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Method for preparing chitin deacetylase

A technology of chitin deacetylase and chitin deacetylase, which is applied in the field of preparation of chitin deacetylase, can solve the problems of environmental pollution, uneven degree of deacetylation, unstable properties of chitosan, etc., and achieve the effect of high titer

Active Publication Date: 2012-09-19
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To prepare chitosan by deacetylation of chitin, 45% NaOH is mostly used at home and abroad to remove acetyl groups at a relatively high temperature. This method consumes a lot of alkali, causes serious pollution to the environment, and the characteristics of the product chitosan are unstable (such as : The degree of deacetylation is not uniform, the molecular weight changes greatly, the position of the acetyl group cannot be fixed, etc.)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 Brachypodophyllum ( Scopulariopsis brevicaulis ) Fermentation of chitin deacetylase

[0022] Pyrosporium brachypodoides ( Scopulariopsis brevicaulis ) ATCC 36840 containing PDA solid medium (preparation and composition of the medium: take 200g of fresh ungerminated potatoes, peel them, cut them into 3mm×3mm blocks, add 800-1000ml of water, boil for 30min, then filter with gauze , add 20g of sucrose (or glucose) and 15-20g of agar to the filtrate, add water to 1000ml after dissolving, natural pH, divide into test tubes (5ml per tube), sterilize at 121°C for 30min, make a slope) on the test tube slope , activated twice at 27°C. Take an activated slant strain and wash the spores with 3-5ml of sterile water, and insert the spore suspension into 80ml of fermentation medium (each 100ml of fermentation medium is composed of: 3,6-O-carboxymethyl carapace Sucrose 2g, sucrose 1g, peptone 0.4g, NaNO3 0.2g, K 2 HPO 4 0.1g, KCl 0.05g, MgSO 4 0.05g, ZnSO 4 ...

Embodiment 2

[0024] Embodiment 2 Brachypodophyllum ( Scopulariopsis brevicaulis ) Separation and purification of chitin deacetylase from fermentation broth

[0025] Centrifuge 1000ml of the above fermentation broth at 10,000×g for 30min at 4°C, collect 800ml of the supernatant, and add 280g (NH 4 ) 2 SO 4 , so that (NH 4 ) 2 SO 4 The concentration reaches 35% (W / V), to be (NH 4 ) 2 SO 4 After completely dissolving, let it stand for 2 hours, centrifuge at 18,000×g for 30 minutes, collect 750ml of supernatant, and continue to add 337.5g (NH 4 ) 2 SO 4 , so that the solution (NH 4 ) 2 SO 4 The concentration reached 80% (W / V). After standing for 18 hours, centrifuge at 20,000×g for 30 minutes to collect the precipitate. Dissolve the precipitate in 200ml of deionized water, and centrifuge at 20,000×g for 30 minutes to remove insoluble solids. The supernatant and supernatant were desalted through a Sephadex G-25 column of 16mm × 80cm, and the column flow rate was 5ml / min. The efflu...

Embodiment 3

[0026] Example 3 Preparation of chitosan from chitin in Aspergillus niger catalyzed by the chitin deacetylase of Cladosporium brevipus

[0027] Disperse 15 g of chitin from Aspergillus niger into 800 ml of 50 mmol Tris–HCl pH 7.5 buffer solution, add 0.15-0.30 g of the above-mentioned chitin deacetylase, and stir continuously at 55°C for 8 hours, and at 10,000×g Centrifuge for 20min to collect the solid, wash the solid with 100ml of deionized water for 3 times, add 100ml of 2% (W / V) acetic acid solution, stir continuously at 30°C for 16h, and centrifuge at 10000×g for 20min to collect Supernatant, add 40% (W / V) NaOH to the supernatant to adjust the pH to 9.0, centrifuge at 12000×g for 20 minutes to collect the solids, wash the solids with 100ml deionized water three times, and then use Chitosan is obtained by washing with 95% (V / V) ethanol for 3 times and freeze-drying. The degree of deacetylation of chitosan products is above 80%.

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PUM

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Abstract

The invention relates to a method for preparing chitin deacetylase. The method includes that Scopulariopsis brevicaulis is utilized as a producing strain of the chitin deacetylase, after an activation, the Scopulariopsis brevicaulis is inoculated to a fermentation container which contains a fermentration medium to be subjected to a fermentation for 90-100 hours on a shaking table, the potential of hydrogen (Ph) value of the shaking table is controlled between 6.5 and 7.0, the temperature of the shaking table is maintained between 27DEG C and 29 DEG C, the rotary speed of the shaking table is 200-240rpm, and a chitin deacetylase product is obtained by separating, salting out and purifying a fermentation liquor. By means of the method, the active unit of one mini liter of the fermentation liquor of the prepared chitin deacetylase can uppermost reach 36 units.

Description

technical field [0001] The present invention relates to a method for preparing chitin deacetylase, in particular to a method of using C. brevisporum ( Scopulariopsis brevicaulis ) A method for preparing chitin deacetylase. Background technique [0002] Chitosan and its derivatives can be widely used in many fields such as medicine, cosmetics, food, chemical industry, agriculture, biotechnology, polymer materials, etc., and they are mainly derived from crustaceans, plants, microorganisms, etc. Chitosan is the product of deacetylation of chitin, and chitin with a degree of deacetylation greater than 70% is generally called chitosan. To prepare chitosan by deacetylation of chitin, 45% NaOH is mostly used at home and abroad to remove acetyl groups at a relatively high temperature. This method consumes a lot of alkali, causes serious pollution to the environment, and the characteristics of the product chitosan are unstable (such as : The degree of deacetylation is not uniform, ...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12R1/645
Inventor 蔡俊王常高郑化
Owner HUBEI UNIV OF TECH
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