Method for preparing magnetic nano carrier immobilized aldolase with high substrate tolerance

A magnetic nanocarrier, resistant technology, applied in the direction of immobilization on or in inorganic carriers, can solve the problem of increased product separation, limited reactant concentration, 2-deoxy-D-ribose-5-phosphate aldol condensation The practical application value of enzymes is reduced, and the effect of improving substrate tolerance, convenient recovery and reuse, and large-scale industrial application value is achieved.

Inactive Publication Date: 2012-06-27
ZHEJIANG UNIV
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Problems solved by technology

Such a high enzyme concentration makes the synthesis expensive and increases the difficulty of product isolation; second, the reaction time takes several days; third, the concentrat

Method used

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  • Method for preparing magnetic nano carrier immobilized aldolase with high substrate tolerance
  • Method for preparing magnetic nano carrier immobilized aldolase with high substrate tolerance

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preparation example Construction

[0015] The steps of the preparation method of the magnetic nanocarrier immobilized aldolase with high substrate tolerance are as follows:

[0016] 1) Add 0.25~0.35mol of ferric salt and 0.15~0.2mol of ferrous salt to 250mL of anaerobic water, adjust to pH = 1~2 with hydrochloric acid, ultrasonically treat for 30-40 min; heat up to 70~85 ℃, Stirring at 1 200~1 400 rpm, N 2 Bubble for 25-30 min, add 9-10 mL of ammonia water with a mass percentage of 25-28% or 10-15 mL of 10mol / L NaOH to make the solution pH = 8-10, after 20-30 min, magnetically separate and wash with anaerobic water 5~10 times, and then washed 3~4 times with 0.010~0.015mol / L ethanol aqueous solution;

[0017] 2) Add 80-100 mL of 50% by volume ethanol aqueous solution, 6-8 mL of silane cross-linking agent, stir at 50-60 °C, 180-220 rpm for 5-6 h, wash with pH = 7 phosphate buffer for 3 ~5 times;

[0018] 3) Add 18-20 mL pH = 7 phosphate buffer solution and 18-20 mL 5% glutaraldehyde aqueous solution, stir at ...

Embodiment 1

[0023] 1) Add 0.25 mol of ferric salt and 0.15 mol of ferrous salt to 250 mL of anaerobic water, adjust to pH = 1 with hydrochloric acid, and sonicate for 30-40 min; 2 Bubble for 25 min, add 9 mL of 25% by mass ammonia water or 10 mL of 10 mol / L NaOH to make the solution pH = 8, after 20 min, magnetically separate, wash with anaerobic water for 5 times, and then wash with 0.010 mol / L ethanol aqueous solution 3 times;

[0024] 2) Add 80 mL of 50% by volume ethanol aqueous solution, 6 mL of silane cross-linking agent, stir at 50°C and 180 rpm for 5 h, and wash with pH = 7 phosphate buffer for 3 times;

[0025] 3) Add 18 mL pH = 7 phosphate buffer and 18 mL 5% glutaraldehyde aqueous solution, stir at 200 rpm for 2 h, magnetically separate, wash 5 times with pH = 7 phosphate buffer, and vacuum dry to obtain Surface-activated magnetic nanoparticle microsphere carrier;

[0026] 4) Prepare 1 mg / ml aldolase protein solution, the mass ratio of aldolase protein to carrier is 1: 1, in...

Embodiment 2

[0029] 1) Add 0.35 mol of ferric salt and 0.2 mol of ferrous salt to 250 mL of anaerobic water, adjust to pH = 2 with hydrochloric acid, and sonicate for 340 min; 2Bubble for 30 min, add 10 mL of 28% by mass ammonia water or 15 mL of 10 mol / L NaOH to make the solution pH = 10, after 30 min, magnetically separate, wash with anaerobic water for 10 times, and then wash with 0.015 mol / L ethanol aqueous solution 4 times;

[0030] 2) Add 100 mL of 50% by volume ethanol aqueous solution, 8 mL of silane crosslinking agent, stir at 60 °C and 220 rpm for 6 h, and wash with pH = 7 phosphate buffer for 5 times;

[0031] 3) Add 20 mL pH = 7 phosphate buffer solution and 20 mL 5% glutaraldehyde aqueous solution, stir at 200 rpm for 3 h, magnetically separate, wash 5 times with pH = 7 phosphate buffer solution, and vacuum dry to obtain Surface-activated magnetic nanoparticle microsphere carrier;

[0032] 4) Prepare 1mg / ml aldolase protein solution, the mass ratio of aldolase protein to car...

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Abstract

The invention discloses a method for preparing magnetic nano carrier immobilized aldolase with high substrate tolerance. The method comprises the following steps of: 1) preparing super-paramagnetic Fe3O4 nano particles by co-precipitation of a mixture of ferrous and ferric iron salts, modifying the particles by using a silane coupling agent, and activating the outer surfaces of the modified particles by using glutaraldehyde to obtain surface activated magnetic nano particles; 2) stirring a purified and desalted aldolase buffer solution and a magnetic carrier at a low temperature, washing, performing freeze drying, and thus obtaining the magnetic nano carrier immobilized aldolase; and 3) catalyzing 2-deoxy-D-ribose-5-phosphoric acid by using the magnetic nano carrier immobilized aldolase as a catalyst to obtain 3-glyceraldehyde phosphate and acetaldehyde. In the reaction of catalyzing the 2-deoxy-D-ribose-5-phosphoric acid by using the immobilized aldolase, the tolerance of the acetaldehyde substrate is remarkably improved, and the recycling operation of enzyme is greatly simplified.

Description

technical field [0001] The invention relates to a method for improving substrate tolerance of an enzyme through immobilization, in particular to a preparation method of a high substrate tolerance magnetic nanocarrier immobilized aldolase. Background technique [0002] 2-Deoxy-D-ribose-5-phosphate aldolase is unique from the other three aldolases in that it can catalyze the complexation of two aldehyde molecules, and the relaxed specificity of the enzyme allows the co-substitution of two substrates , some aldehydes with a chain length of less than four hydrogen atoms can be used as acceptors. When the first acceptor is substituted acetaldehyde, it can undergo two consecutive condensations with acetaldehyde, and the product is rearranged to obtain a stable ring Acetals can continue to be used to synthesize some useful drug intermediates. For example, 2-deoxy-D-ribose-5-phosphate aldolase can be used to catalyze the aldol condensation reaction of two molecules of acetaldehyde ...

Claims

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Application Information

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IPC IPC(8): C12N11/14
Inventor 费辉徐刚吴坚平杨立荣
Owner ZHEJIANG UNIV
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