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Method for crosslinking modification of microfiber collagen by multistep continuous enzyme catalysismicrofibril

A collagen cross-linking and microfiber technology, applied in chemical instruments and methods, animal/human proteins, connective tissue peptides, etc., can solve problems such as research blanks, achieve improved mechanical properties, heat-resistant stability, and cross-linking uniform effect

Active Publication Date: 2019-02-01
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Although microfibrillar collagen is also a kind of natural collagen, the literature reports on the cross-linking modification of microfibril collagen are relatively rare, and there is no literature report on the cross-linking modification of microfibril collagen by enzymatic method. Research is blank

Method used

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  • Method for crosslinking modification of microfiber collagen by multistep continuous enzyme catalysismicrofibril
  • Method for crosslinking modification of microfiber collagen by multistep continuous enzyme catalysismicrofibril

Examples

Experimental program
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Effect test

Embodiment 1

[0053] The selected microfibril collagen raw material is a common product made according to existing literature. The content of short fibers (fiber length≤1mm) in the microfibrous collagen is 36.3%.

[0054] Add 2000g of microfibrous collagen raw materials into deionized water to prepare a mixed solution with a solid content of 2.5%. Fully disperse the microfibrous collagen raw materials evenly by shaking, and no precipitation will occur after standing for 15 minutes. Adjust the pH of the mixed solution to 6.0~ 7.0; then 50g of transglutaminase (specific activity: 100U / g) weighed at 2.5U / g by the dry weight of microfibril collagen was added to the mixed solution and catalyzed for 2.5h at 28°C; the catalytic reaction time reached Finally, the pH of the mixed solution was adjusted to be 5.0 to 5.5, and then 1 g of tyrosinase (specific activity of 500 U / mg) weighed at 250 U / g in terms of the dry weight of microfibril collagen was added to the mixed solution, and the following Th...

Embodiment 2

[0057] The selected microfibril collagen raw material is a common product made according to existing literature. The content of short fibers (fiber length≤1mm) in the microfibrous collagen is 27.2%.

[0058] Add 700g of microfibrous collagen raw materials into deionized water to prepare a mixed solution with a solid content of 2.5%. Fully disperse the microfibrous collagen raw materials evenly by shaking, and no precipitation will occur after standing for 15 minutes. Adjust the pH of the mixed solution to 6.0~ 7.0; Then 14g of transglutaminase (specific activity is 100U / g) weighed in microfibril collagen dry weight 2.0U / g was added to the mixed solution, and 0.1% and mol Concentration of 1mol / L CaCl 2 Catalyze the reaction for 3h at 28°C; after the catalytic reaction time is reached, adjust the pH of the mixed solution to be 6.5 to 7.0, and then take the horseradish peroxidase 1.09g (specifically Activity is 160U / mg) was added to the mixture and continued to catalyze the rea...

Embodiment 3

[0061] The selected microfibril collagen raw material is a common product made according to existing literature. The content of short fibers (fiber length≤1mm) in the microfibrous collagen is 58.6%.

[0062] Add 3200g of microfibrous collagen raw materials into deionized water to prepare a mixed solution with a solid content of 2.5%. Fully disperse the microfibrous collagen raw materials evenly by shaking, and no precipitation will occur after standing for 20 minutes. Adjust the pH of the mixed solution to 6.0~ 7.0; then the transglutaminase 32g (specific activity is 100U / g) weighed by microfibril collagen dry weight 1.0U / g is added in the mixed solution, and 0.1% and mol Concentration of 0.5mol / L CaCl 2 The solution was catalyzed for 2 hours at 25°C; after the catalyzed reaction time was up, the pH of the mixed solution was adjusted to be 5.0 to 5.5, and then 1.28g of tyrosinase (specific activity 500U / mg) was added to the mixed solution to continue the catalytic reaction a...

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Abstract

The invention provides a method for crosslinking modification of microfiber collagen by multistep continuous enzyme catalysis, comprising the following steps: dispersing a microfiber collagen raw material in deionized water to prepare mixed liquor, weighing transglutaminase, adding the transglutaminase into the mixed liquor and carrying out a catalytic reaction for 0.5-3 h, weighing enzyme A and adding the enzyme A into the mixed liquor to continue the catalytic reaction for 0.5-2 h, and finally centrifuging the mixed liquor to obtain a precipitate and postprocessing the obtained precipitate to obtain cross-linked modified microfiber collagen. By the method, the microfiber collagen is cross-linked in a short time through multistep enzyme catalysis. Thereby, modified microfiber collagen with improved mechanical properties, heat stability and enzymolysis resistance is prepared, and the product has a biological activity and is safe and non-toxic.

Description

technical field [0001] The present invention relates to the technical field of microfibril collagen crosslinking modification, in particular to a multi-step continuous enzymatically catalyzed method for microfibril collagen crosslinking modification, especially for microfibril collagen whose fiber content is at least 50% and whose length is less than 1mm. Multi-step continuous enzyme-catalyzed cross-linking modification method. Background technique [0002] Microfibrous collagen is a kind of collagen, which usually refers to fibers with a length of less than 12 mm obtained by depolymerizing and fluffing collagen fiber bundles. Microfibrous collagen completely retains the biological activity and triple helical structure of collagen, and is widely used in biomedical fields such as hemostatic materials and cartilage tissue engineering scaffold materials. At present, microfibrous collagen is mainly extracted from bovine collagen, which can promote platelets to gather on the sur...

Claims

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Application Information

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IPC IPC(8): C12P21/02C07K14/78
CPCC07K14/78C12P21/02
Inventor 李国英黄雨琳唐平平刘思聪
Owner SICHUAN UNIV
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