Extraction method of nereis kinase
An extraction method and technology of clam worm, which is applied in the field of preparation of clam worm kinase, can solve the problems of low product titer, low salting-out yield of clam worm kinase, high viscosity of initial extract, etc.
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Embodiment 1
[0059] 1. Sample pretreatment: Weigh 10Kg of fresh clam worms, place them in filtered flowing sea water and starve them for 12 hours, place the clam worms in filtered sea water after starvation treatment, and fill the water with ozone using an ozone generator , so that the concentration of ozone in water is 0.3mg / L, and maintain this concentration for 13 minutes.
[0060] 2. Sample pulverization and filtration: put the sterilized clamworm into 22L pre-cooled to 9°C pH 8.0, add 0.15mM phosphate buffer solution to the clamworm, the tissue pulverization time is 15s, and then use 6 Squeeze and filter through a layer of gauze, and collect the filtrate.
[0061] 3. Enzyme hydrolysis and enzyme activation: put the filtered liquid at room temperature, add alkaline lipase with an optimum pH of 7.0 to 9.0 to the filtered liquid, the added amount is 0.15% of the weight of the clamworm, and put it under the condition of 32 ℃ , incubating for 2.5 hours, and centrifuging at 10,000 rpm for ...
Embodiment 2
[0071] 1. Sample pretreatment: Weigh 10Kg of fresh clam worms, place them in filtered flowing sea water and starve them for 12 hours, place the clam worms in filtered sea water after starvation treatment, and fill the water with ozone using an ozone generator , so that the concentration of ozone in water is 0.4 mg / L, and maintain this concentration for 10 minutes.
[0072] 2. Sample crushing and filtration: put the sterilized clamworm into 20L pre-cooled to 8°C pH 7.5, add 0.14mM phosphate buffer solution to the clamworm, tissue crushing time is 10s, and then use 6 layers immediately Gauze was squeezed and filtered, and the filtrate was collected.
[0073] 3. Enzyme hydrolysis and enzyme activation: put the filtered liquid at room temperature, add alkaline lipase with an optimum pH of 7.0 to 9.0 to the filtered liquid, the added amount is 0.1% of the weight of the clamworm, and put it under the condition of 30 ℃ , incubating for 2.0 hours, and centrifuging at 10,000 rpm for 2...
Embodiment 3
[0080] 1. Sample pretreatment: Weigh 10Kg of fresh clam worms, place them in filtered flowing sea water and starve them for 12 hours, place the clam worms in filtered sea water after starvation treatment, and fill the water with ozone using an ozone generator , so that the ozone concentration in water is 0.3mg / L, and maintain this concentration for 15min.
[0081] 2. Sample crushing and filtration: put the sterilized clamworm into 25L of pH8.5 pre-cooled to 10°C in advance, add 0.16mM phosphate buffer solution to the clamworm, the tissue crushing time is 20s, and then use 6 Squeeze and filter through a layer of gauze, and collect the filtrate.
[0082] 3. Enzyme hydrolysis and enzyme activation: put the filtered liquid at room temperature, add alkaline lipase with an optimum pH of 7.0 to 9.0 to the filtered liquid, the amount added is 0.2% of the weight of the clamworm, and put it under the condition of 35 ℃ , heat preservation and enzymatic hydrolysis for 3.0h, and after enz...
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Abstract
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