A chitosanase csna1 derived from deep-sea microorganisms and its application

A technology of chitosanase and chitosan, applied to microorganisms, methods based on microorganisms, applications, etc., can solve the problems of high price, low activity, and single variety, and achieve the effects of simple preparation, high yield, and novel sequences

Active Publication Date: 2021-07-09
广东噢美丽大健康产业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the chitosanase currently sold on the market is expensive, has low activity, and has a single variety, which cannot meet the application under specific conditions. Therefore, it is urgent to develop a new chitosanase

Method used

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  • A chitosanase csna1 derived from deep-sea microorganisms and its application
  • A chitosanase csna1 derived from deep-sea microorganisms and its application
  • A chitosanase csna1 derived from deep-sea microorganisms and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1 Sequence Analysis of Chitosanase CsnA1

[0025] The enzyme-producing gene csnA1 of the chitosanase CsnA1 in the invention is an artificially synthesized sequence. In previous studies, we found that the deep-sea bacterium Vibrio sp. A1-09 has high chitosanase activity. When it was sequenced, it was found to contain a predicted chitosanase sequence, which was named csnA1. Under the condition that the amino acid sequence remains unchanged, we optimized the base sequence of the gene sequence according to the codon preference of the host (Escherichia coli), so as to facilitate its high-efficiency expression in Escherichia coli. The chitosanase CsnA1 of the present invention contains 828 base sequences and encodes 276 amino acid sequences. Using the Conserved Domain (CDD) analysis in NCBI, it was found that the sequence contained a conserved region of the 46th family of polysaccharide hydrolase (GH-46). Multiple sequence comparison Basic Local Alignment Search Too...

Embodiment 2

[0034] The preparation and purification method of embodiment 2 chitosanase CsnA1

[0035] Culture the recombinant strain BL21(DE3) / pET22b-CsnA1 in 100 mL of LB liquid medium (50 μg / mL ampicillin) in a shaker at 37°C at 180 rpm until OD 600 =0.6, add the inducer isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.1 mM, and induce at 20°C for 20 h. The standard assay method for chitosanase CsnA1 activity is: add 900 μL 0.3% chitosan substrate (20 mM acetic acid-sodium acetate, pH=6.0) to 100 μL enzyme solution, react at 25°C for 10 min, add 750 μL DNS reagent was reacted in boiling water for 10 min for color development, and its absorbance was detected at OD520. Enzyme activity is defined as 1 U is the amount of enzyme required to produce 1 μM reducing sugar per min. After testing, the chitosanase activity in the fermentation broth can reach 379.9 U / mL.

[0036] After the fermentation stopped, centrifuge at 12000 rpm for 10 min, discard the bacteria, and collec...

Embodiment 3

[0037] Embodiment 3 The influence of temperature on chitosanase CsnA1

[0038] The purified chitosanase CsnA1 obtained in Example 2 was tested for enzyme activity under different conditions to detect the effects of different temperatures and pH on the enzyme activity. React at different temperatures (0-50°C) for 10 min, detect the effect of different reaction temperatures on enzyme activity, and calculate the relative enzyme activity of chitosanase CsnA1 at different temperatures with the highest enzyme activity as 100%. Such as figure 2 As shown, the optimum reaction temperature of chitosanase CsnA1 is 25℃. The chitosanase CsnA1 of the present invention has psychrophilic properties, and the detection activity at 0°C, 10°C, and 15°C can reach 67.6%, 81.5% and 89.3% of the optimum reaction temperature, which can meet the requirements of low temperature applications. Social needs.

[0039] The chitosanase CsnA1 purified in Example 2 was incubated at different temperatures (0...

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Abstract

The invention relates to a chitosanase CsnA1 derived from deep-sea microorganisms and an application thereof. The amino acid sequence of the chitosanase CsnA1 is shown in SEQ ID NO.1. The invention provides a fermentation and purification method of chitosanase CsnA1, the enzyme activity of the fermentation liquid is as high as 379.9 U / mL, the purity of one-step purification is >95%, and the recovery rate is 93.7%. The chitosanase CsnA1 of the present invention has psychrophilic properties, the optimum reaction temperature is 25°C, and the detection activity at 0°C, 10°C, and 15°C can reach 67.6%, 81.5% and 89.3% of the optimum reaction temperature . The chitosanase CsnA1 degrades chitobiose and chitotriose as main products, and has good application potential.

Description

technical field [0001] The invention relates to a chitosanase CsnA1 derived from deep-sea microorganisms and an application thereof, belonging to the field of biotechnology. Background technique [0002] Chitosan (chitosan) is obtained by deacetylation of chitin (chitin), which exists widely in nature, and its chemical name is polyglucosamine (1-4)-2-amino-B-D glucose. Oligochitosan, the degradation product of chitosan with small molecules, has extensive antibacterial, anti-inflammatory and anti-tumor effects, and is recognized as an effective health-care functional food, and is known as "the sixth element of life". Oligochitosan is very soluble in water, and has greater application value than macromolecular chitosan in the fields of medicine, health care products, and functional foods. [0003] Compared with the traditional acid hydrolysis method, the use of chitosanase to degrade macromolecular chitosan has the advantages of mild reaction conditions, single degradation pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12P19/26C12R1/19
CPCC12N9/2434C12N15/70C12P19/26C12Y302/01132
Inventor 杨文钰
Owner 广东噢美丽大健康产业有限公司
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