Quinone-dependent dehydrogenase and application thereof in degradation of deoxynivalenol

Abstract

The invention relates to quinone-dependent dehydrogenase and application thereof in degradation of deoxynivalenol, and belongs to the technical field of bioengineering. The amino acid sequence of the quinone-dependent dehydrogenase is as shown in SEQ ID NO. 1. The quinone-dependent dehydrogenase can realize heterologous high-efficiency expression in a prokaryotic expression system, is easy for later-stage purification of detoxification enzyme and preparation of enzyme preparations, has higher degradation efficiency on a substrate DON, and is low in commercialized production cost. In addition, the invention finds that quinone-dependent dehydrogenase has a dependency relationship with the concentration of a cofactor pyrroloquinoline quinone (PQQ) in the degradation and conversion process of deoxynivalenol.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to quinone-dependent dehydrogenases and their application in the degradation of deoxynivalenols by Fusarium deoxynivalensis. Background Technology

[0002] Deoxynivalenol (DON), also known as vomitoxin, is a sesquiterpene secondary metabolite produced by fungi of the genus *Fusarium*, belonging to the trichothecene type B toxins. DON and its derivatives are among the most widely distributed and influential trichothecene compounds produced by the moldy spoilage of grain crops, found worldwide. They are generally found in higher concentrations in barley, wheat, corn, and oats, and in lower concentrations in rye, sorghum, and rice. Improper planting, harvesting, storage, or processing of grains can lead to mold and spoilage, resulting in the production of vomitoxin. Due to climatic factors and planting and storage methods, DON contamination of major grains in my country is widespread, especially in the hot and humid southern regions and during the wheat flowering season when Fusarium head blight outbreaks are particularly conducive to *Fusarium* growth and vomitoxin production.

[0003] Ingestion of grains or feed contaminated with fungal toxins can cause acute or chronic poisoning in humans and animals. When humans and animals consume food contaminated with DON, they may experience a series of poisoning reactions, including loss of appetite, diarrhea, vomiting, and fainting. DON is chemically stable, possesses strong heat resistance and acid resistance, and can synergistically interact with other toxins, making poisoning symptoms more complex. Therefore, DON poses a safety hazard in grain production and processing. The use of biological detoxification enzymes to degrade and transform contaminated grain food raw materials has advantages such as high efficiency, high specificity, and no secondary pollution, and has experienced rapid development in recent years.

[0004] Currently, commonly used methods for reducing DON content in food ingredients or feed include physical, chemical, and biological detoxification methods. Physical detoxification methods include physical adsorption, heat treatment, high-pressure extrusion, radiation, and plasma technology. However, multiple physical detoxification methods must be combined to achieve good detoxification efficiency. Chemical detoxification methods for DON mainly use strong oxidants, such as strong acids, strong alkalis, or ozone, to convert DON into less toxic substances. Traditional chemical detoxification methods have limitations, including nutrient loss, reagent residues, and secondary environmental pollution.

[0005] Biological detoxification utilizes microorganisms or enzyme preparations to detoxify mycotoxins. It has rapidly developed due to its advantages such as mild reaction conditions, high degradation and conversion efficiency, strong specificity, and no secondary pollution. Degradation and conversion of quinoline quinone (DON) into low-toxicity or even non-toxic metabolites through biological detoxification enzymes is the most promising method for detoxifying mycotoxins in feed and food ingredients. Pyrroloquinoline quinone (PQQ), as a cofactor in the degradation and conversion of DON by oxidoreductases, has a dose-response relationship with the dosage of specific PQQ-dependent dehydrogenases and the efficiency of DON degradation and conversion. This is an important factor of interest in DON biological detoxification, but current research is still limited. Existing reports on PQQ-dependent dehydrogenases have not determined the dose-response relationship between PQQ and the degradation and conversion by detoxification enzymes, failing to fully utilize the conversion efficiency of detoxification enzymes while minimizing the use of PQQ and reducing biological detoxification costs. Quinone-dependent dehydrogenases can oxidize substrates by reducing electron acceptors, promoting the conversion of alcohols or aldehydes. They are a widely distributed subclass of oxidoreductases in organisms and have important biological functions. However, the number of DON biological detoxification enzymes discovered is small, and their degradation and conversion efficiency is not high. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a quinone-dependent dehydrogenase and its application in the degradation of deoxynivalenols by *Fusarium oxysporum*. The quinone-dependent dehydrogenase of this invention is derived from *Devrosella dysporium*, which can be heterologously and efficiently expressed in prokaryotic expression systems, facilitating subsequent purification of the detoxification enzyme and preparation of enzyme formulations. This enzyme exhibits high degradation efficiency for the substrate DON, and its commercial production cost is low. Furthermore, this invention reveals a concentration dependence of the quinone-dependent dehydrogenase on the cofactor pyrroquinone (PQQ) during the degradation and conversion of deoxynivalenols by *Fusarium oxysporum*.

[0007] This invention is achieved through the following technical solution:

[0008] The first objective of this invention is to provide a quinone-dependent dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] A second objective of this invention is to provide a gene encoding the quinone-dependent dehydrogenase.

[0010] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0011] A third objective of this invention is to provide a recombinant plasmid carrying the gene.

[0012] A fourth objective of this invention is to provide a microbial cell expressing the quinone-dependent dehydrogenase.

[0013] Furthermore, the host of the microbial cells is Escherichia coli, Pichia pastoris, Bacillus subtilis, or Saccharomyces cerevisiae.

[0014] Furthermore, the microbial cells use Escherichia coli as the host and pGEX-4T-1 as the expression vector to express the quinone-dependent dehydrogenase.

[0015] A fifth object of the present invention is to provide a product containing the quinone-dependent dehydrogenase or the microbial cells.

[0016] The sixth objective of this invention is to provide the application of quinone-dependent dehydrogenase in the degradation of deoxynivalenzyme toxins, wherein the quinone-dependent dehydrogenase or the microbial cells are added to a reaction system containing deoxynivalenzyme toxins for reaction.

[0017] Furthermore, the reaction system also includes the cofactor pyrroloquinoline quinone.

[0018] This invention provides a quinone-dependent dehydrogenase and its encoding gene, as well as the optimal pH of this dehydrogenase and its application in the degradation of deoxynivalenol by Fusarium oxysporum. The quinone-dependent dehydrogenase rABP1 (crude enzyme) achieves a degradation conversion rate of over 95% for vomitoxin in a buffer system. This invention also provides the optimal dosage of cofactor PQQ for the degradation of vomitoxin by the quinone-dependent dehydrogenase and clarifies its dose-response relationship.

[0019] The technical solution of the present invention has the following advantages compared with the prior art:

[0020] (1) This invention provides a quinone-dependent dehydrogenase and its application in the degradation of deoxynivalenol by Fusarium deoxynivalensis. This invention provides a highly efficient detoxifying enzyme preparation for degrading and transforming vomitoxin, and its encoding gene or engineered bacteria, which can complete the degradation and detoxification of 5-10 mg / L DON within 6 hours.

[0021] (2) The optimal amount of cofactor PQQ required for the degradation of devomiting toxin by the quinone-dependent dehydrogenase provided by the present invention is conducive to accurately controlling the amount of cofactor added and achieving the best degradation and transformation effect, which greatly saves the cost of biological detoxification.

[0022] (3) The biological detoxification of this invention is more environmentally friendly than physical detoxification, does not produce secondary pollution, and ensures the nutrition and flavor of grains and food. Attached Figure Description

[0023] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0024] Figure 1 Construction of the expression plasmid for the degradation and transformation gene abp1 in this invention;

[0025] Figure 2 SDS-PAGE protein electrophoresis of prokaryotic expression of quinone-dependent dehydrogenase rABP1 in this invention;

[0026] Figure 3 This is an SDS-PAGE electrophoresis image of the purified recombinant protein rABP1 in this invention; where M: standard molecular weight of protein; 1: supernatant after pGEX-4T-1 / abp1 induction; 2: pGEX-4T-1 / abp1 flow-through peak; 3-5: pGEX-4T-1 / abp1 washing peaks; 6-8: pGEX-4T-1 / abp1 elution peaks;

[0027] Figure 4 This invention serves as a validation of the degradation activity of crude dehydrogenase rABP1 on DON; Ctl is a blank control.

[0028] Figure 5 The degradation and transformation effect of purified rABP1 on DON in this invention is shown; where PGEX is the empty vector control.

[0029] Figure 6 This invention illustrates the dose-response relationship between the quinone-dependent dehydrogenase rABP1 and the cofactor PQQ during DON degradation. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0031] The key point of this invention is that it provides an amino acid sequence as shown in SEQ ID NO.1 and a nucleotide sequence as shown in SEQ ID NO.2. Given that the amino acid sequence and nucleotide sequence are known, it is obvious to those skilled in the art that the amino acid sequence and nucleotide sequence, as well as the related vector and host cell, can be obtained.

[0032] The present invention provides a quinone-dependent dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.1.

[0033] Due to the specificity of the amino acid sequence, any fragment or variant of a peptide protein containing the amino acid sequence shown in SEQ ID NO.1, such as its conserved variants, bioactive fragments, or derivatives, is within the scope of protection of this invention, provided that the fragment or variant of the peptide protein has more than 95% homology with the aforementioned amino acid sequence.

[0034] This invention also relates to a gene encoding the quinone-dependent dehydrogenase as shown in SEQ ID NO.2. Alternatively, the gene may have a nucleotide sequence with more than 70% homology to the nucleotide shown in SEQ ID NO.2. Due to the specificity of nucleotide sequences, any variant of the polynucleotide shown in SEQ ID NO.2, provided it has more than 70% homology to that polynucleotide, is within the scope of protection of this invention.

[0035] The inventors of this application searched and compared the amino acid and nucleotide sequences of the quinone-dependent dehydrogenase of this invention in protein and nucleotide databases respectively, and found that it belongs to a type of dehydrogenase, named ABP1 (gene name abb1).

[0036] The polynucleotide sequence encoding the quinone-dependent dehydrogenase of the present invention can be obtained by a variety of methods. For example, the DNA fragment sequence of the present invention is obtained by the following methods: (1) isolating a double-stranded DNA sequence from genomic DNA; (2) chemically synthesizing a DNA sequence to obtain the double-stranded DNA of the dehydrogenase.

[0037] The present invention also relates to a recombinant vector containing the said gene, and genetically engineered host cells obtained by transformation, transduction or transfection using the said recombinant vector.

[0038] In this invention, the nucleotide sequence encoding a quinone-dependent dehydrogenase can be inserted into a vector to construct a recombinant vector containing the polynucleotides described in this invention. "Vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Vectors applicable in this invention also include, but are not limited to: T7 promoter-based expression vectors for bacterial expression; pcDNA3.1 vectors for mammalian cell expression; and baculovirus-derived vectors for insect cell expression. In short, any plasmid and vector can be used to construct recombinant expression vectors, provided they can replicate and remain stable in the host, preferably the pET and PGEX vector series and other prokaryotic expression vector series. An important characteristic of expression vectors is that they typically contain a replication origin, a promoter, a marker gene, and translational regulatory elements.

[0039] Methods well known to those skilled in the art can be used to construct expression vectors containing DNA sequences encoding dehydrogenases and suitable transcription / translation regulatory elements. These methods include in vitro recombinant DNA sequencing, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis.

[0040] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein for eukaryotic cell culture, or kanamycin or ampicillin for Escherichia coli.

[0041] In this invention, a polynucleotide encoding a quinone-dependent dehydrogenase or a recombinant vector containing such a polynucleotide can be transformed or introduced into a host cell to form a genetically engineered host cell containing the nucleotide or the recombinant vector. "Host cell" refers to a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.

[0042] This invention also relates to the use of the gene in the preparation of recombinant quinone-dependent dehydrogenase.

[0043] Using conventional recombinant DNA techniques, the polynucleotide sequence of this invention can be used to express or produce recombinant quinone-dependent dehydrogenases. Generally, the following steps are involved:

[0044] (1) Transform or transfect suitable host cells with the polynucleotide (or variant) encoding the quinone-dependent dehydrogenase of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0045] (2) Culture the host cells in a suitable culture medium;

[0046] (3) Isolate and purify proteins from culture media or cells, or use crude enzyme solution after cell disruption.

[0047] In step (2), the culture medium used in the culture can be selected from various suitable media, depending on the host cells used. The cells are cultured under conditions suitable for the host cells. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method, and the cells are cultured for a further period of time.

[0048] In step (3), the recombinant peptide protein may be encapsulated inside the cell, expressed on the cell membrane, or secreted outside the cell.

[0049] Based on the study of quinone-dependent dehydrogenases with the amino acid sequence shown in SEQ ID NO.1, this invention also explores the dose-response relationship between the concentration of pyrroloquinoline quinone (PQQ) and the catalytic efficiency of the detoxification enzyme during the degradation of vomitoxin by quinone-dependent dehydrogenase.

[0050] This invention relates to the dose-response relationship between the degradation rate of vomitoxin and the concentration of the cofactor pyrroloquinoline quinone (PQQ) in quinone-dependent dehydrogenases. Since quinone-dependent dehydrogenases require the addition of PQQ to degrade vomitoxins, this invention provides reference values ​​for the dose-response relationship between the amount of quinone-dependent dehydrogenase and the amount of PQQ added, offering a reference for cost control in subsequent applications of detoxification enzymes.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0052] LB liquid medium: Dissolve 10.0g NaCl, 10.0g tryptone, and 5.0g yeast powder in a small amount of deionized water, bring the volume to 1.0L, and sterilize at 121℃ for 15-20 minutes.

[0053] Example 1: cDNA synthesis and cloning of quinone-dependent dehydrogenase gene

[0054] The *Devros* strain obtained in the laboratory was analyzed by gene sequencing to determine the open reading frame (ORF) nucleotide sequence of the quinone-dependent dehydrogenase gene. Primers encoding the ORF were designed to amplify the upstream primer (abp1-F): 5'- CGCGGATCC CGCAACGTGGTGGGC-3'; Downstream primer (abp1-R): 5'- CCGCTCGAG The gene GCTGCGATCATAGCCGTT-3' incorporates BamHI and XhoI restriction sites on its upstream and downstream primers, respectively, and these sites are underlined. The dehydrogenase gene encoding the amino acid shown in SEQ ID NO.1 was obtained via in vitro amplification, and its nucleotide sequence is shown in SEQ ID NO.2. The recombinant expression plasmid pGEX-4T-1 / abp1 was constructed while ensuring correct reading frames. Figure 1 The enzyme was then transferred into E. coli BL21(DE3). Sequence analysis revealed that the theoretical isoelectric point of this quinone-dependent dehydrogenase was 4.13, and the theoretical molecular weight was 45.23 kDa.

[0055] Example 2: Protein expression and purification of quinone-dependent dehydrogenase gene abp1

[0056] The *Ec. li* BL21(DE3) transformant containing the recombinant plasmid pGEX-4T-1 / abp1 obtained in Example 1 was cultured overnight on a shaker in 100 mL of LB broth containing 100 μg / mL ampicillin. 1.0 mL of the seed culture was inoculated into 100 mL of fresh LB broth (containing 100 μg / mL ampicillin) and cultured at 37°C with shaking at 180 rpm. When the bacterial culture OD... 600 When the concentration reached 0.6, 0.2 mmol / mL IPTG was added and expression was induced at 16°C for 20 h. The cells were centrifuged at 8000 rpm for 10 min, and the supernatant was discarded. The cells were resuspended in 10 mL of 1× phosphate-buffered saline (PBS), and the cells were sonicated on ice, followed by centrifugation. SDS-PAGE electrophoresis showed that the enzyme was present in both the supernatant and the precipitate (inclusion bodies) after cell lysis, with an apparent molecular weight (including the GST tag) of approximately 71 kDa. Figure 2 The molecular weight is consistent with the theoretical molecular weight. After affinity chromatography purification, the crude enzyme solution yielded the rABP recombinant protein as shown in the figure. Figure 3 As shown, the purified protein has a purity greater than 95%.

[0057] Example 3: Application of recombinant detoxification enzyme in the degradation of alcohol toxins by Fusarium deoxynivalensis

[0058] 1. Experimental materials

[0059] The enzyme preparation was the re-enzyme solution after expression of recombinant plasmid pGEX-4T-1 / abp1 in E. coli BL21(DE3) transformant. All other reagents were analytical grade chemicals.

[0060] 2. Experimental Methods

[0061] 10 μL of recombinant enzyme (final concentration 86 mg / mL) was placed in a 2 mL centrifuge tube and added to the reaction system, which contained deoxynivalenzyme toxin (prepared using disodium hydrogen phosphate, pH 6.2) at a final concentration of 3 mg / L (5 mg / L for crude enzyme experiments), pyrroloquinoline quinone solution (prepared using pure water, pH 7.2) at a final concentration of 5 μM, and CaCl2 solution (prepared using pure water, pH 7.2) at a final concentration of 1 mM. The reaction was allowed to proceed for 6 h. 0.8 mL of the reaction solution was then added to 0.8 mL of ethyl acetate to stop the reaction. After centrifugation, the supernatant was collected, and the precipitate was extracted twice more with 0.8 mL of ethyl acetate as described above. The collected 2.4 mL of ethyl acetate extract was dried under nitrogen and reconstituted with 0.8 mL of a 20% methanol / 80% water solution. The sample was centrifuged at 12000 rpm and filtered through a 0.22 μM organic filter membrane. The residual amount of vomitoxin was determined by high-performance liquid chromatography (HPLC). The results are as follows: Figure 4The experimental results showed that after 6 hours of reaction, the supernatant after cell lysis almost completely completed the degradation and transformation of deoxynivalenol by Fusarium nivale. The blank control group (pGEX) was the supernatant of E. coli BL21(DE3) containing the pGEX-4T-1 empty plasmid after cell lysis under the same culture and induction conditions. The crude enzyme solution of the control group after cell lysis did not show any activity in deoxynivalenol degradation and transformation.

[0062] The degradation results of DON by purified quinone-dependent dehydrogenase rABP1 showed that, under the same conditions, the degradation conversion rate of 3 mg / L DON by purified quinone-dependent dehydrogenase rAPB reached over 95%. Figure 5 ).

[0063] Example 4: Relationship between the amount of heterologous recombinant detoxification enzyme and the amount of cofactor pyrroloquinoline quinone added in the degradation of deoxynivalenzyme by Fusarium nivale alcohol toxins

[0064] 1. Experimental materials

[0065] The enzyme preparation was the re-enzyme solution after expression of recombinant plasmid pGEX-4T-1 / abp1 in E. coli BL21(DE3) transformant. All other reagents were analytical grade chemicals.

[0066] 2. Experimental Methods

[0067] Take 10 μL of the supernatant after expression of pGEX-4T-1 / abp1 transformant and place it in a 2 mL centrifuge tube. Add the supernatant to the reaction system containing deoxynivalenzyme toxin at a final concentration of 5 mg / L (prepared with disodium hydrogen phosphate, pH 6.2) and CaCl2 solution at a final concentration of 1 mM (prepared with pure water, pH 7.2). Add 0, 2 μM, 5 μM, 7 μM, 10 μM, 15 μM, 20 μM, and 30 μM pyrroloquinoline quinone solution (prepared with pure water, pH 7.2) respectively. React for 6 h. Take 0.8 mL of the reaction solution and add 0.8 mL of ethyl acetate to stop the reaction. Centrifuge and collect the supernatant. Extract the precipitate twice with 0.8 mL of ethyl acetate as above. Dry the collected 2.4 mL of ethyl acetate under nitrogen and reconstitute it with 0.8 mL of 20% methanol and 80% water solution. After centrifugation at 12000 rpm, the sample was filtered through a 0.22 μM organic filter membrane, and the residue of deoxynivalenol was determined by high-performance liquid chromatography (HPLC). The results are as follows: Figure 6 As shown in the figure. The experimental results show that after 6 hours of reaction, the amount of deoxynivalenol in the supernatant after decomposition increased in a gradient with the increase of the amount of pyrroloquinoline quinone added. That is, when the dehydrogenase rABP1 reaches the highest degradation rate at a concentration of 100 mg / mL during the degradation and conversion of DON, the amount of pyrroloquinoline quinone required is 4.86 μM.

[0068] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A quinone-dependent dehydrogenase, characterized in that, The amino acid sequence of the quinone-dependent dehydrogenase is shown in SEQ ID NO.

1.

2. A gene encoding the quinone-dependent dehydrogenase of claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. A recombinant plasmid carrying the gene of claim 2 or 3.

5. A microbial cell expressing the quinone-dependent dehydrogenase of claim 1.

6. The microbial cell according to claim 5, characterized in that, The host of the microbial cells is Escherichia coli, Pichia pastoris, Bacillus subtilis, or Saccharomyces cerevisiae.

7. The microbial cell according to claim 5, characterized in that, The microbial cells use Escherichia coli as the host and pGEX-4T-1 as the expression vector to express the quinone-dependent dehydrogenase of claim 1.

8. A product containing the quinone-dependent dehydrogenase of claim 1, or the microbial cells of any one of claims 5-7.

9. The application of quinone-dependent dehydrogenases in the degradation of deoxynivalenzyme alcohol toxins, characterized in that, The reaction is carried out by adding the quinone-dependent dehydrogenase of claim 1 or the microbial cells of any one of claims 5-7 to a reaction system containing deoxynivalenol toxin.

10. The application according to claim 9, characterized in that, The reaction system also includes the cofactor pyrroloquinoline quinone.

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