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Method for preparing 5'-nucleotides using malt root complex phospho-esterase

A technology of compound phosphatase and malt root, applied in the biological field, can solve the problems of insufficient research, low nucleotide yield, low product purity, etc.

Inactive Publication Date: 2007-08-08
北京燕京中科生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The process of enzymatically hydrolyzing yeast RNA to prepare 5'-nucleotides using compound phosphatase prepared from malt root has been reported [1, 2], but the research is not deep enough, the process is not very mature, and the activity of the extracted enzyme is low (200 ~300u / ml), low enzymolysis rate (about 50%), low nucleotide yield, low product purity and other defects

Method used

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  • Method for preparing 5'-nucleotides using malt root complex phospho-esterase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1. Pretreatment and enzymatic hydrolysis

[0024] ①Pretreatment: liquid RNA, in OD 260 Absorbance calculation OD 260 The reading is 0.375×2000 (representing the reading after diluting the liquid RNA by 2000 times, the same below), which is equivalent to feeding 3750g of pure RNA, adjusting the pH to 9.0; 6kg of malt root, adding 60L of water, crushing in a colloid mill, extracting for 4 hours, and centrifuging to obtain the enzyme solution 40L. Heat the RNA solution to 93°C, heat the enzyme solution to 40°C for later use;

[0025] ②Enzymolysis conditions: After mixing the enzyme solution and RNA, control the temperature at 74°C, pH 5.2, the final concentration of RNA at 2.5%, the final enzyme activity of 120u / ml, and keep warm for 2 hours;

[0026] ③ Termination reaction: After the enzymatic hydrolysis is completed, raise the temperature to 90°C-100°C and keep it warm for 30 minutes;

[0027] ④Methamidophos: 5'-nucleotide 21mg / ml, enzymolysis rate 84%;

[0028] 2. ...

Embodiment 2

[0034] 1. Pretreatment and enzymatic hydrolysis

[0035] ①Pretreatment: liquid RNA, in OD 260 Absorbance calculation OD 260 The reading is 0.453×2000, which is equivalent to feeding 3500g of pure RNA, adjusting the pH to 9.0; adding 70L of water to 7kg of malt root, crushing in a colloid mill, extracting for 4h, and centrifuging to obtain 55L of enzyme solution. Heat the RNA solution to 95°C, heat the enzyme solution to 40°C for later use;

[0036] ②Enzymolysis conditions: After mixing the enzyme solution and RNA, control the temperature at 72°C, pH 5.2, the final concentration of RNA at 2.5%, the final enzyme activity of 100u / ml, and keep warm for 2 hours;

[0037] ③ Termination reaction: After the enzymatic hydrolysis is completed, raise the temperature to 90°C-100°C and keep it warm for 30 minutes;

[0038] ④Methamidophos: 5'-nucleotide 18mg / ml, enzymolysis rate 72.8%;

[0039] 2. Microfiltration: Inorganic ceramic membrane is used to separate the solid and liquid of th...

Embodiment 3

[0045] 1. Pretreatment and enzymatic hydrolysis

[0046] ①Pretreatment: liquid RNA, in OD 260 Absorbance calculation OD 260 The reading is 0.412×2000, which is equivalent to feeding 3000g of pure RNA, adjusting the pH to 8.0; 8kg of malt root, adding 100L of water, crushing in a colloid mill, extracting for 8h, and centrifuging to obtain 70L of enzyme solution. Heat the RNA solution to 70°C, heat the enzyme solution to 30°C for later use;

[0047] ②Enzymolysis conditions: After mixing the enzyme solution and RNA, control the temperature at 65°C, pH 4.5, the final RNA concentration of 2.0%, the final enzyme activity of 150u / ml, and keep warm for 3 hours;

[0048] ③ Termination reaction: After the enzymatic hydrolysis is completed, raise the temperature to 90°C-100°C and keep it warm for 30 minutes;

[0049] ④Methamidophos: 5'-nucleotide 15.3mg / ml, enzymolysis rate 76.7%;

[0050] 2. Microfiltration: Inorganic ceramic membrane is used to separate the solid and liquid of the ...

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Abstract

The invention discloses a making method of 5'-nucleic acid through enzymolyzing RNA by composite phospho-esterase of malt root, which is characterized by the following: adopting liquid RNA as substrate; inhibiting the activity of phosphomonoesterase and 3'-phosphodiesterase through the activity of composite phospho-esterase; obtaining the production of 5'-nucleic acid more than 15mg / ml.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing 5'-nucleotides by using malt root complex phosphatase. Background technique [0002] The research on nucleotides at home and abroad has reached the stage of secondary industrial production. The industrial production of nucleotides is mainly based on enzymatic hydrolysis and fermentation. In recent decades, the focus is mainly on enzymatic hydrolysis. 1 Enzymatic degradation, as well as compound phosphatase enzymatic hydrolysis using malt root extraction, a by-product of beer production, in recent years. Nucleic acid P 1 Compared with the compound phosphatase prepared from malt root, enzyme preparation has the problems of long microbial culture period, large equipment investment and high cost; 1 Compared with enzymes, it has the advantages of comparable activity, easy preparation, and low cost. The utilization of malt root and yeast RNA solution, ...

Claims

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Application Information

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IPC IPC(8): C12P19/28
Inventor 赵红玲贾乃堃刘朵花黎高沃
Owner 北京燕京中科生物技术有限公司
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