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Preparation and regeneration method of algal toxin degrading bacterium protoplast

A technology of protoplasts and degrading bacteria, applied in biochemical equipment and methods, bacteria, microorganisms, etc., can solve the problems of single population structure and low degradation efficiency, and achieve simple operation, low equipment requirements, enzymatic hydrolysis time and regeneration cycle. short effect

Active Publication Date: 2014-07-09
ANHUI HUANGHE WATER RESOURCE POLYTRON TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the fact that there are not many types of MC-LR biodegrading bacteria, the population structure is relatively single, and the degradation efficiency is not high, the study of protoplast preparation and regeneration of MC-LR degrading bacteria has important theoretical and theoretical implications for the subsequent construction of highly efficient MC-LR degrading engineering bacteria. Practical value

Method used

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  • Preparation and regeneration method of algal toxin degrading bacterium protoplast
  • Preparation and regeneration method of algal toxin degrading bacterium protoplast

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Pick a ring of bacteria from the slant medium of the preserved strain R3 (Lysinibacillus fusiformis) and put it into a conical flask filled with 30mL of fresh bacterial liquid medium, in a shaking incubator at a temperature of 28°C and a speed of 130r / min After activating and culturing for 15 hours, the seed culture solution of R3 was obtained. Take 4mL and centrifuge at 4300r / min for 12min; add 4mL of SMM buffer to the precipitated cells, centrifuge and wash mycelium 3 times; resuspend with 1.8mL of SMM buffer to obtain R3 mycelia suspension. Add 0.2 mL of lysozyme with a concentration of 10 mg / mL and 2 mL of EDTA solution with a concentration of 0.8 mg / mL heated to 32 °C, and perform enzymatic hydrolysis at 32 °C for 1 h in a water bath to obtain an enzymatic hydrolysis solution of R3. Centrifuge at a speed of 3200r / min for 8min, add 4mL of NaCl solution with a concentration of 0.55mol / L to the precipitated protoplasts, wash by centrifugation for 3 times, and suspend ...

Embodiment 2

[0043] Pick a ring of bacteria from the slant medium of the preserved strain R3 (Lysinibacillus fusiformis) and put it into a conical flask filled with 30mL of fresh bacterial liquid medium, in a shaking incubator at a temperature of 28°C and a speed of 130r / min After activating and culturing for 12 hours, the seed culture solution of R3 was obtained. Take 4mL and centrifuge at 4300r / min for 12min; add 4mL of SMM buffer to the precipitated bacteria, centrifuge and wash mycelium 3 times; resuspend with 1.6mL of SMM buffer to obtain R3 mycelia suspension. Add 0.4 mL of lysozyme with a concentration of 10 mg / mL and 2 mL of EDTA solution with a concentration of 0.8 mg / mL heated to 32 °C, and perform enzymatic hydrolysis at 32 °C for 1 h in a water bath to obtain an enzymatic hydrolysis solution of R3. Centrifuge at a speed of 3200r / min for 8min, add 4mL of NaCl solution with a concentration of 0.55mol / L to the precipitated protoplasts, wash by centrifugation for 3 times, and suspe...

Embodiment 3

[0045] Pick a ring of bacteria from the slant medium of the preserved strain R3 (Lysinibacillus fusiformis) and put it into a conical flask filled with 30mL of fresh bacterial liquid medium, in a shaking incubator at a temperature of 28°C and a speed of 130r / min After activating and culturing for 12 hours, the seed culture solution of R3 was obtained. Take 4mL and centrifuge at 4300r / min for 12min; add 4mL of SMM buffer to the precipitated cells, centrifuge and wash mycelium 3 times; resuspend with 1.8mL of SMM buffer to obtain R3 mycelia suspension. Add 0.2 mL of lysozyme with a concentration of 10 mg / mL and 2 mL of EDTA solution with a concentration of 0.8 mg / mL heated to 32 °C, and perform enzymatic hydrolysis at 32 °C for 1 h in a water bath to obtain an enzymatic hydrolysis solution of R3. Centrifuge at a speed of 3200r / min for 8min, add 4mL of NaCl solution with a concentration of 0.55mol / L to the precipitated protoplasts, wash by centrifugation for 3 times, and suspend ...

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Abstract

The invention relates to the technical field of water treatment microorganisms, and in particular relates to preparation and regeneration method of an algal toxin degrading bacterium protoplast. The bacterium is separated from blue-green algae dehydration filtrate and is identified to be a capitatus lysine bacillus and is preserved in the China general microbiological culture collection center, the preservation number is China general microbiological culture collection center (CGMCC) NO.6107, and the preservation name is microcystin-LR (MC-LR) degrading bacterium R3. The preparation and regeneration method mainly comprises bacterium seed culture solution preparation, hypha suspension preparation, cell wall enzymolysis, protoplast suspension preparation and protoplast regeneration. The protoplast preparation and regeneration are adopted, the preparation rate of the protoplast for degrading the capitatus lysine bacillus R3 of the MC-LR can reach 85.10%, and the regeneration rate can reach 38.40%; and moreover, the preparation and regeneration processes are low in equipment requirement and are simple to operate, the enzymolysis time and regeneration period are short, and the subsequent fusant preparation and engineering bacteria structure of R3 are benefited.

Description

technical field [0001] The invention relates to the technical field of water treatment microorganisms, in particular to the preparation and regeneration of protoplasts of algae toxin-degrading bacteria. Background technique [0002] In recent years, with the development of society and economy, a large amount of sewage containing nutrients such as nitrogen and phosphorus has entered the natural water body, leading to the increasingly serious problem of eutrophication in water bodies. In some areas of my country, such as Taihu Lake, Chaohu Lake and Dianchi Lake, blue-green algae blooms have broken out every year. The occurrence of cyanobacterial blooms in lakes or reservoirs not only reduces their ecological regulation and water self-purification ability, but also releases a large amount of algae toxins into the water during the process of algal accumulation and death, and algal cell rupture and decomposition. Microcystin-LR (MC-LR) is a type of algal toxin with the highest fr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12R1/07
Inventor 张文艺李仁霞戴如娟郑泽鑫占明飞
Owner ANHUI HUANGHE WATER RESOURCE POLYTRON TECH INC
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