μM PQQ, 2 mM CaCl2, 2 mM PMS, 200 μg / mL protein, ddH2O (up to 6 mL).
[0035] Incubation system without PMS: final concentration of 100 mM Tris-HCl buffer (pH 7.4), 20 μM PQQ, 2 mM CaCl2, 200 μg / mL protein, ddH2O (up to 6 mL).
[0036] Add 0.5 g polyethylene glycol and 4 mL ultrapure water, vortex for 3 min, sonicate for 20 min, and centrifuge at 6000 rpm for 10 min. Filter the supernatant using a 0.45 μm aqueous syringe filter. Bring the immunoaffinity column, previously stored at low temperature, to room temperature. After the original liquid in the immunoaffinity column has drained, accurately transfer 2 mL of supernatant A into a syringe. Connect an air pressure pump to the syringe and adjust the drip rate to control the sample solution to flow through the immunoaffinity column at a rate of 1 drop per second until air enters the column. Drain the column with 5 mL of PBS buffered saline and 5 mL of water. Accurately add 2 mL of methanol to the affinity column, controlling the drop rate to 1 drop per second, collect all the eluent into a test tube, and slowly blow the eluent to near dryness with nitrogen at 50 °C. Add 1 mL of the initial mobile phase, vortex for 30 s to dissolve the residue, filter with a 0.22 μm organic phase syringe filter, and detect the residual DON content in the system by high performance liquid chromatography.
[0037] The results are as follows Figure 4 As shown in a and b, under the condition of adding the exogenous cofactor PMS, both SDH enzyme variants exhibited highly efficient and similar DON degradation capabilities in four common moldy grain raw materials, with degradation rates all exceeding 75%. However, under conditions without dependence on the toxic cofactor PMS, the single mutant SDH... F103A No effective DON degradation activity was detected in any of the moldy feeds, and the triple mutant SDH was also found to be effective. F103A / S454E / T492E All of them exhibited highly efficient DON degradation activity, with degradation rates ranging from 75% to 100%. The SDH provided by this invention... F103A / S454E / T492E The three mutants, without the addition of PMS, exhibit efficient and stable degradation capabilities for DON in various actual moldy grain raw materials, and have the industrial potential for direct application in the biological detoxification of feed and food raw materials.
[0038] sequence list amino acid sequence MQTAITDEMLANPPAGEWISYGQNQENYRHSPLTQITTENVGQLQLVWARGMQPGKVQVTPLIHDGVMYLANPGDVIQAIDAKTGDLIWEHRRQLPNIATLNSAGEPTRGMALYGTNVYFVSWDNHLVALDMGTGQVVFDVDRGQGDERVSNSSGPIVANGTIVAGSTCQYSPFGCFVSGHDSATGEELWRNYFIPRAGEEGDETWGNDYESRWMTGAWGQITYDPVTNLVHYGSTAVGPASETQRGTPGGTLYGTNTRFAVRPDTGEIVWRHQTLPRDNWDQECTFEMMVTNVDVQPSTEMEGLQSINPNAATGERRVLTGVPCKTGTMWQFDAETGEFLWARDTNYQNMIESIDENGIVTVNEDAILKELDVEYDVCPTFLGGRDWPSAALNPDSGIYFIPLNNVCYDMMAVDQEFTSMDVYNTSNVTKLPPGKDMIGRIDAIDISTGRTLWEVERAAANYSPVLSTGGGVLFNGGTDRYFRALSQETGEELWQTRLATVASGQAISYEVDGMQYVAIAGGGVSYGSGLNSALAGERVDSTAIGNAVYVFALPQLE Nucleotide sequence
Claims
1. A highly efficient deoxynivalenol-degrading enzyme mutant of Fusarium nivale that does not rely on exogenous mediators, characterized in that, The amino acid sequence of the mutant is as follows: based on the sequence shown in SEQ ID NO.1, the phenylalanine at position 103 is mutated to alanine, the serine at position 454 is mutated to glutamic acid, and the threonine at position 492 is mutated to glutamic acid; or the mutant contains an amino acid sequence that has at least 90% identity with SEQ ID NO.1 and simultaneously has the mutations at positions 103, 454, and 492.
2. The deoxynivalenol degrading enzyme mutant according to claim 1, characterized in that, The mutant has alanine at position 103, glutamic acid at position 454, and glutamic acid at position 492.
3. A recombinant expression vector, characterized in that, The recombinant expression vector contains a nucleotide sequence encoding the mutant of claim 1 or 2.
4. A recombinant bacterium, characterized in that, The recombinant bacteria express the deoxynivalenol-degrading enzyme mutant of Fusarium nivale as described in claim 1 or 2.
5. A deoxynivalenol biodegradable agent, characterized in that, It includes the mutant of claim 1 or 2, the recombinant expression vector of claim 3, and the recombinant bacteria of claim 4.
6. The application of the deoxynivalenol degrading enzyme mutant according to claim 1 or 2 in the degradation of deoxynivalenol in cereal samples.