Microwave-assisted immobilization method of aldolase

An aldolase, microwave technology, applied in microorganism-based methods, biochemical equipment and methods, immobilized on or in inorganic carriers, etc., can solve the problem of poor stability, high cost of kit carriers, and process details are not disclosed. and other problems to achieve the effect of improving temperature stability

Inactive Publication Date: 2010-11-24
HANGZHOU NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

After decades of research and development, a variety of immobilization carriers and methods with various properties have been developed successively. Immobilization technology has made great progress, but it still cannot meet all the requirements of an ideal biocatalyst. There are not many enzymes, the main reason is that the cost of the kit carrier used for immobilization is high, followed by low immobilization efficiency and poor stability; and due to commercial confidentiality, many process details are not disclosed

Method used

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  • Microwave-assisted immobilization method of aldolase
  • Microwave-assisted immobilization method of aldolase
  • Microwave-assisted immobilization method of aldolase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Preparation of aldolase free enzyme:

[0045] Take the recombinant strain E.Coil BL21 / pET 303CT / DERA thermo bacteria in our research group, that is, the SEQ ID NO 1 in the Chinese Patent Publication No. CN 101565697A, in the LB liquid medium (Oxoid LTD) of 100ug / ml ampicillin (sigma) ., Basingstoke, Hampshire, England) were cultivated at 37°C for 20 hours to obtain a seed liquid, and 10ml of the seed liquid was added to LB liquid medium of 1L 100ug / ml ampicillin and expanded at 37°C until the bacterial liquid Add the inducer IPTG when the OD600 reaches 0.6, and make the final concentration of the inducer IPTG 0.5mM, and then induce at 30C for 4 hours, collect the bacteria by centrifugation, break the cells of the bacteria, and purify the protein with a nickel column to obtain aldehyde Aldolase free enzyme, measuring enzyme concentration, the aldolase free enzyme is preserved with PH6.5, concentration is the sodium phosphate buffer of 0.01M, and the free enzyme of measur...

Embodiment 2

[0047] Aldolase free enzyme made into immobilized aldolase:

[0048] (1) Activate the carrier: select 3ml of p-benzoquinone solution so that the final concentration in the reaction solution is 1.5mM and 20mg of MCFs-NH 2 Shake at 150rpm in a constant temperature water bath shaker at 20°C for 2 hours, centrifuge, wash the precipitate with pH 7.0, 0.01M PBS solution, and redisperse the washed precipitate in 2.445mL of pH 8.0, 0.1M PBS In the solution, because the solid-liquid separation is realized during the centrifugation process, it is considered that the weight of the carrier remains unchanged, which is still 20 mg.

[0049] (2) get the activated carrier liquid 2.445ml that step (1) obtains, then add the aldolase free enzyme sodium phosphate buffer 0.55mL of the 1.8mg / ml that embodiment 1 obtains according to the mass ratio of 50mg enzyme / g carrier, Mix evenly to obtain a mixture;

[0050] (3) Take the mixed solution of step (2) and vibrate for 20 hours under the condition...

Embodiment 3

[0052] Prepare aldolase free enzyme into immobilized aldolase by microwave method:

[0053] (1) Activate the carrier: select 3ml of p-benzoquinone solution to make the final concentration of 1.5mM and 20mg MCFs-NH 2 Shake at 150rpm in a constant temperature water bath shaker at 20°C for 2 hours, centrifuge, take the precipitate and wash it with a pH 7.0, 0.01M PBS solution, and redisperse the washed precipitate in 2.445mL of a pH 8.0, 0.1M PBS solution Among them, because the solid-liquid separation is achieved during the centrifugation process, the weight of the carrier is considered to remain unchanged, which is still 20 mg.

[0054] (2) In the carrier mixed solution of the activation that step (1) obtains, according to the mass ratio of 50mg enzyme / g carrier, add the aldolase free enzyme sodium phosphate buffer solution 0.55mLml of 1.8mg / ml that embodiment 1 obtains , and mix well to obtain a mixture.

[0055] (3) The mixture in step (2) was irradiated for 1.5 minutes at ...

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Abstract

The invention relates to a method for preparing immobilized aldolase, in particular to a microwave-assisted immobilization method of aldolase. The method comprises the following steps of: (1) activating a carrier; (2) taking an activated carrier liquid obtained in the step (1), adding aldolase free enzyme according to an adding amount of 50mg of aldolase free enzyme/ gMCFs-NH2 to obtain a mixed liquid; and (3) irradiating the mixed solution obtained in the step (2) for 1.5 to 7.5 minutes under the conditions of 5 DEG C and 10 to 50W of microwave, centrifuging the mixed solution, and washing precipitate with 0.01 M of PBS solution with the pH of 7.0 to obtain the microwave immobilized aldolase. The immobilized aldolase prepared by the method has higher temperature stability than that of the aldolase free enzyme under the conditions of 70 DEG C of preserved temperature, and particularly the catalytic effect of the microwave-immobilized aldolase immobilized by microwaves under the low substrate concentration can reach that of the aldolase free enzyme and the immobilized aldolase obtained by the conventional immobilization method under the action of high substrate concentration.

Description

(1) Technical field [0001] The invention relates to a process for immobilizing enzymes in biochemical industry, in particular to an efficient covalent immobilization method for aldolase in amino functionalized mesoporous foam silicon under single-mode microwave radiation. (2) Background technology [0002] As a kind of biocatalyst, enzyme is widely used in food processing, medicine and fine chemical industry because of its high selectivity, mild catalytic reaction conditions and no pollution. However, natural enzymes have poor stability, are easily inactivated, cannot be reused, and are mixed into products after the reaction, making purification difficult, making it difficult to be more widely used in industry. In addition, the isolation and purification of enzymes and their one-time use also greatly increase their cost as catalysts. Under these conditions, the concept and technology of immobilized enzymes have been proposed and developed, and have become the focus of enzym...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12N13/00C12N9/88C12R1/19
Inventor 王安明谢恬王秋岩王梅章
Owner HANGZHOU NORMAL UNIVERSITY
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