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Keto group reduction catalytic system construction and cyclic operation method

A catalytic system and cycle operation technology, applied in the direction of fermentation, etc., can solve the problems of no effective recovery of coenzyme I, complicated and difficult recycling, etc., and achieve the effects of reducing production costs, solving technical bottlenecks, and simple and effective recovery means

Inactive Publication Date: 2018-05-08
SHANDONG RUIYING PIONEER PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In summary, the recovery and utilization of coenzyme I and coenzyme II is a complex and difficult technology, which is the fundamental reason why no relevant literature reports on the effective recovery of coenzyme I and coenzyme II have been found so far.

Method used

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  • Keto group reduction catalytic system construction and cyclic operation method

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Experimental program
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Effect test

Embodiment 1

[0021] (1) Escherichia coli ketoreductase engineering strain ¸Escherichia coli glucose dehydrogenase engineering strain was expanded and cultured separately, and 100ml of seed solution was prepared for each, and inoculated in 15L sterilized TB medium respectively, and the pH was controlled at 7.0, and the culture temperature was 37°C, the maximum speed of the fermenter is 500 rpm; after the dissolved oxygen rebounds, add 60% glycerin solution at a rate of 55ml / h; about 8 hours, the OD600 reaches 20, cool down to 30°C, and press into the sterile-filtered IPTG solution (content 1.2g) for induction, after 6 hours of induction, put it into the tank. The enzyme activity of the fermentation broth was detected, which were 16.5ug / ml and 22.3ug / ml respectively.

[0022] (2) Collect the cells by centrifugation at 8000 rpm, and obtain 1246g and 1356g cells respectively; take 600g of ketoreductase cells and 400g of glucose dehydrogenase cells respectively, and weigh them with 4L of 50mM p...

Embodiment 2

[0033] (1) Escherichia coli glucose dehydrogenase engineering strain was expanded and cultivated, and 100 ml of seed solution was prepared, and 1450 ml of glucose dehydrogenase enzyme solution was prepared according to the method provided in Example 1.

[0034] (2) Expand the cultivation of Pichia ketoreductase engineering strains, prepare 300ml of seed liquid, inoculate in 15L of sterilized medium, medium ratio: phosphoric acid 26.7g / L, calcium sulfate 0.93g / L, Potassium sulfate 18.2g / L, magnesium sulfate 14.9g / L, potassium hydroxide 4.31g / L, glycerin 40.0g / L, defoamer 0.4g / L, trace elements 5ml / L, adjust pH to 5.6 with ammonia water; Control pH 5.0, culture temperature 30°C, maximum speed 500 rpm; start to add 50% glycerin solution after dissolved oxygen rebounds, and the rate should be controlled at about 30% dissolved oxygen; about 6 hours, the wet weight of cells reaches 160g / L, stop supplementing glycerin; after the dissolved oxygen rebounds, add methanol, the total dos...

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Abstract

The invention discloses a keto group reduction catalytic system construction and cyclic operation method. The method comprises the following technical implementation steps that a carrier is used for conducting co-immobilization treatment on reductase for catalyzing a keto group reaction and glucose dehydrogenase for catalyzing regeneration of coenzyme to prepare immobilized multi-enzyme; a keto group reduction cyclic catalysis system composed of an immobilized multienzyme reactor, a product extraction device and a 300-600Da nanofiltration device is adopted; in the immobilized multienzyme reactor, coenzyme is free in a reaction solution and cooperates with the immobilized multi-enzyme, and the keto group reduction reaction and the cyclic regeneration of the coenzyme are completed. After thereaction is completed, the immobilized enzyme is filtered and recycled, and a product is extracted from filtrate; mother liquor is extracted, calcium chloride is added to remove gluconic acid, then inorganic salt is removed by the 300-600Da nanofiltration device, and meanwhile the coenzyme is recycled, so the cyclic operation of the keto group reduction catalysis system is achieved. The method has the advantages that the cost is low, the efficiency is high, the operation is simple, the product quality is high, and few impurities are generated.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a method for constructing and circulating a ketone reduction catalytic system. Background technique [0002] Drugs and drug intermediates are mostly macromolecular organic substances. Many of these substances have optical activity, and there are enantiomers with the same chemical composition and different stereostructures, so they are called chiral compounds. In the past, most chiral drugs were sold in the form of racemates, but it has been found that the enantiomers of many chiral drugs have different pharmacological effects. Many tragic incidents of teratogenicity have been produced, so many drugs or drug intermediates containing chiral factors are required to be provided in the form of a single enantiomer. [0003] Chemical synthesis of organic compounds with optical rotation factors is generally a racemic compound. In order to obtain a single enantiomer, chemical or enzymat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/58
CPCC12P7/58
Inventor 彭继先于海勤刘艳平李辉吴桂平乔久生
Owner SHANDONG RUIYING PIONEER PHARMA
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