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Method for preparing chitosan oligosaccharide, chitosanase as well as gene, enzyme preparation and application of chitosanase

A chitosanase enzyme and chitosanase technology, applied in the field of hydrolase, can solve the problems of product destruction, high equipment requirements, difficult application, etc., and achieve good temperature stability, good enzymatic hydrolysis effect, and high enzymatic activity. Effect

Pending Publication Date: 2022-04-12
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The physical degradation method is to use microwaves, rays, etc. to break the glycosidic bonds in chitosan, but the process requires high equipment and the degree of polymerization of the obtained chitosan oligosaccharide product is uncertain, so it is difficult to apply in industry; the chemical degradation method uses oxidants or The acid solution breaks the glycosidic bonds under high temperature and high pressure conditions, but a large amount of acid-base reagents are needed in the process, which is easy to cause environmental pollution, and some of the products are destroyed in the reaction; the biological enzyme degradation method uses the catalytic action of biological enzymes to break the glycosides It has the advantages of environmental protection in the reaction process, controllable polymerization degree of the product, and high biological safety of the product. It is the preferred method for the production of chitosan oligosaccharides

Method used

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  • Method for preparing chitosan oligosaccharide, chitosanase as well as gene, enzyme preparation and application of chitosanase
  • Method for preparing chitosan oligosaccharide, chitosanase as well as gene, enzyme preparation and application of chitosanase
  • Method for preparing chitosan oligosaccharide, chitosanase as well as gene, enzyme preparation and application of chitosanase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Enzyme Discovery and Sequence Analysis

[0032] For finding chitosanase gene, researchers of the present invention from Methanothrix ( Methanothrixsp.) (According to literature reports, this bacterium belongs to archaea and mostly lives in extreme ecological environments. Researchers speculate that the potential chitosanase contained in this bacterium may have a relatively special activity) and discovered a segment of the encoded protein The potential chitosanase protein containing 1109 amino acids, whose amino acid sequence is shown in SEQ ID NO.1, is named MS-1109. According to the protein sequence comparison analysis, the highest sequence similarity between the protein and the reported glycoside hydrolase is only 34%, indicating that it is a new type of chitosanase.

Embodiment 2

[0033] Embodiment 2 Expression vector construction, expression and enzyme protein purification of enzyme gene

[0034] The codon-optimized nucleotide sequence of the MS-1109 enzyme is shown in SEQ ID NO.2. After the sequence is artificially synthesized, it is connected to the pET-21a(+) vector using seamless junction technology to obtain the recombinant vector pET-21a- MS-1109. The recombinant vector was transformed into Escherichia coli DH5α competent cells by heat shock method. Spread the heat-shock treated cells onto the LB medium plate containing ampicillin, culture overnight at 37°C, select positive clones that can grow on the plate for colony PCR verification, purify the PCR products and send them for sequencing. The correct clones verified by sequencing are the strains containing the successfully recombined plasmid vectors. The strain was picked and cultured in LB liquid medium at 37°C for 20 h. The plasmid was extracted using a plasmid extraction kit. The extracted p...

Embodiment 3

[0036] Example 3 Research on enzymatic properties

[0037] Enzyme activity assay method: use DNS chromogenic method to detect chitosan enzyme activity, mix 20 μL enzyme solution with 80 μL chitosan (2%) solution, and add 200 μL buffer solution to form a 300 μL reaction system. Put the reaction system in a water bath at 50°C for 10 minutes. After the reaction, quickly boil the water bath for 10 minutes to inactivate the enzyme, then add 300 μL of DNS color development solution, and boil the mixture for 5 minutes. After the color development is completed, cool it down rapidly. And add 1 mL of deionized water, use a microplate reader to detect its absorbance at 540 nm, and calculate the enzyme activity according to the absorbance.

[0038] Determination of optimal reaction pH: Mix 20 μL of enzyme solution with 80 μL of chitosan (2%) solution, add 200 μL of different system buffers with pH 4.0-9.0 to adjust the pH of the system, and place the reaction system in a 50°C water bath ...

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Abstract

The invention discloses a method for preparing chitosan oligosaccharide, chitosanase as well as a gene, an enzyme preparation and application of the chitosanase, and belongs to the technical field of hydrolase. The method for preparing the chitosan oligosaccharide comprises the following step: carrying out enzymolysis on chitosan or colloid chitin by adopting chitosanase as shown in SEQ ID NO.1 under the conditions that the pH is 4.0-8.0 and the temperature is 50-80 DEG C to produce the chitosan oligosaccharide. The amino acid sequence of the chitosanase disclosed by the invention is as shown in SEQ ID NO. 1, and the gene for coding the chitosanase is as shown in SEQ ID NO. 2. The enzyme preparation provided by the invention contains the chitosanase. The application of the chitosanase and the enzyme preparation in degradation of chitosan or colloid chitin and the application in preparation of chitosan oligosaccharide are provided. The chitosan oligosaccharide is prepared from specific chitosanase, the catalytic activity is high, and the method can be carried out under a high-temperature condition. The chitosanase disclosed by the invention can be used for carrying out enzymolysis on chitosan under a high-temperature condition, and chitosan oligosaccharide is prepared by efficiently carrying out enzymolysis on the chitosan.

Description

technical field [0001] The invention relates to a method for preparing chitosan oligosaccharide, chitosanase and its gene, enzyme preparation and application, and belongs to the technical field of hydrolytic enzymes. Background technique [0002] my country has abundant shrimp and crab resources, and chitin is a kind of animal polysaccharide from shrimp and crab resources, which has a wide range of applications in the fields of chemical industry, medicine and materials. However, due to its large molecular weight and poor water solubility, it is difficult to be absorbed and utilized by the human body, which limits its use in the field of functional foods. [0003] After deacetylation of chitin, chitosan can be obtained, which has a certain solubility in acid solution. After further degradation, it can be obtained with small molecular weight, good water solubility, absorption and utilization, and the ability to lower blood sugar. Chitooligosaccharides with functional activiti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/26C12P19/14C12P19/12C12P19/00C12N9/42C12N15/56C12N15/70
Inventor 孙建安毛相朝王永臻苏海鹏贾真荣
Owner OCEAN UNIV OF CHINA
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