Immobilization method of proteinase molecule by using nano-material and application thereof

A technology of nanomaterials and proteases, applied in the direction of immobilized on or in inorganic carriers, nanotechnology, nanomedicine, etc., can solve the problems of low catalytic efficiency and achieve the effect of simple production process, low price and high efficiency

Inactive Publication Date: 2012-03-14
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The collision frequency between the immobilized enzyme and the substrate on the nanoparticles is between the free enzyme and the traditional micron-scale carrier immobilized enzyme, which not only stabilizes the structure of the enzyme through enzyme immobilization, overcomes the shortcomings of the instability of the free enzyme, but also overcomes the The problem of low catalytic efficiency of micron-sized supports

Method used

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  • Immobilization method of proteinase molecule by using nano-material and application thereof
  • Immobilization method of proteinase molecule by using nano-material and application thereof
  • Immobilization method of proteinase molecule by using nano-material and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Activation of multi-walled carbon nanotubes (MWNTs) and immobilization of SOD enzyme.

[0023]Pour 0.5g of commercial multi-walled carbon nanotubes into the mixed acid of 15ml of concentrated nitric acid and 45mL of concentrated sulfuric acid, seal the container, vibrate with ultrasonic wave for 3h respectively; Multi-walled carbon nanotubes were washed repeatedly with deionized water until neutral, dried in vacuum at 100°C and weighed. Take 50 mg of dried acid-treated MWNTs, add them into 15 ml of phosphate buffer solution with pH=7.0, and disperse them evenly by ultrasonic; then add 200 mg of carbodiimide (EDC) and N-hydroxysuccinimide (NHS ) 250 mg, sonicate for 30 min; add a certain amount of enzyme solution to the above solution and shake at room temperature for 24 h; then filter with a 0.22 μm polycarbonate membrane; the obtained solid is washed with phosphate buffer, and then dried in vacuum to obtain the final fixation Catase.

[0024] First use mix...

Embodiment 2

[0029] Example 2: Functionalized multi-walled carbon nanotubes are used for lipase immobilization.

[0030] Carboxyl groups are attached to the surface of the multi-walled carbon nanotubes after the mixed acid treatment of concentrated nitric acid and concentrated sulfuric acid at a certain ratio (volume ratio 1:2~1:5), and then thionyl chloride and N,N-dimethylformamide Acyl chlorination is carried out to obtain chlorine-containing acyl multi-walled carbon nanotubes, and then the chlorine-containing acyl multi-walled carbon nanotubes are reacted with hexamethylenediamine in chloroform to obtain polyamino-terminated multi-walled carbon nanotubes, namely CNT-NH. Acidified carbon nanotubes react with hexadecyl bromide to obtain CNT-NH under certain conditions.

[0031] Functionalized carbon nanotubes were sonicated in Tris-HCl buffer solution, then added lipase solution prepared by genetic engineering and centrifuged by shaking. The amount of immobilized enzyme was determined b...

Embodiment 3

[0033] Example 3: Immobilization of SOD enzyme by single-walled carbon nanotubes (SWNTs).

[0034] Suspend 6mg of SWNTs in 6mlBMIM-BF4 (an ionic liquid) to obtain liquid A; then add 60mg of N-hydroxysuccinimide ester 1-pyrenebutyric acid to 12ml of DMF (dimethylformamide) to obtain liquid B; add liquid A to Liquid B was mixed and shaken at room temperature for 12 hours; then 30ml of methanol was added, and the functionalized SWNTs would precipitate; the precipitate was washed three times with pure methanol, 50ml each time, to remove excess reducing agent; dried under vacuum to obtain functionalized SWNTs; 5mg of functionalized SMNTs was suspended in 5ml of BMIM-BF4 and then added to SOD solution (54mg of enzyme was dissolved in 10ml of 20mM pH 7.2 phosphate buffer); the above heterogeneous mixture was shaken at room temperature for 24h; Filtration; the finally obtained solid was washed with phosphate buffer (25mlx2), then dried in vacuo to obtain the final immobilized enzyme. ...

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Abstract

The invention discloses an immobilization method of a proteinase molecule by using a nano-material, namely a carbon nano-tube. The method comprises the following steps: (1) dispersing the carbon nano-tube to form a uniform solution; (2) acidifying the carbon nano-tube, wherein the carbon nano-tube obtained in the step (1) is acidified with mixed acid of concentrated nitric acid and concentrated sulfuric acid at a volume ratio of 1:2-1:5; and (3) functionalizing the carbon nano-tube, wherein a -NH2 functional group is functionally connected to the wall of the carbon nano-tube by utilizing NHS (N-hydroxy-succinamide) and EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), and reaction is carried out with -NH2 of enzyme to immobilize the enzyme on the tube wall. The preparation process has convenient and quick procedure, stable and firm bonding and high efficiency. The carbon nano-tube for bio-catalysis or transformation in energy, environmental and biochemical pharmacy and other fields has low price, is easy for scale utilization, and is a nano-enzyme catalysis means with practical application prospect.

Description

technical field [0001] The invention relates to the fields of bio-nano preparations and bio-nano-enzyme catalysis, more specifically, the invention relates to a method for immobilizing protease molecules on nano-materials and its application. Background technique [0002] For the study of protein stability, as early as the mid-1990s, people began to conduct systematic research on protein stability. At present, the improvement of protein stability at home and abroad is mainly to modify it and add various stabilizers to the solution. The former is mainly used to modify pharmaceutical proteins due to its high cost, such as polyethylene glycol (PEG) to modify various stabilizers. Cytokines and other protein drugs, while the latter is mainly used in various enzyme preparations. Stabilizing the protein with a stabilizer is simpler and cheaper than chemical modification; and after the protein is modified, its structure, activity and antigenicity have changed, but this change often...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14B82Y5/00
Inventor 马兴元高原张倩刘洋
Owner EAST CHINA UNIV OF SCI & TECH
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