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Genetic engineering bacterium for degrading chitin absent from chitobiose enzyme

A kind of technology of genetic engineering bacteria and succinase, applied in the field of chitin degrading bacteria

Inactive Publication Date: 2006-02-01
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, so far, the use of microbial chitinase to produce chitin oligosaccharides has not been successfully industrialized.

Method used

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  • Genetic engineering bacterium for degrading chitin absent from chitobiose enzyme
  • Genetic engineering bacterium for degrading chitin absent from chitobiose enzyme
  • Genetic engineering bacterium for degrading chitin absent from chitobiose enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] figure 1 Comparison of the ability of Q2 and CB101 to degrade chitinobiosaccharase substrate 4-MU-chitobioside. Q2 (3 clones on the upper part of the plate) and CB101 (one clone on the bottom of the plate) were spotted on the LB plate and cultured at 37 degrees for 12 hours, then sprayed with the chitinobiosacidase substrate 4-MU-chitobioside, and incubated at 37 degrees in the dark Observations were performed under UV light after 30 minutes.

[0014] Q2 cannot degrade 4-MU-chitobioside, and the colonies do not fluoresce under ultraviolet light excitation; CB101 can effectively degrade 4-MU-chitobioside, and the colonies emit strong fluorescence under ultraviolet light excitation.

Embodiment 2

[0016] The ability and degradation products of Q2 to degrade chitin were analyzed:

[0017] 1. Qualitative Analysis of Chitin Degradation Ability Using Solid Chitin Plates

[0018] Pick single clones of Q2 and wild bacteria CB101 and spread them on a chitin solid plate, culture them at 37 degrees for about 12 hours, and observe the diameter of the transparent circle after degrading chitin. like figure 2 As shown, Q2 retains the ability to degrade chitin efficiently, indicating that although Q2 is mutated to produce chitinobiosaccharase, its ability to degrade chitin has not been reduced.

[0019] figure 2 Qualitative comparison of Q2 and CB101 for degradation of colloidal chitin. Q2 (3 clones on the upper part of the plate) and CB101 (one clone on the bottom of the plate) were spotted on the chitin plate and incubated at 37 degrees for 12 hours to observe the size of the transparent circle after chitin degradation.

Embodiment 3

[0021] Analysis of Degraded Chitin Products

[0022] Q2 was inoculated with basic medium containing 1% colloidal chitin, and the colloidal chitin was completely degraded after the shake flask was cultured at 37 degrees for 24 hours. Take 1ul solution for TLC product analysis, such as image 3 As shown, there are mainly chitinodisaccharides and a small amount of chitinotriose in the fermentation supernatant of Q2; while there are only chitinomonoses in the fermentation product of CB101 wild bacteria. image 3 Thin layer chromatography. G1 is the monosaccharide standard, G2 is the chitobiose standard, Q2 is the fermentation supernatant of strain Q2, and CB101 is the fermentation supernatant of CB101.

[0023] The chitin-degrading genetically engineered bacterium lacking chitinobiosaccharase-Aeromonas caviae, known in Latin civilization as: Aeromonas caviae, was preserved in the China Type Culture Collection Center (Address: Wuhan, Wuhan, China) on December 14, 2003 University...

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Abstract

A chitin degradating bacterium is deformed by genetic engineering means to obtain the chitobiose deficient efficient genetically engineered chitin-degradating bacterium. It can effectively degradate the crystalline and colloidal chitin. After the chitin substrate is directly added to the fermented liquid containing them cultured in shake flask, the chitin oligose product without chitin monose can be directly obtained.

Description

technical field [0001] The invention relates to chitin-degrading bacteria, especially the transformation of chitinase system. Background technique [0002] Chitin is a linear polymer of N-acetylglucosamine connected by β-1,4-glycosidic bonds. It is the second most abundant polysaccharide in nature except cellulose. It mainly exists in the shells of shrimps and crabs, fungi cell walls and insect shells. Many microorganisms can secrete chitinase, which can degrade chitin to provide carbon and nitrogen sources for growth. The degradation and utilization of chitin by microorganisms is an important part of the cycle of chitin in nature. Chitinase (EC3.2.1.14) can degrade the β-1,4-glycosidic bond of chitin, and can be divided into endonuclease and exonuclease. Under the joint action of endonuclease and exonuclease, chitin produces chitobiose, which is finally degraded by chitinobiose into chitin monosaccharide, which is absorbed by microorganisms as a nutrient source. [0003]...

Claims

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Application Information

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IPC IPC(8): C12N1/00C12N9/24C12P19/14
Inventor 肖湘王风平陈贞茂李强
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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