Magnetic composite microsphere immobilized laccase and preparation method thereof

A technology of magnetic composite microspheres and immobilized laccase, which is applied in the direction of immobilization on or in the inorganic carrier, can solve the problems of large loss of enzyme activity, large loss of enzyme activity, and shedding, so as to avoid chemical damage and benefit Effects of recovery and reuse, improvement of operational stability

Inactive Publication Date: 2011-10-05
BEIJING NORMAL UNIVERSITY
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AI-Extracted Technical Summary

Problems solved by technology

Among them, the cross-linking method and the covalent bond method have relatively severe reaction conditions, and the loss of enzyme activity is relatively large; the encapsulation method has a large loss of enzyme activity because the substrate binding site and active center are blocked by the carrier; the adsorption method ha...
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Abstract

The invention relates to a magnetic composite microsphere immobilized laccase and a preparation method thereof, and belongs to the field of environmental microbiology. In the immobilized laccase, metal-chelated magnetic composite microspheres are taken as a carrier, and laccase is immobilized on the carrier through coordination bonding action. The preparation method comprises two steps of: preparing the carrier, namely synthesizing magnetic ferroferric oxide nanoparticles by using a chemical precipitation process, preparing core-shell magnetic silica by taking the nanoparticles as cores through a sol-gel method, grafting polymer containing carbonyl or amino on the surface of the magnetic silica by taking gamma-chloropropyltrimethoxysilane as a medium, and preparing a functional carrier capable of immobilizing the laccase by chelating transition metal ions (such as Cu2+. Zn2+, Ni2+ or Co2+); and immobilizing the laccase, namely performing coordination adsorption of the laccase on the surface of the carrier, washing, and drying to obtain the finished product of immobilized laccase. The immobilized laccase prepared by the method has high stability and activity and quick magnetic response, is easily recovered from a reaction system and can be repeatedly used; and the preparation process is simple and the cost is low.

Application Domain

On/in inorganic carrier

Technology Topic

ChemistryCarbonyl group +15

Examples

  • Experimental program(3)

Example Embodiment

[0022] Example 1
[0023] A preparation method of immobilized laccase on magnetic composite microspheres mainly includes two steps:
[0024] Vector preparation
[0025] (1) Take FeCl with a molar ratio of 1:2 2 ·4H 2 O and FeCl 3 ·6H 2 The mixed powder of O was dissolved in 50 mL of a 0.5% sodium dodecyl sulfonate solution, and a 2 mol/l NaOH solution was added dropwise with stirring to adjust the pH to about 12. After the obtained product is matured in a water bath at 80°C for 30 minutes, it is repeatedly washed with distilled water to neutrality, dried and ground at 50°C to obtain ferroferric oxide powder;
[0026] (2) Take the ground Fe 3 O 4 1.5g was ultrasonically dispersed in 60ml of secondary water, while 6.4ml of tetraethoxysilane was ultrasonically dispersed in 120ml of methanol, and the methanol solution of tetraethoxysilane was added to Fe under mechanical stirring. 3 O 4 After stirring vigorously for 15 minutes, add 6ml of concentrated ammonia water, continue to stir the reaction at room temperature for 4 hours, wash the solid precipitate with methanol and secondary water until it is neutral, and dry it at 50℃ to obtain the core-shell structure magnetic two Silicon oxide powder;
[0027] (3) Disperse 1.5g of magnetic silica powder in 37.5ml of methanol, add 37.5ml of glycerin after ultrasonic dispersion for 20 minutes, then add 37.5ml of glycerin after ultrasonic dispersion for 10 minutes, then put it into a 250ml three-necked flask; at the same time add 5ml of γ-chloropropyltrimethylsilane Disperse in 50ml methanol, mix well and add to the three-necked flask, mix well with magnetic silica, stir and react at 80℃ for 3 hours, wash the solid precipitate 6 times with methanol and twice water, and bake at 50℃ Dry to obtain silanized magnetic silica;
[0028] (4) 1.4g of silanized magnetic silica and 175mL of 2mg/L polyacrylamide solution were stirred and reacted at 50℃ for 3 hours, then dried at 50℃; 0.5g of the dried powder and 75mL 0.2mol/L The copper sulfate solution was shaken at 30°C for 10 hours, and dried at 50°C to obtain the carrier product.
[0029] Laccase immobilization
[0030] Take 0.1 g of the finished carrier product and mix it thoroughly with 5 mL of free laccase dissolved in disodium hydrogen phosphate-citric acid buffer solution, stir for 1 hour at pH 4.0 and temperature at 25°C, and stand for 8 hours at 4°C. Wash with a pH of 3.0 disodium hydrogen phosphate-citric acid buffer solution for many times until no protein is detected in the washing solution, and then dry the immobilized laccase by low-temperature vacuum drying. The mixing ratio of free laccase and carrier product is 35mg/1g.

Example Embodiment

[0031] Example 2
[0032] The preparation method of the carrier is as in Example 1, except that the concentration of polyacrylamide is 0.5 mg/L, the concentration of copper sulfate solution is 0.1 mol/L, and the shaking time is 12 hours. The laccase immobilization method is the same as in Example 1, except that 0.1g of the carrier product and 5mL of free laccase dissolved in disodium hydrogen phosphate-citric acid buffer solution are thoroughly mixed, and then stirred at pH 3.0 and temperature 5℃. hour. The mixing ratio of free laccase and carrier product is 20mg/1g.

Example Embodiment

[0033] Example 3
[0034] The carrier preparation method is as in Example 1, except that the polyacrylamide concentration is 5 mg/L, the copper sulfate solution concentration is 0.05 mol/L, and the shaking time is 8 hours; the laccase fixation method is the same as in Example 1, the difference is 0.1 After the finished product of g carrier is fully mixed with 5 mL of laccase dissolved in disodium hydrogen phosphate-citric acid buffer solution, the mixture is stirred for 5 hours under the conditions of pH 5.0 and temperature 15°C. The mixing ratio of free laccase and carrier product is 50mg/1g.

PUM

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