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Screening and application of beer microzyme containing CMP kinase and CDP kinase

A technology of brewer's yeast and brewer's yeast, which is applied in the field of bioengineering, can solve the problems of high enzyme inactivation rate, inability to completely separate the glycolysis system, and low conversion rate, and achieve high purity, stable process, and long maintenance time Effect

Active Publication Date: 2008-09-17
CHINA PHARM UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the 1980s, the domestic production method of CTP was mainly to transform CMP by isolating its own enzyme system in yeast. The production process is relatively mature, but this process cannot completely separate all the enzymes in the glycolysis system, and the enzyme loss High activity rate, low conversion rate, (Ethan SSimon, 1990)

Method used

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  • Screening and application of beer microzyme containing CMP kinase and CDP kinase
  • Screening and application of beer microzyme containing CMP kinase and CDP kinase
  • Screening and application of beer microzyme containing CMP kinase and CDP kinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] (1) After the commercially available brewer's yeast to be screened is activated on a slant medium, it is transferred to a shaking table for culture. The temperature of the shaking table is 30°C and the rotation speed is 200rpm. Bed culture to the logarithmic growth phase, centrifuge to recover the bacteria, add 5ml of phosphate buffer solution with pH 7.0 to make the bacteria suspension.

[0033] (2) Weigh 20mg of NTG, dissolve it in acetone, add distilled water to 10ml, which is 2mg / ml NTG solution, mix 1ml of bacterial suspension with 1ml of nitrosoguanidine (NTG) solution, shake and mix Afterwards, bathe in a constant temperature water bath at 30°C for 30 minutes, and then dilute the mixture to 10 -3 Stop mutagenesis. After the mutagenesis was terminated, the stop solution was diluted to 10 -5 、10 -6 Afterwards, cytidine diphosphate-ethanolamine (300 μg / ml) plates were coated with the structural analogue of cytidine triphosphate, and cytidine diphosphate-ethanolam...

Embodiment 2

[0036] (1) Weigh 200g of κ-carrageenan sodium salt, 100g of refined konjac powder, dissolve in 5L of deionized water, heat to 121°C with steam, keep warm for 60min, pour it into a 20L reactor with stirring, and let it cool naturally to 40 °C, keep the glue solution warm for later use.

[0037] (2) Weigh 4 kg of washed beer yeast wet cells and add 1 kg of physiological saline, stir evenly, and keep warm at 35°C for later use.

[0038] (3) Pour the insulated yeast suspension into the glue, stir at 40°C for half an hour to mix evenly, and set aside.

[0039] (4) Add 60L soybean oil into a 100L stirred reactor, preheat to 40°C, control the stirring speed at 125rpm, pour the mixture of yeast and glue into the soybean oil, stir for half an hour until the particles are formed, filter, and the soybean oil After recovery, let it stand still, and discard the water in the lower layer, and then reuse it.

[0040] (5) Soak the particles filtered in step (4) for one hour with 3% potassium...

Embodiment 3

[0053] In the reaction system for the synthesis of CTP by immobilized yeast, in addition to generating a large amount of CTP, there are also impurities such as unreacted CMP and by-product CDP, phosphate, etc. In order to separate CTP from the reaction solution, HZ-1 ion exchange resin is used for Ion exchange chromatography, and use 0.4mol / L sodium chloride first, and then use 1.0mol / L sodium chloride solution to carry out gradient elution with a column volume of 0.2-0.5 per hour. Concentrate the eluent under reduced pressure in a water bath at 50-60°C, add 3 times the volume of 95% ethanol, let it stand overnight in the refrigerator, filter it with suction, and dry the filter cake. The finished product is detected by high-performance liquid chromatography. Chromatographic conditions and implementation Example 1 is the same. For the continuous response of immobilized yeast to CTP, see figure 2 , it can be seen from the figure that more than ten batches of immobilized spheri...

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Abstract

The invention relates to a method for screening Saccharomyces cerevisiae with high-activity CMP kinase and CDP kinase, and the use of the Saccharomyces cerevisiae in the production of cytidine triphosphate, and belongs to the technology field of biological engineering. The Saccharomyces cerevisiae strains is preserved in the China General Microbiological Culture Collection Center (CGMCC) with accession number of CGMCC NO.2448. The Saccharomyces cerevisiae strains with high-activity CMP kinase and CDP kinase are screened by means of nitrosoguanidine (NTG) mutagenesis. In the production of the cytidine triphosphate, the Saccharomyces cerevisiae strains are used to be prepared into immobilized cells of various grain diameters with carrageenan as carriers by different controlled stirring speeds realized through embedding. Compared with manual cutting, the screening method of the invention has the advantages of small cell leakage, regular grains with high strength, low labor intensity, simple grain-producing procedure, no complicated equipment and instrument, and is suitable for industrialized production. Furthermore, bean oil used in the invention is safe and nontoxic solvent, suitable for various requirements and easy to recycle.

Description

technical field [0001] The invention relates to the screening of brewer's yeast containing high-activity CMP kinase and CDP kinase and the application of the brewer's yeast in the production of cytidine triphosphate, belonging to the technical field of bioengineering. Background technique [0002] Cytidine triphosphate (abbreviation: CTP) is a normal component in the cells of the body. It participates in the synthesis and metabolism of nucleic acids and phospholipids in the body, promotes protein synthesis, and provides energy. It can also regulate and promote nerve cells and glia. Synthesis and construction of plasma cells and vascular wall cell membrane structure, can resist nerve cell damage caused by excitatory amino acids and free radicals, thus playing an anti-vascular sclerosis effect. The main function of cytidine monophosphate kinase (CMP kinase) and cytidine diphosphate kinase (CDP kinase) is responsible for activating cytidine triphosphate synthetase (CTP syntheta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N11/04C12Q1/04C12P19/30C12R1/865
Inventor 周长林罗众球闫蓬勃邱蔚然
Owner CHINA PHARM UNIV
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