Zearalenone degrading enzyme mutant as well as coding gene and application thereof
A technology of zearalenone and coding genes, which is applied in the fields of application, hydrolase, genetic engineering, etc., can solve the problems such as few researches on enzymes, and achieve the effect of high activity and good stability
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Embodiment 1
[0047] Embodiment 1, preparation and purification of protein and gene
[0048] 1. Artificial synthesis of gene sequence
[0049] The nucleotide sequence shown in SEQ ID NO.2 was entrusted to Wuhan Jinkairui Bioengineering Co., Ltd. to artificially synthesize the gene according to the conventional technology in the field, and the gene was inserted into the plasmid vector pUC57, and stored for future use.
[0050] 2. Gene sequence amplification
[0051] According to the nucleotide sequence shown in SEQ ID NO.2, the primer pair is designed as follows:
[0052] Forward primer: 5′-CGC GGATCC ATGCGCACCAGGTCCAATATCACC-3', as shown in SEQ ID NO.5;
[0053] Reverse primer: 5′-CCG CTCGAG TTACAAGTACTTTCGAGTCTTTTCC-3', as shown in SEQ ID NO.6;
[0054] The underlined part of the forward primer is the restriction site of BamHI, and the underlined part of the reverse primer is the restriction site of XhoI.
[0055] PCR reaction system:
[0056]
[0057] PCR reaction conditions: ...
Embodiment 2
[0073] Example 2. Verification of protein function using zearalenone as a substrate
[0074] The enzyme activity unit is defined as the amount of enzyme required to degrade 1 μg of the substrate zearalenone within 1 min as an enzyme activity unit U.
[0075] (1) Optimum temperature
[0076] The ZhdAY3 pure enzyme solution in Step 5 of Example 1 was diluted with 50 mM glycine-NaOH buffer solution of pH 9.5, and the enzyme activity was measured with the diluted enzyme solution. The diluted enzyme solution was recorded as the diluted enzyme solution.
[0077] The composition of solution A: it is composed of 50mM, pH9.5glycine-NaOH buffer solution and zearalenone solution; the final concentration of the substrate zearalenone in the reaction system 0.5mL is 20.0μg / ml.
[0078] Experimental group: The activity assay reaction system is 0.5mL, diluted with 0.45mL solution A and 0.05mL enzyme solution; the pH value of the reaction system is 9.5; mL of chromatographic grade methanol ...
Embodiment 3
[0114] Example 3. Verification of protein function using β-zearalenol as a substrate
[0115] The enzyme activity unit is defined as the amount of enzyme required to degrade 1 μg of the substrate zearalenone within 1 min as an enzyme activity unit U.
[0116] The diluted enzyme solution described below is obtained by diluting the ZhdAY3 pure enzyme solution in Step 5 of Example 1 with 50 mM glycine-NaOH buffer solution.
[0117] Experimental group: β-zearalenol was used as the substrate (the final concentration of the substrate in the reaction system was 20.0 μg / ml), the activity assay reaction system was 0.5mL, and 0.45mL substrate solution and 0.05mL diluted enzyme solution; the pH value of the reaction system was 9.5; after the reaction system was reacted at an optimum temperature of 40° C. for 10 min, 0.5 mL of chromatographic grade methanol was used to terminate the reaction, and after cooling, the amount of degradation of the substrate was measured using high performance...
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