CN116042558A - Degradation agent containing aldehyde ketone reductase mutant and eucommia ulmoides leaf extract and application thereof - Google Patents

Degradation agent containing aldehyde ketone reductase mutant and eucommia ulmoides leaf extract and application thereof Download PDF

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CN116042558A
CN116042558A CN202211721650.6A CN202211721650A CN116042558A CN 116042558 A CN116042558 A CN 116042558A CN 202211721650 A CN202211721650 A CN 202211721650A CN 116042558 A CN116042558 A CN 116042558A
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ketone reductase
akrh
degradation agent
mutant
aldehyde
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娄倩倩
张西雷
籍立民
曹爱智
孙恩江
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Shandong Longchang Animal Health Product Co ltd
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Shandong Longchang Animal Health Product Co ltd
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Abstract

The invention provides a degradation agent containing aldehyde ketone reductase mutant and eucommia ulmoides leaf extract and application thereof. The invention makes mutation on aldehyde-ketone reductase from methyl capsule a sp, constructs mutant library, uses Trichoderma reesei as expression host, and obtains aldehyde-ketone reductase mutant AkrH with obviously improved vomit toxin degradation effect through screening. The mutant AkrH has an approximately 3-fold increase in the level of degradation of vomitoxin compared to the original gene. The degradation agent prepared by compounding the spray dried powder prepared by the recombinant bacteria containing the aldehyde ketone reductase mutant AkrH, the eucommia ulmoides leaf extract, the montmorillonite powder and other adsorbents can degrade vomit toxin in feed raw materials to more than 95 percent on average, has good application prospect in the fields of preventing mycotoxin, grains, feed additives and the like, and is beneficial to animal cultivation.

Description

Degradation agent containing aldehyde ketone reductase mutant and eucommia ulmoides leaf extract and application thereof
Technical Field
The invention belongs to the field of enzyme engineering, and in particular relates to a degradation agent containing aldehyde ketone reductase mutants and eucommia ulmoides leaf extracts and application thereof.
Background
Vomitoxin, also known as deoxynivalenol toxin (DON), is a B-type trichothecene toxin, and is a secondary metabolite produced mainly by Fusarium graminearum (Fusarium graminearum) and Fusarium flavum (Fusarium culmorum), which easily contaminates grains and feeds. Vomitoxin can seriously affect the yield and quality of grains, can generate strong stimulation and reflection on the mucous membrane of the digestive tract of animals after entering a food chain to act on vomit centers, and affects the production performance, immune cell proliferation, apoptosis and the generation of immune cytokines, and has cytotoxicity. The ingestion of certain amounts of grains contaminated with DON by humans and animals poses a serious threat to their health, and research into the control of DON is therefore urgent.
The prior detoxification methods of DON mainly comprise a physical method, a chemical method and a biological degradation method. However, the structure of DON is very stable, the detoxification effect of physical method is not ideal, while the chemical treatment method can achieve better detoxification effect, but introduces other harmful substances and causes nutrition loss of grains, while the biodegradation method is to degrade mycotoxin under mild conditions by screening microorganisms in nature, convert DON into non-toxic or low-toxic substances and can not cause nutrition loss. Biodegradation is therefore considered to be the best method for removing mycotoxins.
Disclosure of Invention
The invention provides a degradation agent containing aldehyde ketone reductase mutant and eucommia ulmoides leaf extract and application thereof. The mutant AkrH with obviously improved vomitoxin degradation effect is obtained through screening, and can obviously degrade vomitoxin in feed raw materials with a degradation agent prepared from eucommia ulmoides leaf extract, montmorillonite powder and the like.
In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:
the invention provides an aldehyde ketone reductase mutant AkrH, the amino acid sequence of which is shown in SEQ ID NO: 3.
Further: the aldehyde ketone reductase mutant AkrH consists of a sequence of amino acid SEQ ID NO:1, from which glutamine at position 64 of the aldehyde-ketone reductase was changed to aspartic acid, leucine at position 131 was changed to serine, and leucine at position 212 was changed to valine.
The invention provides a coding gene of the aldehyde ketone reductase mutant AkrH, and the nucleotide sequence of the coding gene is shown as SEQ ID NO: 4.
The invention provides a recombinant expression vector containing the coding gene.
The invention provides a recombinant bacterium containing the coding gene, wherein the recombinant bacterium is bacillus subtilis, trichoderma reesei or pichia pastoris.
The invention provides a degradation agent, which comprises aldehyde ketone reductase mutant AkrH, eucommia ulmoides leaf extract, montmorillonite powder, glucose oxidase and bacillus subtilis.
Further: the degradation agent comprises 55% -70% of aldehyde ketone reductase mutant AkrH,8% -10% of montmorillonite powder, 0.1% -3% of glucose oxidase, 1% -3% of bacillus subtilis and the balance of eucommia ulmoides leaf extract by weight.
The invention provides application of the degradation agent in degrading vomitoxin in feed raw materials.
Further: the dosage of the degradation agent is 0.1% -0.5% of the weight of the feed raw material.
Further: the feed raw materials comprise wheat bran, DDGS and corn steep liquor.
Compared with the prior art, the invention has the advantages and technical effects that:
the invention takes aldehyde ketone reductase (WP_ 158815623.1) from methyl capsule a sp as basic raw material to mutate and improve, and a mutant AkrH with obviously improved effect on vomitoxin degradation is obtained by screening, and the mutation site is Q64D/L131S/L212V. Compared with the original gene, the degradation level of the mutant AkrH obtained by the invention on vomitoxin is improved by about 3 times.
According to the invention, the degradation agent prepared by carrying out compound treatment on the fermented liquid obtained by fermenting recombinant bacteria containing the aldehyde ketone reductase mutant AkrH, the spray-dried powder obtained after spray drying, the adsorbent such as eucommia ulmoides leaf extract, montmorillonite powder and the like can degrade vomitoxin in feed raw materials, and the degradation rate can reach more than 95% on average. Therefore, the aldehyde ketone reductase mutant AkrH and the degradation agent thereof can obviously degrade vomitoxin, reduce toxin pollution, have good application prospects in the fields of preventing mycotoxin, food, feed additives and the like, and are beneficial to animal cultivation.
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FIG. 1 shows the amplification result of the aldehyde-ketone reductase gene.
FIG. 2 is the effect of the aldehyde ketone reductase mutant on the degradation of vomitoxin.
FIG. 3 shows the results of 30L fermentation of the aldehyde ketoreductase mutant.
FIG. 4 shows the effect of degrading vomitoxin after the aldehyde ketone reductase mutant broth and montmorillonite are compounded.
Detailed Description
The present invention will be described more fully hereinafter with reference to the accompanying drawings and examples, in which the invention is shown, but the scope of the invention is not limited to the specific examples.
The molecular biology experimental methods not specifically described in the following examples can be performed with reference to the specific methods listed in the "molecular cloning Experimental guidelines (third edition) J.Sam Brookfield, or according to the kit and product instructions. Reagents and biological materials used in the specific examples are commercially available unless otherwise specified.
1. The formula of the culture medium used in the invention is as follows:
LB medium: 1% tryptone, 0.5% yeast extract, 1% nacl;
trichoderma reesei conversion medium (g/L):
conversion of the upper layer: glucose 10g, mgS0 4 ·7H 2 O 1g，KH 2 PO 4 10g，(NH 4 ) 2 SO 4 6g, sodium citrate 3g, feSO 4 ·7H 2 O 0.005g，MnSO 4 ·H 2 O 0.0016g，ZnSO 4 ·7H 2 O 0.0014g,CoCl 2 ·2H 2 0.002g of O, 182.18g of sorbitol, 6g of agarose and sterilizing for 30min at 115 ℃;
the lower layer was transformed: glucose 10g, mgS0 4 ·7H 2 O 1g，KH 2 PO 4 10g，(NH 4 ) 2 SO 4 6g, sodium citrate 3g, feSO 4 ·7H 2 O 0.005g，MnSO 4 ·H 2 O 0.0016g，ZnSO 4 ·7H 2 O 0.0014g,CoCl 2 ·2H 2 0.002g of O, 12g of agar powder and sterilizing for 30min at 115 ℃.
In the above culture medium, 10mM uracil was added to culture uracil-deficient cells.
PDA medium: 20% of potato extract and 2% of glucose.
MM medium: glucose 20g, mgS0 4 ·7H 2 O 0.6g,CaCl 2 0.6g，KH 2 PO 4 15g，
(NH 4 ) 2 SO 4 5g，Peptone 2g，FeSO 4 ·7H 2 O 0.005g，MnSO 4 ·H 2 O 0.0016g，ZnSO 4 ·7H 2 O0.0014g,CoCl 2 ·2H 2 O 0.002g。
Fermentation medium: 1-3% of soybean meal, 1000U/g of neutral protease, 3-5% of glucose, 0.1-0.5% of ammonium sulfate, 0.01-0.1% of magnesium sulfate, 0.01-0.1% of calcium chloride, 0.2-0.8% of potassium dihydrogen phosphate, 0.02-0.05% of tween and 3-10% of corn steep liquor.
2. Conversion-related reagents:
solution 1 (200 mL): 43.72g of sorbitol, 2.72g of potassium dihydrogen phosphate and adjusting the pH to 5.5 with sodium hydroxide solution.
Solution 2 (100 mL): sorbitol 18.22g, calcium chloride dihydrate 0.730 g,10mM Tris-HCl, pH 7.48.
Solution 3 (100 mL): PEG6000 25g, calcium chloride dihydrate 0.730 g,10mM Tris-HCl pH adjusted to 7.5.
3. Vomitoxin assay kit was purchased from Beijing Hua' an Mytilidae biotechnology Co., ltd, and was prepared according to the assay method described in the specification.
4. Trichoderma reesei conversion method
(1) Experiment preparation: PDA plate, sterile physiological saline (0.9% sodium chloride, 0.05% Tween-20), funnel with four layers of mirror cleaning paper or filter paper (sterilized), and glassine paper, cutting glassine paper into culture dish, placing one filter paper in the middle of each layer, placing in culture dish, and sterilizing.
(2) Culturing the bacterial cells: QP4 spores are taken on a plate of PDA (containing uracil) and cultured at 30 ℃ until the spores are grown; taking fresh spores, adding 5ml of sterile physiological saline, and washing out the spores to obtain spore suspension; a layer of cellophane was applied to a PDA (uracil containing) plate and spread with a spreading bar, 100ul of spore suspension was applied to the cellophane, spread evenly, and incubated at 30℃until more hyphae developed.
(3) Protoplast preparation and transformation: taking a clean plate, adding 0.1g of lyase, adding 20mL of solution 1, and uniformly mixing; washing the cultured mycelium into a plate containing lyase, and performing cleavage for 90 minutes at 30 ℃; after lysis, the mycelium is filtered by a funnel containing filter paper (mirror paper), and protoplasts are collected by centrifugation; resuspension with 4mL of solution 2, centrifugation at 2500rpm at 4 ℃ for 10min, discarding supernatant; sub-packaging after re-suspending with solution 2, adding 15ul of transformed plasmid into each tube, and ice-bathing for 5 min; then 2mL of solution 3 and 4mL of solution 2 are added respectively, and 30mL of transformation upper layer is added to the plate; culturing at 30 deg.C until the transformant grows.
Example 1: construction and screening of aldehyde ketone reductase mutant gene
Referring to the amino acid sequence of aldehyde ketone reductase (WP_ 158815623.1) of methyl capsule a sp, translating into a corresponding nucleotide sequence, and artificially synthesizing the amino acid sequence after gene sequence optimization, wherein the obtained amino acid sequence is shown as SEQ ID NO:1, the nucleotide sequence is shown as SEQ ID NO: 2. Primers were designed with BamHI restriction sites at the 5 'end and Sal I restriction sites at the 3' end.
Akr-F1：CGCGGATCCATGACAAAACCTACAGCTGC(SEQ ID NO：5)
Akr-R1：ACGCGTCGACTTATTTAGGACGAGCTTGAGCAT(SEQ ID NO：6)。
The random mutation is carried out by using the GeneMorph II random mutation PCR kit and using the artificially synthesized gene as a template and the primers, and the PCR amplification result is shown in figure 1.
The amplified random mutation PCR product is digested with BamHI and SalI, purified and recovered, and then connected to a pWB980 carrier, and positive clones are screened by referring to a bacillus subtilis transformation method created by Spizizin, bacillus subtilis WB600 is transformed and kanamycin-resistant LB plates, so that pAkrx is obtained. The synthesized original gene was ligated into the pWB980 vector in the same manner and transformed into Bacillus subtilis to give pAkr0.
The screened single colonies were inoculated into 96-well deep well plates. 2 single colonies expressing pAkr0 were inoculated per plate as controls. Each well was filled with 300uL of LB liquid medium (containing 20. Mu.g/mL kanamycin), and after shaking culture at 37℃and 200rpm for 12 hours, 50uL of the bacterial liquid was transferred to a new 96-well plate for seed preservation, and the effect of vomitoxin degradation was measured in the remaining bacterial liquid of the plate. Screening out mutants with degradation effect higher than that of a control group, and carrying out sequencing analysis to obtain an aldehyde ketone reductase mutant AkrH, wherein the amino acid sequence of the aldehyde ketone reductase mutant AkrH is shown as SEQ ID NO:3, the corresponding nucleotide sequence is shown as SEQ ID NO:4, the mutation site is Q64D/L131S/L212V.
Example 2: verification of the expression of Aldone reductase mutant AkrH in Trichoderma reesei
The excellent mutant AkrH obtained by screening in example 1 was amplified using the following primers Akr-F2 and Akr-R2, cloned into the Nco I and Sac I sites of plasmid pCBH, and transformed into Trichoderma reesei QP4 according to the genetic transformation method of Trichoderma reesei to obtain recombinant bacteria. And (3) carrying out shake flask fermentation on the recombinant bacteria in a fermentation medium at 30 ℃ for 4-5 days, and centrifuging the culture solution to obtain a supernatant for later use.
Akr-F2：CATGCCATGGATGACAAAACCTACAGCTGC(SEQ ID NO：7)；
Akr-R2：CGAGCTCTTATTTAGGACGAGCTTGAGCAT(SEQ ID NO：8)。
According to the determination method in the kit, the supernatant after the fermentation liquor is centrifuged is taken, and the degradation activity of each mutant fermentation liquor supernatant on vomitoxin is determined, and the result is shown in figure 2, the degradation effect of mutant AkrH recombinant bacteria on vomitoxin is about 3 times that of a control group, and the degradation rate is increased from 25.5% to 75%.
Example 3: akrH recombinant bacteria are fermented and prepared in a 30L fermentation tank
The AkrH recombinant bacteria are streaked on a PDA plate, cultured at 30 ℃ until the spores are generated, the spores are cultured for about 7 days, the spores are washed out by sterile water, and monospore separation is carried out, namely, the spores are diluted and coated into MM culture medium (containing Triton-X100) until the colonies grow out, then the spores are cultured, and the third-generation pure seeds are separated. The pure spores were washed with 100mL of sterile water and used as seed solution for inoculating the fermenter.
The fermentation production process comprises the following steps: the fermentation medium has natural pH, 30 ℃, stirring speed of 600rpm and ventilation rate of 1.5 (v/v), and dissolved oxygen is controlled to be more than 20%. After 24h fermentation, the enzyme activity is measured, and after the fermentation is finished (generally 48 h), the fermentation broth is used for application test.
The fermentation process curve is shown in fig. 3: sampling every 8h, measuring enzyme production level, fermenting for 48h, and reaching the highest enzyme activity level.
Example 4: preparation and application of degradation agent containing AkrH
1. Preparation of degradation agent
After the AkrH recombinant bacterium fermentation broth of example 3 was centrifuged, a supernatant was obtained and spray-dried to obtain AkrH spray-dried powder. By weight, 10% of montmorillonite powder, 1.5% of glucose oxidase, 1% of bacillus subtilis, 22% of eucommia ulmoides leaf extract and 65.5% of AkrH spray-dried powder are uniformly mixed to prepare the degradation agent, and the degradation agent is used according to the dosage of 0.1-0.5%.
2. Application of degradation agent
(1) The degradation agent degrades wheat bran:
weighing 20g of wheat bran, adding 20mL of water, then adding 0.1g of degradation agent, and vibrating at 37 ℃ for different times to detect the residual content of vomitoxin by a kit method.
(2) Degradation agent degradation DDGS (distillers dried grains with solubles):
weighing 20g of raw material (DDGS), adding 0.1g of degradation agent, adjusting pH to 8.0 with buffer solution, treating at 37deg.C for different times, and detecting the residual content of vomitoxin by using the kit method.
(3) Degradation agent degrading corn steep liquor:
weighing 20mL of corn steep liquor, adding 0.1g of degradation agent, adjusting pH to 8.0 with buffer solution, treating at 37 ℃ for different time, and detecting the residual content of vomitoxin by a kit method.
As shown in the figure 4, the degradation agent is used for treating wheat bran for 5 hours, the vomitoxin degradation rate is 75%, and the vomitoxin degradation rate reaches 96.5% after the treatment is continued for 5 hours; after DDGS is treated for 5 hours, the degradation rate of vomitoxin is 55%, the treatment is continued for 5 hours, the degradation rate is increased to 80%, and after the treatment is carried out for 15 hours, the degradation rate reaches 94.7%; corn steep liquor was treated for 5h and 10h at 59% and 94.5%, respectively. The results show that the degradation agent has remarkable effect of degrading vomitoxin in different feed raw materials.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Claims (10)

1. An aldehyde ketone reductase mutant AkrH, characterized in that the amino acid sequence is as shown in SEQ ID NO: 3.
2. The aldehyde ketone reductase mutant AkrH according to claim 1, characterized in that it consists of an amino acid sequence of SEQ ID NO:1, from which glutamine at position 64 of the aldehyde-ketone reductase was changed to aspartic acid, leucine at position 131 was changed to serine, and leucine at position 212 was changed to valine.
3. The aldehyde ketone reductase mutant AkrH encoding gene of claim 1, having a nucleotide sequence as set forth in SEQ ID NO: 4.
4. A recombinant expression vector comprising the coding gene of claim 3.
5. A recombinant bacterium comprising the coding gene of claim 3, wherein the recombinant bacterium is bacillus subtilis, trichoderma reesei, or pichia pastoris.
6. A degradation agent, characterized in that the degradation agent comprises the aldehyde ketone reductase mutant AkrH, eucommia ulmoides leaf extract, montmorillonite powder, glucose oxidase and bacillus subtilis according to claim 1.
7. The degradation agent according to claim 6, wherein the degradation agent comprises 55% -70% of aldehyde ketone reductase mutant AkrH,8% -10% of montmorillonite powder, 0.1% -3% of glucose oxidase, 1% -3% of bacillus subtilis and the balance of eucommia ulmoides leaf extract by weight.
8. Use of the degradation agent of claim 6 for degrading vomitoxin in feed stocks.
9. The use according to claim 8, wherein the degradation agent is used in an amount of 0.1% -0.5% by weight of the feed material.
10. The use according to claim 8, wherein the feed stock comprises wheat bran, DDGS and corn steep liquor.
CN202211721650.6A 2022-12-30 2022-12-30 Degradation agent containing aldehyde ketone reductase mutant and eucommia ulmoides leaf extract and application thereof Pending CN116042558A (en)

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Citations (4)

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