Preparation method of magnetic nanoparticle immobilized amino-acylase as well as product and application of preparation method

A technology of magnetic nanoparticles and aminoacylase, which is applied in the direction of immobilization on or in inorganic carriers, chemical industry, climate sustainability, etc., can solve the problems of poor practicability and high price, and achieves simple operation and low cost. , the effect of improving stability

Inactive Publication Date: 2015-04-01
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the industrial fermentation production process, the temperature in the fermentation tank is difficult to reach a low temperature of 37°C, or requires expensive cooling equipment to achieve it, so the above patents are less practical

Method used

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  • Preparation method of magnetic nanoparticle immobilized amino-acylase as well as product and application of preparation method
  • Preparation method of magnetic nanoparticle immobilized amino-acylase as well as product and application of preparation method
  • Preparation method of magnetic nanoparticle immobilized amino-acylase as well as product and application of preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Preparation of Magnetic Ferric Oxide Nanoparticles Modified by Amino Surface

[0041] (1) Weigh 6.76g of ferric chloride hexahydrate and 3.48g of ferrous sulfate heptahydrate and dissolve them in 100ml of deionized water so that the molar ratio of ferric ions to ferrous ions is equal to two to one. Nitrogen deoxygenation, add 5mol / L NaOH drop by drop while stirring mechanically until the solution pH = 10, to obtain a black iron ferric oxide nanoparticle solution, put this solution at 60°C for 30min, and the obtained iron ferric oxide nanoparticle Rinse 3 times with deionized water and ethanol, and redisperse in 50% (v / v) ethanol.

[0042] (2) Ultrasonic disperse 1.5g of magnetic iron ferric oxide nanoparticles into 150ml of 50% (v / v) ethanol, add 500μl of 3-aminopropyltriethoxysilane (APTES), nitrogen protection, 40 ℃ mechanical After stirring for 24 hours, the amino-modified Fe3O4 nanoparticles were collected by a magnet, washed three times with deionized water and ab...

Embodiment 2

[0045] Amino-modified iron ferric oxide nanoparticles are mixed with an aminoacylase solution to obtain the magnetic nanoparticle-immobilized aminoacylase.

[0046] (1) Dissolve a certain amount of amino-modified magnetic ferric iron tetroxide nanoparticles in 0.1mol / L, pH 7.0 phosphate buffer to make the final concentration 8mg / ml, sonicate for 15min, add a certain amount of glutaraldehyde to make The final concentration was 1.0%, stirred at 30°C for 1 h, collected by a magnet, and washed with 0.1 mol / L, pH 7.0 phosphate buffer to remove residual glutaraldehyde.

[0047] (2) The collected nanoparticles were mixed with a certain concentration of aminoacylase solution for 1.5 h, collected by a magnet, washed with 0.1 mol / L, pH 7.0 phosphate buffer to remove residual free enzyme.

Embodiment 3

[0049] Determination of the enzyme activity and enzyme reaction temperature of the aminoacylase immobilized on the magnetic nanoparticles prepared in Example 2 and the free enzyme.

[0050] Measure 40mL 0.1mol / L pH7.0 phosphate buffer solution into a 250mL Erlenmeyer flask, add 5mL deionized water and 5mL 0.2mol / L N-acetyl-DL-theanine, respectively at 29°C, 42°C, and 52°C , 62°C, 72°C for 5 minutes, add 20 mg of free aminoacylase or the magnetic nanoparticle-immobilized aminoacylase obtained in Example 2, shake for 30 minutes, immediately stop the reaction by boiling water for 5 minutes, and dilute to a certain concentration after cooling , take 1mL of the sample to be tested for ninhydrin reaction, shake well, measure its OD570, and compare the standard curve to obtain the output of L-theanine. The enzyme activity unit is defined as: at pH 7.0, at 42°C, within 1 hour, the enzyme catalyzes the reaction to produce 1 μmol L-theanine, which is an activity unit (U). The relative ...

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Abstract

The invention relates to a preparation method of a magnetic nanoparticle immobilized amino-acylase as well as a product and application of the preparation method, and belongs to the field of nanomaterial preparation technologies and the biocatalysis technologies. The preparation method of the magnetic nanoparticle immobilized amino-acylase comprises the following steps: preparing ferroferric oxide nanoparticles; after ultrasonic dispersion of the ferroferric oxide nanoparticles, reacting the ferroferric oxide nanoparticles with 3-aminopropyltriethoxysilane in the presence of nitrogen, thereby obtaining amino-modified ferroferric oxide nanoparticles; and mixing the amino-modified ferroferric oxide nanoparticles with an amino-acylase solution for reacting, thereby obtaining the magnetic nanoparticle immobilized amino-acylase. The prepared magnetic nanoparticle immobilized amino-acylase is used for resolving theanine. Compared with the prior art, the preparation method of the magnetic nanoparticle immobilized amino-acylase is capable of greatly increasing the optimum catalysis temperature and reutilization efficiency of the amino-acylase on the premise of ensuring the enzyme activity; besides, the product is convenient to recover and can be reutilized; the preparation method is suitable for large-scale industrial production.

Description

technical field [0001] The invention belongs to the field of nanomaterial preparation technology and biocatalysis technology, and in particular relates to a preparation method of magnetic nanoparticle-immobilized aminoacylase and its product and application. Background technique [0002] Theanine is a non-protein amino acid, which belongs to the amide small molecular compound. It is only found in the camellia plant and a kind of mushroom body, and it is a biologically active secondary metabolite. Studies have shown that theanine can be used as a safe and reliable food and health product additive to improve flavor, lower blood pressure, help relieve anxiety and promote learning and memory, protect the nervous system and antioxidant effects. [0003] There are two enantiomers of L-type and D-type theanine, and most of theanine naturally occurring in nature is L-type. Efficient resolution of theanine enantiomers is the key to in-depth study of the effects of L- and D-theanine ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12P41/00C12P13/04
CPCY02P20/50
Inventor 李平冯骏晨莫婷
Owner TONGJI UNIV
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