Zearalenone hydrolase zhd101 mutant and method for hydrolyzing zearalenone using the mutant
A technology of zearalenone and mutants, applied in hydrolytic enzymes, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of irreversible inactivation of enzymes, unsatisfactory catalytic efficiency, etc., and achieve improved catalytic activity , high expression activity, and the effect of improving enzyme activity
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Embodiment 1
[0064] Example 1. Preparation and purification of ZHD101 mutants
[0065] 1. Construction of ZHD101 mutant encoding gene and expression vector
[0066] The coding sequence (SEQ ID NO.2) of wild-type zearalenone hydrolase was replaced by the DNA sequence (SEQ ID NO.17) composed of Escherichia coli preferred codons, and corresponding mutation was introduced to obtain as SEQ ID NO. 4. The nucleotide sequences shown in SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14 and SEQ ID NO.16, 5' of the above sequence The Nde I restriction site CATATG was added to the end, and the DNA sequence CACCATCACCACCATCAC (SEQ ID NO. 18) encoding the hexahistidine tag, the stop codon TAA and the Xho I restriction site CTCGAG were sequentially added to the 3' end, chemical synthesis The resulting DNA sequence (Invitrogen, USA). A histidine tag 6×His was added to the C-terminal end of the encoded protein sequence to facilitate subsequent purification steps. The obtained DNA sequen...
Embodiment 2
[0080] Example 2. Detection of catalytic activity of ZHD101 mutants
[0081] The target protein obtained in step 3 of Example 1 was analyzed by HPLC (LC-20AT, Shimadzu Corporation) to determine the catalytic activity and kinetic parameters of the enzyme mutant. HPLC chromatogram such as image 3 shown (exemplarily showing experimental results of ZHD101M3).
[0082] Specifically, the selected chromatographic column was a Hypersil C18 reversed-phase column (Elite, 5 μm, 4.6 mM×250 mM). The ratio of the reaction system is: 4850μL sodium acetate (pH 5.5), 50μL substrate (dissolved in acetonitrile, the concentration changes in a gradient, the highest concentration is 2mg / mL), 100μL enzyme solution (stored in 100mM Tris buffer, pH 8.0 , concentration of 100 μg / mL), and reacted in a constant temperature water bath at 37 °C for 10 min. After the reaction was completed, the reaction was terminated by boiling in boiling water for 1 min.
[0083] The mobile phase ratio (v / v) of HPLC ...
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