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Preparation method of palmitoyl coenzyme A

A palmitic acid and coenzyme technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problem of high cost, achieve high-efficiency expression, and reduce costs

Active Publication Date: 2013-05-01
BEIJING LEADMAN BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The technical problem to be solved by the present invention is to overcome the high cost defect of existing enzyme conversion method to prepare palmitic acid coenzyme A, and to provide a method for preparing palmitic acid coenzyme A with lower cost

Method used

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  • Preparation method of palmitoyl coenzyme A
  • Preparation method of palmitoyl coenzyme A
  • Preparation method of palmitoyl coenzyme A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Preparation of palmitic acid coenzyme A

[0081] 1) Construction of experimental strains

[0082] ① Gene source strain: Pseudomonas stutzeri (ATCC 17588), purchased from ATCC.

[0083] ② Medium

[0084] Solid medium for culturing Pseudomonas stutzeri: glucose: 2%, peptone: 1%, yeast extract: 0.5%, agar: 2%, sterilized at 121° C. for 20 minutes.

[0085] Liquid medium for Pseudomonas stutzeri culture: glucose: 2%, peptone: 1%, yeast extract: 0.5%, sterilized at 121° C. for 20 minutes.

[0086] ③ activation

[0087] Activation of Pseudomonas stutzeri was cultured in a 30°C incubator for 36 hours, and then the colonies were inoculated into YPD liquid medium; cultured at 30°C for 24 hours.

[0088] ④ Genomic DNA extraction

[0089] Single colonies of Pseudomonas stutzeri cells were picked and inoculated in 5 ml of YPD medium for overnight shaking at 30°C. The overnight culture was transferred to a 1.5ml EP tube and centrifuged at 10000rpm for 5min. Discard ...

Embodiment 2

[0123] Example 2 Preparation of palmitic acid coenzyme A

[0124] 1) Construction of experimental strains

[0125] ① Gene source strain: Pseudomonas stutzeri (ATCC 17588), purchased from ATCC.

[0126] ② Medium

[0127] Solid medium for culturing Pseudomonas stutzeri: glucose: 2%, peptone: 1%, yeast extract: 0.5%, agar: 2%, sterilized at 121° C. for 20 minutes.

[0128] Liquid medium for Pseudomonas stutzeri culture: glucose: 2%, peptone: 1%, yeast extract: 0.5%, sterilized at 121° C. for 20 minutes.

[0129] ③ activation

[0130] Activation of Pseudomonas stutzeri was cultured in a 30°C incubator for 36 hours, and then the colonies were inoculated into YPD liquid medium; cultured at 30°C for 24 hours.

[0131] ④ Genomic DNA extraction

[0132] Single colonies of Pseudomonas stutzeri cells were picked and inoculated in 5 ml of YPD medium for overnight shaking at 30°C. The overnight culture was transferred to a 1.5ml EP tube and centrifuged at 10000rpm for 5min. Discard ...

Embodiment 3

[0166] Example 3 Preparation of palmitic acid coenzyme A

[0167] 1) Construction of experimental strains

[0168] ① Gene source strain: Pseudomonas stutzeri (ATCC 17588), purchased from ATCC.

[0169] ② Medium

[0170] Solid medium for culturing Pseudomonas stutzeri: glucose: 2%, peptone: 1%, yeast extract: 0.5%, agar: 2%, sterilized at 121° C. for 20 minutes.

[0171] Liquid medium for Pseudomonas stutzeri culture: glucose: 2%, peptone: 1%, yeast extract: 0.5%, sterilized at 121° C. for 20 minutes.

[0172] ③ activation

[0173] Activation of Pseudomonas stutzeri was cultured in a 30°C incubator for 36 hours, and then the colonies were inoculated into YPD liquid medium; cultured at 30°C for 24 hours.

[0174] ④ Genomic DNA extraction

[0175] Single colonies of Pseudomonas stutzeri cells were picked and inoculated in 5 ml of YPD medium for overnight shaking at 30°C. The overnight culture was transferred to a 1.5ml EP tube and centrifuged at 10000rpm for 5min. Discard ...

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Abstract

The invention discloses a preparation method of a palmitoyl coenzyme A. The preparation method comprises the following steps of 1, carrying out clone coding of an acetylcoenzyme A synthetase gene, transforming the acetylcoenzyme A synthetase gene into an escherichia coli cell, and carrying out expression to obtain a gene engineering strain for expression of an acetylcoenzyme A synthetase, 2, inoculating a fermentation initial medium with the gene engineering strain, and carrying out initial culture, 3, after the initial culture, adding an induction feed-supplement medium into the culture products, and carrying out induction culture to obtain the acetylcoenzyme A synthetase, and 4, after the induction culture, adding SDS into the culture products, carrying out pre-conversion culture, carrying out fed-batch of a conversion feed-supplement medium into the culture products, and carrying out conversion culture to obtain the palmitoyl coenzyme A. The preparation method utilizes acetylcoenzyme A synthetase recombinant bacteria to realize conversion of ATP and coenzyme A produced by organisms into the palmitoyl coenzyme A having a high additional value under mild conditions, and has a cost greatly lower than a cost of the existing enzymatic conversion method for preparation of the palmitoyl coenzyme A.

Description

technical field [0001] The invention relates to an enzyme preparation method, in particular to a method for preparing palmitic acid coenzyme A by fermentation, and belongs to the field of palmitic acid coenzyme A preparation. Background technique [0002] Palmitic acid coenzyme A is the synthesis product of palmitic acid and coenzyme A, which can be used in multiple fields such as biochemistry. The existing production methods of palmitic acid-CoA mainly include chemical synthesis and enzymatic conversion. The chemical synthesis method has the disadvantages of multiple synthesis steps and low yield. The enzymatic conversion method needs to use coenzyme A (CoA) and ATP. Because coenzyme A (CoA) and ATP are expensive, the cost of the enzymatic conversion method of palmitic acid coenzyme A is very high. High, room for improvement. Contents of the invention [0003] The technical problem to be solved by the present invention is to overcome the defect of high cost in the prepa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/32C12N15/52C12N15/63C12N1/21C12N9/00C12R1/19
Inventor 不公告发明人
Owner BEIJING LEADMAN BIOCHEM
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