A kind of immobilized nuclease p1 and its preparation method and application
A nuclease and resin technology, applied in the field of bioengineering, can solve the problems of double inhibition of product substrates, difficulty in product separation and purification, low utilization rate of free enzymes, etc., and achieve the effects of stable properties, obvious effects and high catalytic performance.
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Embodiment 1
[0040] Example 1: Concanavalin A (ConA) modified amino resin immobilized nuclease P 1 preparation.
[0041] 1. Preparation of ConA modified amino resin.
[0042] (1) ConA activation: Dissolve a certain mass of ConA in 0.1mol / LKCl, 0.01mol / LCaCl 2 , 0.01mol / LMnCl 2 In 0.1mol / L phosphate buffer solution with pH 7.0, activate at 4°C for 6h;
[0043] (2) Amino resin activated by glutaraldehyde: add 400mL, 0.1mol / L pH 8.0 phosphate buffer to 100g of amino resin, stir for 15 minutes, measure pH, maintain pH 7.8~8.2, filter and drain after 1 hour, this step It is for cleaning amino resins. Then, add 400mL of 2% (w / w) glutaraldehyde phosphate buffer to the obtained amino resin, stir for 1 hour at 25°C, filter, wash the carrier with deionized water until the water is clear to obtain glutaraldehyde-activated amino Resin HA-GA;
[0044] (3) ConA modified amino resin: take 2g of the amino resin obtained in step (2) in a conical flask, add activated ConA solution, place it in a cons...
Embodiment 2
[0058] Example 2: Polyethylene glycol (PEG) modified amino resin immobilized nuclease P 1 preparation.
[0059] 1. PEG modified amino resin
[0060] (1) Dissolve PEG with a molecular weight of 10KDa in 0.1mol / L phosphate buffer to prepare a PEG solution;
[0061] (2) Take 2g of amino resin in a Erlenmeyer flask, add a certain amount of PEG solution, shake it in a constant temperature shaker, separate after a certain period of time, and obtain PEG surface-modified amino resin by suction filtration, and store in a refrigerator at 4°C.
[0062] 2. PEG-modified amino resin immobilized nuclease P 1 preparation of
[0063] Take 1 g of the above-mentioned blank carrier in a Erlenmeyer flask, add 100 mL, 0.1 mg / mL enzyme solution, and adjust the pH of the enzyme solution to 5.5, stir at 100 rpm at room temperature, separate the supernatant after 6-8 hours, wash with water to remove loosely bound proteins on the surface, and pump Filter and dry to obtain PEG-modified amino resin as...
Embodiment 3
[0068] Example 3: Polydopamine (pDopa) Modified Sulfonated Resin Immobilization of Nuclease P 1 preparation.
[0069] 1. Polydopamine modified sulfonated resin
[0070] (1) Add a certain amount of dopamine hydrochloride to the Tris buffer to adjust the pH of the solution to 7.8~8.2.
[0071] (2) Take 2g of sulfonated resin in a conical flask, add 50mL of dopamine hydrochloride Tris buffer solution, place it in a constant temperature shaker at 25°C and 180rmp, shake it, separate the resin for 3~5h, wash away unbound dopamine hydrochloride, pump The polydopamine surface-modified sulfonated resin was obtained by filtration, and stored in a refrigerator at 4°C.
[0072] 2. Polydopamine modified sulfonated resin immobilized nuclease P 1 preparation of
[0073] Take 1g of the above carrier in a Erlenmeyer flask, add 50mL, 0.1mg / mL enzyme solution, adjust the pH of the enzyme solution to 5.5~6.5, stir at 120rpm at room temperature, separate the supernatant after a certain period ...
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