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Microbial transglutaminase and daily bleeding stopping products prepared thereby

A technology of transglutaminase and microorganisms, which is applied in the field of medicine, can solve the problems affecting the medical use of transglutaminase proenzyme, the hemostatic effect and anti-adhesion effect need to be improved, and achieve good healing effect and fast onset of action , Ease of use

Active Publication Date: 2014-05-28
李肯
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the mutant enzyme has a good hemostatic effect, it needs to be used in conjunction with gelatin, and its own hemostatic effect and anti-adhesion effect need to be improved, thus affecting the medical use of the transglutaminase zymogen to a certain extent

Method used

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  • Microbial transglutaminase and daily bleeding stopping products prepared thereby
  • Microbial transglutaminase and daily bleeding stopping products prepared thereby
  • Microbial transglutaminase and daily bleeding stopping products prepared thereby

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Enzyme Activity Determination

[0053] 1) Preparation of reagents for measuring enzyme activity

[0054] Solution A (0.5L): Add 0.015mol (5.06g) of substrate Na-CBZ-Gln-Gly, 0.05mol (3.475g) of hydroxylamine hydrochloride, 0.005mol (1.536g) of reduced glutathione to 400ml of distilled water in a beaker After stirring with a magnetic stirrer for 20 minutes, add 0.1mol (12.11g) Tris, adjust the pH to 6.0 with 6mol / L (or 1mol / L) hydrochloric acid, transfer the solution to a 500ml volumetric flask, and wash the beaker with distilled water Pour into the volumetric flask 3 times and make the volume to 500ml.

[0055] Solution B: 3mol / L HCl, 12% trichloroacetic acid (W / V), 5% ferric chloride (W / V, dissolved in 0.1mol / L hydrochloric acid, then filtered) according to the volume ratio of 1:1:1 mix.

[0056] 2) Determination of enzyme activity

[0057] The actinomycetes fermentation broth was filtered, and the filtrate was diluted 5 times. Experimental group: Take 0...

Embodiment 2

[0058] The mutagenesis of embodiment 2 wild strains

[0059]The wild-type mTG strain is Streptomyces mobaraensis (such as the strain ATCC number 29032 of the American ATCC or the strain ATCC number 27441 of the American ATCC, or the strain of the China Microorganism Conservation Center into CGMCC number 4.1719 and CGMCC number 4.5591).

[0060] Medium configuration: Gaoshi No. 1 medium (g / L): soluble starch 20, KNO 3 1, mg 8O 4 ·7H 2 O0.5,K 2 HPO 4 ·3H 2 O0.5, NaC10.5, FeSO 4 ·7H 2 O0.01, agar 20, pH7.2-7.4. Fermentation medium (g / L): glycerin 20, yeast extract 6, fish meal peptone 25, mg 8O 4 ·7H 2 o 2 , K 2 HPO 4 ·3H 2 o 2 , pH7.4.

[0061] Add 10ml of cold sterile water to Gao's No. 1 medium, use an inoculation needle to fully scrape the surface hyphae, break up the spores, and filter with sterile filter paper.

[0062] The operation is carried out under red light or dark conditions, and the ultraviolet light is turned on 0.5h in advance to stabilize the lig...

Embodiment 3

[0064] The acquisition of embodiment 3 mutant mTG gene

[0065] Genome extraction: inoculate the fresh mycelia of the mutant strain with the highest enzyme activity in liquid culture in Example 2 in fresh medium, and cultivate for about 24 hours; 10ml thalline is collected by centrifugation; fresh thalline is placed in a mortar (-20°C) (pre-cooled), grind repeatedly with liquid nitrogen until fine powder, quickly and evenly distribute into two 1.5mL centrifuge tubes; add 550 μL of TE buffer solution, add 30 μL of 20% SDS solution preheated at 65 °C, vortex for 5 seconds, add 20 μL of 20 mg / mL proteinase K, mix gently, and incubate at 37°C for 1 h; add an equal volume of Tris-saturated phenol / chloroform / isoamyl alcohol (25:24:1) for extraction, mix by inverting slightly, and centrifuge at 10,000 r / min for 10 min; Carefully draw the supernatant into a new centrifuge tube, add an equal volume of chloroform / isoamyl alcohol (24:1) for extraction, centrifuge at 10,000 r / min for 5 mi...

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Abstract

The invention discloses microbial transglutaminase and rapid bleeding stopping products for traumatic blood loss in daily life. The products adopt mutant microbial transglutaminase as a main active substrate or an effective component. The raw material microbial transglutaminase used in the invention is a mutant enzyme obtained through gene mutation in the circumstance of not changing the functions and the original low toxicity of traditional microbial transglutaminase, wherein compared with wild type enzymes, the mutant enzyme has amino acid mutagenesis at 4 loci. The enzyme has the advantages of excellent heat resistance and good stability at normal temperature. The rapid bleeding stopping products for the traumatic blood loss in daily life, which are prepared through the mutant enzyme, comprise patches, creams (ointments and plasters), pastes and liniments. Proved by tests, the products disclosed by the invention have the effects of rapidly stopping bleeding, easing pain, preventing wound adhesion, blocking bacteria and the like and have the advantages of low toxic or side effects and convenience in use.

Description

technical field [0001] The invention belongs to the field of medicine, and in particular relates to a band-aid containing microbial transglutaminase and a preparation method and application thereof. Background technique [0002] In daily life, people often suffer from small traumas that cause bleeding. If the bleeding is not stopped in time, it may cause infection and aggravate the trauma. At present, an ideal hemostatic material should have the following characteristics: rapid and long-lasting hemostasis; no toxic side effects, no induction of thrombus formation; no increase in infection chances, no influence on tissue healing; simple and practical. [0003] Daily trauma hemostatic products mainly include patches, dressings, medicinal gauze or bandages, gels or blocks, among which patch hemostatic products (such as band-aids, etc.) are the most commonly used surgical drugs in people's lives , commonly known as "hemostatic plaster", has the functions of hemostasis and skin ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54A61K38/45A61P7/04A61P17/02A61K38/39
CPCA61K38/00A61L15/44A61L29/048C12N9/1044C12Y203/02013
Inventor 易正芳吕方裴正培刘明耀
Owner 李肯
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