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Method for preparing and regenerating sphingomonas sp protoplast

A technology of protoplast and welan gum, applied in the field of microbial engineering, can solve the problems of complex operation, difficult conditions for wall removal and regeneration, etc., and achieve the effects of simple operation, strong repeatability and high activity

Active Publication Date: 2017-04-26
TIANJIN AGRICULTURE COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the field of protoplast fusion of bacteria, compared with Gram-positive bacteria, the operation of Gram-negative bacteria is more complicated, mainly because the cell wall of negative bacteria is more complex, and after the extracellular secretion of polysaccharide products by bacteria, It will be more difficult to master the conditions of wall removal and regeneration

Method used

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  • Method for preparing and regenerating sphingomonas sp protoplast
  • Method for preparing and regenerating sphingomonas sp protoplast
  • Method for preparing and regenerating sphingomonas sp protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Take the preserved strain Sphingomonas sp, streak it on the slant of the test tube under aseptic conditions, and incubate at a constant temperature of 30°C for 72h. Rinse the bacteria on the slant of the test tube with 5 mL of sterile water, inoculate it into 100 mL of liquid medium for the inoculum, cultivate it on a shaker at 30 ° C for 10 h at 180 r / min, and add 12 mmol / L EDTA for treatment. The bacterial solution was taken and centrifuged at 8000r / min for 5min, the supernatant was discarded, the bacterial cells were washed with phosphate buffer, and the cell morphology was observed under a microscope ( figure 1 ). Suspend the bacteria in SMM buffer, then dilute with sterile water, spread the medium on the plate, and culture at 30°C for 4 days to obtain the number of colonies A (total number of colonies). Under the same conditions, centrifuge again and discard the supernatant, suspend the cells with 45μg / mL lysozyme, incubate at 28°C for 25min, centrifuge at 4000r / m...

Embodiment 2

[0033] Take the preserved strain Sphingomonas sp, streak it on the slant of the test tube under aseptic conditions, and incubate at a constant temperature of 30°C for 72h. Rinse the bacteria on the slant of the test tube with 5 mL of sterile water, inoculate it into 100 mL of liquid medium for the inoculum, cultivate it on a shaker at 30 ° C for 10 h at 180 r / min, and add 12 mmol / L EDTA for treatment. The bacterial solution was centrifuged at 8000r / min for 5min, the supernatant was discarded, and the bacterial cells were washed with phosphate buffer. Suspend the bacteria in SMM buffer, suspend the cells with 125μg / mL lysozyme, incubate at 37°C for 30min, centrifuge at 4000r / min for 10min, discard the supernatant, wash with hypertonic buffer to remove the enzyme, and the protoplasts obtained during this process The body formation rate was 88.0%, and the regeneration rate was 25.0%.

Embodiment 3

[0035] Take the preserved strain Sphingomonas sp, streak it on the slant of the test tube under aseptic conditions, and incubate at a constant temperature of 30°C for 72h. Rinse the bacteria on the slant of the test tube with 5 mL of sterile water, inoculate it into 100 mL of liquid medium for the inoculum, cultivate it on a shaker at 30 ° C for 10 h at 180 r / min, and add 12 mmol / L EDTA for treatment. The bacterial solution was centrifuged at 8000r / min for 5min, the supernatant was discarded, and the bacterial cells were washed with phosphate buffer. Suspend the bacteria in SMM buffer, suspend the cells with 45μg / mL lysozyme, incubate at 37°C for 30min, centrifuge at 4000r / min for 10min, discard the supernatant, wash with hypertonic buffer to remove the enzyme, and the protoplasts obtained during this process The body formation rate was 73.0%, and the regeneration rate was 31.1%.

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Abstract

The invention provides a method for preparing and regenerating sphingomonas sp protoplast, and belongs to the technical field of microbial engineering. According to the method, sphingomonas sp is used as an original strain; the sphingomonas sp protoplast is prepared by an enzymolysis method; the formation rate of the obtained protoplast is 97.3 percent; and the regeneration rate is 33.9 percent. The method has the advantages that the operation is simple and convenient; the preparation rate of the protoplast is high; the activity is high; and the further breeding of the excellent sphingomonas sp strain by using protoplast fusion and genome shuffling is facilitated.

Description

technical field [0001] The invention belongs to the field of microbial engineering, and in particular relates to a method for preparing and regenerating protoplasts of Welan gum-synthesizing bacteria. Background technique [0002] Welan gum is a kind of microbial metabolic glue, which is a polysaccharide product synthesized by Sphingomonas strains. It has excellent rheological properties, and its viscosity is relatively stable to temperature, pH and metal salt ions, especially It has good high temperature resistance, and the viscosity of Welan gum solution changes little in the range of 25-100°C. As a thickener, suspending agent, emulsifier, stabilizer and lubricant, Welan gum is widely used in food, concrete, petroleum, chemical and other fields. However, compared with the well-developed microbial metabolic gums such as xanthan gum, there are still some problems in the fermentation production of Welan gum, especially the low fermentation yield, less gum content in the ferm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12R1/01
Inventor 张芃黄海东李晓雁周明明任梦楠
Owner TIANJIN AGRICULTURE COLLEGE
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