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Novel chitosanase CsnM with thermal recovery characteristic and application thereof

A technology of chitosanase and chitosan, applied in the direction of application, enzyme, lyase, etc., can solve the problems of limiting industrial application prospects, low activity of chitosanase, thermal stability and storage stability, etc., to achieve Good industrial application prospects, simple purification method, and excellent physical and chemical properties

Active Publication Date: 2019-03-19
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the reported chitosanase activity is low, and the thermal stability and storage stability are poor, which seriously limits its industrial application prospects.

Method used

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  • Novel chitosanase CsnM with thermal recovery characteristic and application thereof
  • Novel chitosanase CsnM with thermal recovery characteristic and application thereof
  • Novel chitosanase CsnM with thermal recovery characteristic and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Sequence analysis and recombinant expression of embodiment 1 chitosanase CsnM

[0026] The enzyme-producing gene csnM of the chitosanase CsnM of the present invention is derived from the marine bacterium Pseudoalteromonasesp.SY39, contains 927 base sequences, and encodes 309 amino acid sequences. Using the conserved domain analysis in the National Center for Biotechnology Information (NCBI) to analyze the Conserved domain (CDD) and the multiple sequence alignment Basic Local Alignment Search Tool (Blast), it was found that the sequence contains a conserved chitosanase of the polysaccharide hydrolase GH family Area. Among the reported chitosanases, the one with the highest amino acid sequence similarity to CsnM is the chitosanase (Genbank EMF00356) of the polysaccharide hydrolase family 46 (GH46), and the amino acid sequence similarity (Identity) between the two is 63%. The chitosanase CsnM described in the invention belongs to the polysaccharide hydrolase (GH46) famil...

Embodiment 2

[0035] The preparation and purification method of embodiment 2 chitosanase CsnM

[0036] Cultivate the recombinant strain BL21(DE3) / pET22b-csnM 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 chitosan enzyme activity assay method is: add 900 μL 0.3% chitosan substrate (20 mM acetic acid-sodium acetate, pH=5.9) to 100 μL enzyme solution, react at 40 ° C for 10 min, add 750 μL DNS reagent, and react in boiling water for 10 min Perform color development and detect its absorbance 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 680.2U / mL.

[0037] After the fermentation stops, centrifuge at 12000rpm for 10min, discard the bacteria, and collect the supernatant; the...

Embodiment 3

[0038] Embodiment 3 temperature and the impact of pH on chitosanase CsnM

[0039] The chitosanase CsnM purified 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 (0-70° C.) for 10 minutes, detect the effect of different reaction temperatures on enzyme activity, and calculate the relative enzyme activity of CsnM at different temperatures with the highest enzyme activity as 100%. Such as figure 2 As shown in A, the optimum reaction temperature of chitosanase CsnM is 40℃.

[0040] The purified chitosanase CsnM obtained in Example 2 was incubated at different temperatures (0-70° C.) for 1 h, and immediately after taking it out, its enzyme activity was detected at its optimum reaction temperature (40° C.) to obtain the activity before incubation. as 100%, as in figure 2 As shown in B, chitosanase CsnM has poor temperature stability, and after incu...

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Abstract

The invention relates to incision chitosanase CsnM with a thermal recovery characteristic and application thereof. The amino acid sequence of the chitosanase CsnM is shown as SEQ ID NO.1. The yield ofthe chitosanase disclosed by the invention can reach 680.2U / mL, the optimal reaction temperature is 40 DEG C, the chitosanase has the thermal recovery characteristic, and after the chitosanase is boiled by 10 minutes and incubated in ice water for 30 minutes, the activity can be restored by 89.1%. The enzyme action is in an incision manner, and main degradation products comprise chitobiose and chitotriose. The chitosanase disclosed by the invention is high in yield and novel in property, and has excellent industrial application potential.

Description

technical field [0001] The invention relates to an endo-type chitosanase CsnM with heat recovery property and application thereof, belonging to the field of biotechnology. Background technique [0002] Oligochitosan is an oligosaccharide with a degree of polymerization of less than 20 and a molecular weight of less than 3900 after hydrolysis of chitosan, which is produced by N-acetyl-D-glucosamine (GLcNAc) and D-glucosamine (GLcN) linked by glycosidic bonds. Due to lower molecular weight and better solubility, chitosan has a wider range of applications than chitosan. In addition, chitosan oligosaccharides also have unique pharmacological functional activities such as anti-tumor, lowering blood pressure, and enhancing immunity, which makes chitosan oligosaccharides have greater application value in the fields of medicine, health care products, food, and breeding. [0003] Chitosan can be hydrolyzed by various enzymes. Including non-specific enzymes: lipase, protease, carbo...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/56C12P19/26C12P19/12
CPCC12N9/88C12P19/12C12P19/26C12Y402/02003Y02P20/59
Inventor 李尚勇韩彦弢周宇
Owner QINGDAO UNIV
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