Microbial transformation method

A microbial transformation and levodopa technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as expensive and unstable

Active Publication Date: 2016-06-01
卓虹超源生物科技(郑州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although 3,4-dihydroxyphenylpyruvate is a more direct substrate, it is expensive and unstable, so levodopa is the best substrate

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Analytical Determination of Transformation Products.

[0023] Prepare medium as follows: yeast extract 5g / L, peptone 10g / L, sodium chloride 5g / L. The liquid volume in the 500ml Erlenmeyer flask is 100ml, a total of 50 bottles, sterilized at 120°C for 20 minutes. Take ProvidenciarustigianiiATCC33673 glycerol tube seed solution 1mL for inoculation, fermentation temperature is 30°C, shaker speed is 200rpm. After culturing for 24 hours, the fermentation broth was centrifuged (10,000 rpm for 10 min), the supernatant was removed to obtain bacterial cells, and the bacterial cells obtained by centrifugation were washed twice with sterile water. Get 20g wet thallus and put into the 1000ml solution that levodopa concentration is 2g / L, mix well. Shake (100 rpm) at 37°C for 24 hours, then heat in a water bath at 100°C for 3 minutes to kill bacteria. Then centrifuge (10000rpm, time 10min) to remove the bacteria, and obtain the transformed supernatant. Liquid phase analysis showe...

Embodiment 2

[0027] Comparison of aerobic and anaerobic cultures. Preparation medium: glucose 30g / L, peptone 20g / L, diammonium hydrogen phosphate 1g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.3g / L, ferrous sulfate 0.3g / L; initial pH and fermentation The pH of the process is natural. The liquid volume in the 500ml Erlenmeyer flask is 100ml, a total of 2 bottles, sterilized at 120°C for 20 minutes.

[0028]Take ProvidenciarettgeriATCC29944 glycerol tube seed solution 1mL to inoculate 1 bottle, culture in an anaerobic incubator at 35°C for 72 hours, and the bacteria obtained by centrifugation are washed twice with sterile water. Take 0.1g of wet bacteria and put it into 4ml of levodopa solution with a concentration of 2g / L, mix evenly, put it in an anaerobic incubator at 35°C for 24 hours, and then heat it in a water bath at 100°C for 3 minutes to kill the bacteria. Then centrifuge (10000rpm, time 10min) to remove the bacteria, and obtain the transformed supernatant. Liquid...

Embodiment 3

[0031] The medium composition is: glucose 1g / L, urea 1g / L, diammonium hydrogen phosphate 1g / L, potassium dihydrogen phosphate 0.1g / L, magnesium sulfate 0.1g / L, ferrous sulfate 0.1g / L. The volume of liquid in a 250ml Erlenmeyer flask is 50ml, sterilized at 120°C for 20 minutes. Take Providenciarettgeri CICC10488 glycerol tube seed liquid 0.5mL to inoculate, the fermentation temperature is 35°C, and the shaker speed is 200rpm. After culturing for 24 hours, the fermentation broth was centrifuged (10,000 rpm for 10 min), the supernatant was removed to obtain bacterial cells, and the bacterial cells obtained by centrifugation were washed twice with sterile water. Take 0.05g of wet bacteria and put it into 2ml of levodopa solution with a concentration of 0.1g / L, adjust the pH to 6, and mix well. Static transformation was carried out in an anaerobic incubator for 24 hours, the transformation liquid was filtered through a microporous membrane, and the concentration of danshensu in th...

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PUM

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Abstract

The invention relates to a method for transforming Providencia microorganisms into levodopa to produce R-(+)-3-(3,4-dihydroxyphenyl)-2-hydracrylic acid. The method is simple in process and has significant industrial application value.

Description

technical field [0001] The invention adopts Providencia bacteria to transform levodopa to produce danshensu, and belongs to the field of industrial microorganisms. Background technique [0002] Danshensu, scientific name R-(+)-3-(3,4-dihydroxyphenyl)-2-hydroxypropionic acid, D-(+)-β-(3,4-dihydroxyphenyl)lactic acid, English Name: Danshensu, (R)-(+)-3-(3,4-Dihydroxyphenyl)-lacticacid, (R)-(+)-3-(3,4-Dihydroxyphenyl)-2-hydroxypropanoic acid, is a Dextrophenolic acid compounds. [0003] Danshensu is an important active ingredient in the water extract of Danshen, and its structure was obtained and identified from the water extract of Danshen in 1980 in China (Study on the water-soluble active ingredients of Danshen, Ⅱ.D(+)β(3,4- The structure of dihydroxyphenyl) lactic acid, Journal of Shanghai First Medical College, 1980, 05 (7), 384-385), various studies have shown that Danshensu has important pharmacological and pharmacological effects, and is used in the treatment of cardi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12R1/01
CPCC12P7/42
Inventor 蔡宇杰徐锦槿邓华祥陈佳君王静钟妮尔白亚军郑晓晖
Owner 卓虹超源生物科技(郑州)有限公司
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