Plate culture medium enzyme activity determination method
A technology for enzyme activity measurement and plate culture medium, which is applied in the field of plate culture medium enzyme activity measurement, can solve the problems of inability to accurately determine enzyme activity and large loss of enzyme activity, and achieve the effect of being beneficial to quality control and improving stability and quality.
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Embodiment 2
[0038] Embodiment dihydroxylamine method is measured β-lactamase activity
[0039] Prepare the solution:
[0040] 0.25mol / L hydroxylamine hydrochloride: Take hydroxylamine hydrochloride 1.7379, dissolve in water, and dilute to 100mL.
[0041]Alkaline buffer solution (pH12.5): Take 4.32g of NaOH and 0.52g of anhydrous sodium acetate to dissolve, and dilute to 500mL.
[0042] 0.125mol / L neutral hydroxylamine (pH6.75): mix equal volumes of 0.25mol / L hydroxylamine hydrochloride and alkaline buffer.
[0043] 0.25mol / L ferric ammonium sulfate: Take 12.05g of ferric ammonium sulfate, suspend it in 0.15mol / L sulfuric acid solution, heat to dissolve it, after cooling, dilute to 100mL with 0.15mol / L sulfuric acid, filter to remove insoluble matter.
[0044] 50mrnol / L sodium citrate-hydrochloric acid buffer solution (pH6.0): Take 7.35g of sodium citrate, dissolve it in about 400mL of water, adjust the pH to 6.0 with 1mol / L hydrochloric acid, and then set the volume to 500mL. This solut...
Embodiment 3
[0055] Embodiment three macrolide enzyme activity assay method
[0056] Using spectrophotometry, the content of p-nitrophenol produced by p-Nitrophenyl butyrate (p-NPB) under the action of azithromycin enzyme is proportional to its absorption value at 405nm, and the molar extinction coefficient ε=24,000M -1 cm-1. Under the conditions of pH 7.5 and 37°C, the increase of absorption value after hydrolysis of p-nitrophenylbutyrate within a certain period of time reflects the enzyme activity.
[0057] The assay buffer is 50mM HEPES pH 7.5, 0.2% Triton X-100), take 1ml of the assay buffer that has been temperature-balanced at 37°C, add p-nitrophenylbutyrate to make the final concentration 1mM, add the sample 10 μl, react at 37°C for 30 minutes, measure the absorbance of the reaction solution at 405 nm, and use the sample buffer without enzyme as a blank.
[0058] Enzyme activity unit (U) is defined as the amount of enzyme required to hydrolyze 1 micromole of p-nitrophenol per minu...
Embodiment 4
[0059] The thermostability test of embodiment four enzymes
[0060] Take 4 tubes each of penicillinase, cephalosporinase, metallo-β-lactamase and macrolide enzyme freeze-dried powder, dissolve them according to the instructions, and place them at 37°C, 50°C, 65°C and 95°C respectively for a certain period of time. The enzyme activity was measured, and the untreated enzyme activity measurement result was taken as 100%, and the residual enzyme activity percentage at each temperature was calculated. The results are shown in Table 1. The results showed that the loss of enzyme activity was no more than 5% when placed at 37°C for 1 hour, about half of the enzyme activity remained after 50°C treatment for 1 hour, and the enzyme deactivation was faster at 65°C, and the enzyme activity did not exceed 30% after 1 hour. At 95°C, the enzyme almost completely loses its activity in a short time, so it is not feasible to measure the enzyme activity after melting the agar plate at a higher te...
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