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Method for producing optically pure L-alanine by one pseudomonas and two enzymes

An alanine and optical technology, applied in the field of enzymatic production of L-alanine, can solve the problems of high cost, many production links, low optical purity of products, etc., achieve high yield, reduce environmental pollution, and reduce production The effect of link and equipment investment

Inactive Publication Date: 2009-11-18
ANHUI HUAHENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0017] As can be seen from the above, the existing research and production reports on the production of L-alanine by enzymatic methods generally utilize Pseudomonas to produce L-aspartic acid-β-decarboxylase, and convert L-aspartic acid to generate L -Alanine, the optical purity of the product produced by this method is low, generally ≤95%, and there are many production links and high cost

Method used

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  • Method for producing optically pure L-alanine by one pseudomonas and two enzymes

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Experimental program
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Effect test

Embodiment 1

[0041]Prepare seed solution 1000ml, seed solution proportioning (mass percentage, hereinafter the same): 1.0% fumaric acid, 1.0% peptone, 0.8% corn steep liquor, 0.02% magnesium sulfate, 0.01% potassium dihydrogen phosphate, and the balance is purified water, Adjust the pH to 7.0 with ammonia water. Pack in five 1000ml Erlenmeyer flasks after sterilization, inoculate the inclined surface at 30°C, incubate on a shaking table for 15 hours at a rotating speed of 90r / min, inoculate 100L of fermentation broth, the ratio of fermentation broth is 2.0% sodium glutamate, 1.0% peptone , 0.8% of corn steep liquor, 0.02% of magnesium sulfate, 0.01% of potassium dihydrogen phosphate, the balance is pure water, and the pH value is adjusted to 7.0 with ammonia water. Fermentation culture conditions: culture temperature 29±2°C, ventilation ratio 1:2, tank pressure 0.05MPa, culture for 26 hours. Take the fermentation broth to prepare 500L conversion liquid, add 25g ammonium fumarate, the conv...

Embodiment 2

[0043] Mix 2000ml of seed solution, the ratio of seed solution is 1.2% fumaric acid, 1.1% peptone, 0.9% corn steep liquor, 0.03% magnesium sulfate, 0.02% potassium dihydrogen phosphate, the balance is pure water, adjust the pH value to 7.0 with ammonia water . Pack in 10 1000ml Erlenmeyer flasks after sterilization, inoculate the inclined surface at 30°C, incubate on a shaking table for 18 hours, the rotating speed of the shaking table is 100r / min, inoculate 100L fermentation broth, the ratio of fermentation broth is 2.2% sodium glutamate, 1.1% peptone , 0.9% of corn steep liquor, 0.03% of magnesium sulfate, 0.02% of potassium dihydrogen phosphate, and the balance is pure water, and the pH value is adjusted to 7.0 with ammonia water. Fermentation culture conditions: culture temperature 29±2°C, ventilation ratio 1:2, tank pressure 0.05MPa, culture for 28 hours. Take the fermentation broth to prepare 500L transformation liquid, add 25g ammonium fumarate, the transformation temp...

Embodiment 3

[0045] Mix 800ml of seed solution, the ratio of seed solution is 0.8% fumaric acid, 0.8% peptone, 0.6% corn steep liquor, 0.01% magnesium sulfate, 0.01% potassium dihydrogen phosphate, the balance is pure water, adjust the pH value to 7.0 with ammonia water . Divide into four 1000ml Erlenmeyer flasks after sterilization, inoculate the slope at 30°C, incubate on a shaking table for 14 hours at a speed of 110r / min, inoculate 100L of fermentation broth, the ratio of fermentation broth is 1.8% sodium glutamate, 0.9% peptone , 0.6% of corn steep liquor, 0.01% of magnesium sulfate, 0.01% of potassium dihydrogen phosphate, and the balance is pure water, and the pH value is adjusted to 7.0 with ammonia water. Fermentation culture conditions: culture temperature 29±2°C, ventilation ratio 1:3, tank pressure 0.05MPa, culture 20h. Take the fermentation broth to prepare 500L of transformation liquid, add 25g of ammonium fumarate, the transformation temperature is 45°C, and the transformat...

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Abstract

The invention discloses a method for producing optically pure L-alanine by one pseudomonad and two enzymes, comprising the following steps of: seed culture in a shake flask and fermentation; utilizing pseudomonas to generate two enzymes: an L-aspartase and an L-aspartic acid-Beta-decarboxylase; taking a fumaric acid ammonium salt as a substrate to directly generate the L-alanine; and obtaining the optically pure L-alanine by changing the system reaction condition and adding the L-alanine racemase inhibitor. The method reduces the whole production link and equipment investment for the L-alanine, lightens the environmental pollution, has high yield of L-alanine, has the optical purity of more than or equal to 99.99% and meets the requirements of factories that have special requirements on the L-alanine with high optical purity.

Description

technical field [0001] The invention relates to a production method of L-alanine, in particular to a method for producing L-alanine by an enzymatic method. Background technique [0002] In recent years, research on chiral drugs is in the ascendant, and there are many domestic companies producing chiral drugs. Different chiral drugs have great differences in efficacy and side effects. L-alanine is a widely used source of chirality for pharmaceuticals, such as the production of L-aminopropanol, VB 6 , S-2-Chloroacetic acid, An Niaotong, etc. [0003] In the process of using L-alanine, domestic and foreign enterprises have higher and higher requirements for its optical purity. L-alanine synthetic drug with low optical purity is found to have low curative effect and large side effects during use. Therefore, it is imperative to study a high optical purity L-alanine production process. [0004] Domestic companies such as Ningbo Kerui Biological Engineering Co., Ltd., Beijing ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P41/00C12P13/06C12R1/38
Inventor 黄建坡张晓斌蒋光玉唐思青
Owner ANHUI HUAHENG BIOTECH
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