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Bacterial with high-yield of nucleoside phosphorylase and method for synthesizing arabinose nucleoside

A technology of arabinoside and nucleoside, which is applied to high-yielding nucleoside phosphorylase strains and the field of enzymatic synthesis of arabinopurine nucleosides, can solve the problems of long reaction steps, difficult to control enzymatic reactions, long steps, etc. Reduce production operation procedures and production costs, the effect of high conversion rate

Inactive Publication Date: 2008-01-30
SHANGHAI WEIPING BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

What (1) method obtains is the mixture of α and β configuration, and steps are long, and yield is low
(2) The reaction steps of the method are also long, and must be separated by layers
[0013] Although some of these enzymatic chemical methods can obtain higher yields, such as more than 90%, the amount of arabouridine used is three times that of purine, which is a serious waste, and the enzymatic conversion time is as long as more than 24 hours, and the enzymatic reaction is difficult to control. shortcoming

Method used

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  • Bacterial with high-yield of nucleoside phosphorylase and method for synthesizing arabinose nucleoside
  • Bacterial with high-yield of nucleoside phosphorylase and method for synthesizing arabinose nucleoside

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Enterobacter aerogenes ATCC13048 was scraped from the slope and inoculated in a sterile medium (medium 1: peptone 10g / L, beef extract 10g / L, yeast powder 5g / L, sodium chloride 5g / L), 30 Cultivate with shaking at ℃ for 16 hours, collect the bacteria, wash three times with sterile normal saline, and dissolve in sterile normal saline, adjust the bacterial density to 10 8 cells / mL, after a series of mutagenesis treatments, spread evenly on a sterile petri dish (medium 2: medium plus 100 μg / mL 5-fluorocytosine and 20 g / L agar), and culture at 30°C For three days, select the strains with better growth and larger colonies, inoculate them in shake flasks containing medium 1, and culture them with shaking for 24 hours. The bacteria were collected, washed three times with phosphate buffer, and the enzyme activity was determined. Finally, a strain DWOQ-58 with high activity of purine and uridine phosphorylase was obtained. Enzyme activity and characteristics are shown in Table 1....

Embodiment 2

[0078] Enterobacter aerogenes DWOQ-58 was scraped from the slope and inoculated in sterile medium 1, cultured with shaking at 35°C for 16 hours, collected the bacteria, washed three times with sterile saline, and refrigerated for later use.

[0079] ATCC13048 also obtains wet thalline by the same method.

[0080] Take 1 gram of DWOQ-58 wet bacteria, add 10mmol / L adenine, 30mmol / L arabouridine, potassium phosphate buffer solution with pH=7.0, the total volume is 10mL, shake and react at 60°C for 6 hours, after the reaction is completed, high-speed centrifugation , take the supernatant for analysis. The molar conversion rate of vidarabine was 92%.

[0081] The same operation was performed on ATCC13048, and the molar conversion rate of adenosine vidarabine was only 11%.

Embodiment 3

[0083] Same as Example 2, but the arabinoside is replaced by arabino-5-methyluridine. DWOQ-58 catalyzes the synthesis of vidarabine with a molar conversion rate of 87%, while the molar conversion rate of ATCC13048 for the synthesis of vidarabine is only 8%.

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Abstract

A compound method of high-yield nucleoside phosphorylase strains and arabinose nucleoside pertains to biochemical engineering field, in particular to the high-yield nucleoside phosphorylase strains and a method for compounding arabinose purine nucleoside with the strains by an enzyme method. The invention aims at solving a technical problem for providing the strains of the high-yield nucleoside phosphorylase and strains of uridine phosphorylase and the method for producing the arabinose purine nucleoside with the strains. The invention discloses enterobacter aerogenes with a preservation number of CGMCC No.2035 and the method for producing the arabinose purine nucleoside with the strains, and the invention comprises steps that (1) the enterobacter aerogenes DWOQ-58 of the invention is cultured and collected, and (2) the enterobacter aerogenes DWOQ-58 is contacted with arabinose donor and receptors of purine base. The strains of the invention are rich in vigor and resists 5-flucytosine with an average conversion rate of more than 80 percent in general and the reaction time of the invention is shortened to less than 12 hours.

Description

technical field [0001] The invention belongs to the field of biochemical engineering, and in particular relates to a high-yield nucleoside phosphorylase strain and a method for enzymatically synthesizing arabinopurine nucleosides using the strain. Background technique [0002] Vidarabine purine nucleosides include vidarabine, diamino vidarabine, vidarabine, etc., whose chemical structural formula is as follows. [0003] [0004] Adenosine Vidarabine Diaminoadenosine Vidarabine [0005] Among them, adenosine vidarabine has a broad-spectrum antiviral effect, and it has obvious effects on herpes simplex virus type I, type II, and herpes zoster virus, and has obvious effects on adenovirus, Epstein-Barr virus, vaccinia virus, cytomegalovirus, and hepatitis B virus (HBV). ) etc. also have inhibitory effect on ribonucleic acid (RNA) virus. Clinically, it can be injected intravenously or applied externally. Vidarabine is also an important intermediate of vidarabine adenosine m...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P19/40
Inventor 魏晓琨丁庆豹欧伶阎蓬勃邱蔚然
Owner SHANGHAI WEIPING BIOLOGICAL TECH
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