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Method for preparing chitooligosaccharide

A technology of chitosan oligosaccharide and chitin, which is applied in the field of nanomaterial preparation and chitosan oligosaccharide preparation, can solve the problems of low yield, pollute the environment, use concentrated acid, etc., and achieves low crystallinity, high enzymatic hydrolysis conversion rate, and easy hydrolysis. Effect

Active Publication Date: 2021-07-09
OCEAN UNIV OF CHINA
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] Aiming at the above-mentioned prior art, the present invention provides a method for preparing chitin oligosaccharides, which overcomes the problems in the prior art that the preparation of colloidal chitin requires the use of concentrated acid, consumes a large amount of water, and pollutes the environment, and solves the problem of enzymatic hydrolysis of chitin. The problem of low vegetarian efficiency and low yield

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  • Method for preparing chitooligosaccharide
  • Method for preparing chitooligosaccharide
  • Method for preparing chitooligosaccharide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 chitinase gene SbChiAJ143 clone

[0035] Streptomyces bacillaris CGMCC 4.1584 genome was used as a template for PCR amplification. The PCR reaction conditions were: pre-denaturation at 95°C for 10 minutes, denaturation at 98°C for 10 seconds, annealing at 58°C for 30 seconds, extension at 68°C for 3 minutes, 30 cycles of reaction, and extension at 68°C 10min.

[0036] The nucleotide sequences of the specific primers used for PCR amplification are as follows:

[0037] Upstream primer: 5'-GGCCGCAAGCTTCGGCAGGCTGCGGAC-3';

[0038] Downstream primer: 5'-CAAATGGGTCGCGGATCCATGGTGCAACGCTCCG-3'.

[0039] The target gene was connected with the pET-28a(+) cloning vector using seamless connection technology, and the connection product was transferred into E.coliDH5α competent cells, and the sequencing was successful after positive verification.

Embodiment 2

[0040] Embodiment 2 recombinant protein expression and purification

[0041] The recombinant plasmid was transferred into the expression strain E.coliBL21(DE3), cultured at 20°C, 220rpm for 48 hours, and the cells were collected (centrifuged at 8000g for 10 minutes at 4°C). The bacteria were resuspended in 50mM Tirs-HCl buffer solution with pH 7.4, sonicated for 50min and then centrifuged at 12000g for 20min, and the supernatant was the crude enzyme solution. Purify the crude enzyme solution by affinity chromatography with a Ni-NTA column, use 10mM imidazole solution (500mM NaCl, 50mM Tris-HCl) to equilibrate the column, and then use 20mM, 40mM imidazole solution (500mM NaCl, 50mM Tris-HCl) to elute the binding Weak foreign protein, 60mM imidazole solution elutes to obtain the target protein, and the obtained solution is subjected to SDS-PAGE detection, such as figure 2 As shown, the molecular weight of the obtained target protein is about 59.2KD, which is equivalent to chit...

Embodiment 3

[0042] Embodiment 3 Recombinant chitinase optimum pH and stability determination

[0043] Method for determining the optimum pH value: 10 μL of enzyme solution (prepared in Example 2) was added to 1% colloidal chitin substrate prepared by 190 μL of different pH buffers, respectively: citric acid buffer (pH 3.0- 6.0), phosphate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 7.0-9.0) and glycine-NaOH buffer (pH 9.0-10.0), react at 55°C for 30min. The DNS method was used to measure the activity of the pure enzyme, and the results were as follows: image 3 As shown in A, it can be seen that the optimum reaction pH is pH 6.0 (phosphate buffer).

[0044] The pH stability test method is: add pure enzyme (prepared in Example 2) to the above pH buffer solution, place it at 25°C for 0, 12, 24, 36, 48, 60, and 72 hours, take out 10 μL, add colloidal chitin 1% substrate, determine the residual enzyme activity, the results are as follows image 3 As shown in B, it can be seen that within the ...

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Abstract

The invention discloses a method for preparing chitooligosaccharide, which comprises the following steps of: firstly, pretreating chitin to obtain nano chitin, and then hydrolyzing the nano chitin by using chitinase SbChiAJ143 to obtain the chitooligosaccharide; wherein the amino acid sequence of the chitinase SbChiAJ143 is as shown in SEQ ID NO. 1. The mode of pretreating the chitin to obtain the nano chitin is that a grinding machine is used for grinding a chitin aqueous solution. The method for preparing chitooligosaccharide combines a physical grinding method and an enzyme method, and is high in efficiency. Compared with a method for preparing chitooligosaccharide through single enzymatic hydrolysis of colloid chitin, the method has the advantages that no chemical reagent is used in chitin treatment, the cost is saved, the steps are simplified, and environmental pollution is avoided. The method for preparing chitooligosaccharide is green, environment-friendly and efficient, and has good industrial application potential.

Description

technical field [0001] The invention relates to a method for preparing chitosan oligosaccharide, which belongs to the technical fields of nanometer material preparation and oligosaccharide preparation. Background technique [0002] Nano-grinding technology is a new type of high-efficiency technology for deep processing and utilization of resources. The development of this technology originates from the progress of electronic information engineering, high-tech ceramics and new energy, and has a profound impact on the realization of high-value utilization of raw materials. At present, the deep processing and utilization of raw materials has become one of the new trends in the development of energy and environmental protection technologies. [0003] Chitin is a biomacromolecular polysaccharide widely present in the shells of crustaceans and the cell walls of fungi, and its yield in nature is second only to cellulose. Its structure is formed by the polymerization of N-acetylgl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/26C12P19/14C12N9/24
CPCC12P19/26C12P19/14C12N9/2402C12Y302/01014
Inventor 毛相朝邢爱佳孙建安薛长湖
Owner OCEAN UNIV OF CHINA
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