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Method for producing chitinase through fermentation of chaetomium globosum and application of chitinase

A technology of chitinase and Chaetomium globosa, applied in the biological field, can solve problems such as poor stability, low enzyme activity, and lack of efficient degradation, and achieve the effects of easy operation, low nutritional requirements, and high yield

Active Publication Date: 2020-11-17
LUDONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Chitinase (Chitinase, EC.3.2.1.14) is a kind of enzyme that specifically degrades chitin, which widely exists in bacteria and eukaryotes, but there is still a lack of efficient chitin degradation in industrial applications. Chitinase, low enzyme activity, poor stability and other characteristics limit the application of chitinase in the preparation of chitooligosaccharides, which in turn limits the application in food, agriculture, medicine, environmental protection and other industries

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0022] Preparation of the colloidal chitin used: Weigh 10 g of chitin, add 40 mL of acetone to fully infiltrate, add 300 mL of concentrated HCl while grinding at 4 °C, and slowly add to 1.5 L of 50% ethanol after standing for 5 h Stir vigorously while adding, and let stand for 2 h to precipitate colloidal chitin. Pour off the supernatant, collect the colloidal chitin precipitate, and wash it repeatedly with distilled water until neutral.

[0023] The configuration of the DNS reagent used: add 91g potassium sodium tartrate into 500 mL hot water, stir and dissolve in a water bath at 45°C, then add 20g NaOH and 3.15g 3,5-dinitrosalicylic acid (DNS) in sequence, and then add 2.5g Crystalline phenol and 2.5 g of anhydrous sodium sulfite, stirred to dissolve, cooled, and distilled water to 1000mL, stored in a brown bottle in a dark place for one week before use.

Embodiment 1

[0024] Example 1: Fermentation of Chaetomium globosa HY1 to produce chitinase on a shaking table (1)

[0025] A ring was picked from the strains preserved on the slant of the test tube and transferred to the activation medium, and activated at 28°C and 200 rpm for 25 h to obtain the turbidity of the bacterial cells to obtain the activated seed solution. Activation medium: 8 g / L glucose, 3 g / L peptone, 1 g / L potassium dihydrogen phosphate, 1 g / L disodium hydrogen phosphate, pH 6.5.

[0026] Using the preferred fermentation medium: 15 g / L soluble starch, 10 g / L glycerol, 5 g / L tryptone, 2 g / L yeast powder, 4 g / L corn steep liquor, 2 g / L NH 4 NO 3 , 0.5 g / L MnSO 4 , 2 g / L MgSO 4 , 1 g / L Na 2 HPO 4 , 1 g / LK 2 HPO 4 , 4 g / L colloidal chitin, initial pH 6.5. The optimal fermentation medium after sterilization was inserted into the activated seed culture solution according to the inoculum amount of 3% (v / v), cultivated in a rotary shaker at 200 rpm, the culture temperature wa...

Embodiment 2

[0027] Example 2: Fermentation of Chaetomium globosa HY1 to produce chitinase on a shaking table (2)

[0028] The strains stored on the slant of the test tube were transferred to the activation medium, and activated at 28°C and 200rpm for 30h until the bacteria became turbid to obtain the activated seed solution. Activation medium: 15 g / L glucose, 6 g / L peptone, 3 g / L potassium dihydrogen phosphate, 2.5 g / L disodium hydrogen phosphate, pH 7.0.

[0029] Optimal fermentation medium: 20 g / L soluble starch, 15 g / L glycerol, 10 g / L tryptone, 4 g / L yeast powder, 8 g / L corn steep liquor, 5 g / L NH 4 NO 3 , 1.5 g / L MnSO 4 , 4 g / L MgSO 4 , 2.5 g / L Na 2 HPO 4 , 3 g / LK 2 HPO 4 , 8 g / L colloidal chitin, initial pH 7.5. The optimal fermentation medium after sterilization was inserted into the activated seed culture solution according to the inoculation amount of 6% (v / v), and cultivated in a rotary shaker at 200 rpm at a temperature of 30°C. After 5 days of fermentation, the cultiva...

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PUM

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Abstract

The invention discloses a method for producing chitinase through fermentation of chaetomium globosum and application of the chitinase. According to the method, chaetomium globosum HY1 is adopted for strain activation, an activation culture medium comprises glucose, peptone, monopotassium phosphate and disodium hydrogen phosphate, then shake cultivation or fermentation tank cultivation are carriedout, a fermentation medium comprises soluble starch, glycerin, tryptone, yeast powder, corn steep liquor, NH4NO3, MnSO4, MgSO4, Na2HPO4, K2HPO4 and colloidal chitin, and chitinase produced through fermentation is used for preparing chitosan oligosaccharide. The culture method is simple, the nutritional requirement is not high, and the chitinase produced by liquid state fermentation is high in activity and has a relatively great industrial application value.

Description

technical field [0001] The invention relates to a method for fermenting and producing chitinase by Chaetomium globosa and its application in the preparation of chitooligosaccharides, belonging to the field of biotechnology. Background technique [0002] Chitin is one of the renewable resources with huge reserves in nature. It is widely distributed in nature and mostly exists in the cell walls of fungi and exoskeletons of arthropods such as shrimps and crabs as the content of biological structures. Chitin is only found in marine organisms. Chitin reserves are about 1 billion tons. Chitosan oligosaccharides are the degradation products of chitin or chitin. Different from chitin, due to the smaller molecular weight and lower degree of polymerization, it has better water solubility and is easily absorbed by the collective. Completely degraded in vivo. Chitosan oligosaccharides have different pharmacological effects and biological activities, and have strong application prospec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12P19/26C12P19/14C12R1/645
CPCC12N9/2442C12P19/14C12P19/26C12Y302/01014
Inventor 程仕伟张萍葛宜和冯志彬毛志远缪静
Owner LUDONG UNIVERSITY
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