Method for improving thermal stability of fumonisins degrading enzyme, mutant and application

Abstract

The invention belongs to the technical field of protein engineering modification, and particularly relates to a method for improving thermal stability of fumonisins degrading enzyme by forming disulfide bonds. The thermal stability of the fumonisins degrading enzyme FumDPS mutants T194V, T194V-P40C / Y173C, T194V-N43C / D83C and T194V-F71C / S117C related to the invention is obviously improved compared with that of the wild type fumonisins degrading enzyme, and the thermal stability of the fumonisins degrading enzyme FumDPS mutants is obviously improved. The fumonisins enzyme is mainly applied to the industries of livestock feed processing, food production and the like so as to reduce the harm of fumonisins to the health of animals and human beings. The obtained fumonisins degrading enzyme mutant with improved thermal stability is convenient to transport and store, so that the investment of industrial cost can be reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein engineering modification, and particularly relates to a mutant of fumonisin degrading enzyme with improved thermal stability and its application. Background Art

[0002] Mycotoxins are secondary fungal metabolites harmful to plants, animals and humans. Mycotoxins widely exist in foods and have become an important public health problem, having a significant impact on society and economy. FUMs are one of the most common mycotoxins, mainly occurring in grains such as corn. These toxins are mainly produced by some strains of the genera Fusarium and Aspergillus. They can be divided into four major categories, A, B, C and P according to their chemical structures, among which B-type fumonisins (FBs) are the most common. Among them, FB1 is the most prevalent and toxic type, accounting for 70-80% of all FB contaminants. Generally speaking, due to the relatively high occurrence frequency and high toxicity of FB1, it has received the most attention.

[0003] At present, the biological enzyme method has been proven to be one of the effective ways to remove fumonisin contamination. Since fumonisin is highly soluble in water and also has strong thermal stability, it is very difficult for traditional physical and chemical methods to safely and thoroughly eliminate fumonisin. The detoxifying enzymes produced by microorganisms can catalyze the degradation of the toxic groups of mycotoxins and convert them into forms with less toxicity or no toxicity. Therefore, the enzyme degradation method is considered to be a high-quality detoxification method with high efficiency, specificity, safety and environmental protection. The fumonisin degrading enzyme with good thermal stability can withstand the high-temperature treatment conditions during the enzyme preparation process, and can increase the use time of the enzyme, thereby reducing the production cost and improving the enzyme use efficiency. Summary of the Invention

[0004] In order to be able to degrade fumonisin by the biodegradation method, the present invention is proposed and completed.

[0005] The purpose of the present invention is to provide a mutant of fumonisin degrading enzyme with improved thermal stability.

[0006] Another purpose of the present invention is to provide a gene of fumonisin degrading enzyme with improved thermal stability.

[0007] Another purpose of the present invention is to provide a recombinant expression vector comprising the above gene.

[0008] Another purpose of the present invention is to provide a recombinant strain comprising the above gene.

[0009] Another purpose of the present invention is to provide a method for preparing fumonisin degrading enzyme.

[0010] Another purpose of the present invention is to provide a method for improving the thermal stability of fumonisin degrading enzyme.

[0011] The fumonisin degrading enzyme mutant T194V according to the specific embodiment of the present invention has an amino acid sequence as shown in SEQ ID NO:1.

[0012] SEQ ID NO:1:

[0013] AGSPGGNPPGEPLPPGIARTSAGLVRGDVGEIVSFKGIPYARPPVGDLRWRPPADAEPWEGVRDALAFGPACIQAGSRVQSEDCLTLNVWVPRESLDRGEKLPVLVWVYGGSFVGGSGDFEAEGLARKGAVVVSMNYRVSTMGFMAHPGLSAESPEG V SGNYGLLDIAQSLKWVRKNIANFGGDAGRVTVWGSSSGASAITALMVSPRSDGLFDQVILDSPGAMRHWKTLAEAEQDGIAIGSDIGQLRKLPADQVPVIQNTGGGTAVRALAEPRVIGPVLDGVVLPLEERPAFERGQARAVPVLVGYNTDEGASFTGGYQIRTVDAYRAYLKDPKIFAEFGDEAFSYYPVSSDSEVQRAISDSFGDNQFVFGTRGIARAMAAQGQPVYRYWFRRKGNGGTGADAVHGAELPYVRADARLDAAPYTADDVKLSRMMNDAWFRFVSTGDPNGGEVTNWPRYDTRSEPVYVFDAATEIVNGPRNDRLDFIGRVDAALNPVP

[0014] The present invention provides a gene encoding the above-mentioned fumonisin degrading enzyme mutant T194V, and the genomic sequence of this gene is as shown in SEQ ID NO:2.

[0015] SEQ ID NO:2:

[0016]

[0017] The fumonisin-degrading enzyme mutant T194V-P40C / Y173C according to the specific embodiment of the present invention has an amino acid sequence as shown in SEQ ID NO:3.

[0018] SEQ ID NO:3:

[0019] AGS C GGNPPGEPLPPGIARTSAGLVRGDVGEIVSFKGIPYARPPVGDLRWRPPADAEPWEGVRDALAFGPACIQAGSRVQSEDCLTLNVWVPRESLDRGEKLPVLVWVYGGSFVGGSGDFEAEGLARKGAVVVSMN C RVSTMGFMAHPGLSAESPEG V SGNYGLLDIAQSLKWVRKNIANFGGDAGRVTVWGSSSGASAITALMVSPRSDGLFDQVILDSPGAMRHWKTLAEAEQDGIAIGSDIGQLRKLPADQVPVIQNTGGGTAVRALAEPRVIGPVLDGVVLPLEERPAFERGQARAVPVLVGYNTDEGASFTGGYQIRTVDAYRAYLKDPKIFAEFGDEAFSYYPVSSDSEVQRAISDSFGDNQFVFGTRGIARAMAAQGQPVYRYWFRRKGNGGTGADAVHGAELPYVRADARLDAAPYTADDVKLSRMMNDAWFRFVSTGDPNGGEVTNWPRYDTRSEPVYVFDAATEIVNGPRNDRLDFIGRVDAALNPVP

[0020] The present invention provides a gene encoding the above-mentioned fumonisin-degrading enzyme mutant T194V-P40C / Y173C, and the genomic sequence of this gene is as shown in SEQ ID NO:4.

[0021] SEQ ID NO:4:

[0022]

[0023] The fumonisin-degrading enzyme mutant T194V-N43C / D83C according to the specific embodiment of the present invention has an amino acid sequence as shown in SEQ ID NO:5.

[0024] SEQ ID NO:5:

[0025] AGSPGG C PPGEPLPPGIARTSAGLVRGDVGEIVSFKGIPYARPPVG C LRWRPPADAEPWEGVRDALAFGPACIQAGSRVQSEDCLTLNVWVPRESLDRGEKLPVLVWVYGGSFVGGSGDFEAEGLARKGAVVVSMNYRVSTMGFMAHPGLSAESPEG V SGNYGLLDIAQSLKWVRKNIANFGGDAGRVTVWGSSSGASAITALMVSPRSDGLFDQVILDSPGAMRHWKTLAEAEQDGIAIGSDIGQLRKLPADQVPVIQNTGGGTAVRALAEPRVIGPVLDGVVLPLEERPAFERGQARAVPVLVGYNTDEGASFTGGYQIRTVDAYRAYLKDPKIFAEFGDEAFSYYPVSSDSEVQRAISDSFGDNQFVFGTRGIARAMAAQGQPVYRYWFRRKGNGGTGADAVHGAELPYVRADARLDAAPYTADDVKLSRMMNDAWFRFVSTGDPNGGEVTNWPRYDTRSEPVYVFDAATEIVNGPRNDRLDFIGRVDAALNPVP

[0026] The present invention provides a gene encoding the above-mentioned fumonisin-degrading enzyme mutant T194V-N43C / D83C, and the genomic sequence of this gene is as shown in SEQ ID NO:6.

[0027] SEQ ID NO:6:

[0028]

[0029] The fumonisin-degrading enzyme mutant T194V-F71C / S117C according to the specific embodiment of the present invention has an amino acid sequence as shown in SEQ ID NO:7.

[0030] SEQ ID NO:7:

[0031] AGSPGGNPPGEPLPPGIARTSAGLVRGDVGEIVSCKGIPYARPPVGDLRWRPPADAEPWEGVRDALAFGPACIQAGSRVQ C EDCLTLNVWVPRESLDRGEKLPVLVWVYGGSFVGGSGDFEAEGLARKGAVVVSMNYRVSTMGFMAHPGLSAESPEGVSGNYGLLDIAQSLKWVRKNIANFGGDAGRVTVWGSSSGASAITALMVSPRSDGLFDQVILDSPGAMRHWKTLAEAEQDGIAIGSDIGQLRKLPADQVPVIQNTGGGTAVRALAEPRVIGPVLDGVVLPLEERPAFERGQARAVPVLVGYNTDEGASFTGGYQIRTVDAYRAYLKDPKIFAEFGDEAFSYYPVSSDSEVQRAISDSFGDNQFVFGTRGIARAMAAQGQPVYRYWFRRKGNGGTGADAVHGAELPYVRADARLDAAPYTADDVKLSRMMNDAWFRFVSTGDPNGGEVTNWPRYDTRSEPVYVFDAATEIVNGPRNDRLDFIGRVDAALNPVP

[0032] The present invention provides a gene encoding the above-mentioned fumonisin-degrading enzyme mutant T194V-F71C / S117C, and the genomic sequence of this gene is as shown in SEQ ID NO:8.

[0033] SEQ ID NO:8:

[0034]

[0035] The present invention also provides a recombinant vector containing the coding gene of the above fumonisin degrading enzyme mutant. The coding gene of the fumonisin degrading enzyme mutant of the present invention is inserted between appropriate restriction enzyme cleavage sites of an expression vector, so that its nucleic acid sequence is ligated with an expression regulatory sequence to express the mutant protein.

[0036] The method for improving the thermal stability of fumonisin degrading enzyme according to the present invention comprises the following steps:

[0037] Perform a single point mutation of T194V on the fumonisin degrading enzyme with the amino acid sequence as shown in SEQ ID NO:9.

[0038] For the fumonisin degrading enzyme with the amino acid sequence as shown in SEQ ID NO:9, mutate the amino acid residues at positions 40 and 173 from proline (P) to cysteine (C) and tyrosine (Y) to cysteine (C), respectively.

[0039] According to the method for improving the thermal stability of fumonisin degrading enzyme of the present invention, when performing the T194V mutation on the fumonisin degrading enzyme with the amino acid sequence as shown in SEQ ID NO:9, the method further comprises the steps of performing T194V-P40C / Y173C, T194V-N43C / D83C, and T194V-F71C / S117C mutations.

[0040] The fumonisin degrading enzyme FumDPS according to the specific embodiment of the present invention has an amino acid sequence as shown in SEQ ID NO:9.

[0041] SEQ ID NO:9:

[0042] AGSPGGNPPGEPLPPGIARTSAGLVRGDVGEIVSFKGIPYARPPVGDLRWRPPADAEPWEGVRDALAFGPACIQAGSRVQSEDCLTLNVWVPRESLDRGEKLPVLVWVYGGSFVGGSGDFEAEGLARKGAVVVSMNYRVSTMGFMAHPGLSAESPEGTSGNYGLLDIAQSLKWVRKNIANFGGDAGRVTVWGSSSGASAITALMVSPRSDGLFDQVILDSPGAMRHWKTLAEAEQDGIAIGSDIGQLRKLPADQVPVIQNTGGGTAVRALAEPRVIGPVLDGVVLPLEERPAFERGQARAVPVLVGYNTDEGASFTGGYQIRTVDAYRAYLKDPKIFAEFGDEAFSYYPVSSDSEVQRAISDSFGDNQFVFGTRGIARAMAAQGQPVYRYWFRRKGNGGTGADAVHGAELPYVRADARLDAAPYTADDVKLSRMMNDAWFRFVSTGDPNGGEVTNWPRYDTRSEPVYVFDAATEIVNGPRNDRLDFIGRVDAALNPVP

[0043] The present invention provides a gene encoding the above-mentioned fumonisin degrading enzyme FumDPS, and the genomic sequence of this gene is shown in SEQ ID NO:10.

[0044] SEQ ID NO:10:

[0045]

[0046] The present invention also provides recombinant strains containing the encoding genes of the above fumonisin-degrading enzyme mutants T194V, T194V-P40C / Y173C, T194V-N43C / D83C, and T194V-F71C / S117C, and preferably, the strain is Escherichia coli.

[0047] The present invention also provides a method for preparing a fumonisin-degrading enzyme, comprising the following steps:

[0048] (1) Transforming a recombinant expression vector carrying the fumonisin-degrading enzyme mutant gene into a host cell to obtain a recombinant strain;

[0049] (2) Culturing the recombinant strain to the logarithmic growth phase and expressing the fumonisin-degrading enzyme under suitable conditions;

[0050] (3) Separating and purifying to obtain the fumonisin-degrading enzyme.

[0051] Among them, preferably, the host cell is an Escherichia coli competent cell, and preferably, the Escherichia coli strain is BL21(DE3).

[0052] The present invention also provides the application of the above fumonisin-degrading enzyme mutants, especially in the application of degrading fumonisins.

[0053] Compared with the properties of the fumonisin-degrading enzyme FumDPS, the thermal stabilities of the fumonisin-degrading enzyme mutants T194V, T194V-P40C / Y173C, T194V-N43C / D83C, and T194V-F71C / S117C of the present invention are all higher than that of the fumonisin-degrading enzyme FumDPS. By using the method of the present invention, fumonisin-degrading enzyme mutants with excellent properties can be obtained. This enzyme can be applied to industries such as agriculture, feed, and food to reduce the harm of fumonisins to animal and human health and overcome the limitations of natural enzymes in industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 It is a protein electrophoresis diagram of the fumonisin-degrading enzyme mutant, wherein, M: Marker; 1: Fumonisin-degrading enzyme wild type FumDPS; 2: Fumonisin-degrading enzyme mutant T194V; 3: Fumonisin-degrading enzyme mutant T194V-P40C / Y173C; 4: Fumonisin-degrading enzyme mutant T194V-N43C / D83C; 5: Fumonisin-degrading enzyme mutant T194V-F71C / S117C;

[0055] Figure 2 Showing the activity of the fumonisin-degrading enzyme mutant.

[0056] Figure 3 Show the thermal stability of fumonisin degrading enzyme mutants. Detailed implementation methods

[0057] The test materials and reagents involved in the following examples:

[0058] 1. Strains and vectors: Escherichia coli expression vector pET28a(+) and strain BL21(DE3).

[0059] 2. Medium: Escherichia coli medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0).

[0060] Example 1 Preparation of recombinant fumonisin degrading enzyme mutants

[0061] Using fumonisin degrading enzyme FumDPS as a template, plasmids pET28a(+)-FumDPS-T194V, pET28a(+)-FumDPS-T194V-P40C / Y173C, pET28a(+)-FumDPS-T194V-N43C / D83C and pET28a(+)-FumDPS-T194V-F71C / S117C of fumonisin degrading enzyme mutants were obtained by site-directed mutagenesis. They were transformed into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli strains BL21(DE3) / FumDPS-T194V, BL21(DE3) / FumDPS-T194V-P40C / Y173C, BL21(DE3) / FumDPS-T194V-N43C / D83C and BL21(DE3) / FumDPS-T194V-F71C / S117C.

[0062] The BL21(DE3) strain carrying the recombinant plasmid was activated overnight, then inoculated into 100 mL of LB liquid medium and cultured with shaking at 37 °C and 220 rpm. When the OD 600 reached 0.6 - 0.8, an IPTG inducer with a final concentration of 0.5 mM was added, and then protein expression was induced at 25 °C for 20 h. After the induction ended, the cells were collected by centrifugation at 4 °C. Through ultrasonic disruption, the supernatant was collected and the target protein was purified by nickel column. The SDS-PAGE results showed that the recombinant fumonisin degrading enzyme mutants were expressed in Escherichia coli. As Figure 1 shown, lane 1: fumonisin degrading enzyme wild type FumDPS; lane 2: fumonisin degrading enzyme mutant T194V; lane 3: fumonisin degrading enzyme mutant T194V-P40C / Y173C; lane 4: fumonisin degrading enzyme mutant T194V-N43C / D83C; lane 5: fumonisin degrading enzyme mutant T194V-F71C / S117C.

[0063] Example 2 Determination of the Activity of Fumonisin Degrading Enzyme Mutants in Degrading FB1

[0064] 2.12.1 Detection of the enzyme activity of fumonisin degrading enzyme mutants by high performance liquid chromatography. The specific method is as follows:

[0065] (1) FB1 standard stock solution: Weigh an appropriate amount of FB1 standard product, dissolve it with 50% acetonitrile water, and prepare an FB1 standard solution with a concentration of 50 μg / mL, and store it at 4°C;

[0066] (2) Preparation of samples: Take 45 μL of the fumonisin degrading enzyme purified by nickel column, mix it with 5 μL of the FB1 standard stock solution, react in a water bath at 37°C in the dark for 20 min, and inactivate it at 100°C for 10 minutes to terminate the reaction after the reaction;

[0067] (3) Sample derivatization: Add 200 μL of 50% acetonitrile water and 250 μL of OPA derivatization solution to the reaction solution to make the final concentration of FB1 10 μg / ml, mix well, filter through a 0.22 μm filter membrane, and detect by HPLC after 5 min. By comparing with the peak pattern of the FB1 standard product, the enzyme activity of the fumonisin degrading enzyme mutant is determined. Figure 2 As shown, there is almost no difference in the activity between the fumonisin degrading enzyme mutant and the wild-type FumDPS, and the activity of the mutant does not decrease.

[0068] 2.2 Determination of the thermal stability of fumonisin degrading enzyme mutants

[0069] Take appropriate amounts of wild-type and mutant fumonisin degrading enzymes and treat them at 45°C for 0 min, 30 min, and 60 min respectively. After the treatment, detect the enzyme activity in the manner of Example 2.1. Three parallel experiments are carried out for each temperature gradient. As Figure 3 shown, the stability of the fumonisin degrading enzyme mutant is significantly improved after being treated at 45°C for one hour compared with the wild-type. Among them, T194V is about 23% higher than the wild-type, the stability of T194V-N43C / D83C is about 28% higher than that of FumDPS, the stability of T194V-P40C / Y173C is about 41% higher than that of FumDPS, and the stability of T194V-F71C / S117C is about 50% higher than that of FumDPS.

[0070] (1) Fumonisin degradation rate: It is expressed by the ratio of the peak area m0 of hydrolytic fumonisin HFB1 to the peak area m of the fumonisin FB1 standard product;

[0071]

[0072] (2) Relative activity: Use the degradation rate w of the mutant oIt is expressed as the ratio to the degradation rate w of the wild type;

[0073]

[0074] The above embodiments are only used to understand the technical solution of the present application and do not limit the protection scope of the present application.

Claims

1. A fumonisin-degrading enzyme mutant with improved thermal stability, characterized in that, The amino acid sequence of the mutant is shown in SEQ NO:

1.

2. A fumonisin degrading enzyme mutant with improved thermal stability, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO:

7.

3. A fumonisin degrading enzyme gene, characterized in that, The gene encodes the fumonisin-degrading enzyme mutant with improved thermal stability as claimed in claim 1 or 2.

4. The fumonisin degrading enzyme gene according to claim 3, wherein The nucleotide sequence of the gene is shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO:

8.

5. A recombinant vector comprising the fumonisin-degrading enzyme gene as claimed in claim 3.

6. A recombinant strain comprising the fumonisin-degrading enzyme gene as claimed in claim 3.

7. Use of the fumonisin-degrading enzyme mutant with improved thermal stability as claimed in claim 1 or 2.

8. Use of the fumonisin-degrading enzyme mutant with improved thermal stability as claimed in claim 1 or 2 for degrading fumonisin.

9. A method for improving the thermal stability of fumonisin-degrading enzyme, characterized in that, The method comprises the following steps: mutating the fumonisin-degrading enzyme FumDPS with the amino acid sequence shown in SEQ ID NO:

9.

10. The method for improving the thermal stability of fumonisin-degrading enzyme according to claim 9, characterized in that, When mutating the fumonisin-degrading enzyme FumDPS with the amino acid sequence shown in SEQ ID NO: 9, the method further comprises the steps of mutating T194V, T194V-P40C / Y173C, T194V-N43C / D83C or T194V-F71C / S117C.

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