Production method of recombinant phospholipase D

A production method, phospholipase technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of more PLD inclusion bodies, less PLD inclusion bodies, and low fermentation enzyme activity, so as to increase the culture density , optimization of fermentation process conditions, and the effect of high-density fermentation

Inactive Publication Date: 2013-04-03
宁夏乙征生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a production method of recombinant phospholipase D, the purpose is to overcome the problems in the prior art that Escherichia coli recombinantly expresses PLD with many inclusion bodies and low fermentation enzyme activity, and provides a kind of Escherichia coli recombinant expression PLD with few inclusion bodies , production method with high fermentation enzyme activity and low cost

Method used

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  • Production method of recombinant phospholipase D
  • Production method of recombinant phospholipase D

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Experimental program
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Effect test

Embodiment 1

[0060] Construct recombinant genetically engineered bacteria, which is recombinant E. coli expressing phospholipase D; the plasmid used is the pET system plasmid pET-32(a) developed by Merck; the host bacteria used is developed by Merck. BL21 series Escherichia coli BL21(DE3)pLysS; the phospholipase D gene used is derived from the microorganism Streptomyces.

[0061] Prepare shake flask seed culture medium and fermentation medium, the composition of shake flask seed culture medium comprises glycerol 10g / L, tryptone 20g / L, yeast extract 10g / L, disodium hydrogen phosphate 5.5g / L and ampicillin sodium 40mg / L L, the above items were dissolved in deionized water, and the volume was adjusted to 1L, and the pH value was adjusted to 7 with hydrochloric acid or sodium hydroxide.

[0062] Activate the constructed recombinant genetically engineered bacteria on the plate for 18 hours, then pick the activated genetically engineered bacteria on the plate and inoculate them into shake flasks...

Embodiment 2

[0067] Production of recombinant PLD.

[0068] According to 10.0% of the volume of the fermentation medium, the seed solution was added, and the composition of the fermentation medium was dextrin 50g / L, fish peptone 30g / L, angel yeast extract 15g / L, basic salt 50mL / L, trace elements 30mL / L, Ampicillin sodium 100mg / L. At 28°C, the pH was controlled to be 6.5, and the dissolved oxygen was 30% for fermentation and cultivation. When the OD600 was 0.5, lactose was added to the fermentation broth until its final concentration in the fermentation broth was 1.0%, and then controlled at 20°C. The pH value is 7.0, the dissolved oxygen is 20%, and the fermentation is continued for 32 hours. The PLD is obtained from the fermentation broth according to the conventional method, and the enzyme activity is 18.05U / mL 发酵液 . Other parts are identical with embodiment 1.

Embodiment 3

[0070] Production of recombinant PLD.

[0071] Add the seed solution according to 5.0% of the volume of the fermentation medium, and the composition of the fermentation medium is dextrin 30g / L, fish peptone 20g / L, angel yeast extract 10g / L, basic salt 30mL / L, trace elements 20mL / L, Ampicillin sodium 50mg / L. At 28°C, the pH was controlled to be 6.5, and the dissolved oxygen was 30% for fermentation and cultivation. When the OD600 was 0.5, lactose was added to the fermentation broth until its final concentration in the fermentation broth was 1.0%, and then controlled at 20°C. The pH value is 7.0, the dissolved oxygen is 20%, and the fermentation is continued for 32 hours. The PLD is obtained from the fermentation broth according to the conventional method, and the enzyme activity is 24.36U / mL 发酵液 . Other parts are identical with embodiment 1.

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Abstract

The invention relates to a method for producing recombinant phospholipase D by genetic engineering bacteria fermentation, and particularly relates to a production method of recombinant phospholipase D. The production method is characterized by comprising the following steps of (1) building recombinant gene engineering bacteria; (2) preparing a shake flask seed culture medium and a fermentation medium; (3) activating the built recombinant genetic engineering bacteria for 16-20h on a plane plate, and inoculating into a shake flasks with the shake flask seed culture medium to obtain seed liquid; (4) inoculating into the fermentation medium for fermenting; adding an inducer until the final volume concentration in fermentation liquor is 1-5%, and continuing to ferment for 32-48h; and (5) obtaining the phospholipase D from culture of the fermentation liquor. Compared with the prior art, the production method disclosed by the invention has the following beneficial effects that carbon source dextrin is utilized as a carbon source at a slow speed; on one hand, the culture density of the cell is increased; and high-density fermentation is achieved; on the other hand, the expression speed of a PLD (programmable logic device) is reduced, and generation of an inclusion body is also reduced.

Description

technical field [0001] The invention relates to a method for fermenting and producing phospholipase D by using genetically engineered bacteria, in particular to a method for producing recombinant phospholipase D. Background technique [0002] Phosphatidylserine (PS) is a member of the phospholipid family. It is the only phospholipid that can regulate the functional state of key proteins in the cell membrane. It has unique physical and chemical properties and nutritional value, and is widely used in food, health products and pharmaceutical industries. PS can affect the transmission of chemical information in the brain, and help brain cells store and read data. It is an important nutrient element to maintain the brain's normal memory, response and healthy mood. Animal experiments and clinical applications at home and abroad in the past 20 years have shown that the main effects of PS are: (1) Improve brain function and improve Alzheimer's disease; (2) Help repair brain damage; ...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12R1/19
Inventor 陈龙王双平成小飞
Owner 宁夏乙征生物工程有限公司
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