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Method for converting escherichia coli to produce 2'-deoxyadenosine

A technology of Escherichia coli and deoxyadenosine, applied in the field of bioengineering, can solve the problems of complex steps, high cost, and low conversion rate, and achieve the effect of mild reaction conditions, low production cost and high conversion rate

Active Publication Date: 2014-12-03
西藏天虹科技股份有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most 2′-deoxyadenosine products come from DNA degradation, but this traditional method of producing deoxynucleosides will become more and more limited to limited natural resources, and it is difficult to separate and extract pure products
There are many problems in the synthesis of 2′-deoxyadenosine by chemical synthesis. Using ribose or deoxyribose as raw material is too expensive, most of the steps are complicated, and the conversion rate is low. Some will produce α and β of 2′-deoxyadenosine. -Isomers, difficult to separate, low yield, and need to protect and deprotect specific groups, catalysts are necessary for those that do not need protection, and catalysts are often toxic and expensive, such as mercury salts, In addition, some by-products produced by chemical synthesis are not easy to decompose, seriously pollute the environment, and it is difficult to realize large-scale industrial production

Method used

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  • Method for converting escherichia coli to produce 2'-deoxyadenosine

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Experimental program
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Effect test

Embodiment 1

[0045] 1) Seed culture

[0046] Take a ring of activated Escherichia coli strains with high nucleoside phosphorylase activity, inoculate them in a 500ml Erlenmeyer flask, and cultivate them on a shaker at 37°C for 28 hours. The seed medium consists of: 5.2g of yeast extract, Peptone 11.5g, NaCl 5.5g and distilled water 1000mL, sterilized at 121°C for 20min, pH value 7.0.

[0047] 2) Fermentation culture and cell preparation

[0048] Use 12L enzyme-producing medium in a 20L automatic fermentation tank, inoculate Escherichia coli seed culture solution according to the inoculum volume of 6% of the volume of the enzyme-producing medium, cultivate at 37°C for 8h, and then centrifuge at 6000rpm for 8min at 4°C , to obtain the Escherichia coli wet thalline, the gained wet thalline is 7.0 with the pH value, the concentration is the potassium phosphate buffer solution washing 2 times of 20mmol / L, centrifugation obtains the required Escherichia coli thalline, in- Store frozen at 20°C ...

Embodiment 2

[0058] 1) Seed culture

[0059] Take a loop of activated Escherichia coli strains with high nucleoside phosphorylase activity, inoculate them in a 500ml Erlenmeyer flask, and culture them on a shaker at 37°C for 36 hours. The seed medium consists of: 6g of yeast extract, peptone 12.5g, NaCl7.5g and distilled water 1000mL, sterilized at 121°C for 20min, pH value 7.2.

[0060] 2) Fermentation culture and cell preparation

[0061] Use 12L enzyme-producing medium in a 20L automatic fermentation tank, inoculate the Escherichia coli seed culture solution according to the inoculum volume of 6% of the volume of the enzyme-producing medium, incubate at 37°C for 9h, and then centrifuge at 5000rpm for 10min at 4°C , to obtain the Escherichia coli wet thalline, the gained wet thalline is 7.0 with the pH value, and concentration is the potassium phosphate buffer solution washing 3 times of 20mmol / L, centrifugation obtains the required Escherichia coli thalline, in- Store frozen at 20°C f...

Embodiment 3

[0071] 1) Seed culture

[0072] Take a ring of activated Escherichia coli strains with high nucleoside phosphorylase activity, inoculate them in a 500ml Erlenmeyer flask, and cultivate them on a shaker at 37°C for 40 hours. The seed medium consists of: 8.2g of yeast extract, Peptone 14.1g, NaCl 7.3g and distilled water 1000mL, sterilized at 121°C for 20min, pH value 7.3.

[0073] 2) Fermentation culture and cell preparation

[0074] Use 12L enzyme-producing medium in a 20L automatic fermentation tank, inoculate Escherichia coli seed culture solution according to the inoculum volume of 7% of the volume of the enzyme-producing medium, cultivate at 37°C for 10h, and then centrifuge at 8000rpm at 4°C for 7min , to obtain the Escherichia coli wet thalline, the gained wet thalline is 7.0 with the pH value, the concentration is the potassium phosphate buffer solution washing 2 times of 20mmol / L, centrifugation obtains the required Escherichia coli thalline, in- Store frozen at 20°C...

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Abstract

The invention discloses a method for converting escherichia coli to produce 2'-deoxyadenosine. Escherichia coli thallus with high nucleoside phosphorylase activity is directly adopted as an enzyme source to mix with deoxythymidine which is taken as a conversion reaction substrate as well as adenine to carry out conversion reaction to synthesize 2'-deoxyadenosine. The method has the advantages of simple process, high conversion ratio, low production cost and gentle reaction conditions; moreover, the by-products are fewer, green and pollution-free, and the products are easy to separate and purify. A conversion ratio of adenine is over 72.5%. Besides, the 2'-deoxyadenosine is separated and purified by adopting a method of crystallization and recrystallization, so that the purity of the 2'-deoxyadenosine is over 99%.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for transforming and producing 2′-deoxyadenosine by Escherichia coli. Background technique [0002] 2'-deoxyadenosine (2-deoxyadenosine) is a natural deoxynucleoside, an organic part of deoxyribonucleic acid (DNA), and an important raw material for genetic medicine and genetic engineering research, with good physiological activity. 2′-Deoxyadenosine itself has certain medicinal value, can inhibit the release of insulin induced by sugar, and can reduce the effect of specific phosphodiesterase inhibitors or adenylyl cyclase activators on promoting insulin secretion. Although it has not been reported that it can be directly used for anti-virus, it can be used as an important raw material. The 2′-deoxyadenosine analogue obtained through chemical structure modification is a good choice for many anti-virus, anti-tumor, and anti-AIDS nucleoside drugs. Good intermediate....

Claims

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Application Information

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IPC IPC(8): C12P19/40C07H19/173C07H1/06C12R1/19
Inventor 张春颖杨旭锦
Owner 西藏天虹科技股份有限责任公司
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