FUMONISIN DEGRADING ENZYME FUMDPS AND GENE AND USE THEREOF

Abstract

The present invention relates to the field of genetic engineering, particularly to a fumonisin degrading enzyme FumDPS, gene and application thereof. The present invention provides a fumonisin degrading enzyme FumDPS from Pigmentiphaga sp. which has the amino acid sequence of SEQ ID NO: 1, has the optimum temperature of 37° C., and the optimum pH of 8 to 9, remains more than 60% of the activity in the range of pH 3 to 7, and has 100% of degradation rate of fumonisin B1 at the optimum temperature and pH. The enzyme of the present invention can be applied to reduce the harm of fumonisin to animals and human health in agriculture, feed and food

Claims

1. (canceled)

2. (canceled)

3. (canceled)

4. (canceled)

5. (canceled)

6. (canceled)

7. (canceled)

8. (canceled)

9. The application of the fumonisin degrading enzyme FumDPS in the degradation of fumonisin wherein said fumonisin degrading enzyme FumDPS comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

10. The application according to claim 9, wherein said fumonisin degrading enzyme FumDPS being encoded by gene comprising a nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4.

11. The application according to claim 9, wherein said fumonisin degrading enzyme FumDPS having the optimum temperature of 37° C. and the optimum pH of 8 to 9, remaining more than 60% of the activity in the range of pH 3 to 7, having 100% of degradation rate of fumonisin B1 at the optimum temperature and pH.

12. The application according to claim 9, wherein said fumonisin degrading enzyme FumDPS being prepared by the method comprising the steps of transforming an isolated host cell with a DNA construct comprising a polynucleotide which comprises a nucleotide sequence encoding said fumonisin degrading enzyme FumDPS comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 to obtain a recombinant host cell; cultivating the recombinant host cell to produce the fumonisin degrading enzyme FumDPS; and recovering the fumonisin degrading enzyme FumDPS.

13. The application according to claim 12, wherein said isolated host cell being Escherichia coli.

Description

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0028] FIG. 1 shows the SDS-PAGE purification of the fumonisin degrading enzyme FumDPS of the present invention, in which M: protein marker; 1: fumonisin degrading enzyme FumDPS;

[0029] FIG. 2 shows the optimum temperature of the fumonisin degrading enzyme FumDPS of the present invention; and

[0030] FIG. 3 shows the optimum pH of the fumonisin degrading enzyme FumDPS of the present invention.

EMBODIMENT

[0031] Test Materials and Reagents

[0032] 1. Strains and vectors: E. coli expression vector pET28a (+) and strain BL21 (DE3).

[0033] 2. Medium:

[0034] E. coli LB medium: 1% peptone, 0.5% yeast extract, and 1% NaCl, pH 7.0.

Example 1 Cloning of the Gene Encoding the Fumonisin Degrading Enzyme FumDPS of Decarboxylase Family

[0035] The artificial nucleotide sequence of the coding region of the gene encoding the fumonisin degrading enzyme FumDPS was chemically synthesized, and was add with the the restriction enzyme sites EcoR I and Xho I in its 5 ‘end and 3’ end respectively.

Example 2 Preparation of Recombinant Fumonisin Degrading Enzyme FumDPS

[0036] The expression vector pET28a(+) and the gene encoding fumonisin degrading enzyme was digested with the restriction enzyme EcoR I and Xho I, respectively, and the obtained gene fragment encoding mature fumonisin degrading enzyme was connected with the expression vector pET28a (+) to obtain the recombinant plasmid pET28a(+)-FumDPS containing FumDPS which was transformed into strain E. coli BL21 (DE3) to obtain the recombinant strain E. coli BL21 (DE3)/FumDPSs.

[0037] The recombinant expression vector comprising the fumonisin degrading enzyme gene with signal peptide sequence was constructed by the same way.

[0038] Furthermore, the strain BL21 (DE3) comprising the recombinant plasmid was inoculated in 100 mL of LB medium, cultured by shaking at 37° C. and 220 rpm for 2 to 3 h, and add with IPTG in a final concentration of 1 mM when OD600 was 0.6 to 0.8, followed by inducing at 25° C. for 20 h, and collecting the cells by centrifugation at 4° C. The supernatant was collected after ultrasonic crushing and the protein was purified by nickel column. The results of SDS-PAGE showed that the recombinant fumonisin degrading enzyme was expressed in E. coli shown as shown by the lane 1 indicating the purified FumDPS of FIG. 1.

Example 3 Determination of the Properties of the Fumonisin Degrading Enzyme FumDPS

[0039] The enzyme activity of the fumarotoxin degrading enzyme was detected by high performance liquid chromatography (HPLC) including the following steps of

[0040] (1) preparing FB1 standard stock solution, wherein 1 mg of standard sample, was dissolve with 10 mL of acetonitrile and water in a ratio of 1:1, to obtain the standard solution in the concentration of 100 ug/mL, being stored at −20° C.

[0041] (2) preparing sample wherein 900 ul of the purified fumonisin degrading enzyme solution was added with 100 ul of FB1 standard stock solution to 10 ug/ml of the final concentration of FB1 followed by being cultured for 20 min at 37° C. and 220 rpm in dark. and

[0042] (3) deriving the sample wherein 400 uL of acetonitrile water at the concentration of 50% was added to 100 uL of sample to obtain 500 uL of OPA derivatization solution which was mixed evenly for 30 s, and derived for 2 min followed by being loaded into the filter membrane to determine the activity of FumDPS by comparing the peak diagram with that of FB1 standard.

[0043] 1. Determination of the Optimum Temperature of Fumonisin Degrading Enzyme

[0044] 100 μL of FB1 as substrate was added with 900 μL of enzyme solution until the final concentration of 10 μg/mL to react for 20 min in citric acid disodium hydrogen phosphate buffer in pH7.0 at different temperatures, and then be inactivated by boiling for 10 min, followed by detecting after cooling to the room temperature and membrane filtration.

[0045] As shown in FIG. 2, the optimum temperature of enzyme FumDPS was 37° C.

[0046] 2. Determination of the Optimum pH of Fumonisin Degrading Enzyme

[0047] The purified recombinant enzyme FumDPS was perform the enzymatic reaction to determine the optimum pH wherein the enzyme reacted with the substrate FB1 at the final concentration of 1 μg/mL min at 37° C. for 30 min in the different pH buffers, and boiled for 10 min followed by high performance liquid chromatography (HPLC), wherein the buffer gradients of the buffer solutions were 100 mM of citric acid disodium hydrogen phosphate from pH 3.0 to 8.0, 100 mM of Tris Hcl from pH 8.0 to 9.0, and 100 mM of glycine-NaOH from pH 9.0 to 12.0.

[0048] As shown in FIG. 3, the optimum pH of FumDPS was between 8.0 and 9.0, and the enzyme FumDPS had a degradation rate more than 58% between pH 3.0 and 7.0.