CN111394333A

CN111394333A - Ochratoxin detoxification enzyme, and coding gene, recombinant vector and application thereof

Info

Publication number: CN111394333A
Application number: CN201910004498.1A
Authority: CN
Inventors: 周育; 王旭; 陈楠; 杜郑君
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2019-01-03
Filing date: 2019-01-03
Publication date: 2020-07-10
Anticipated expiration: 2039-01-03
Also published as: CN111394333B

Abstract

The invention discloses ochratoxin detoxification enzyme, and a coding gene, a recombinant vector and application thereof, and belongs to the technical field of enzyme engineering. The invention provides ochratoxin detoxification enzyme and a coding gene thereof, and also provides the optimum pH value of the ochratoxin detoxification enzyme and application of the ochratoxin detoxification enzyme in degradation of ochratoxin and biological detoxification of ochratoxin in foods. The ochratoxin detoxification enzyme treats ochratoxin A in a buffer system with pH 7.3 at 37 ℃ for 2min, and the degradation rate of the ochratoxin A reaches 100%, which is unprecedented in the field.

Description

Ochratoxin detoxification enzyme, and coding gene, recombinant vector and application thereof

Technical Field

The invention relates to ochratoxin detoxification enzyme, and a coding gene, a recombinant vector and application thereof, and belongs to the technical field of enzyme engineering.

Background

The aspergillus mycotoxins are mainly secondary metabolites produced by aspergillus strains producing toxin such as aspergillus flavus, aspergillus parasiticus, aspergillus ochraceus and the like, more than twenty structural analogs have been separated and identified, wherein the aflatoxin B1(AFB1) and the aspergillus ochraceus A (OTA) have the highest toxicity, and the aflatoxins are most widely polluted in agricultural production and food industry. In 1993, the World Health Organization (WHO) cancer research institute classified aflatoxins as class I carcinogens and ochratoxins as class II B carcinogens. Studies have shown that ochratoxins are the major cause of human balm's disease and have become the focus of growing public health and scientific attention due to their strong nephrotoxicity, as well as possible carcinogenesis, teratogenicity and mutagenicity. Contamination of cereal and oil crops with aspergillus mycotoxins has become a global problem, in animal husbandry, aspergillus mycotoxins can harm animal health by contaminating feed, resulting in low animal husbandry productivity, causing severe economic losses, and affecting human health by contaminating food either directly or indirectly (animal product transmission). With the gradual deepening of people's understanding of the harmfulness of the aspergillus mycotoxins, research on the detection, pollution prevention and control and detoxification technology of the mycotoxins is continuously carried out, and the research is relevant to the quality of life, health and economic benefits of people.

The detoxification method of ochratoxin commonly adopted at present comprises the following steps: physical adsorption and chemical decomposition methods can effectively reduce or degrade the pollution concentration of ochratoxin in food and feed, but the detoxification methods still have the problems of high cost, difficult thorough detoxification, sensory changes of the food and feed caused by the detoxification methods in the treatment process, and loss of nutrient components.

Biological detoxification is considered to be one of the most promising methods for removing ochratoxin A pollution in recent years due to the advantages of low cost, safety, high efficiency and the like, biological detoxification enzymes and genes thereof are screened, the biological functions of the detoxification enzymes are perfected, critical biological materials and matching technologies are accumulated for biological detoxification methods, and the research on the biological detoxification technology in China is effectively promoted. Ochratoxin detoxification enzymes are a general term for enzymes that degrade ochratoxin, and most of them belong to the class of hydrolases. Widely present in fungi, plants, insects and bacteria, and have been a research hotspot in the fields of biological, chemical and environmental science since the discovery. However, the existing enzyme for degrading ochratoxin has low degradation efficiency or is not suitable for industrial application, and no enzyme which has high degradation rate of ochratoxin and is suitable for industrial production exists at present.

Disclosure of Invention

In order to solve the problems, the invention provides the ochratoxin detoxification enzyme derived from the lysobacter strain of xanthomonas, which realizes heterologous high expression in a prokaryotic expression system, is soluble protein, is easy to purify in the later period and has low commercial production cost.

The first object of the present invention is to provide an ochratoxin detoxification enzyme, the amino acid sequence of which is shown in (a) or (b):

(a) an amino acid sequence shown as SEQ ID NO. 1;

(b) and (b) an amino acid sequence which has 95% or more homology with the amino acid sequence shown in (a) and has ochratoxin detoxification enzyme activity.

Further, the ochratoxin detoxification enzyme includes any peptide protein fragment containing the amino acid sequence shown in SEQ ID NO.1 or its variant, such as conservative variant, bioactive fragment or derivative thereof, as long as the peptide protein fragment or peptide protein variant has a homology of more than 95% with the amino acid sequence, and the peptide protein fragment or peptide protein variant is within the protection scope of the present invention. Particular such alterations may include deletions, insertions or substitutions of amino acids in the amino acid sequence; where conservative changes to a variant are made, the substituted amino acid has similar structural or chemical properties as the original amino acid, e.g., replacement of isoleucine with leucine, and the mutant may also have non-conservative changes, e.g., replacement of glycine with tryptophan. The fragment, derivative or analogue of the polypeptide protein of the present invention refers to a peptide protein that substantially retains the same biological function or activity as the ochratoxin detoxification enzyme of the present invention, and may be: (I) one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), and the substituted amino acid may or may not be encoded by the genetic code; (II) one or more of the amino acid residues is substituted with another group; (III) the mature peptide protein is fused to another compound (such as a compound that extends the half-life of the peptide protein, e.g., polyethylene glycol); (IV) peptide protein sequences formed by fusing additional amino acid sequences to the mature peptide protein (e.g., sequences used to purify the peptide protein or proprotein sequence).

Further, the ochratoxin detoxification enzymes may be recombinant, natural or synthetic, pure natural purified products, or chemically synthesized products, or produced by recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect and mammalian cells). The peptide proteins of the invention may be glycosylated depending on the host used in the recombinant production scheme. The ochratoxin detoxification enzymes of the invention may or may not also include the initial methionine residue.

It is a second object of the present invention to provide a gene encoding the ochratoxin detoxification enzyme.

Furthermore, the gene has a nucleotide sequence shown as SEQ ID NO.2, or a nucleotide sequence which has more than 70 percent of homology with the nucleotide shown as SEQ ID NO. 2.

Due to the specificity of the nucleotide sequence, any variant of the polynucleotide shown in SEQ ID NO.2 is within the scope of the present invention as long as it has more than 70% homology with the polynucleotide. A variant of the polynucleotide refers to a polynucleotide sequence having one or more nucleotide changes. Variants of the polynucleotide include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of a polynucleotide, without substantially altering the function of the peptide protein encoded thereby.

Furthermore, polynucleotides which hybridize to the polynucleotide shown in SEQ ID No.2 (having at least 50% homology, preferably 70% homology) are also within the scope of the present invention, in particular polynucleotides which hybridize under stringent conditions to the nucleotides described herein. The "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2SSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum, 0.1% Ficoll, 42 deg.C; or (3) hybridization occurs only when the homology between two sequences is at least 95% or more, preferably 97% or more. And, the peptide protein encoded by the hybridizable polynucleotide hybridizes to SEQ ID NO: the peptide protein shown in 1 has the same biological functions and activities.

The polynucleotide sequence encoding the ochratoxin detoxification enzyme of the invention can be obtained in a number of ways. For example, polynucleotides are isolated using hybridization techniques well known in the art. These techniques include (but are not limited to): (1) hybridization of probes to gene or cDNA libraries to detect homologous polynucleotide sequences and (2) activity screening of expression libraries, which may include environmental metagenomic libraries or libraries of clones created from a pure culture of the strain, to detect cloned polynucleotide fragments having common structural features. The DNA fragment sequences of the present invention can also be obtained by the following methods: (1) isolating double-stranded DNA sequences from genomic DNA; (2) chemically synthesizing a DNA sequence to obtain double-stranded DNA of the ochratoxin detoxification enzyme.

The genes of the present invention can be screened from these cNDA libraries by conventional methods. These methods include (but are not limited to):

(1) DNA-DNA or DNA-RNA hybridization; (2) the appearance or loss of function of a marker gene; (3) the protein product of gene expression is detected by measuring biological activity. The above methods can be used singly or in combination of multiple methods.

The polynucleotide sequences of the gene of the present invention or various DNA fragments obtained as described above can be determined by the dideoxy chain termination method according to a conventional method. Such polynucleotide sequencing may also be performed using commercial sequencing kits and the like. Sequencing was repeated to obtain a full-length cDNA sequence. Sometimes it is necessary to sequence the cDNA of multiple clones to splice the full-length cDNA sequence.

The third purpose of the invention is to provide a recombinant vector carrying the gene.

Further, the vector is a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus or a mammalian cell virus.

In the present invention, the nucleotide sequence encoding ochratoxin detoxification enzyme may be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention. "vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors well known in the art. Vectors suitable for use in the present invention also include, but are not limited to: expression vectors based on the T7 promoter for expression in bacteria; pcDNA3.1 vector expressed in mammalian cells and baculovirus-derived vector expressed in insect cells. In general, any plasmid or vector can be used to construct recombinant expression vectors, preferably the pET vector series as well as other prokaryotic expression vector series, so long as it is replicable and stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation regulatory elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing DNA sequences encoding ochratoxin detoxification enzymes and appropriate transcription/translation regulatory elements, including in vitro recombinant DNA sequences, DNA synthesis techniques, in vivo recombinant techniques, etc., which can be operably linked to appropriate promoters in the expression vector to direct the synthesis of mRNA, representative examples of these promoters are the lac or trp promoter of E.coli, the P L promoter of bacteriophage, eukaryotic promoters including the CMV early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, L TRs of retrovirus, and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein for eukaryotic cell culture, or kanamycin or ampicillin for E.coli, and the like.

The fourth purpose of the invention is to provide a recombinant bacterium for expressing the ochratoxin detoxification enzyme.

Further, bacterial, fungal, plant, insect or animal cells are used as host cells.

In the present invention, a polynucleotide encoding ochratoxin detoxification enzyme or a recombinant vector containing the polynucleotide may be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the nucleotide or recombinant vector. "host cell" refers to prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells such as salmonella typhimurium; fungal cells such as yeast; a plant cell; insect cells such as Drosophila S2 or Sf 9; animal cells such as CHO, COS or Bowes melanoma cells.

Transformation of a host cell with a DNA sequence according to the invention or a recombinant vector containing said DNA sequence may be carried out by conventional techniques well known to those skilled in the art. When the host is prokaryotic, such as E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase and treated by the CaCl2 method, as is well known in the art, alternatively with MgCl 2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, or conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The recombinant ochratoxin detoxification enzymes can be expressed or produced by conventional recombinant DNA techniques using the polynucleotide sequences of the invention. Generally, the following steps are performed:

(1) transforming or transfecting a suitable host cell with the polynucleotide encoding ochratoxin detoxification enzyme of the invention, or with a recombinant expression vector containing the polynucleotide;

(2) culturing the host cell in a suitable medium;

(3) separating and purifying protein from culture medium or cell.

In step (2), the medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is carried out under conditions suitable for the host cell. After the host cells have been grown at the appropriate cell density, the selected promoter is induced by suitable means and the cells are cultured for an additional period of time.

In step (3), the recombinant enzyme may be encapsulated inside the cell, or expressed on the cell membrane, or secreted outside the cell, if desired, the recombinant protein may be isolated and purified by various separation methods using its physical, chemical, and other properties, which are well known to those skilled in the art.

The fifth purpose of the invention is to provide the application of the ochratoxin detoxification enzyme in degradation of ochratoxin A.

Further, the application comprises degrading ochratoxin A in oil, grains or tea.

It is a sixth object of the present invention to provide an enzyme preparation comprising the ochratoxin detoxification enzyme.

The invention has the beneficial effects that:

the invention provides ochratoxin detoxification enzyme and a coding gene thereof, and also provides the optimum pH value of the ochratoxin detoxification enzyme and application of the ochratoxin detoxification enzyme in degradation of ochratoxin and biological detoxification of ochratoxin in foods. The ochratoxin detoxification enzyme treats ochratoxin A in a buffer system with pH 7.3 at 37 ℃ for 2min, and the degradation rate of the ochratoxin A reaches 100%, which is unprecedented in the field.

Drawings

FIG. 1 shows the electrophoresis of the Haematitum toxin detoxification enzyme SDS-PAGE protein expressed by pronucleus;

FIG. 2 shows the effect of ochratoxin detoxification enzyme on the degradation of ochratoxin A in food products (wheat flour);

FIG. 3 shows the effect of ochratoxin detoxification enzyme on the degradation of ochratoxin A in Fuzhuan tea.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The method for measuring the activity of ochratoxin detoxification enzyme comprises the following steps:

the transformant E.coli B L (DE)3 containing pGEX-4T-1/adh239 expression vector expresses under the optimal condition, the crushed supernatant of 10 mu L expression bacterial liquid is placed in a 2m L centrifuge tube, added into 490 mu L ochratoxin A standard buffer (the pH value is 7.3 by using 1 PBS), the final concentration of ochratoxin A is about 40 mu g/L, the reaction is stopped for 2min, 1.5m L acetonitrile is added into the reaction system to stop the reaction, the residual amount of OTA is detected by using high performance liquid chromatography, and the experimental result shows that after the reaction is 2min, the recombinase crude enzyme liquid can completely degrade the substrate in the reaction system.

Example 1: synthesis and cloning of ochratoxin detoxification enzyme gene cDNA

The strain is derived from a flavomonads bacterium lysobacter strain obtained in the early stage of a laboratory, an open reading frame nucleotide sequence of ochratoxin A hydrolase gene is obtained through analysis by gene sequence determination, and a primer upstream primer (adh239-F) (SEQ ID NO.3) of a complete coding reading frame is designed and amplified: 5' -ATCTGGTTCCGCGTGGATCCATGACCGTCCGCCTGGTCCG-3'; downstream primer (adh239-R) (SEQ ID NO. 4): 5' -TCACGATGCGGCCGCTCGAGTCATGGCGACGTCCCGGGC-3', upstream and downstream primersRestriction endonuclease sites (determined by the selected vector, BamHI and XhoI restriction sites are added in the invention, underlined and italic sequences are pGEX-4T-1 vector downstream terminal homologous sequences) are underlined, italic sequences are pGEX-4T-1 vector downstream terminal homologous sequences are underlined, an ochratoxin detoxification enzyme gene encoding the amino acid shown in SEQ ID NO.1 is obtained by an in vitro amplification technology, the sequence of the ochratoxin detoxification enzyme is shown in SEQ ID NO.2, an expression recombinant plasmid pGEX-4T-1/ADH239 is constructed on the premise of ensuring correct reading frame, and then the expression recombinant plasmid is transferred into E.Coil B L21 (DE3), and sequence analysis shows that the theoretical isoelectric point of the ADH239 enzyme is 6.10 and the theoretical molecular weight is 46.1 kDa.

Example 2: heterologous expression of ochratoxin detoxification enzyme genes

The E.coil B L21 (DE3)/pGEX-4T-1/adh239 transformant obtained in example 1 was subjected to shake cultivation overnight in L B liquid medium containing 100. mu.g/m L ampicillin at 100m L, 1.0m L seed broth was inoculated into fresh 100m LL B liquid medium containing 100. mu.g/m L ampicillin, and shake cultivation was carried out at 37 ℃ at 180r/min, when the OD is reached₆₀₀When the molecular weight reaches 0.6, 0.2mmol/m L of IPTG is added to induce expression for 4h under the condition of 16 ℃, the bacterial strain is frozen and centrifuged for 10min under the rotating speed of 7000r/min, the supernatant is discarded, the bacterial strain is suspended by 1 × Phosphate Buffer Solution (PBS) of 10m L, the bacterial strain is crushed by ultrasonic waves under the ice bath condition, the centrifugal treatment is carried out, and the precipitate is subjected to inclusion body renaturation, and the SDS-PAGE electrophoresis result shows that the enzyme exists in the supernatant and the precipitate (inclusion body) after the bacterial strain is crushed, and the apparent molecular weight (including GST tag 26kDa) of the enzyme is about 72kDa (figure 1) and is consistent with the theoretical molecular weight.

Example 3: application of heterologous recombinant ochratoxin detoxification enzyme in ochratoxin degradation

1. Experimental Material

The enzyme preparation is crude enzyme solution of a broken supernatant after expression of the pGEX-4T-1/adh239 transformant, and other reagents are analytically pure chemical reagents.

2. Experimental methods

And (3) placing the crushed supernatant after 10 mu L pGEX-4T-1/adh239 transformant expression into a 2m L centrifuge tube, adding 490 mu L ochratoxin A standard buffer (the pH value is 7.3 by using 1 x PBS), wherein the final concentration of the ochratoxin A is about 40 mu g/L, reacting for 2min, adding 1.5m L acetonitrile into the reaction system to stop the reaction, detecting the residual amount of the OTA by using high performance liquid chromatography, and the experiment result shows that the crushed supernatant completely degrades the OTA after the reaction is 2 min.

Example 4: application of heterologous recombinant ochratoxin detoxification enzyme in detoxification of ochratoxin-polluted grains

1. Experimental Material

The enzyme preparation is crude enzyme solution of broken supernatant after pGEX-4T-1/adh239 transformant expression, and the experimental material is wheat flour. All other reagents are analytical pure chemical reagents.

2. Experimental methods

Adding a proper amount of OTA standard stock solution into 50m L phosphate buffer solution, pouring into 500g of crushed wheat flour sample immediately after mixing, stirring uniformly to enable the final concentration of OTA to reach 80.0 mu g/kg, airing in a cool and ventilated place for standby application, taking 200m L enzyme preparation, mixing with 200g of prepared toxin-contaminated wheat flour uniformly, culturing the prepared sample at 37 ℃, sampling for 0h, 1h, 3h and 6h, setting 3 times for each degradation experiment, mixing 200m L fresh buffer solution and 200g of prepared toxin-contaminated wheat flour uniformly as negative control treatment, sampling 20g of treatment group and blank control group each time, and extracting, purifying and analyzing immediately according to an ochratoxin standard detection method.

Accurately weighing 10g of cultured sample in a triangular flask with a plug of 150m L, adding 1g of NaCl and 20m of L methanol-water mixed solution (volume ratio is 4:1), shaking and extracting at normal temperature for 30min, taking down the plug, filtering in a clean cup by a cano-line filter paper (or centrifuging at 3000r/min at normal temperature for 5min), accurately transferring 5.0m of L supernatant, adding 20m of L pure water, diluting and mixing uniformly, filtering by glass fiber filter paper for 1-2 times until the filtrate is clarified, and purifying by using an immunoaffinity column.

10.0m L (representing 1.0g of sample) of the clarified filtrate was removed accurately and injected into a glass syringe, the pressure was adjusted to allow the solution to pass slowly through the immunoaffinity column at a flow rate of 1-2 drops/sec until a portion of the air passed through the column, the column was rinsed with 10m L pure water (repeated once), all the effluent was discarded and a portion of the air passed through the column, 1.5m L methanol was added accurately and eluted at a flow rate of 1 drop/sec, and the eluate was collected in a glass vial for HP L C-F L D assay.

The dynamic degradation curve of ochratoxin detoxification enzyme on aflatoxin is shown in figure 2, and the experimental result shows that the 3-hour degradation rate of ochratoxin detoxification enzyme-treated toxin-contaminated wheat flour (with the final concentration of 40 mug/L) is 100%.

Example 5: application of heterologous recombinant ochratoxin detoxification enzyme in detoxification of ochratoxin-polluted dark green tea

1. Experimental Material

The enzyme preparation is crude enzyme liquid of a supernatant fluid after the disruption of the expression of the pGEX-4T-1/adh239 transformant, and the experimental material is Fuzhuan tea. All other reagents are analytical pure chemical reagents.

2. Experimental methods

Adding a proper amount of OTA standard stock solution into 50m L phosphate buffer solution, pouring into 500g of crushed tea powder sample immediately after mixing, stirring uniformly to enable the final concentration of OTA to reach 80.0 mu g/kg, airing in a cool and ventilated place for standby application, taking 200m L enzyme preparation, mixing with 200g of prepared toxin-contaminated tea powder uniformly, culturing the prepared sample at 37 ℃, sampling for 0h, 1h, 3h and 6h, setting 3 times of repetition for each degradation experiment, simultaneously mixing 200m L fresh buffer solution and 200g of prepared toxin-contaminated tea powder uniformly as negative control treatment, sampling 20g of treatment group and blank control group each time, and extracting, purifying and analyzing immediately according to an ochratoxin standard detection method.

A dynamic degradation curve of ochratoxin detoxification enzyme on ochratoxin in tea powder is shown in figure 3, and an experimental result shows that the degradation rate of ochratoxin detoxification enzyme-treated tea powder (with a final concentration of 40 mug/L) polluted by toxin for 6 hours is 100%.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

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Claims

1. An ochratoxin detoxification enzyme, characterized in that the amino acid sequence is as shown in (a) or (b):

(a) an amino acid sequence shown as SEQ ID NO. 1;

2. A gene encoding ochratoxin detoxification enzyme according to claim 1.

3. The gene as claimed in claim 2, wherein the gene has a nucleotide sequence shown in SEQ ID No.2, or a nucleotide sequence having 70% or more homology with the nucleotide sequence shown in SEQ ID No. 2.

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

5. The recombinant vector according to claim 4, wherein the vector is a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus or a mammalian cell virus.

6. A recombinant bacterium expressing ochratoxin detoxification enzyme according to claim 1.

7. The recombinant bacterium according to claim 6, wherein the host cell is a bacterial, fungal, plant, insect or animal cell.

8. Use of an ochratoxin detoxification enzyme according to claim 1 in the degradation of ochratoxin a.

9. Use according to claim 8, which comprises degrading ochratoxin A in oil, cereal or tea.

10. An enzyme preparation comprising an ochratoxin detoxification enzyme according to claim 1.