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Chitin deacetylase

A deacetylase and chitin technology, applied in the field of protease, can solve the problems of insignificant increase in enzyme activity of genetically engineered bacteria, narrow substrate spectrum, and low enzyme production activity.

Inactive Publication Date: 2020-05-19
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the currently reported CDA production strains are fungi, and most of these CDA derived from fungi are glycoproteins, which must be glycosylated to be active; currently reported bacterial CDAs are less, and most of them are glycoproteins. It is a chitosan oligosaccharide deacetylase, which can act on a narrow spectrum of substrates, and has problems such as low activity of the produced enzyme, decreased activity of the purified enzyme, and insignificant improvement of the enzyme activity of genetically engineered bacteria. It has not yet achieved industrialization

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Substrate Spectrum

[0022] The substrate spectrum of RCDA was studied, and large molecular chitin powder, colloidal chitin, ethylene glycol chitin, chitosan with a deacetylation degree of 85%, and chitooligosaccharides with small molecular weight and bands were selected. Several amino acids of the acetyl group were used as substrates, and 5 mL of RCDA crude enzyme solution was added to the excess substrate solution. Under stirring, the mixture was allowed to react at 37°C and pH 4.0, and stirred for 12 hours. Then the sample was boiled for 5 minutes, and the deacetylation ability of its different substrates was characterized by the content of acetic acid generated by deacetylation by high performance liquid chromatography, and the substrate activity (157.6U / mL) of 4-nitroacetanilide was set is 100%. The results are shown in Table 1. RCDA has activity on most of the tested substrates, including low molecular weight chitosan oligosaccharides, colloidal chitin,...

Embodiment 2

[0026] Embodiment 2 enzymatic properties

[0027] (1) In order to determine the optimal reaction temperature of the enzyme, the enzyme was reacted at different temperatures (30-60°C) and pH 7.0 for 60 minutes to detect the enzyme activity;

[0028] (2) In order to study the temperature stability of the enzyme, the enzyme was pre-incubated at different temperatures (25-60°C) for 30 minutes, pH 7.0, and reacted at 37 degrees for 60 minutes to detect the remaining enzyme activity of the enzyme;

[0029] (3) In order to determine the optimal pH value, the enzyme activity of the enzyme was measured for 60 minutes at 37°C under different pH conditions;

[0030] (4) In order to study the pH value stability of the enzyme, the enzyme was placed in various pH2-10 buffers for pre-incubation overnight, and then reacted at pH7.0, 37 degrees for 60 minutes to detect the remaining enzyme activity;

[0031] (5) Simultaneously studied RCDA and different metal ions Zn 2+ , Fe 2+ , Mg 2+ , M...

Embodiment 3

[0042] Embodiment 3 Application of RCDA

[0043]Chitin powder was first pretreated with 48% choline aqueous solution at 90°C for 12 hours, the amount of choline added was 10mL per 5g of material, filtered after pretreatment and the ionic liquid remaining in the solid phase was washed with deionized water to pH6 .8-7.0, then dry at 105°C to constant weight. Weigh 1 g of the dried material and add 10 mL of the chitin deacetylase crude enzyme solution of the present invention (enzyme activity is 157.6 U / mL) to react at 37°C for 12 hours, and use liquid chromatography to detect the total amount of acetic acid generated in the liquid after the deacetylation reaction. The amount reaches 127mg / g chitin powder.

[0044] In this example, the role of choline is to destroy the crystal structure of chitin and increase the area of ​​action of the enzyme. After pretreatment, it is the pretreated chitin powder that is dried to constant weight. The addition of RCDA produced The deacetylatio...

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Abstract

The invention belongs to the technical field of protease, and particularly relates to chitin deacetylase. The chitin deacetylase RCDA derives from Rhodococcus equi, has an amino acid sequence represented by SEQ ID NO.1 in a sequence table, is a novel protein sequence reported for the first time at present, has relatively low similarity with a protein sequence of the reported chitin deacetylase andhas a relatively wide substrate spectrum; and the chitin deacetylase has the characteristics that metal ion influence is low, and the like.

Description

Technical field: [0001] The invention belongs to the technical field of protease, and in particular relates to a chitin deacetylase. Background technique: [0002] Chitin, also known as chitin, scientifically named (1,4)-2-acetylamino-2-deoxy-β-D-glucan, is an amino acid contained in natural organisms that is second only to cellulose Polysaccharides, mainly found in invertebrates, such as shrimp, insects, algae, fungi and yeast. However, chitin is insoluble in water, acid, alkali and organic solvents, so it has little commercial value, and the product chitosan after deacetylation is widely used in medicine, food, chemical industry, cosmetics and other industries. There are many problems in the chemical method currently used in the production of chitosan, such as long reaction time, high energy consumption, unstable product quality, and especially the discharge will cause huge environmental pollution. [0003] Chitin deacetylase (Chitin deacetylase, CDA) can hydrolyze and r...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/55C12P19/26
CPCC12N9/80C12P19/26C12Y305/01041
Inventor 王敏马钦元申雁冰屠琳娜毕心宇闫文芹
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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