Means and methods for detoxifying ochratoxin A
A technology of ochratoxin and antidote, which is applied in the field of new peptides and can solve the problems of insufficient activity, stability, recombination and reduction ability, etc.
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Embodiment 1
[0246] Example 1: Cloning of genes and production of recombinant polypeptides
[0247] Genes encoding any of the polypeptide sequences SEQ ID NOs: 1-4 were synthesized by the commercial supplier TWIST Bioscience (https: / / www.twistbioscience.com / ) with codons optimized for expression in E. coli. The genes were originally designed to further encode a hexahistidine tag C-terminally fused to either of the polypeptide sequences to facilitate subsequent purification. Subsequent polypeptide production and enzyme characterization were also performed with unlabeled enzyme, yielding essentially the same results.
[0248] Genes were cloned into expression vectors using common tools known in the art for expression in E. coli. In particular, the T7 promoter system with the lac operator (Dubendorf and Studier Studier 1991, J. Mol. Biol. 219, 45-59) was used to regulate gene expression. Escherichia coli BL21 (DE3) strain was transformed with the expression vector. A kanamycin resistance m...
Embodiment 2
[0251] Embodiment 2: the mensuration of specific activity
[0252] To determine the specific activity in units per liter (U / L) of a recombinant polypeptide of interest produced eg as described above in Example 1, an ochratoxin A (OTA) hydrolytic activity assay was performed. One unit is defined as the amount of enzyme required to hydrolyze 1 μmol or OTA in 1 min when using a starting OTA concentration of 0.495 μM.
[0253] The OTA hydrolytic activity assay was set up with a final volume of 200 μL of a reaction mixture containing a recombinant enzyme preparation (preferably diluted to produce a volumetric activity of 1-10 mU / L) and 200 ng / L in 100 mM sodium phosphate buffer. mL OTA.
[0254] Specific activity was determined at pH 6.0 and pH 7.5. Once all components of the reaction mixture are mixed, follow OTA hydrolysis at 37°C on a fluorescence spectrophotometer (BioTek Synergy H1 MFD multimode microplate reader) by following the decrease in fluorescence at ex / em 390nm / 450n...
Embodiment 3
[0258] Embodiment 3: the mensuration of thermal stability
[0259] To determine the kinetic stability of the produced enzymes against temperature, a thermostability assay was performed. For this, the purified protein samples were diluted in 20 mM Tris-HCl buffer pH 7.5 to a protein concentration of 0.1 mg / mL and aliquots were cycled in a thermal cycler at 45, 55, 65, 75, or 85 °C. Incubate for 10 minutes. After this incubation, aliquots were stored at 4°C until further analysis. Reference aliquots were stored at 4 °C throughout the incubation period. All aliquots were then assayed for residual OTA hydrolytic activity using the assay described in Example 2. Residual enzyme activity was calculated relative to a reference aliquot. The temperature range at which 50% of the initial activity was lost after incubation was determined relative to a reference aliquot. The results are shown in Table 2.
[0260] Table 2: Temperature range for loss of 50% of initial activity.
[026...
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