Aldehyde-ketone reductase and use thereof

Abstract

The present application belongs to the technical field of bioengineering, and particularly relates to a kind of aldehyde ketone reductase and its application.The aldehyde ketone reductase is a protein consisting of the amino acid sequence shown in SEQ ID NO.1;the coding gene of the aldehyde ketone reductase, nucleotide sequence is shown in SEQ ID NO.2.The aldehyde ketone reductase constructed in the present application has high catalytic performance, in the reaction system with aldehyde ketone reductase as biological catalyst, PAT as substrate, NADPH as coenzyme and MES buffer, 15ug / mL of patulin can be completely degraded in 4h;and 0.5ug / mL of patulin in fresh pear juice can be completely degraded in 4h.Simultaneously, the present application is safe and environmentally friendly, and the expressed and purified aldehyde ketone reductase can be effectively used to control patulin pollution in self-made pear juice, and has broad application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to an aldehyde-ketone reductase and application thereof in degrading patulin. BACKGROUND

[0002] Patulin is a strong virulence fungal secondary metabolite, and its molecular formula is C7H6O4, and its chemical name is 4-hydroxy-4H-furo(furol)(3,2c)pyran-2(6H)-one. In an alkaline environment, the biological activity of patulin is severely destroyed, but it is extremely stable in an acidic environment; it is easily soluble in water, chloroform, acetone, ethanol and ethyl acetate, slightly soluble in diethyl ether and benzene, and insoluble in petroleum ether; and it is mainly produced by fungi such as Aspergillus, Byssochlamys, Paecilomyces and Penicillium. Related toxicology tests show that patulin can affect fertility, has toxicological effects such as carcinogenesis and immunization, and is also a neurotoxin. In addition, patulin also has teratogenicity and can damage the respiratory and urinary systems.

[0003] Patulin is widely distributed and mainly exists in various types of fruits and their products, seriously affecting people's health, polluting the environment and causing economic losses. Therefore, many countries have formulated a series of limit standards for the content of patulin in food. In China, the limit standard of patulin in apple and hawthorn semi-finished products is 100 μg / kg, and the limit standard of patulin in fruit juice, jam, fruit wine, canned food and preserved fruit is 50 μg / kg. The European Union stipulates that the maximum limit standard of patulin in apple juice and alcoholic beverages containing apple juice is 50 μg / kg; the maximum limit standard of patulin in solid apple products is 25 μg / kg, and the maximum limit standard of patulin in apple juice for infants is 10 μg / kg. The U.S. Food and Drug Administration stipulates that the maximum content of patulin in apple juice is 50 μg / L.

[0004] Physical methods, chemical methods and biological methods are the main control methods of patulin in fruits and their products at present. Physical methods mainly use manual sorting, high-pressure water washing, clarification, filtration, adsorption, heating, radiation, microwave treatment and ultraviolet irradiation to treat raw fruits and contaminated fruit juice. These methods not only require a large amount of manpower, material resources and financial resources, but also cannot completely remove patulin. Chemical methods mainly include treating apples with related chemicals to prevent them from being infected by pathogenic bacteria, thereby preventing the production of patulin from the source, and treating fruit juice with chemicals to remove the patulin already produced in the fruit juice. For example, benzimidazole fungicides are often used to prevent patulin pollution caused by postharvest infection of pathogenic fungi in apples, but they can cause drug residues, resistance and environmental pollution. Ammoniation and potassium permanganate oxidation can also reduce the content of patulin in fruit juice by more than 99%, but the product quality will be damaged to a certain extent.

[0005] In view of the shortcomings of the above-mentioned methods for controlling patulin, biological methods, especially the use of biological enzymes to degrade patulin, show good application prospects. However, the number of biological degradation enzymes reported so far is limited, so there is an urgent need to explore new and efficient biological degradation enzymes for removing patulin. SUMMARY

[0006] In view of the defects and shortcomings of the prior art, the present application provides a new aldehyde-ketone reductase, a coding gene, a recombinant vector and a genetically engineered bacterium. In addition, the application also provides a new use of aldehyde-ketone reductase in degrading patulin.

[0007] The technical scheme adopted by the present application is as follows:

[0008] An aldehyde-ketone reductase, wherein the amino acid sequence of the aldehyde-ketone reductase is shown in SEQ ID NO. 1.

[0009] Any polypeptide fragment or mutant thereof obtained by deleting, inserting or replacing any amino acid in the amino acid sequence shown in SEQ ID NO. 1, as long as it has more than 90% homology with the amino acid, belongs to the protection scope of the present application.

[0010] The present application also provides a coding gene of the aldehyde-ketone reductase, wherein the nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO. 1 is shown in SEQ ID NO. 2.

[0011] Any nucleotide sequence obtained by substituting, deleting or inserting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 2, as long as it has more than 90% homology with the nucleotide sequence shown in SEQ ID NO. 2, belongs to the protection scope of the present application.

[0012] The present application also relates to a recombinant vector constructed by the aldehyde ketone reductase gene and a genetically engineered bacterium obtained by transformation of the vector.

[0013] The obtaining step of the recombinant expression vector is inserting the coding gene of the aldehyde ketone reductase (denoted as MgAKR) into the plasmid pET-30a after enzyme digestion by BamHI and XhoI to obtain the expression recombinant expression vector, denoted as pET-30a-MgAKR.

[0014] The obtaining step of the recombinant engineered bacterium is introducing the recombinant vector pET-30a-MgAKR into the competent cell of Escherichia coli Rosetta (DE3) to obtain the recombinant engineered bacterium, denoted as pET-30a-MgAKR / Rosetta (DE3).

[0015] The present application also provides the use of the aldehyde ketone reductase coding gene in the preparation of aldehyde ketone reductase, which is constructing the recombinant vector containing the aldehyde ketone reductase gene, transforming the recombinant vector into Escherichia coli to obtain the genetically engineered bacterium, inducing expression of the genetically engineered bacterium, separating the culture solution to obtain the bacterium cell containing the aldehyde ketone reductase, and further separating to obtain the aldehyde ketone reductase.

[0016] The specific steps for obtaining the aldehyde ketone reductase are as follows:

[0017] (1) inoculating the recombinant engineered bacterium into the LB liquid medium containing kanamycin, wherein the final concentration of kanamycin is 50 μg / mL; culturing at 37℃, 180 rpm for 12-16 h to obtain the seed liquid;

[0018] (2) transferring the obtained seed liquid into the LB liquid medium according to the inoculation volume of 1:100, and the medium contains 50 μg / mL of kanamycin; culturing at 37℃, 180 rpm until the OD value reaches 0.5-0.6; then adding isopropyl-β-D-thiogalactopyranoside into the LB liquid medium to make the final concentration of isopropyl-β-D-thiogalactopyranoside in the medium reach 0.5 mM, and culturing at 16℃, 120 rpm for 12 h to obtain the Escherichia coli culture solution; 600

[0019] Then centrifuging at 4℃, 8000 rpm for 5 min, discarding the supernatant and retaining the bacterium; washing the bacterium with the phosphate buffered saline solution with pH 7.4 by centrifugation, and the washed bacterium is the genetically engineered bacterium containing the aldehyde ketone reductase gene; resuspending the bacterium by adding the phosphate buffered saline solution again, wherein the usage ratio of the buffer solution to the bacterium is 2-3 ml: 1 g to obtain the cell suspension;

[0020] ​(3) using cell ultrasonic cell disrupter to break the cells in the cell suspension; the cell suspension is placed in ice, the amplitude rod of the disrupter is inserted into the cell suspension without sticking to the wall; the working power is set to 30%, the disruption is 3s, the rest is 3s, and the process is continued for 30min; after the disruption, centrifugation is performed at 12000rpm and 4℃ for 20min, and the supernatant is collected;

[0021] (4) finally, the supernatant is purified by a nickel column affinity, and an aldonoketol reductase is obtained, which is denoted as MgAKR.

[0022] The LB culture medium is composed of 10g / L of tryptone, 5g / L of yeast powder and 10g / L of sodium chloride, and the pH is natural.

[0023] The phosphate buffered saline solution is composed of 8.0g of NaCl, 0.2g of KCl, 1.44g of Na2HPO4·2H2O and 0.24g of KH2PO4, which are dissolved in distilled water, and the solution is adjusted to pH 7.4 by hydrochloric acid, and then distilled water is used to make up to 1L to obtain a phosphate buffered saline solution with a concentration of 0.01M.

[0024] The application also provides a use of the aldonoketol reductase in degrading patulin, and the use comprises the following steps:

[0025] The reaction system is composed of the aldonoketol reductase as a biological catalyst, patulin as a substrate, NADPH as a coenzyme and 2-morpholinoethanesulfonic acid buffer or fruit juice as a reaction medium; the pH of the reaction system is controlled to be 3-6.6, and the temperature is controlled to be 4-45℃, so that the degradation of patulin can be realized.

[0026] The concentration of patulin in the reaction system is 1-20μg / mL, the amount of the aldonoketol reductase is 50-300μg / mL, and the molar concentration of the NADPH is 0.01-0.5mM.

[0027] More preferably, the concentration of patulin is 15μg / mL, the amount of the aldonoketol reductase is 300μg / mL, and the molar concentration of the NADPH is 0.5mM.

[0028] More preferably, the pH of the 2-morpholinoethanesulfonic acid buffer is 6.0, the pH of the reaction system is 6-6.6, the temperature is 16-25℃, and the reaction time is 2-4h.

[0029] Beneficial effects:

[0030] The present application provides a new aldehyde ketone reductase, which has higher catalytic performance, and can completely degrade patulin with a final concentration of 15 μg / mL in a reaction system comprising the aldehyde ketone reductase as a biological catalyst, patulin as a substrate, NADPH as a coenzyme and 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.0) as a reaction medium; and can completely degrade patulin with a final concentration of 0.5 μg / mL in fresh-pressed pear juice within 4 h.

[0031] The present application is safe and environmentally friendly, and the expressed and purified aldehyde ketone reductase can be effectively used for controlling patulin pollution in self-made pear juice, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 A recombinant plasmid map of aldehyde ketone reductase;

[0033] Figure 2 SDS-PAGE results of aldehyde ketone reductase;

[0034] Figure 3 Effect of coenzyme NADPH concentration on degradation of patulin by MgAKR;

[0035] Figure 4 Effect of temperature, pH, substrate concentration and enzyme amount on degradation of patulin by MgAKR;

[0036] Figure 5 Effect of MgAKR on degradation of patulin in fresh-pressed pear juice. DETAILED DESCRIPTION

[0037] The various illustrative embodiments of the present application will now be described in detail in connection with the accompanying drawings. This description is made for the purpose of demonstrating certain aspects of the present application and should not be taken as a limitation on the present application. It is to be understood that the detailed description is merely exemplary of the present application and that the application is to be given full scope for modification and equivalence thereof.

[0038] It is to be understood that the terminology used in the present application is merely for the purpose of describing particular embodiments and is not intended to limit the present application. In addition, for numerical ranges in the present application, it is to be understood that each intermediate value between the upper limit and the lower limit of the range is specifically disclosed. Each smaller range between any stated value or stated range and any other stated value or stated range is also encompassed within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains.

[0040] Although preferred methods and materials are described, any method and materials similar or equivalent to those described herein can be used in the practice or testing of the present application.

[0041] All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. The citation of any document is not construed as an admission that it is prior art with respect to the present application.

[0042] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application, which will be apparent to those skilled in the art.

[0043] Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0044] Meyerozyma guilliermondii from the China Center for Type Culture Collection (CCTCC) of Wuhan University, China, has a preservation number of CCTCC NO: M2017270, and is a publicly disclosed strain.

[0045] Example 1:

[0046] Construction of genetically engineered bacteria:

[0047] Based on transcriptome data, an aldehyde-ketone reductase base sequence from Meyerozyma guilliermondii was screened, and the nucleotide sequence is shown as SEQ ID NO. 2.

[0048] First, MgAKR with 15bp homologous arms and enzyme digestion sites BamHI and Xhol sequences was amplified by PCR, then the vector pET30a was digested with BamHI and Xhol, after gel electrophoresis, the DNA product was recovered using an agarose DNA product recovery kit, finally the above target gene and the digested vector were ligated using ClonExpress II One Step Cloning Kit (Novagen), the ligation liquid was transformed into E. coli DH5α competent cells by chemical transformation method, and plated on LB solid plates containing 50 μg / mL kanamycin. The suspected transformants were verified by colony PCR and sent to Shanghai Biosciences for sequencing. The recombinant plasmid pET30a-MgAKR was successfully constructed; the recombinant plasmid after integration was extracted and transformed into E. coli Rosetta (DE3) to establish an aldehyde-ketone reductase genetically engineered strain, and the recombinant engineering bacteria pET-30a-MgAKR / Rosetta (DE3) was obtained.

[0049] The amplification primer of the aldehyde-ketone reductase coding gene MgAKR is:

[0050] Forward amplification primer pET30a-04009-F: gccatggctgatatcggatccATGACTTCCCTCACCATGAA;

[0051] Reverse amplification primer pET30a-04009-R: gtggtggtggtggtgctcgagGTCCTGGTACGTCGTGGG.

[0052] The recombinant plasmid map is shown in Figure 1. Figure 1 .

[0053] Example 2:

[0054] Culture of recombinant E. coli and purification of aldehyde ketone reductase:

[0055] (1) The recombinant engineering bacteria obtained in Example 1 were inoculated into 30 mL of LB medium (10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of sodium chloride, pH natural) containing kanamycin sulfate (50 μg / mL), and cultured at 37°C, 180 rpm for 12-16 h in a shaker to obtain a seed solution;

[0056] (2) Then, the seed solution was transferred to a flask containing 100 mL of LB medium at an inoculation amount of 1%, and cultured under the same conditions (37°C, 180 rpm, for 12-16 h). When the OD 600 of the bacteria reached 0.5-0.6, isopropyl-β-D thiogalactoside (IPTG) was added to a final concentration of 0.5 mM, and the culture solution was further induced to express at 16°C for 12 h, then centrifuged at 4°C, 8000 rpm for 5 min to collect the bacteria, which were washed twice with phosphate buffered saline solution to obtain the washed bacteria.

[0057] The washed bacteria were the genetically engineered bacteria containing the aldehyde ketone reductase gene; the bacteria were resuspended in phosphate buffered saline solution, and the ratio of the amount of buffer to the amount of bacteria was 2 ml: 1 g to obtain a cell suspension;

[0058] (3) The cell suspension was treated by cell ultrasonic disruption instrument; the cell suspension was placed in ice, the amplitude rod of the disruption instrument was inserted into the cell suspension without sticking to the wall; the working power was set to 30%, and the disruption was performed for 3 s, followed by 3 s of rest, for a total of 30 min; after the disruption, the supernatant was collected by centrifugation at 11000 rpm, 4°C for 20 min;

[0059] (4) Finally, the supernatant was purified by nickel column affinity to obtain the aldehyde ketone reductase, which was denoted as MgAKR;

[0060] As shown in Figure 2 Figure 3, the SDS-PAGE results of aldo-keto reductase.

[0061] The phosphate buffered saline solution is composed of 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4·2H2O, and 0.24 g KH2PO4, dissolved in distilled water, and the solution is adjusted to pH 7.4 with hydrochloric acid, and then diluted to 1 L with distilled water to obtain a phosphate buffered saline solution with a concentration of 0.01 M.

[0062] Example 3:

[0063] Effect of coenzyme NADPH concentration on degradation of expacillin by aldo-keto reductase:

[0064] MES buffer containing 0, 0.01, 0.05, 0.1, and 0.5 mM NADPH was mixed with expacillin (final concentration 15 μg / mL) and aldo-keto reductase purified in Example 2 (final concentration 300 μg / mL) in 1.5 mL centrifuge tubes to form five groups of systems.

[0065] Control group: the difference lies in the absence of NADPH and MgAKR in the system.

[0066] All samples were placed in a shaking incubator at 20°C and 180 rpm, and after 0, 2, and 4 hours of incubation, the samples were centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm pore size filter before detection, and the concentration of expacillin was detected by high performance liquid chromatography.

[0067] The liquid chromatography detection conditions are as follows: Agilent 1260 is used; Zorbax SB-C 18 chromatographic column (250 mm x 4.6 mm, 5 μm) is used; mobile phase: water: acetonitrile (90:10, v / v), flow rate: 1.0 mL / min, column temperature: 30°C, injection volume: 20 μL, detection time: 15 min, detection wavelength: 276 nm.

[0068] Figure 3 Effect of coenzyme NADPH concentration on degradation of expacillin by MgAKR;

[0069] The results are shown in Figure 3 MgAKR requires NADPH to provide hydrogen ions for the degradation of expacillin, and as the concentration of NADPH increases, the effect of MgAKR on the degradation of expacillin is enhanced.

[0070] Example 4:

[0071] Effect of temperature, pH, substrate concentration, and enzyme amount on degradation of expacillin by aldo-keto reductase:

[0072] The patulin, NADPH and MgAKR were added into the MES buffer to the final concentration of 10 μg / mL, 0.5 mM and 300 μg / mL respectively, mixed well in a 1.5 mL centrifuge tube, and then reacted for 4 h at different temperatures (4, 10, 16, 20, 25, 37, 45 ℃);

[0073] The patulin, NADPH and MgAKR were added into the citric acid-sodium citrate buffer at different pH (3, 4, 5, 6, 6.6) to the final concentration of 10 μg / mL, 0.5 mM and 300 μg / mL respectively, mixed well in a 1.5 mL centrifuge tube, and then reacted for 4 h at 20 ℃;

[0074] The patulin, NADPH and MgAKR were added into the MES buffer to the final concentration of 1, 5, 10, 15, 20 μg / mL, 0.5 mM and 300 μg / mL respectively, mixed well in a 1.5 mL centrifuge tube, and then reacted for 4 h at 20 ℃;

[0075] The patulin, NADPH and MgAKR were added into the MES buffer to the final concentration of 10 μg / mL, 0.5 mM and 0, 50, 100, 150, 200, 250, 300 μg / mL respectively, mixed well in a 1.5 mL centrifuge tube, and then reacted for 4 h at 20 ℃;

[0076] All samples were centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm pore size filter before detection. The concentration of patulin was detected by high performance liquid chromatography (see Fig. 1). Figure 4

[0077] The liquid phase detection conditions were as follows: Agilent 1260 was used; Zorbax SB-C 18 chromatographic column (250 mm x 4.6 mm, 5 μm) was used; mobile phase: water: acetonitrile (90:10, v / v), flow rate: 1.0 mL / min, column temperature: 30 ℃, injection volume: 20 μL, detection time: 15 min, detection wavelength: 276 nm.

[0078] Figure 4 Effects of temperature, pH, substrate concentration and enzyme amount on the degradation of patulin by MgAKR;

[0079] The results are shown in Fig. 2. The degradation rate of patulin increased with the increase of the initial enzyme amount (50-300 μg / mL). When the MgAKR concentration reached 300 μg / mL, the degradation rate of patulin reached 100%. Figure 4

[0080] ​​The initial concentration of patulin was 1-5 μg / mL, which was completely removed after 4 h. The initial concentration of patulin was 10-20 μg / mL, and the degradation rate was about 80% after 4 h. The optimal reaction temperature of MgAKR was 16°C, and the degradation rate was as high as 99%. The degradation rates at 20°C and 25°C were 96% and 82%, respectively.

[0081] The pH had a great influence on the degradation of patulin by MgAKR. When the pH was in the range of 3-4, the degradation efficiency of patulin by MgAKR was less than 5%. When the pH was increased to 5, the degradation efficiency of patulin by MgAKR reached 22%. When the pH was increased to 6-6.6, the degradation rate of patulin by MgAKR was more than 70%.

[0082] Example 5:

[0083] Effect of MgAKR on the degradation of patulin in fresh pear juice:

[0084] In fresh pear juice, 0.5 μg / mL of patulin, 0.5 mM of NADPH, and 300 μg / mL of MgAKR were added, respectively, and then mixed thoroughly in a 1.5 mL centrifuge tube. The mixture was placed at 20°C for 4 h. All samples were centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm pore size filter before detection. The concentration of patulin was detected by high performance liquid chromatography.

[0085] The liquid phase detection conditions were as follows: Agilent 1260 was used; a Zorbax SB-C 18 chromatographic column (250 mm x 4.6 mm, 5 μm) was used; the mobile phase was water:acetonitrile (90:10, v / v), the flow rate was 1.0 mL / min, the column temperature was 30°C, the injection volume was 20 μL, the detection time was 15 min, and the detection wavelength was 276 nm.

[0086] Figure 5 Effect of MgAKR on the degradation of patulin in fresh pear juice.

[0087] The results are shown in Table 1. Figure 5 As shown in Table 1, MgAKR can significantly degrade patulin in fresh pear juice. The initial concentration of patulin was 0.5 μg / mL, which was completely degraded by MgAKR within 4 h.

[0088] It should be understood by those skilled in the art that the technical solutions described in the present application can be modified or replaced equivalently, and all technical solutions and improvements that do not deviate from the spirit and scope of the present application should be covered within the scope of the claims of the present application.

[0089] SEQ ID NO. 1

[0090] MTSLTMNSKLTLNNGLKIPVIAMGVYMTPAGVASQVAYNALKVGYRHIDSAEFYKNEKEV

[0091] GEGISKWLQESPNNKREDVFYVTKIFDFNHGYEKAKRAIDECLQKVKDLQYIDMVLIHSPQ

[0092] SNRTKRLETWKALQEAVKSGKIKSIGVSNYGVHHMKELLEWDGLEIKPVVNQVELNPWL

[0093] MRTHIVDYAKANGIVMEAYSPLTRGKNFHDPTLVQLAKKYQKTPAQVLIRWSLQQGFVVLP

[0094] KSEKKDRAVENINVFDFDILSQDMKLLSHPESSQRFTNWDPTTYQDSEQ ID NO. 2

[0095] ATGACTTCCCTCACCATGAACTCAAAGCTCACTCTTAACAATGGCTTAAAAATCCCCGTGATTGCAAT

[0096] GGGAGTTTACATGACACCCGCCGGTGTAGCTTCACAAGTCGCATACAATGCTTTGAAGGTTGGGTATA

[0097] GGCACATTGATTCGGCGGAATTTTACAAGAATGAAAAGGAAGTTGGTGAAGGAATTAGTAAATGGCTT

[0098] CAGGAAAGTCCCAACAACAAGAGAGAGGACGTTTTTTACGTCACCAAGATTTTCGACTTTAACCATG

[0099] GATATGAAAAAGCCAAGCGAGCCATTGACGAATGCTTGCAAAAAGTCAAGGATTTGCAATATATTGAC

[0100] ATGGTGTTGATCCACTCGCCTCAATCGAACAGAACGAAGAGATTGGAGACGTGGAAAGCATTGCAAG

[0101] AAGCTGTCAAGAGTGGAAAAATCAAGTCGATTGGTGTATCGAATTATGGTGTTCACCACATGAAAGA

[0102] GTTGTTGGAATGGGACGGATTGGAGATCAAGCCTGTGGTTAACCAAGTTGAATTGAATCCATGGTTGA

[0103] TGCGGACCCATATAGTTGACTATGCCAAGGCCAATGGTATCGTTATGGAAGCATATAGTCCACTTACTC

[0104] GAGGCAAAAATTTTCACGATCCTACCTTGGTCCAATTGGCCAAAAAGTATCAAAAAACTCCAGCTCA

[0105] GGTGTTGATCAGATGGTCATTGCAACAAGGGTTTGTTGTGTTGCCCAAGAGTGAGAAGAAGGACCGT

[0106] GCCGTTGAGAACATTAATGTCTTTGATTTTGACATTCTGAGCCAAGACATGAAGCTTCTCTCGCATCCT

[0107] GAGTCGAGCCAGCGGTTTACCAACTGGGACCCCACGACGTACCAGGACTAG.

Claims

1. Use of an aldehyde-ketone reductase for degrading patulin, characterized in that, The aldonolactone reductase is a protein consisting of the amino acid sequence shown in SEQ ID NO. 1; the nucleotide sequence of the coding gene of the aldonolactone reductase is shown in SEQ ID NO.

2.

2. Use according to claim 1, characterized in that, The steps are as follows: taking the aldonolactone reductase as a biological catalyst, taking patulin as a substrate, taking NADPH as a coenzyme, taking 2-morpholinoethanesulfonic acid buffer or fruit juice as a reaction medium to form a reaction system; the pH of the reaction system is controlled at 6-6.6, and the temperature is controlled at 4-45℃, so that the degradation of PAT can be realized; the concentration of PAT in the reaction system is 1-20 μg / mL, the dosage of the aldonolactone reductase is 50-300 μg / mL, and the molar concentration of the NADPH is 0.01-0.5 mM.

3. Use according to claim 2, characterized in that, The concentration of PAT is 15 μg / mL; the dosage of the aldonolactone reductase is 300 μg / mL; the molar concentration of the NADPH is 0.5 mM; the pH of the 2-morpholinoethanesulfonic acid buffer is 6.0; the temperature of the reaction system is 16-25℃, and the reaction time is 2-4 h.

4. Use according to any one of claims 1 to 3, characterized in that, The preparation method of the aldonolactone reductase comprises the following steps: (1) inoculate the recombinant engineering bacteria carrying the aldonolactone reductase coding gene in claim 1 into LB liquid medium containing kanamycin sulfate, wherein the final concentration of kanamycin is 50 μg / mL; cultivate at 37℃, 180 rpm for 12-16 h to obtain a seed liquid; (2) The obtained seed liquid is transferred to LB liquid medium in a volume ratio of 1:100, and the medium contains 50 μg / m of kanamycin; it is cultured at 37°C, 180 rpm until the OD 600 value reaches 0.5-0.6; then isopropyl-β-D-thiogalactoside is added to the LB liquid medium, so that the final concentration of isopropyl-β-D-thiogalactoside in the medium is 0.5 mM, and it is cultured at 16°C, 120 rpm for 12 h to obtain an E. coli culture solution; then centrifuge at 8000 rpm for 5 min at 4℃, pour off the supernatant, and reserve the bacterial cells; then wash the bacterial cells with pH 7.4 phosphate buffered saline solution, and the washed bacterial cells are the genetically engineered bacteria containing the aldonolactone reductase gene; resuspend the bacterial cells by adding phosphate buffered saline solution again, wherein the dosage ratio of the buffer solution to the bacterial cells is 2-3 ml: 1 g, to obtain a cell suspension; (3) use a cell ultrasonic cell disrupter to treat the obtained cell suspension; place the cell suspension in ice, and make the amplitude rod of the disrupter penetrate into the cell suspension without sticking to the wall; set the working power to 30%, and disrupt for 3 s, rest for 3 s, and continue for 30 min; after the disruption, centrifuge at 11000 rpm and 4℃ for 20 min, and collect the supernatant; (4) finally, purify the supernatant through a nickel column affinity, and obtain the aldonolactone reductase, which is denoted as MgAKR; The composition of the LB medium is: 10 g / L of tryptone, 5 g / L of yeast powder, and 10 g / L of sodium chloride, with natural pH; the composition of the phosphate buffered saline solution is: 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4-2H2O, and 0.24 g of KH2PO4, dissolved in distilled water, and adjusted to pH 7.4 with hydrochloric acid, and then diluted to 1 L with distilled water to obtain a phosphate buffered saline solution with a concentration of 0.01 M.

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