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Enzyme-macromolecule conjugate and preparing method and application thereof

A technology of macromolecular conjugates and macromolecular compounds, which is applied in the direction of being fixed on/in organic carriers, can solve the problems of difficulty in recycling and utilization of enzyme preparations, and achieves easy industrial implementation and amplification, mild reaction conditions, and easy acquisition. Effect

Inactive Publication Date: 2013-12-18
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In nano-biocatalysis systems, while improving the dispersion of enzymes in the organic phase and thus improving the activity, the recycling of enzyme preparations is usually difficult

Method used

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  • Enzyme-macromolecule conjugate and preparing method and application thereof
  • Enzyme-macromolecule conjugate and preparing method and application thereof
  • Enzyme-macromolecule conjugate and preparing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The enzyme is Candida antarctica lipase B (enzyme activity is 9U / mg) which is 10 parts by weight, and the polymer compound is a compound with an average molecular weight of 12,600 60 parts by weight, 10 parts by weight of the activator Dess-Martin oxidant, and 6 parts by weight of sodium cyanoborohydride as the reducing agent.

[0063] 1) Dissolve the above-mentioned polymer and polymer activator in dichloromethane, carry out oxidation reaction at room temperature for 15 hours, oxidize the terminal hydroxyl group to aldehyde group, and then add cold ether to precipitate to obtain the activated polymer product, vacuum drying;

[0064] 2) Dissolve the activated polymer compound and enzyme obtained in step 1) in 10mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH value of 7, carry out the coupling reaction of aldehyde group and amino group at room temperature for 2 hours, then add The reducing agent carries out the reduction reaction of...

Embodiment 2

[0079] According to the steps of Example 1, only the raw materials of steps 1) and 2) are replaced as follows to obtain the enzyme-polymer conjugate provided by the present invention:

[0080] The enzyme is Candida rugosa lipase (enzyme activity: 700-1000U / mg) is 10 parts by weight, and the polymer is the average molecular weight of 12,600 It is 72 parts by weight, the polymer activator is 12 parts by weight of Dess-Martin oxidant, and the reducing agent is 7 parts by weight of sodium cyanoborohydride.

[0081] The enzyme-polymer conjugate SDS-PAGE results are as follows figure 2 shown.

[0082] After dispersing in toluene, the particle size is 25nm ( image 3 b).

[0083] Evaluation:

[0084]1. Determination of the enzyme activity in the water phase as follows: First, dissolve p-nitrophenol palmitate in acetone, and then slowly add hydrogen phosphate containing 1.25% (w / v) Triton X-100 under stirring A 0.5 mM substrate solution was prepared in disodium-sodium dihydrog...

Embodiment 3

[0089] The enzyme is cytochrome C (enzyme activity is 12U / mg), 10 parts by weight, and the polymer compound is F-127 is 300 parts by weight, activator is 50 parts by weight of Dess-Martin oxidant, and reducing agent is 30 parts by weight of sodium cyanoborohydride.

[0090] Subsequent steps are the same as in Example 1.

[0091] The conjugate SDS-PAGE results are as follows figure 2 As shown, its particle size after dispersion in toluene is 30nm ( image 3 c).

[0092] Evaluation:

[0093] 1. Measure the enzyme activity in the water phase as follows: mix 960 μL ABTS aqueous solution (2.8 mg / mL) with 20 μL enzyme-polymer conjugate (enzyme content is 0.1 mg / ml), add an equivalent amount of natural enzyme aqueous solution to the control group (Cytochrome C, purchased from Sigma-Aldrich Company, the product number is C7752). Add 20 μL of hydrogen peroxide aqueous solution (0.3% v / v) to initiate the reaction, measure the absorbance at 415 nm versus time (a straight line wit...

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Abstract

The invention discloses an enzyme-macromolecule conjugate and a preparing method and application thereof. The conjugate is formed by the fact that a macromolecule active end group and an amino group on the surface of an enzyme react to form covalent linkage. Under the conventional reaction temperature (37-40 DEG C), the conjugate can form nanoscale dispersing in common organic solvents, and the organic phase catalytic activity is 1-2 magnitude order higher than that of natural enzyme powder with the same quantity. Meanwhile, nanoparticles of the conjugate have thermosensitivity in organic phase and can be separated out in an agglomeration mode under low temperature, and separating and recycling of enzyme catalysts are convenient. The nanoparticles have wide application prospect in the field of organic phase enzymatic synthesis. The invention further discloses the preparing method of the nanoparticles, namely a macromolecule active end group is activated first, and then the macromolecule active end group is in covalent linkage with an enzyme molecule. According to the method, reaction raw materials are easy to obtain, reaction conditions are mild, and preparing scale can be easily enlarged.

Description

technical field [0001] The invention relates to an enzyme-macromolecule conjugate, a preparation method and application thereof. Background technique [0002] The enzyme-catalyzed process can be carried out not only in the aqueous phase system, but also in the organic phase. The use of organic solvents is usually beneficial to dissolve the substrate, inhibit the reverse reaction, and eliminate the side effects of water on the reaction. At the same time, it may also retain or increase the advantages of enzyme-catalyzed chemoselectivity, regioselectivity, and stereoselectivity. However, most natural enzymes with high catalytic activity in vivo have very limited activity in organic solvents. One of the main reasons for this problem is the extremely low solubility of enzymes in organic solvents, which greatly limits the access of substrates to the active sites of enzymes. The study of increasing the solubility (dispersion) of enzyme preparations in organic solvents by physical...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/08
Inventor 朱晶莹戈钧张一飞卢滇楠刘铮
Owner TSINGHUA UNIV
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