A kind of preparation method of Mortierella alpina protoplast

A technology of Mortierella alpina and protoplasts, applied in the field of preparation of Mortierella alpina protoplasts, can solve problems such as uncontrollable and achieve high yield and stable protoplasts

Inactive Publication Date: 2017-10-13
GUANGDONG PROVINCIAL BIOENGINEERING INST (GUANGZHOU SUGARCANE IND RES INST)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Utilizing traditional bioengineering techniques, such as mutagenesis selection and fermentation process control, etc., it is impossible to effectively regulate the biosynthetic metabolic pathway of ARA to break through this bottleneck problem

Method used

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  • A kind of preparation method of Mortierella alpina protoplast

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Experimental program
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Effect test

Embodiment 1

[0023] 1) Expanded culture of spore suspension: 1ml of the above spore suspension was placed in a conical flask with a liquid volume of 50ml / 250ml, cultured with shaking at 28°C and 150 r / min for 15-16 hours, centrifuged at 3000r / min to collect mycelium, and used Bacteria water and osmotic agent were washed twice respectively for the preparation of protoplasts;

[0024] 2) Preparation of enzymatic solution: Mix the three enzyme solutions according to the following ratio, 300U / ml cellulase, 15mg / ml helicase and 2500U / ml lysozyme, use 0.8mol / L NaCl as osmotic pressure stabilization Prepare the reagent and adjust the pH to 5.8; filter and sterilize through a 0.22µm microporous membrane, and store in a refrigerator at 4°C for later use;

[0025] 3) Protoplast preparation: Take 5ml of enzyme solution and add 1g of wet bacteria, shake well, incubate at 30°C and shake gently, after enzymatic hydrolysis for 4 hours, filter with four layers of high-grade lens paper to remove mycelium f...

Embodiment 2

[0027] 1) Expanded culture of spore suspension: 1ml of the above spore suspension was placed in a conical flask with a liquid volume of 50ml / 250ml, cultured with shaking at 28°C and 150 r / min for 15-16 hours, centrifuged at 3000r / min to collect mycelium, and used Bacteria water and osmotic agent were washed twice respectively for the preparation of protoplasts;

[0028] 2) Preparation of enzymatic solution: Mix the three enzyme solutions according to the following ratio, 300U / ml cellulase, 15mg / ml helicase and 2500U / ml lysozyme, use 0.8mol / L NaCl as osmotic pressure stabilization Prepare the reagent and adjust the pH to 5.8; filter and sterilize through a 0.22µm microporous membrane, and store in a refrigerator at 4°C for later use;

[0029] 3) Protoplast preparation: Take 5ml of enzyme solution and add 1g of wet bacteria, shake well, incubate at 35°C and shake gently, after enzymatic hydrolysis for 4 hours, filter with four layers of high-grade lens paper to remove mycelium f...

Embodiment 3

[0031] 1) Expanded culture of spore suspension: 1ml of the above spore suspension was placed in a conical flask with a liquid volume of 50ml / 250ml, cultured with shaking at 28°C and 150 r / min for 16 hours, centrifuged at 3000r / min to collect mycelium, and washed with sterile water Wash twice with the osmotic agent for the preparation of protoplasts;

[0032] 2) Preparation of enzymatic solution: Mix the three enzyme solutions according to the following ratio, 375U / ml cellulase, 20mg / ml helicase and 5000U / ml lysozyme, use 0.8mol / L NaCl as osmotic pressure stabilization Prepare the reagent and adjust the pH to 5.8; filter and sterilize through a 0.22µm microporous membrane, and store in a refrigerator at 4°C for later use;

[0033] 3) Protoplast preparation: Take 5ml of enzyme solution and add 1g of wet bacteria, shake well, incubate at 30°C and shake gently, after enzymatic hydrolysis for 4 hours, filter with four layers of high-grade lens paper to remove mycelium fragments, in...

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Abstract

The invention discloses a preparation method for mortierella alpina protoplast. An enzymatic hydrolysate consists of active enzyme, an osmotic pressure stabilizing agent and water, wherein the active enzyme comprises the following components according to mixing ratio: 300-375 U / ml cellulase, 15-20 mg / ml helicase and 2500-5000 U / ml lysozyme. The preparation method comprises the following steps: inoculating activated mortierella alpina into a liquid medium, shaking culturing, collecting mycelium, and washing to be clean; adding the sterile enzymatic hydrolysate into mycelium, conducting full enzymolysis, removing mycelium scraps, collecting, and washing, so as to obtain the protoplast. The enzymatic hydrolysate can efficiently conduct enzymolysis on the cell wall of the mycelium of the mortierella alpina, so that the high-quality mortierella alpina protoplast can be prepared, and the yield of the protoplast can reach 5.5*10<7> per millilitre.

Description

technical field [0001] The invention relates to a preparation method of Mortierella alpina protoplast. Background technique [0002] Arachidonic acid (ARA) is an essential omega-6 polyunsaturated fatty acid. ARA not only exists as an important structural lipid (phospholipid) widely in mammalian organs, muscles and blood tissues, but also is the direct precursor of many eicosanic acid derivatives with special activity, the pharmaceutical value of ARA, nutrition It has excellent health care functions and has been widely used in medicine, food, feed and other fields. [0003] Mortierella alpina ( Mortierella alpina ) is one of the most potential strains for producing ARA, but it was found in the research process that the ARA yield and the percentage of total fatty acids in oils are low, making it one of the technical bottlenecks for the production of ARA by microbial fermentation. Utilizing traditional bioengineering techniques, such as mutagenesis selection and fermentation...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/14C12R1/645
CPCC12N1/14
Inventor 班甲陈骏佳王帆班雯婷徐晓燕周正雄
Owner GUANGDONG PROVINCIAL BIOENGINEERING INST (GUANGZHOU SUGARCANE IND RES INST)
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