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New preparation method for chitin deacetylase

The technology of a deacetylase and a new method, which is applied in microorganism-based methods, biochemical equipment and methods, hydrolase, etc., can solve the problem of low enzyme activity, strict substrate requirements, and difficult industrial large-scale fermentation of fungi, etc. question

Inactive Publication Date: 2009-12-23
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The ability of these strains to produce CDA is low, the enzyme activity is not high, and the fungus is difficult to be used for industrial large-scale fermentation, so it must be modified by genetic engineering before it can be applied
Second, there are currently only 2 strains of bacteria that can produce CDA (Colletotrichum lindemuthia-num ATCC56676, Colletotrichum indemuthianum DSM63144) reported in the literature, both of which belong to the genus Alcaligenes. and its derivatives as substrates

Method used

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  • New preparation method for chitin deacetylase
  • New preparation method for chitin deacetylase
  • New preparation method for chitin deacetylase

Examples

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example 1

[0035] Example 1: (1) Flat plate coating: Soak the shrimp shell culture soil (obtained after burying freshwater shrimp shells in the pumpkin field and decomposing for 31 days) with 3 times the volume of sterile saline (containing 300ppm carbendazim) for 6 hours The supernatant was collected by filtration, spread on a beef extract-peptone plate (0.9% beef extract, 3% peptone, 0.15% NaCl, 2% agar, pH 7.2), and incubated at 37°C for 36 hours.

[0036] (2) Plate streaking: Select single colonies with different colony forms from the beef extract peptone plate, and separate them by streaking on the beef extract peptone plate.

[0037] (3) CDA activity verification: select a single colony with different colony morphology from the streaked plate to the PN-casein plate (0.5% casein, 0.1% K 2 HPO 4 , 0.1% KH 2 PO 4 , 0.05% MgSO 4 , 2% agar, 0.1% PN), and cultured at 37°C for 48h. Select the colony that makes the medium turn yellow (the colorless PN acetyl group is removed and then co...

example 2

[0038] Example 2: (1) liquid fermentation: the bacterial strain obtained by purification is inoculated into casein culture fluid (0.5% casein, 0.1% K 2 HPO 4 , 0.1% KH 2 PO 4 , 0.05% MgSO 4 ), and cultured with shaking at 37°C for 48h, centrifuged (3000g×15min) to collect the supernatant.

[0039] (2) Verification of CDA activity: transfer an appropriate amount of supernatant to small wells on a PN-casein plate, and after incubating at a constant temperature at 37°C for 48 hours, it is found that yellow is formed around the small wells.

[0040] (3) Determination of CDA activity: Refer to the Araki and Ito (1975) method to measure the CDA activity of the supernatant: 1 unit of enzyme activity is defined as the amount of enzyme required to remove 1 μmol of acetyl from acetylated EGC per minute, which is equivalent to The amount of enzyme required to produce 1 μmol of glucosamine per minute; use glucosamine hydrochloride as a standard curve to quantitatively measure the abil...

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Abstract

The invention relates to a new preparation method for chitin deacetylase (chitin deacetylase, E.C.3.5.1.41, CDA), which comprises the following steps: shrimp-shell cultivated soil is taken as screened raw material; antifungal drug is used for restraining the growth of fungus; methods such as spread-plate, plate streaking and the like are used for purification; simultaneously N-paranitroacetanilide (PN)-casein plate is used for CDA activity verification; and finally bacillus cereous which can secrete CDA is obtained by separation. CDA is prepared by liquid fermentation; the CDA vitality of fermentation liquor supernatant is 0.598 to 0.912U / ml; the CDA has enzyme properties which are different from that reported in literatures. Bacillus cereous is used for secreting CDA to ectoenzyme; products are more easily obtained; and bacterium is suitable for large-scale industrial fermentation and can be applied without needing processing and transformation.

Description

technical field [0001] The invention relates to a new preparation method of chitin deacetylase (Chitindeacetylase, E.C.3.5.1.41, CDA), specifically, the extracellular secretion of CDA by bacillus cereus. Background technique [0002] Chitin is composed of N-acetylamino-D-glucose monomers linked by β-1,4-glucosidic bonds. It is a natural high molecular polymer that is abundant, easy to obtain, and renewable. The outer shell, or cuticle, of vertebrates, also found in the cell walls of many fungi and some algae. Due to the poor solubility of chitin, the scope of application is limited; while the deacetylation product of chitin - the degradation product of chitosan, chitosan oligosaccharide is not only good in water solubility and easy to absorb, but also has anti-tumor, anti-bacterial, immune activation and moisturizing properties. Moisture absorption and other characteristics. Therefore, the deacetylation of chitin is of great significance for the development and application...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12R1/085
Inventor 李晓红李潇潇邓如伟张涛虞宝中
Owner SICHUAN UNIV
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