A sorbose dehydrogenase and its mutant for degrading vomitoxin

Abstract

The present invention discloses a sorbose dehydrogenase SDH with the property of degrading vomitoxin, wherein the nucleotide sequence and amino acid sequence thereof are SEQ ID NO.1 and SEQ ID NO.2 respectively. And a directed modified enzyme SDH with improved enzyme activity F103L , whose nucleotide sequence and amino acid sequence are SEQ ID NO.3 and SEQ ID NO.4, respectively. Compared with the wild type, the mutant enzyme of the present invention has higher enzyme activity characteristics, and the enzyme can degrade deoxynivalenol (DON) into its weak toxic products. The present invention belongs to the field of agricultural biotechnology, can be applied to the preparation of DON detoxification preparations, and has broad application prospects in the field of DON detoxification.

Description

Technical Field

[0001] The invention belongs to the fields of agricultural biotechnology and genetic engineering technology, and specifically relates to a sorbose dehydrogenase SDH and a mutant thereof for use in degrading vomitoxin. Background Art

[0002] Deoxynivalenol (DON), also known as vomitoxin, is one of the most widespread and influential mycotoxins in the world. About 25% of agricultural products in the world are contaminated by mold and mycotoxins, and mycotoxin contamination has become a global problem. Eating food contaminated with DON toxin can lead to a decrease in human immunity and cause pathological reactions such as anemia, headache, nausea and abdominal pain. Animals that eat it may suffer from adverse reactions such as food refusal, vomiting, growth retardation and reproductive disorders.

[0003] At present, the main methods for detoxification of vomitoxin include physical adsorption and microbial detoxification. The physical adsorption method is not specific and will cause the loss of nutrients during the adsorption process. There have been many reports on the effectiveness of microbial detoxification, but this method has potential risks of microbial contamination in food processing and feed production, so there are certain limitations in its application. Enzyme is a green catalyst with the characteristics of strong specificity and high efficiency. In actual feed detoxification applications, it does not affect the palatability and nutrients of feed, and has high research value. However, there are currently few enzyme resources that can degrade vomitoxin. It has been reported that manganese peroxidase has the effect of degrading vomitoxin, but its metabolites cannot be determined at present. In addition, the only known products are dehydrogenases (DepA and QDDH) and aldehyde-keto reductases (DepB, AKR13B2, and AKR6D1) from Devoserella, which can detoxify vomitoxin by step-by-step isomerization, and DepA and QDDH, as well as DepB and AKR13B2, respectively encode the same amino acid sequence.

[0004] Food and feed contamination caused by DON toxin pollution has always attracted much attention. The development of enzyme preparations with high-efficiency degradation activity has great economic value for practical production applications. Summary of the invention

[0005] The purpose of the present invention is to explore the resources of deoxynivalenol degrading enzymes and find that sorbose dehydrogenase SDH derived from Ketogulonigenium vulgare also has the potential to degrade deoxynivalenol.

[0006] The second object of the present invention is to further modify SDH based on its potential to degrade vomitoxin, and provide a mutant encoding gene with improved vomitoxin degradation activity.

[0007] The third object of the present invention is to provide a vector containing a gene encoding SDH and its mutants.

[0008] The fourth object of the present invention is to provide a recombinant strain or cell containing the above encoding gene.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The nucleotide sequence and amino acid sequence of the detoxifying enzyme SDH with detoxification function of the present invention may be: SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0011] The nucleotide sequence and amino acid sequence of the mutant of the present invention are SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0012] In one embodiment of the present invention, the amino acid at position 103 is mutated.

[0013] In one embodiment, the vector is a pET22b vector.

[0014] In one embodiment, the recombinant bacteria can be constructed using Escherichia coli, Bacillus, or yeast as hosts.

[0015] The present invention also provides SDH and SDH F103L Application in metabolism of DON.

[0016] Specifically, the application is to degrade DON into 3-keto-DON.

[0017] Preferably, when used to metabolize DON, SDH F103L It has more efficient degradation efficiency.

[0018] Preferably, SDH F103L The incubation temperature is 10℃~40℃ and the pH is 6~9.

[0019] The present invention has the following advantages and effects:

[0020] The present invention discovered that a sorbose dehydrogenase SDH has the potential to degrade vomitoxin, and a mutant enzyme with greatly improved degradation activity was transformed through semi-rational design. The degradation rate of this mutant enzyme can reach more than 75% within 1 hour, and it has a wide temperature and pH applicability, maintaining high activity in the temperature range of 10-40°C and pH6-9. In addition, the mutant enzyme also has extremely strong thermal stability. After being treated at 95°C for 10 minutes, the degradation rate can still reach more than 50%, and the metabolic effect is basically unchanged after being placed at 4°C for 2 months. The present invention is the first enzyme that has been transformed through enzyme engineering to efficiently degrade vomitoxin. Based on its wide range of applicable conditions and strong stability, the enzyme has good application prospects in the field of biological detoxification of vomitoxin. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 For SDH and SDH F103L SDS-PAGE diagram of expression and purification of Pet22b-SDH. Among them: M, protein molecular weight standard, the band sizes from top to bottom are 97.2KD, 66.4KD, 44.3KD, 29.0KD, 20.1KD, 15.0KD; lanes 1-5 are Pet22b-SDH samples, 1, whole bacteria before induction; 2, whole bacteria after induction; 3, supernatant after bacterial crushing; 4, precipitate after bacterial crushing; 5, purification of expression product; lanes 6-10 are Pet22b-SDH F103L Sample, 6. Whole bacteria before induction; 7. Whole bacteria after induction; 8. Supernatant after bacterial disruption; 9. Precipitation after bacterial disruption; 10. Purification of expression product;

[0022] Figure 2 For SDH F103L Catalytic effect on DON (a is inactivated SDH F103L For the DON treatment group, b is SDH F103L For the DON-treated group, Compond A and Compond B are degradation products of DON);

[0023] Figure 3 For different time SDH and SDH F103L Changes in the degradation rate of DON;

[0024] Figure 4 Temperature for SDH and SDH F103L Effect of DON degradation activity;

[0025] Figure 5 pH for SDH and SDH F103L Effect of DON degradation activity;

[0026] Figure 6 For SDH F103LEffect of heat treatment time on DON degradation rate;

[0027] Figure 7 For SDH F103L Effects of different time periods at 4°C on the degradation rate of DON;

[0028] Figure 8 For SDH F103L After treatment, the toxicity of DON to HEK293T cells can be reduced. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0030] Example 1 Sorbitol dehydrogenase SDH and SDH F103L Expression vector construction

[0031] The pET22b-SDH plasmid was synthesized by Shanghai Sangon Biotechnology Co., Ltd., wherein the nucleotide sequence of sorbose dehydrogenase SDH in the plasmid pET22b-SDH is shown as SEQ ID NO: 1 (the amino acid sequence is shown as SEQ ID NO: 2).

[0032] Recombinant plasmid pET22b-SDH F103L To construct the site-directed mutagenesis construct, primers for site-directed mutagenesis were designed and synthesized based on the amino acid site 103. Forward primer: 5′-ACATTGCAACCCTGAACTCCCTGGGTGAACCGACCCGTGG-3′, reverse primer: 5′-CCACGGGTCGGTTCACCCAGGGAGTTCAGGGTTGCAATGT-3′.

[0033] The plasmid pET22b-SDH containing the SDH gene was used as a template and the above primers were used for PCR. The reaction conditions were: 94°C for 2 min; 98°C for 30 s, 54°C for 30 s, 68°C for 7 min, and 30 cycles. The PCR product was digested with DnPI enzyme and transformed into DH5α competent cells. The plasmid of the positive clone was extracted by antibiotic screening and sent for sequencing. The expression plasmid pET22b-SDH with correct sequencing was F103L By transforming into the expression strain BL21, the expression engineering bacteria of the SDH mutant can be successfully constructed.

[0034] Example 2 SDH and SDH F103L Expression and purification

[0035] The engineered bacteria were inoculated into 10 mL of LB medium containing 50 μg / mL of ampicillin, and shaken at 37°C and 200 rpm. When the bacterial solution became turbid, it was inoculated into 500 mL of fresh LB medium containing 50 μg / mL of ampicillin at a ratio of 1:100, and shaken at 37°C and 200 rpm. When the OD600 value of the bacterial solution reached 0.5-0.6, the inducer IPTG was added at a final concentration of 0.5 mM, and the bacteria were harvested after induction at 25°C and 150 rpm for 24 hours according to the preliminary experimental results. The induced bacterial solution was precooled on ice for half an hour, centrifuged at 4°C and 5000 rpm for 10 minutes, and the bacteria were collected. The collected bacteria were resuspended in precooled ddH2O, centrifuged at 4°C and 5000 rpm for 10 minutes, and the supernatant was discarded to retain the bacteria. Resuspend each 1g of bacterial cells in 20mL buffer (50mM Tris-HCl, 1mM EDTA, 2mM DTT, 0.4mM PMSF), and disrupt the cells by ultrasonication at low temperature. After the bacterial solution is broken until clear, centrifuge at 4℃, 5000rpm for 30min, and take the supernatant through a 0.22μm filter.

[0036] Purification was performed using a protein purifier FPLC system and a GE His TrapTMHP (5mL) column. The formula of the equilibrium buffer was: 50mM Tris-Hcl, 50mM NaCl, 0.5mM EDTA, 15mM imidazole, pH 8.0; the formula of the elution buffer was 50mM Tris-Hcl, 50mM NaCl, 0.5mM EDTA, 500mM imidazole, pH 8.0. The collected protein was dialyzed overnight to reduce the imidazole concentration. The formula of the dialysate was: 50mM Tris-Hcl, 0.5mM EDTA, 2mM DTT, 20% glycerol, pH 8.0. After dialysis, ultrafiltration and concentration were performed using a 10kDa ultrafiltration tube. The expression and purification results of the target protein were detected by SDS-PAGE electrophoresis.

[0037] The results are as follows Figure 1 As shown, the left side of the marker is the result of SDH expression and purification, and the right side is SDH F103L Expression and purification results: The protein expressed by the recombinant strain is at 66.4KD, which is consistent with the theoretical molecular weight.

[0038] Example 3 SDH and SDH at different times F103L Degradation rate of DON

[0039] The incubation experiment was performed with a 200 μL reaction system as follows: final concentrations of 50 mM Tris-HCl buffer (pH 7.0), 10 μM PQQ, 1 mM CaCl2, 1 mM PMS, SDH / SDH F103LProtein (40 μg), 15 μg / mL DON. The system without protein was used as a control. The reaction was carried out at 25°C for 15 min, 1 h, 4 h, and 16 h. Then 600 μL of ethyl acetate was added for full shaking extraction, centrifuged at 12000 rpm for 1 min, the upper layer of ethyl acetate was dried with nitrogen, resuspended with 200 μL of 50% methanol, filtered with a 0.22 μm filter membrane, and the residual DON content in the system was detected by high performance liquid chromatography.

[0040] The substrate and product were detected using Agilent 1260HPLC with water and acetonitrile as the mobile phases. The elution procedure 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% to 12%; and re-equilibration for 4 min.

[0041] Degradation rate of DON (%) = (1-remaining DON amount in the treatment group / DON amount in the control group) × 100%

[0042] The results are as follows Figure 2 As shown, it is SDH F103L The liquid phase diagram of DON metabolism clearly shows the decrease in DON level and the generation of products A and B.

[0043] The results are as follows Figure 3 As shown in Figure 2, after SDH was incubated with DON for 4 hours, the degradation rate of DON toxin was about 17%, while after the phenylalanine at position 103 was mutated to leucine, the mutant SDH F103L When incubated with DON toxin for 1 hour, the degradation rate can reach more than 75%, and the enzyme activity is greatly improved.

[0044] Example 4 Effect of Temperature on SDH and SDH F103L Effect of DON degradation activity

[0045] SDH and SDH F103L Incubate with DON toxin at different temperatures (10, 15, 20, 25, 30, 35, 40, 45°C) for 4 hours. Use the reaction system and detection method used in Case Implementation 2.

[0046] The results of SDH were based on the enzyme activity at 20°C as 100%. F103L The results were expressed as relative activities at other temperatures, with the enzyme activity at 35°C as 100%.

[0047] The results are as follows Figure 4 As shown in the figure, the optimum temperature for SDH to degrade DON is 20℃. F103L The optimum temperature for degradation of DON is 35℃, SDHF103L It has high activity in the range of 10-40℃ and has a wide temperature application range.

[0048] Example 5 Effect of pH on SDH and SDH F103L Effect of DON degradation activity

[0049] SDH and SDH F103L Incubate with DON toxin for 4 hours under different pH conditions (3, 4, 5, 7, 8, 9, 10, 11). Glycine-HCl buffer was used for pH (3, 4, 5), Tris-HCl buffer was used for pH (7, 8), and glycine-NaOH buffer was used for pH (9, 10, 11). Other components and detection methods in the system are as shown in Case Implementation 2.

[0050] SDH vs SDH F103L The results are expressed as relative activity at other pH conditions, with the enzyme activity at pH 7 as 100%. Figure 5 As shown, SDH and SDH F103L The optimum pH for degradation of DON is 7, SDH F103L It has high activity in the pH range of 4-9 and has a wide pH application range.

[0051] Example 6 SDH F103L Effect of heat treatment time on DON degradation rate

[0052] SDH F103L Treat at 95°C for different time periods (10 min, 30 min, 1 h, 2 h), place on ice for cooling, and then incubate with DON for 4 h at 20°C. The reaction system and detection method used in Case Implementation 2 were used.

[0053] The results are as follows Figure 6 As shown, treated at 95℃ for 10min, SDH F103L The degradation rate of DON is about 52%. After treatment at 95℃ for 30min, SDH F103L The degradation rate of DON was 26%. After treatment at 95℃ for 1h, SDH F103L Completely lost its degradation activity.

[0054] Example 7 SDH F103L Effect of storage time at 4°C on DON degradation rate

[0055] SDH F103L Place at 4°C for a long time and test the enzyme activity every month. Use the reaction system and detection method used in Case Implementation 2.

[0056] The results are as follows Figure 7 As shown, SDHF103L There was no significant change in enzyme activity after storage at 4°C for 2 months.

[0057] Example 8 Metabolite Cytotoxicity Detection

[0058] Take the same amount of DON in the same system, add SDH to one portion F103L Processing, one copy without SDH F103L Treatment, incubation at 20℃ for 16 hours. At the same time, after extraction with ethyl acetate, add an equal amount of DMSO to resuspend, and take 1μL of liquid phase detection for each of the two samples. SDH F103L Processing and without SDH F103L The DON in the treated samples was reduced by 86.4%. After HEK 293T cells were attached and grown in 96-well plates, DMSO resuspensions of equal dilutions were added to HEK 293T cells. The final concentrations in the culture medium were 60, 120, 240, and 480 ng / mL, calculated according to the original DON content. After culturing for 24 hours, CCK8 detection solution was added, and the culture was continued for 1-2 hours. The absorbance at 450 nm was detected by a microplate reader.

[0059] The results are as follows Figure 8 As shown, SDH F103L Compared with the untreated group, the cytotoxicity of the treated group was greatly reduced, about 1 / 8 of that of the untreated group. Since 13.6% DON remained after treatment, it showed that the product may be close to non-toxic.

Claims

1. A sorbose dehydrogenase mutant SDH with improved deoxynivalenol degradation activity F103L , characterized in that, The amino acid sequence of the mutant is based on the amino acid sequence shown in SEQ ID NO.2, with the phenylalanine at position 103 mutated to leucine. The nucleotide sequence and amino acid sequence of the mutant are SEQ ID No.3 and SEQ ID No.4, respectively.

2. A method comprising the sorbose dehydrogenase mutant SDH as claimed in claim 1 F103L The recombinant expression vector is characterized in that The recombinant expression vector is used for detoxification of vomitoxin.

3. A recombinant bacterium capable of degrading vomitoxin, characterized in that: The recombinant bacteria express the sorbose dehydrogenase mutant SDH according to claim 1 F103L .

4. A biodegradation agent for vomitoxin, characterized in that: The biodegradation agent comprises the sorbose dehydrogenase mutant SDH as claimed in claim 1 F103L .

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