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Method for preparing and regenerating lysine bacillus protoplast capable of degrading MC-LR

A protoplast and bacillus technology, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of aggravating cyanobacterial bloom algal toxin pollution, etc., and achieve strong repeatability, high preparation rate, and convenience. effect of operation

Active Publication Date: 2013-04-03
安徽水韵环保股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, alginolytic bacteria have been found to release algae toxins into the water while dissolving algae cells, which intensifies the algae toxin pollution caused by cyanobacteria blooms, among which MC is the most serious hazard

Method used

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  • Method for preparing and regenerating lysine bacillus protoplast capable of degrading MC-LR
  • Method for preparing and regenerating lysine bacillus protoplast capable of degrading MC-LR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The preserved strain T1 was retransferred to the plate and streaked and separated for 3 times, a single colony was picked and inoculated into fresh bacterial liquid medium, and placed in a shaking incubator at a temperature of 30 °C and a rotation speed of 120 r / min for pure cultivation until Logarithmic phase, about 10 8 -10 9 individual / mL. Take 4 mL of activated bacterial liquid, centrifuge at 4500 r / min for 10 min, collect the bacterial cells, wash with SMM buffer three times, and resuspend with 1.2 mL of SMM buffer; add 0.1 mg / mL lysozyme 0.8 mL and 2 mL of EDTA solution heated to the enzymatic hydrolysis temperature was enzymatically hydrolyzed for 1 h at a water bath temperature of 35°C; after the enzymolysis was completed, the protoplasts were collected by centrifugation at 3000 r / min, washed three times with 0.55 mol / L NaCl solution, and washed away After the enzymolysis solution was suspended with 4 mL of 0.55 mol / L NaCl solution for later use, the preparati...

Embodiment 2

[0035] The preserved strain T1 was retransferred to the plate and streaked and separated for 3 times, a single colony was picked and inoculated into fresh bacterial liquid medium, and placed in a shaking incubator at a temperature of 30 °C and a rotation speed of 120 r / min for pure cultivation until Logarithmic phase, about 10 8 -10 9 individual / mL. Take 4 mL of activated bacterial liquid, centrifuge at 4500 r / min for 10 min, collect the bacterial cells, wash 3 times with SMM buffer, and resuspend with 1.8 mL of SMM buffer; add 0.2 mL of 1 mg / mL lysozyme and 2 mL of EDTA solution heated to the enzymatic hydrolysis temperature was enzymatically hydrolyzed for 1 h at a water bath temperature of 35°C; after the enzymolysis was completed, the protoplasts were collected by centrifugation at 3000 r / min, washed three times with 0.55 mol / L NaCl solution, and washed away After the enzymolysis solution was suspended with 4 mL of 0.55 mol / L NaCl solution for later use, the protoplast p...

Embodiment 3

[0037] The preserved strain T1 was retransferred to the plate and streaked and separated for 3 times, a single colony was picked and inoculated into fresh bacterial liquid medium, and placed in a shaking incubator at a temperature of 30 °C and a rotation speed of 120 r / min for pure cultivation until Logarithmic phase, about 10 8 -10 9 individual / mL. Take 4 mL of activated bacterial liquid, centrifuge at 4500 r / min for 10 min, collect the bacterial cells, wash 3 times with SMM buffer, and resuspend with 1.6 mL of SMM buffer; add 0.4 mL of 1 mg / mL lysozyme and 2 mL of EDTA solution heated to the enzymatic hydrolysis temperature was enzymatically hydrolyzed for 1 h at a water bath temperature of 35°C; after the enzymolysis was completed, the protoplasts were collected by centrifugation at 3000 r / min, washed three times with 0.55 mol / L NaCl solution, and washed away After the enzymolysis solution was suspended with 4 mL of 0.55 mol / L NaCl solution for later use, the protoplast p...

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Abstract

The invention discloses a method for preparing and regenerating a lysine bacillus protoplast capable of degrading MC-LR and relates to the technical field of methods for preparing and regenerating protoplasts in bioengineering. According to the method, the protoplast fusion technology for establishing engineering bacteria is taken as the basis, and the lysine bacillus protoplast is prepared from the separated MC-LR high-efficiency degrading bacteria T1(Bacillus sphaericus) protoplast. According to the research, an inactivated protoplast fusion method is selected, so that the workload is reduced, and the quality of a fusant in the subsequent research is guaranteed. The method for preparing and regenerating the protoplast is simple in process, convenient to operate and high in repeatability; and the obtained protoplast preparation rate can be up to 96.64 percent, the regeneration rate can be 83.95 percent, the protoplast is high in preparation rate and high in activity, and the preparation and regeneration of the subsequent fusant are facilitated.

Description

technical field [0001] The invention relates to the technical field of protoplast preparation and regeneration methods in bioengineering, in particular to the preparation and regeneration of lysine bacillus protoplasts that degrade MC-LR. Background technique [0002] In recent years, with the aggravation of eutrophication in water bodies, blue-green algae multiply rapidly in large areas in summer and autumn, frequent outbreaks of blue-green algae blooms, a large number of blue-green algae cover the surface of water bodies, seriously hinder the flow of water bodies, reduce the self-purification ability of water bodies, The rate of pollution decreases, the transparency decreases, the water quality is foul, and the plankton such as fish and shrimp in the water body die due to the deterioration of the living environment. At present, the most commonly used method to control cyanobacteria is still the traditional mechanical salvage. However, after all, manpower and material resou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12R1/07
Inventor 张文艺陈雪珍李仁霞李秋艳
Owner 安徽水韵环保股份有限公司
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