Biosynthesis method of L-carnitine

A biosynthesis and carnitine technology, applied in the field of medicine, can solve the problems of electrodialysis desalination treatment technology, such as electricity consumption, high water consumption, shortening the use time of instruments, and strict turbidity requirements, so as to reduce toxic effects, The effect of short conversion time and low catalyst dosage

Inactive Publication Date: 2018-05-11
武汉佰施达生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The shortcoming that the preparation method of above-mentioned L-carnitine exists is: one, splitting method, no matter be chemical splitting or enzymatic splitting, research and development cycle is all longer, and splitting process route is more complicated, and the screening workload of enzyme is also relatively long. Larger, and most of them need to use more toxic cyanide as raw material
2. The asymmetric chemical synthesis of L-carnitine, the reaction process produces a large amount of sodium chloride. At present, the electrodialysis desalination process is widely used to remove sod

Method used

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preparation example Construction

[0032] A biosynthetic method for L-carnitine, comprising the following steps:

[0033] 1) Dissolve the substrate ethyl 4-chloroacetoacetate in MOPS buffer solution, then add whole cells of engineering bacteria containing ketoreductase, coenzymes, butanediol, surfactants and additives, and react to control the pH value to 6.5 -7.5, generating (R)-4-chloro-3-hydroxyl-butyric acid ethyl ester;

[0034] 2) Dissolving sodium hydroxide in an aqueous trimethylamine solution with a mass ratio of 25% to form a mixed solution, and then slowly adding the mixed solution dropwise to the (R)- In 4-chloro-3-hydroxy-butyric acid ethyl ester, react at -5-5°C for 20-30 hours, stop the reaction, adjust the pH to 5-7, and then obtain L-carnitine through purification.

[0035] Preferably, the coding gene of the engineering bacterium containing ketoreductase is the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing.

[0036]Further, the concentration of whole cells of engineering b...

Embodiment 1

[0042] The biosynthetic method of L-carnitine, comprises the steps:

[0043] 1) The reaction was carried out in a 1L shake flask, and the reaction system was controlled to 300mL, including 27.27mL ethyl acetate and 272.73mL MOPS buffer solution. First, 15g of 4-chloroacetoacetate substrate was injected, and 60g / L of ketone-containing The entire cell of the enzyme engineering bacteria is used as a catalyst, and 0.135g of coenzyme NAD is added + , 14.78g (0.164mol) of butanediol, then add surfactant and additive to establish biosynthesis system and carry out conversion reaction, the pH of the control reaction system is 7, with FeCl 3 Chromogenic method to instantly determine the time point of feeding and the end point of conversion: during the reaction, take a sample, extract it with ethyl acetate of the same volume as the sample, centrifuge, take the upper organic phase, and the colorimetric system of the cuvette is 3ml ethanol, 10ul Ethyl acetate extract phase, 35ul of 1M FeC...

Embodiment 2

[0047] The biosynthetic method of L-carnitine, comprises the steps:

[0048] 1) The reaction is carried out in a 50L fermenter, and the reaction system is controlled to 30L, including 2.7273L ethyl acetate and 27.27L MOPS buffer solution. The whole cell of the reductase engineered bacteria is used as a catalyst, and 18g of coenzyme NAD is added + , 1971.16g (21.8726mol) of butanediol, then add surfactant and additive to establish biosynthesis system and carry out conversion reaction, the pH of the control reaction system is 7, with FeCl 3 Chromogenic method to instantly determine the time point of feeding and the end point of conversion: during the reaction, take a sample, extract it with ethyl acetate of the same volume as the sample, centrifuge, take the upper organic phase, and the colorimetric system of the cuvette is 3ml ethanol, 10ul Ethyl acetate extract phase, 35ul of 1M FeCl 3 Carry out the color reaction, write down the absorbance value of the ultraviolet spectroph...

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Abstract

The invention discloses a biosynthesis method of L-carnitine. The biosynthesis method comprises: 1) dissolving a substrate ethyl 4-chloroacetoacetate in a MOPS buffer solution, adding ketoreductase-containing engineering bacteria whole cells, coenzyme, butylene glycol, a surfactant and an additive, and carrying out a reaction to generate (R)-4-chloro-3-hydroxy-butyric acid ethyl ester, wherein thepH value is controlled at 6.5-7.5; and 2) dissolving sodium hydroxide in a trimethylamine aqueous solution with a mass ratio of 25% to form a mixed solution, slowly adding the mixed solution to the (R)-4-chloro-3-hydroxy-butyric acid ethyl ester generated in the step 1) at a temperature of -5-5 DEG C in a dropwise manner, carrying out a reaction for 20-30 h at a temperature of -5-5 DEG C, stopping the reaction, adjusting the pH value to 5-7, and purifying to obtain L-carnitine. According to the present invention, the biosynthesis method has characteristics of strong stereoselectivity, mild reaction conditions, low cost, low pollution, high efficiency, high yield and high optical purity of the product.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to a biosynthesis method of L-carnitine. Background technique [0002] L-carnitine is a kind of amino acid that promotes the conversion of fat into energy. Red meat is the main source of L-carnitine, which has no toxic and side effects on the human body. Different types of daily diets already contain 5-100 mg of L-carnitine, but the average person can only take in 50 mg a day from meals, and vegetarians take in even less. The main physiological function of L-carnitine is to promote the conversion of fat into energy. Taking L-carnitine can reduce body fat and weight without reducing water and muscle. In 2003, L-carnitine was recognized as The safest weight loss nutritional supplement without side effects. In recent years, L-carnitine has been widely used in the fields of nutrition and health care, weight loss and bodybuilding, improving athletic ability, fatigue recovery, food add...

Claims

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Application Information

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IPC IPC(8): C12P13/00
CPCC12P13/007
Inventor 陈建华胡磊徐灿
Owner 武汉佰施达生物技术有限公司
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