PEG modified methyl parathion hydrolase (MPH) and application thereof
A technology of methyl parathion and hydrolase, applied in the biological field, can solve the problems of high cost and poor product stability, and achieve the effects of controllable stability, food safety guarantee and cost reduction.
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Embodiment 1
[0046] Fermentation and purification of methyl parathion hydrolase:
[0047] Add antibiotics and trace elements at the beginning of fermentation, let it grow naturally at 37°C, use a lower initial medium concentration, so that the fermentation process can proceed according to the expected specific growth rate, the growth rate is natural during the initial culture, and the wet weight reaches 50g / L Then start to add ammonia water, the flow acceleration rate is determined based on the specific growth rate = 0.1, and no longer add yeast extract. Add trace elements into the sugar solution and add them together with the feed. When the wet weight is between 50g / L and 70g / L, induce at 30°C. Induced by low concentration IPTG (0.1mM), the specific growth rate was controlled at 0.05 at the initial stage of induction. When the wet weight > 120, the rate of ammonia supplementation will not be changed. The rotational speed was set at 500 rpm. The pH is set at 6.8-7.0.
[0048] The amou...
Embodiment 2
[0050] Add methyl parathion hydrolase to buffer I to a protein concentration of 2 mg / ml, adjust the pH to 7, 8, and 9, add a polyethylene glycol modifier, and the molar ratio of protein to polyethylene glycol modifier is 1: 5. 1:10, 1:20, 1:30, 1:40, 1:50. The buffer I is 0.02-0.2mol / L Tris-Cl or phosphate buffer (PB), pH 7-9, modified at 4°C, room temperature, or 37°C under constant temperature conditions. Quickly dissolve the modifier within 10 seconds and fully shake and mix evenly, and then quickly place it at the specified temperature for 16 hours.
[0051]According to the content of PEG-modified methyl parathion hydrolase with different modification degrees in the reaction solution, choose the combination of hydrophobic chromatography, ion exchange chromatography and gel filtration chromatography to purify different modified products. The chromatographic column is butyl Hydrophobic chromatography column Butyl Sepharose6FF, anion-cation exchange chromatography column CM / ...
Embodiment 3
[0054] Activity and Stability Determination: Activity detection uses the pesticide methyl parathion as a substrate. The specific method is: 200ul system, 20mM mol / L PB pH7.0 as the reaction buffer, containing the substrate methyl parathion with a final concentration of 0.125mM, adding a certain amount of enzyme solution to react, and reacting in the UV / VIS spectrophotometer Absorbance was detected at 405 nm. Definition of enzyme activity unit: at room temperature, the amount of enzyme required to catalyze the production of 1uM p-nitrophenol per minute is an activity unit. The stability test adopts the classic constant temperature method. The principle is to store the sample at a constant temperature of 37°C according to the Arrhenius formula, take samples regularly, observe the change of its activity, and extrapolate to 4°C and room temperature according to the Arrhenius relationship diagram, so that the activity of the target protein under the constant temperature of 37°C ca...
PUM
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