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Combination of bacterial agents with high yield of chitin deacetylase and its application

A technology of oxo-deacetylase and oxo-deacetylase is applied in the field of bacterial agent combination for high-yield chitin deacetylase, which can solve the problems of misfolding of recombinase and no literature related to co-fermentative production of chitin deacetylase, etc. To achieve the effect of long fermentation cycle

Active Publication Date: 2021-10-15
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

3. Construction of genetically engineered bacteria, Carole Gauthier et al. introduced three chitin deacetylase genes (RC, D2, and 13 / 2) from Rhizopus circinans into Pichia pastoris for expression, and found that only one recombinase ( RC) is active, presumably because the secretion signal cleavage sites of the three enzyme proteins are different (RC and I3 / 2 are upstream), and the wrong selection of this site leads to misfolding of the latter two recombinases
[0008] In the literature that has been reported so far, there is no relevant literature on the production of chitin deacetylase by synergistic fermentation

Method used

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  • Combination of bacterial agents with high yield of chitin deacetylase and its application
  • Combination of bacterial agents with high yield of chitin deacetylase and its application
  • Combination of bacterial agents with high yield of chitin deacetylase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment one: the breeding of bacterial classification

[0028] (1) strain

[0029] Rhodococcus HQcdag (Rhodococcus sp.)

[0030] (2) culture medium

[0031] Each 1L of the enriched medium contains: NaCl 0.5g, K 2 HPO 4 1g, KH 2 PO 4 1g, MgSO 4 0.1g, 5g of colloidal chitin, the pH of the liquid medium is natural;

[0032] The screening medium per 1L contains: NaCl 0.5g, K 2 HPO 4 1g, KH 2 PO 4 1g, MgSO 4 0.1g, colloidal chitin 5g, p-nitroacetanilide 0.2g, agar 20g, the pH of the plate solid medium is natural;

[0033] The seed medium on each 1L of the shaker flask contains: 10g of peptone, 3g of beef extract, 5g of sodium chloride, and the pH of the liquid medium is natural;

[0034] Each 1L of the solid slant medium contains: 10g of peptone, 3g of beef extract, 5g of sodium chloride, 20g of agar, sterilized at 0.1MPa for 20min, and the pH of the slant solid medium is natural;

[0035] The re-sieved fermentation medium on each 1L shaker flask conta...

Embodiment 2

[0044] Embodiment two: the seed solution ratio optimization of double strain synergistic fermentation

[0045] (1) Strains: Rhodococcus HQcdag (Rhodococcus sp.) and Bacillus cereus CJPE209 (Bacillus cereus) screened in Example 1, deposit number CCTCC NO: M 2015734.

[0046] (2) Method steps:

[0047] The medium is seed medium (peptone 10g / L, beef extract 3g / L, sodium chloride 5g / L). The culture conditions are: natural pH, inoculum size 1%, solution volume 100ml / 300ml, 37°C, 180r / min.

[0048] a. Optimization test of double strain mixing time

[0049] The growth curves of the two strains were determined separately, such as figure 1 and figure 2 Shown, find out its logarithmic growth phase.

[0050] b. Optimization experiment of the inoculum ratio of double strains

[0051] In the growth cycle, the number of viable bacteria was measured at the 20th hour in the logarithmic growth period of the two strains, and then the number of live bacteria was measured according to a ce...

Embodiment 3

[0052] Embodiment three: the culture condition optimization of dual-strain synergistic fermentation

[0053] (1) Bacterial strains: Rhodococcus HQcdag (Rhodococcus sp.) and Bacillus cereus CJPE209 (Bacillus cereus) screened in Example 1, the ratio of viable bacteria between Bacillus cereus and Rhodococcus is 2:8.

[0054] (2) Method steps:

[0055] The fermentation medium is the initial medium (yeast extract powder 2.5g / L, peptone 2.5g / L, colloidal chitin 5g / L, NaCl 5g / L, K 2 HPO 4 1g / L, KH 2 PO 4 1g / L, MgSO 4 0.5g / L). The initial fermentation conditions are: natural pH, inoculum size 1%, liquid filling volume 50ml / 250ml, 37°C, 180r / min shake flask fermentation for 24h.

[0056] A single factor test was used to investigate the effects of the fermentation period, total inoculum size, fermentation speed, liquid volume and fermentation temperature on the production of chitin deacetylase in the double-strain synergistic fermentation.

[0057] a. Optimization test of ferm...

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Abstract

The invention discloses a combination of bacterial agents for high-yielding chitin deacetylase, including Rhodococcus HQcdag ( Rhodococcus sp . ), deposit number CCTCC NO: M 2020336, and Bacillus cereus CJPE209 ( Bacillus cereus ), deposit number CCTCC NO:M 2015734. Compared with the single fermentation of Rhodococcus, the two strains increased the activity of chitin deacetylase by about 14 times through collaborative fermentation, and significantly shortened the fermentation time.

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a bacterial agent combination with high yield of chitin deacetylase and its application. Background technique [0002] Chitin (C8H13O5N)n, also known as chitin, is an insoluble N-acetyl-D-glucosamine polymer that exists in the shells of various crustaceans and in the cell walls of fungi. When the deacetylation rate of chitin reaches 55% and above, it is chitosan. Chitosan is widely used in industry (cloth, clothing, dyes, paper and water treatment, etc.), agriculture (pesticides, plant antiviral agents), fishery (fish feed, cosmetic beauty agent, hair protection, moisturizer, etc.), medical industry (contact lens, artificial skin, suture, artificial dialysis membrane and artificial blood vessel, etc.), especially in the field of biomedicine, due to its good Biocompatibility has good application prospects in tissue engineering, wound healing, and biosen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/80C12P19/26C12R1/01C12R1/085
CPCC12N1/20C12N9/80C12P19/26C12Y305/01041
Inventor 蔡俊郭依依王常高杜馨赵泽鑫
Owner HUBEI UNIV OF TECH
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