Fumonisin degrading enzyme FumDXA as well as gene and application thereof
A fumonisin and enzyme-degrading technology, applied in the field of agricultural biology, can solve the problems of changing food quality and flavor, high requirements, and large limitations of chemical methods, and achieve the effect of reducing harm
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Embodiment 1
[0035] Example 1 Cloning of Fumonisin Degrading Enzyme FumDXA
[0036] The gene fragment of fumonisin degrading enzyme FumDXA derived from Xenophilus azovoran was obtained by artificial chemical synthesis, and the endonuclease site EcoR I was introduced at the 5' end, and the endonuclease site Xho I was introduced at the 3' end.
Embodiment 2
[0037] Example 2 Preparation of Recombinant Fumonisin Degrading Enzyme FumDXA
[0038] The expression vector pET28a was subjected to double enzyme digestion (EcoR I+Xho I), and at the same time, the gene FumDXA encoding fumonisin detoxification enzyme was double enzyme digested (EcoR I+Xho I), and the gene fragment encoding the detoxification enzyme was cut out and the expression vector pET28a After connection, the recombinant plasmid pET28a-FumDXA containing the degrading enzyme FumDXA was obtained and transformed into Escherichia coli BL21(DE3) to obtain the recombinant Escherichia coli strain BL21(DE3) / FumDXA.
[0039] Take the BL21(DE3) strain containing the recombinant plasmid, inoculate it in 100mL LB culture medium, shake it at 220rpm at 37℃ for 2-3h, and the OD 600 When the temperature is 0.6-0.8, add IPTG with a final concentration of 1 mM, induce for 20 hours at 25°C, and after the induction is over, collect the cells by centrifugation at 4°C. The supernatant was co...
Embodiment 3
[0040] Example 3 Determination of the properties of fumonisin degrading enzyme FumDXA
[0041] The enzyme activity of fumonisin degrading enzyme is detected by high performance liquid chromatography, and the specific method is as follows:
[0042] (1) FB1 standard stock solution: Weigh 1mg of the standard product, dissolve it in 10ml of acetonitrile and water (1:1), prepare a standard solution with a concentration of 100ug / mL, and store at -20°C.
[0043] (2) Sample preparation: Take 900ul of purified fumonisin degrading enzyme enzyme solution, add 100ul of FB1 standard stock solution to make the final concentration of FB1 10ug / ml, incubate at 37°C, 220rpm, in the dark for 20min.
[0044] (3) Sample derivatization: take 100 ul of the sample to be tested, add 400 ul of 50% acetonitrile water, 500 ul of OPA derivatization solution, mix for 30 s, inject the sample within 2 min of derivatization, and filter the membrane for testing. The enzymatic activity of the fumonisin degradi...
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