Application of kasugamycin and derivatives thereof as chitinase inhibitor
A technology of chitinase and kasugamycin, applied in the application, biocide, animal repellent and other directions
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Embodiment 1
[0023] Using chitinase OfChtI as the target, 556 microbial secondary metabolites were screened for inhibitors. Specific steps are as follows:
[0024] Positive control: Set up 2 parallel positive controls. Under the condition of 30℃ reaction temperature and 100μL reaction system, 2nmol / L glycosyl hydrolase OfChtI and 50μmol / L substrate (MU-(GlcNAc) 2 ) in 20mmol / L pH 6.0 phosphate buffer for 30min, then add 100μL 0.5mol / L sodium carbonate solution to terminate the reaction, and measure the absorbance value at 450nm wavelength after the reaction solution is excited with 360nm wavelength excitation light.
[0025] Experimental group: set up 3 parallel experimental groups. Under the condition of 30℃ reaction temperature and 100μL reaction system, 2nmol / L glycosyl hydrolase OfChtI and 50μmol / L substrate (MU-(GlcNAc) 2 ) and 50 μM compound were incubated in 20 mmol / L pH 6.0 phosphate buffer for 30 min, then 100 μL 0.5 mol / L sodium carbonate solution was added to terminate the re...
Embodiment 2
[0030]1) Half inhibitory concentration IC 50 determination
[0031] OfChtI: MU-(GlcNAc) 2 As a substrate, the substrate concentration was 30 μM. Under the same substrate concentration, eight groups of compound concentration gradients with final concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 μM were used to determine the inhibitory activity. The reaction system was 100 μL, the buffer environment was 20 mM phosphate buffer, pH 6.0, the final enzyme concentration was 2 nM, the reaction temperature was 30 ° C, the reaction time was 30 min, and then 100 μL of 0.5 M sodium carbonate solution was added to terminate the reaction, and the released MU The absorbance value was measured at a wavelength of 450 nm after being excited by 360 nm excitation light. IC obtained after data fitting 50 =14μM, the result is as figure 2 shown.
[0032] 2) Determination of applicable objects of compounds
[0033] GH18: 18 family chitinases from 7 different species were selec...
Embodiment 3
[0035] The specific steps of the insecticidal activity evaluation of the compound Kasugamycin are as follows:
[0036] In the experiment, healthy larvae on the fourth day of the fifth instar were selected as experimental materials, and a control group and an experimental group were set up. Compound Kasugamycin with H 2 O was dissolved at a concentration of 500 μM. Control group larvae were injected with 2 μL of H 2 O, the larvae of the experimental group were injected with 2 μL of the compound Kasugamycin at a concentration of 500 μM. The injected larvae were cultured at 26°C, with a relative humidity of 70%-90%, 16 hours of light per day, and 8 hours of darkness until all of them pupated. During this period, normal larvae, dead larvae, normal pupae, and abnormal pupae Quantities and phenotypes were counted. The statistical results are plotted as image 3 , where A is the control group and B is the experimental group. The results showed that the development of larvae in ...
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