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Preparation method of core-shell nano polymer and application of core-shell nano polymer in cyclic regeneration of coenzyme

A technology of recycling and polymers, applied in multi-enzyme systems, immobilized on or in the direction of inorganic carriers, can solve the problems of low efficiency and inability to fold correctly, and achieve the effect of increased conversion rate

Pending Publication Date: 2021-05-28
HENAN UNIVERSITY OF TECHNOLOGY
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Problems solved by technology

One of the limitations of multi-enzyme co-immobilization is the inability to properly fold large multi-domain proteins and is therefore generally less efficient

Method used

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  • Preparation method of core-shell nano polymer and application of core-shell nano polymer in cyclic regeneration of coenzyme
  • Preparation method of core-shell nano polymer and application of core-shell nano polymer in cyclic regeneration of coenzyme
  • Preparation method of core-shell nano polymer and application of core-shell nano polymer in cyclic regeneration of coenzyme

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Embodiment 1

[0026] Preparation of coenzyme polymer: Weigh 2g of carboxyl-modified mesoporous silicon nanoparticles, sonicate in 30mL deionized water for 8-15 minutes, then add a certain amount of coenzyme NAD + , adjust the pH to 4.5-5.5 with 0.5M hydrochloric acid solution, and stir magnetically at 25-30°C. Add 50 mg of water-soluble carbodiimide EDC continuously within 5 minutes, continue to add hydrochloric acid solution dropwise during the addition process, keep the pH at 4.5-5.5, and stir magnetically at 25-30°C for 25-30h. After the reaction, centrifuge to remove unreacted NAD + and other impurities, washed with 0.2M NaCl solution until NAD was not detected in the washing solution + . Finally, freeze-dry to obtain immobilized coenzyme.

[0027] In situ co-immobilization of multiple enzymes: 0.5-1mg alcohol dehydrogenase, 0.5-1mg lactate dehydrogenase, 1-2mg MSN-NAD + Add 5-10mL zinc acetate (40mM) solution, quickly add 5-10mL (2.8mM) 2-methylimidazole under stirring at room temp...

Embodiment 2

[0031]Weigh 2 g of group-modified mesoporous silicon nanoparticles, ultrasonicate in 30 mL of deionized water for 8-15 minutes, add a certain amount of coenzyme NADPH, and stir magnetically at 25-30°C. Add 50 mg of water-soluble carbodiimide EDC continuously within 5 minutes, control the pH to maintain at 7.4-8 during the addition, and stir magnetically at 25-30° C. for 25-30 h. After the reaction, unreacted NADPH and other impurities were removed by centrifugation, and washed with 0.2M NaCl solution until no NADPH was detected in the washing solution. Finally, freeze-dry to obtain immobilized coenzyme. Glutamate dehydrogenase and glucose-6-phosphate dehydrogenase were co-immobilized and coenzyme polymer encapsulated by in situ growth of ZIF-67 and ZIF-8 materials, respectively. Adding a certain amount of glutamic acid and glucose 6-phosphate initiates the coenzyme cycle and multi-enzyme cascade reaction in the system. In the presence of glutamate dehydrogenase and NADPH in ...

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Abstract

The invention discloses a preparation method of a core-shell nano polymer and application of the core-shell nano polymer in cyclic regeneration of coenzyme. The polymer with a core-shell structure is prepared by co-immobilizing a coenzyme polymer and two coenzyme dependent enzymes in situ through a metal framework compound ZIF material. According to the method, a ZIF shell is utilized to limit a multi-enzyme biological catalytic reaction area, and cyclic regeneration of coenzyme and double-enzyme cascade reaction are realized by utilizing double enzymes under the action of an initiator and a substrate respectively. During use of the core-shell polymer, no additional coenzyme needs to be added, and after the core-shell polymer is repeatedly used for five times, the ratio of the substrate conversion rate to the initial conversion rate is still 70-80%. Therefore, the core-shell nano polymer has a very wide prospect in biosynthesis and conversion in which coenzyme participates.

Description

technical field [0001] The invention relates to the field of nanometer materials, and also relates to the fields of multi-enzyme co-immobilization and coenzyme cycle regeneration. Background technique [0002] Oxidoreductases are widely used to catalyze the preparation of chiral alcohols, hydroxy acids, amino acids and so on. Most oxidoreductase catalyzed reactions require coenzymes, among which NAD(P) + -NAD(P)H system accounts for about 90%. However, these coenzymes are expensive, which limits the application of oxidoreductases. The spatial organization of enzymes in constrained structures provides a general approach for designing biocatalytic cascades, using the product of one enzyme as the substrate of another enzyme to achieve intercommunication between multiple enzymes, taking advantage of this The principle can be utilized to realize multi-enzyme catalytic cascades and cyclic regeneration of coenzymes at the nanoscale. [0003] Co-immobilization of multiple enzyme...

Claims

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Application Information

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IPC IPC(8): C12N11/18C12N11/14
CPCC12N11/18C12N11/14
Inventor 王乐孙朋雪杨翌昱
Owner HENAN UNIVERSITY OF TECHNOLOGY
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