MANGANESE PEROXIDASE, GENE THEREOF, AND USE THEREOF IN DETOXIFICATION OF MYCOTOXIN

Abstract

The present invention provides use of a manganese peroxidase in the detoxification of mycotoxins, and specifically, the present invention provides five manganese peroxidases (MnP-1, MnP-2, MnP-4, MnP-5, and MnP-6), genes thereof, and uses thereof. The present invention provides five manganese peroxidases (MnP-1, MnP-2, MnP-4, MnP-5, and MnP-6) derived from lignocellulose degradation bacteria, the amino acid sequences thereof being as set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 13.

Claims

1. Application of manganese peroxidase to detoxification of mycotoxin.

2. The application according to claim 1, being characterized in that said manganese peroxidase is selected from a) a polypeptide having the amino acid sequence as set in forth in SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 10, or SEQ ID NO. 13; b) a polypeptide comprising the amino acid sequence obtained by substituting, deleting, and or inserting one or more amino acid residues in the amino acid sequence depicted in SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 10 or SEQ ID NO. 13, and maintaining the ability of detoxifying mycotoxin; or c) a polypeptide having at least 70% identity to the amino acid sequence depicted in SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 10 or SEQ ID NO. 13, and maintaining the ability of detoxifying mycotoxin.

3. The application according to claim 1 being characterized in that said manganese peroxidase is a mature protein selected from a) a polypeptide having the amino acid sequence as set in forth in SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12, or SEQ ID NO. 15; b) a polypeptide comprising the amino acid sequence obtained by substituting, deleting, and or inserting one or more amino acid residues in the amino acid sequence depicted in EQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12, or SEQ ID NO. 15, and maintaining the ability of detoxifying mycotoxin; or c) a polypeptide having at least 70% identity to the amino acid sequence defined by “a)”, and maintaining the ability of detoxifying mycotoxin.

4. Manganese peroxidase being characterized in that said manganese peroxidases are selected from a) a polypeptide having the amino acid sequence as set in forth in SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 10 or SEQ ID NO. 13, and the ability of detoxifying mycotoxin; b) a polypeptide comprising the amino acid sequence obtained by substituting, deleting, and or inserting one or more amino acid residues in the amino acid sequence depicted in SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 10 or SEQ ID NO. 13, and maintaining the ability of detoxifying mycotoxin; or c) a polypeptide having at least 70% identity to the amino acid sequence defined by “a)”, and maintaining the ability of detoxifying mycotoxin.

5. The manganese peroxidase according to claim 5 being characterized in that said manganese peroxidase is a mature protein selected from a) a polypeptide having the amino acid sequence as set in forth in SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12, or SEQ ID NO. 15, and the ability of detoxifying mycotoxin; b) a polypeptide comprising the amino acid sequence obtained by substituting, deleting, and or inserting one or more amino acid residues in the amino acid sequence depicted in EQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12, or SEQ ID NO. 15, and maintaining the ability of detoxifying mycotoxin; or c) a polypeptide having at least 70% identity to the amino acid sequence defined by “a)”, and maintaining the ability of detoxifying mycotoxin.

6. Gene encoding the manganese peroxidase of claim 4.

7. The gene according to claim 6, being characterized of a) having the nucleotide sequence as set in forth in SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22, SEQ ID NO. 25 or SEQ ID NO. 28; b) having the nucleotide sequence as set in forth in SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 27 or SEQ ID NO. 30; or c) having the nucleotide sequence at least 70% identity to the nucleotide sequence defined by “a)” or “b)”, and encoding the protein having the same function as that encoded by gene defined in “a)” or “b)”.

8. (canceled)

9. (canceled)

10. A method for preparing the manganese peroxidase of claim 4, being characterized in that said method includes the steps of 1) transforming a host cell with the recombinant vector containing the gene of claim 6 or 7 to obtain a recombinant strain; 2) culturing the said recombinant strain to induce express recombinant manganese peroxidase; and 3) purifying the said manganese peroxidase.

Description

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0051] FIG. 1 shows degradation rates of aflatoxin by recombinant manganese peroxidases MnP-1, MnP-2, MnP-4, MnP-5 and MnP-6.

[0052] FIG. 2 shows degradation rates of zearalenone by recombinant manganese peroxidases MnP-1, MnP-2, MnP-4, MnP-5 and MnP-6.

[0053] FIG. 3 shows degradation rates of vomitoxin by recombinant manganese peroxidases MnP-1, MnP-2, MnP-4, MnP-5 and MnP-6.

[0054] FIG. 4 shows HPLC analysis results of the degradation of aflatoxin by recombinant manganese peroxidases MnP-1, MnP-2, MnP-4, MnP-5 and MnP-6 FIG. 5 shows HPLC analysis results of the degradation of zearalenone by recombinant manganese peroxidases MnP-1, MnP-2, MnP-4, MnP-5 and MnP-6.

[0055] FIG. 6 shows HPLC analysis results of the degradation of vomitoxin by recombinant manganese peroxidases MnP-1, MnP-2, MnP-4, MnP-5 and MnP-6.

EMBODIMENT

[0056] The present invention is further illustrated with reference to the following Examples and the appended drawings, which should by no means be construed as limitations of the present invention.

[0057] Test Materials and Reagents

[0058] 1. Strains and vectors: Irpex lacteus from which the five genes encoding manganese peroxidases MnP-1, MnP-2, MnP-4, MnP-5 and MnP-6 were cloned respectively, the E coli expression vectors pET-28a(+) and strain BL21(DE3) purchased from Invitrogen.

[0059] 2. Enzymes and other biochemical reagents: restriction endonucleases (Fermentas), ligase (Invitrogen), aflatoxin (Aladdin), zearalenone and vomitoxin (Sigma-Aldrich), the other reagents available purchased.

[0060] 3. Medium:

[0061] (1) Irpex lacteus producing enzyme medium: 1% of lignocellulose, 0.2 g/L of ammonium tartrate, 2 g/L of KH.sub.2PO.sub.4, 0.71 g/L of MgSO.sub.47H.sub.2O, 0.1 g/L of CaC.sub.2, 70 mL of macroelements concentrate.

[0062] (2) Microelement solution: 1 g/L of NaCl, 0.184 g/L of CoCl.sub.2.6H.sub.2O, 0.1 g/L of FeSO.sub.4.7H.sub.2O, 0.1 g/L of ZnSO.sub.4.7H.sub.2O, 0.1 g/L of CuSO.sub.4, 0.01 g/L of H.sub.3BO.sub.3, 0.01 g/L of Na.sub.2MoO.sub.4.2H.sub.2O, 0.01 g/L of KAl(SO.sub.4).sub.2.12H.sub.2O, 1.5 g/L of nitrilotriacetic acid. [0063] (3)E. coli. LB medium: 1% of peptone, 0.5% of yeast extract, and 1% of NaCl, natural pH.

[0064] Suitable biology laboratory methods not particularly mentioned in the examples as below can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other kit laboratory manuals.

Example 1 Cloning Gene Encoding Manganese Peroxidase MnP-1, MnP-2, MnP-4, MnP-5 and MnP-6 from Irpex lactus

[0065] Isolating the total RNA of Irpex lactus

[0066] First, bacteria cells cultured in enzyme-producing medium for 3 days were collected on the filter paper and pressed dry, followed by adding liquid nitrogen to a high-temperature sterilized mortar and quickly ground the bacteria into powder. Then, the grounded powder was transferred to a centrifuge tube with 800 μL of Trizol, blended well and left in the room temperature for 5 min. 200 L of chloroform was added, shaken violently for 15 s, placed at room temperature for 3 min, and centrifuged at 4° C. at 12,000 RPM for 15 min. The supernatant was obtained, and isopropanol of the equal volume was added to be mixed well, placed at room temperature for 10 min and centrifuged at 4° C. at 12,000 RPM for 10 min. The supernatant was removed and the precipitation was washed twice with 70% of ethanol followed by drying in the air for 5 min, and an appropriate amount of DNase/Rnase-free deionized water was added to dissolve RNA.

[0067] The specific primers for manganese peroxidase gene were synthesized as below.

TABLE-US-00023 MnP-1: P1:5′-CGCGGATCCGCACCCTCTTCTAGAGTGACATGCAGT-3′; P2:5′-TAAAGCGGCCGCTTACACAGGAACGATGGAGGTGGCG-3′. MnP-2: P3:5′-CGCGGATCCGCAATCACCAAGCGTGTTGCTTGTCCT-3′; P4:5′-CCGCTCGAGTTACGAGGGAGGGACAGGGGCGACAGA-3′. MnP-4: P5:5′-CGCGGATCCGCTCCCCAAGACGTTACTGCCGC-3′; P6:5′-CCGCTCGAGTTACGACGGAGGTACTGGAGGAATCG-3′. MnP-5: P7:5′-CGGAATTCGCCGTCGTCAGGCGTGTCACTTG-3′; P8:5′-CCGCTCGAGTTAGGACGGAGGGACAGGAGCGAC-3′. MnP-6: P9:5′-CGGAATTCGCTATCACCAGACGTGTTGCGTGC-3′; P10:5′-ATTTGCGGCCGCTTAAGACGGGGGAACAGGGGCAAC-3′.

[0068] PCR amplification was performed with cDNA obtained by RT-PCR using the total RNA of Irpex lacteu. PCR reaction parameters were denaturation at 95° C. for 5 min, followed by 35 cycles of denaturing at 94° C. for 30 sec, annealing at 55° C. for 30 sec, and extending at 72° C. for 1 min, and being kept at 72° C. for 10 min. After electrophoresis on 1% of agarose gel, the target fragment was cut, recovered and connected with vector pEASY-T3 for sequencing.

Example 2 Preparing the Recombinant Manganese Peroxidases

[0069] The expression vectors pET28a-MnP-1, pET28a-MnP-2, pET28a-MnP-4, pET28a-MnP-5 and pET28a-MnP-6 were constructed by connecting the gene encoding the mature manganese peroxidases MnP-1,MnP-2,MnP-4,MnP-5 and MnP-6 with the expression vector ET-28a(+), both of which were digested with restriction enzymes, and were transformed to E coli strain BL21(DE3) to obtain the recombinant strains BL21(DE3)/MnP-1, BL21(DE3)/MnP-2, BL21(DE3)/MnP-4, BL21(DE3)/MnP-5 and BL21(DE3)/MnP-6.

[0070] The strain D3 containing the recombinant plasmid was planted into 40 mL of LB culture medium for culturing at 37° C. for 12 h, followed by being planted into 300 mL of LB culture medium at a ratio of 2% for culturing for 4 h at 37° C. with 250 rpm, with addition of inducer IPTG in the final concentration of 1 mM to induce for 4 h when reaching to 0.8 of OD.sub.600, and collecting bacteria by centrifuging. The bacteria cells were lysed by Lysozyme using 8M of urea to dissolve inclusion body protein, and the refolding system prepared with 50 mM of Tris-HCl buffer with 9.5 of pH, 0.5 m M of urea, 0.5 mM of GSSG, 0.1 mM of DTT, 10 μM of hemin, 5 mM of CaC.sub.2, and 0.1 mg/mL of protein solution, for renaturing for 10 h at 15° C. After the renaturated manganese peroxidase was purified, the content of protein reached to more than 95% of the total protein.

Example 3 Degradation of Aflatoxin by the Recombinant Manganese Peroxidase

[0071] Aflatoxin was dissolved into 50 mg/L of mother liquor of dimethyl sulfoxide to react for 10 h at 30° C. in the reaction system of 70 μl of malonic acid buffer (0.2 m, pH 5.0), 20 μl of aflatoxin solution, 5 μl of manganese sulfate (40 mM), 100 μl of manganese peroxidase (1000 U/L), 5 μl of hydrogen peroxide (4 mM), taking the system without manganese peroxidase as control, wherein each manganese peroxidase was set three repeats. The reaction was terminated by adding DMSO in three times of volume, to measure the degradation rate of aflatoxin in wavelength of 365 nm by high performance liquid chromatography (HPLC) using Nexera UHPLC system of which the chromatographic column is Zorbax sb-c18 (4.6×250.5 um), the mobile phase A was 0.06% of TFA water, and the mobile phase B was 0.05% TFA acetonitrile, and eluted with gradient content of 30% of solution B for 4 min, 30%-100% of solution B for 15 min, and 100% of solution B for 5 min.

Example 4 Degradation of Zearalenone by the Recombinant Manganese Peroxidase

[0072] Zearalenone was dissolved into 50 mg/L of mother liquor of dimethyl sulfoxide to react for 10 h at 30° C. in the reaction system of 70 μl of malonic acid buffer (0.2 m, pH 5.0), 20 μl of aflatoxin solution, 5 μl of manganese sulfate (40 mM), 100 μl of manganese peroxidase (1000 U/L), 5 μl of hydrogen peroxide (4 mM), taking the system without manganese peroxidase as control, wherein each manganese peroxidase was set three repeats. The reaction was terminated by adding DMSO in three times of volume, to measure the degradation rate of zearalenone in wavelength of 365 nm by high performance liquid chromatography (HPLC) using Nexera UHPLC system of which the chromatographic column is Zorbax sb-c18 (4.6×250.5 um), the mobile phase A was 0.06% of TFA water, and the mobile phase B was 0.05% TFA acetonitrile, and eluted with gradient content of 30% of solution B for 4 min, 30%-100% of solution B for 15 min, and 100% of solution B for 5 min.

Example 5 Degradation of v by the Recombinant Manganese Peroxidase

[0073] Vomitoxin was dissolved into 50 mg/L of mother liquor of dimethyl sulfoxide to react for 10 h at 30° C. in the reaction system of 70 μl of malonic acid buffer (0.2 m, pH 5.0), 20 μl of aflatoxin solution, 5 μl of manganese sulfate (40 mM), 100 μl of manganese peroxidase (1000 U/L), 5 μl of hydrogen peroxide (4 mM), taking the system without manganese peroxidase as control, wherein each manganese peroxidase was set three repeats. The reaction was terminated by adding DMSO in three times of volume, to measure the degradation rate of vomitoxin in wavelength of 365 nm by high performance liquid chromatography (HPLC) using Nexera UHPLC system of which the chromatographic column is Zorbax sb-c18 (4.6×250.5 um), the mobile phase A was 0.06% of TFA water, and the mobile phase B was 0.05% TFA acetonitrile, and eluted with gradient content of 30% of solution B for 4 min, 30%-100% of solution B for 15 min, and 100% of solution B for 5 min.