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Novel chitosanase chi1, its coding gene and its application

A technology of chitosan enzyme and coding gene, which is applied in the field of bioengineering, can solve the problems of a large amount of waste water, waste, non-compliance with energy saving and emission reduction, and consumption of large water resources, etc., and achieve the effect of high enzyme activity

Active Publication Date: 2021-04-13
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the physical method and chemical method will generate a lot of waste water and waste in the production process, and consume a lot of water resources, it does not meet the national policy of energy conservation and emission reduction.

Method used

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  • Novel chitosanase chi1, its coding gene and its application
  • Novel chitosanase chi1, its coding gene and its application
  • Novel chitosanase chi1, its coding gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Construction of metagenomic library of intestinal contents of small yellow croaker

[0054] 1.1 Extraction of metagenomic DNA from intestinal contents of small yellow croaker

[0055] (1) Sample pretreatment and DNA extraction

[0056] Fish samples were processed on a sterile operating table. First, 75% alcohol was used to wipe the surface of the fish and tweezers and other utensils, and then the anus was cut upwards and forwards in an arc with dissecting scissors. The outer wall of the digestive tract was wiped with 75% alcohol, and rinsed several times with sterile flushing solution (0.9% sterile saline). The alimentary canal was isolated with sterile scissors, rinsed with sterile irrigating solution and the contents of the alimentary canal were collected. Next, the metagenomic DNA of the digestive tract contents of the small yellow croaker was extracted by CTAB method, and the extracted metagenomic DNA was detected by electrophoresis. The results are show...

Embodiment 2

[0082] Example 2 Screening of small yellow croaker intestinal metagenomic library chitosanase

[0083] Screening method: Select white positive colonies from many clones in the library, and spot them on the LB (Amp+IPTG+X-gal) medium plate containing 1% colloidal chitosan, culture at 37°C for 1-2d , observe whether there is a hydrolysis circle around the colony. After the colony grows, it will be stained with 1 mg / mL Congo red solution for 10-15 minutes and then decolorized. The colony with a transparent circle is the target colony. Purify the target colony by streaking to obtain a single colony of the target bacterium. The above-mentioned clones with hydrolysis circles were extracted with plasmids, and the plasmid sequencing was completed by Shanghai Sangon Bioengineering Co., Ltd.

[0084]Experimental results: After a large number of screenings, it was found that there were many different library clones that could produce transparent circles, and the one with the best effect...

Embodiment 3

[0085] The bioinformatics analysis of embodiment 3 chitosanase gene CHI1

[0086] Carry out various biological information analysis of chitosanase gene CHI1 according to different tools in the following table 1:

[0087] Table 1 Bioinformatics analysis tools

[0088]

[0089]

[0090] 3.1 Multiple sequence alignment results

[0091] The sequences of CHI1 and chitosanases of family 8 were compared, and the results of the comparison can be found in Image 6 . The comparison results show that the protein sequence information of chitosanase CHI1 can be found in SEQ ID No.1 in the sequence table. This enzyme belongs to the glycoside hydrolase 8 family. The primary structure comparison of chitosanase CHI1 shows that the most similar sequence is WP -053425757.1 is a chitosanase from Rheinheimera sp., with a similarity of 72%. The above results indicate that CHI1 has a low similarity with the known sequence and is a new chitosanase gene.

[0092] 3.2 Analysis results of prot...

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Abstract

The invention discloses a new chitosanase CHI1, its coding gene and application thereof, the new chitosanase has an amino acid sequence as shown in Seq ID No: 1 or at least 95% sequence identity and has substantially the same enzyme live amino acid sequence. The new chitosanase is derived from the metagenome of the digestive tract contents of the small yellow croaker. The coding gene of the above-mentioned novel chitosanase, the coding gene has a nucleotide sequence such as Seq ID No:2. The method for producing the above-mentioned new chitosanase CHI1 comprises the following steps: under conditions suitable for the production of the new chitosanase, ferment the above-mentioned culture capable of expressing the new chitosanase, and separate the fermentation product and purification to obtain chitosanase. The novel chitosanase provided by the invention has high activity and high stability, and the enzyme can be mass-produced by fermentation of engineered bacteria, so that the chitosanase can be applied to industrial oligosaccharide production.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a new chitosanase CHI1, its coding gene and its application. Background technique [0002] Chitosan (chitosan), also known as chitosan, deacetylated chitin, etc., the chemical name is β-(1,4)-2-amino-2-deoxy-β-D-glucose, it is a kind of The natural polymer compound obtained by the deacetylation of the substance is composed of glucosamine or a small amount of acetylglucosamine residues connected by β-(1,4)-glycosidic bonds, and the relative molecular mass ranges from hundreds of thousands to several million. The molecular formula is (C6H11NO4)n. The main source of chitin is the epidermis of arthropods and the shells of molluscs, and it also exists in the cell walls of fungi, bacteria, and lower algae. Chitin is also the organic renewable resource with the highest nitrogen content except protein, and its abundance is second only to cellulose. However, because chitin has a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/26C12P19/14A01N63/50A01P3/00C12N1/06C12R1/19
CPCC12N9/2434C12Y302/01132C12N15/70C12P19/26C12P19/14A01N63/50C12N1/06
Inventor 程凡升
Owner QINGDAO AGRI UNIV
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