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Method for synthesizing ademetionine by biological catalytic process

A technology of adenosylmethionine and biocatalysis, which is applied in the field of biopharmaceuticals, can solve the problems of complex separation and purification procedures of final products, long product production cycle, low conversion rate of raw materials, etc., and achieves improved yield, reduced separation and purification difficulty, high active effect

Active Publication Date: 2014-12-03
ZHEJIANG ZHENYUAN PHARMA CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The invention provides a method for synthesizing adenosylmethionine by biocatalysis and extracting adenosylmethionine. Firstly, Escherichia coli is used instead of yeast, and a special process is adopted to express the required SAM synthetase, and then SAM is obtained by enzymatic production. To solve the problems of low raw material conversion rate, long product production cycle, complicated separation and purification procedures of final product and high price in the production of adenosylmethionine by yeast fermentation method

Method used

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  • Method for synthesizing ademetionine by biological catalytic process
  • Method for synthesizing ademetionine by biological catalytic process
  • Method for synthesizing ademetionine by biological catalytic process

Examples

Experimental program
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Effect test

Embodiment 1

[0029] 1) Take 1 mL of the culture solution of Escherichia coli (A 550=1.0 ~ 1.2) After expressing SAM synthetase, centrifuge at 4°C and 5000 r / min to harvest 40 kg of wet cells, add 9.6 liters of 50% ethyl acetate aqueous solution to the wet cells, 500 Vigorously stirred at r / min for 30 minutes, then treated with 20 liters of 0.175 mol / L H 2 S0 4 After continuing the treatment for 1.5 hours, centrifuge at 5000 r / min for 20 minutes to remove cell debris and obtain 40 liters of extract containing SAM, wherein the content of SAM in the extract is 8.5 g / L.

[0030] 2) Adjust 40 liters of extract containing SAM to pH 5.0 with 5mol / l sodium hydroxide solution, and pass through 6.7 liters of JK110 ion exchange resin column at a flow rate of 2 times the resin bed volume per hour for sample loading. After the end, the column was washed with 30 liters of deionized water, and then eluted with PBS solution to obtain 25 L of SAM eluent. The SAM concentration in the eluent was 13.2 g...

Embodiment 2

[0034] 1) After Escherichia coli fermentation and cultivation, centrifuge at 4°C and 5000r / min to harvest 40 kg of wet bacteria, add 12 liters of 20% ethyl acetate aqueous solution to the wet bacteria, and treat at 500r / min for 60 minutes. Then use 16 liters of 0.15mmol / LH 2 SO 4 Continue processing for 2 hours. Centrifuge at 5000r / min for 20 minutes to remove cell debris to obtain 38 liters of extract containing SAM, wherein the content of SAM is 8.7g / l.

[0035] 2) Adjust 38 liters of the extract containing SAM to pH 4.5 with 5mol / L sodium hydroxide solution, and load the sample through a 9.5 liter D113 ion exchange resin column at a flow rate of 1 times the resin bed volume per hour. After loading the sample, the column was washed with 30 liters of deionized water, and then eluted with PBS solution to obtain 25 L of SAM eluent, the SAM concentration was 13.2 g / l, and the SAM recovery rate was 96%.

[0036] All the other follow-up treatments are the same as in Example...

Embodiment 3

[0037] Embodiment 3: Catalyst recovery

[0038] After the expression, take the filtrate after bacteriostasis and the filtrate after enzymatic catalysis, and put it on the separation column of weakly acidic macroporous 1AJ enoic acid cation exchange resin (D11:3). Check the eluate every 30 minutes to measure its OD value, and when the absorption of the solution at a wavelength of 204nm is greater than 0.4, wash with a sulfuric acid aqueous solution of pH 2.0 for 4 hours, and stop washing with water. The recovery rate reaches 80%.

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Abstract

The invention relates to a method for synthesizing ademetionine by a biological catalytic process, belonging to the technical field of biological pharmacy. The method comprises the following steps: inoculating Escherichia coli into a seed culture medium, carrying out shake culture over night, adding the over-night culture material into an LB (Langmuir-Blodgett) liquid culture medium, carrying out shake culture to mid-log phase, adding IPTG (isopropyl-beta-D-thiogalactopyranoside) into the bacterium liquid to fix the concentration, continuing carrying out shake culture and SAM synthetase expression, centrifuging to obtain wet bacteria, stirring for extraction treatment, loading to an ion exchange resin column, concentrating by nanofiltration, crystallizing, and carrying out vacuum drying to obtain the crystal. The technical scheme has the advantages of high conversion rate, short period, high purity and the like.

Description

technical field [0001] The invention relates to a method for synthesizing adenosylmethionine by a biocatalytic method, belonging to the technical field of biopharmaceuticals. Background technique [0002] S-adenosylmethionine (S.adenosyl-L.methionine, SAM) is an important metabolic intermediate substance widely present in animals, plants and microorganisms, and is also an important physiologically active substance in the human body. The effect of base, transsulfur and transaminopropyl group has good curative effect on arthritis, depression, liver dysfunction, pancreatitis and so on. SAM is also a high-level healthcare drug for cancer prevention, cardiovascular disease and anti-aging. In 1999, the US FDA approved SAM as a health product, and quickly became one of the best-selling nutritional products in the United States. With the improvement of people's quality of life and the renewal of health concepts, the demand for SAM will also increase. [0003] The national annual ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/40C12R1/19
Inventor 傅小峰沈剑锋姚黎栋王刚周军贤胡纯琦
Owner ZHEJIANG ZHENYUAN PHARMA CO LTD
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