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Method for preparing L-5-calcium methyl tetrahydrofolate through enzymic method

A technology for the preparation of calcium methyltetrahydrofolate and enzymatic method, which is applied in organic chemistry, fermentation, etc., can solve the problems of inability to obtain L-tetrahydrofolate, difficulty in industrialization, and large pollution, and achieve good industrialization prospects and environmental protection. Friendly, high-yield effect

Inactive Publication Date: 2019-04-16
ZHEJIANG SHENGDA BIO PHARM
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  • Abstract
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  • Claims
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Problems solved by technology

The methods for chemically synthesizing L-5-methyltetrahydrofolate have many problems such as difficult separation, low yield, heavy pollution, and difficulty in industrialization. Therefore, it is of great practical significance to find a method other than chemical synthesis. explore
[0004] In the prior art related to the preparation of L-5-methyltetrahydrofolic acid calcium by enzymatic method, the document "Methods in Enzymology", 1971,18(6):728-731A New Preparation of Tetrahydrofolic Acid introduces that dihydrofolate reductase catalyzes the production of folic acid Dihydrofolate, and then use NADPH, the dialysis extract of Streptococcus faecalis, and glue-linked glucose-G25 to obtain racemic tetrahydrofolate, but this method cannot obtain L-tetrahydrofolate

Method used

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  • Method for preparing L-5-calcium methyl tetrahydrofolate through enzymic method

Examples

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Effect test

Embodiment 1

[0028] Example 1: Preparation of dihydrofolate reductase wet bacteria.

[0029] Get Escherichia coli genetically engineered bacteria with high expression of dihydrofolate reductase, inoculate them on LB plate for pre-culture (LB medium: 5g yeast extract per liter, 10g peptone, 10g sodium chloride, 20g agar powder), and then inoculate to Continue culturing in TB shake flasks (TB medium: 24g yeast extract per liter, 12g peptone, 2g potassium dihydrogen phosphate, 16g dipotassium hydrogen phosphate trihydrate, 5g glycerin). The supernatant and bacterial precipitates were collected into ziplock bags and frozen in a -20°C refrigerator for later use.

Embodiment 2

[0030] Example 2: Preparation of glucose dehydrogenase wet cells.

[0031] Take Escherichia coli genetically engineered bacteria with high glucose dehydrogenase expression, inoculate them on LB plate for pre-cultivation (LB medium: 5g yeast extract per liter, 10g peptone, 10g sodium chloride, 20g agar powder), and then inoculate into TB plate Continue culturing in shake flasks (TB medium: 24g yeast extract per liter, 12g peptone, 2g potassium dihydrogen phosphate, 16g dipotassium hydrogen phosphate trihydrate, 5g glycerin), collect the bacteria by centrifugation after the culture is completed, and discard the supernatant solution, and bacterial precipitates were collected in ziplock bags, and frozen in a -20°C refrigerator for later use.

Embodiment 3

[0033] (1) Add 100g of folic acid and 1000ml of water into a 2000ml reaction bottle, and add the pre-prepared NaOH solution (60gNaOH+165ml of water) dropwise under stirring. After the dropwise addition, add 50g of zinc powder, react at 10°C for 1 hour, add phosphoric acid to adjust the pH of the reaction solution to 7.0, and filter to obtain a dihydrofolic acid solution;

[0034] (2) Add 10g glucose dehydrogenase fermented wet cells and 20g dihydrofolate reductase wet cells to the filtrate, 45g glucose, 0.2g NADP, 10g disodium EDTA, react at 10°C for 1 hour, filter after the reaction, adjust with hydrochloric acid pH to 3.3, filtered to obtain L-tetrahydrofolic acid wet powder.

[0035] (3) Add the above-mentioned L-tetrahydrofolic acid wet powder into a 5000ml reaction bottle, add 600ml of water, adjust the pH to 7.5 with NaOH, and add 6.3ml of formaldehyde (1.0eq, calculated as folic acid) dropwise. Lower the temperature to 10°C, add sodium borohydride solution (25.7g (3eq,...

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Abstract

The invention belongs to the technical field of enzymatic synthesis, and relates to a method for preparing L-5-calcium methyl tetrahydrofolate through an enzymic method. The method comprises the following steps that 1, folic acid serves as the raw material, and zinc powder is used for making zinc powder reduced to be dihydrofolic acid; 2, dihydrofolic acid reductase and glucose dehydrogenase are added in dihydrofolic acid, and a reaction is conducted to obtain L-tetrahydrofolic acid; 3, formaldehyde and hydroboron are added in L-tetrahydrofolic acid, and a reaction is conducted for obtaining L-5-methyltetrahydrofolate; 4, L-5-methyltetrahydrofolate is salified, and L-5-calcium methyl tetrahydrofolate is obtained. Accordingly, the effective method for preparing L-5-calcium methyl tetrahydrofolate through the enzymic method is provided, the preparation method is free of splitting, mild in reaction, high in yield, high in product purity and friendly to environment, and has a very good industrial prospect.

Description

technical field [0001] The invention belongs to the technical field of enzymatic synthesis, and relates to a method for enzymatically preparing calcium L-5-methyltetrahydrofolate. Background technique [0002] Calcium L-5-methyltetrahydrofolate, also known as (6S)-5-calcium tetrahydrofolate. (6S)-5-Methyltetrahydrofolate is the main form of tissue and blood folic acid, it does not need to go through tedious enzymatic steps in the human body, and can directly participate in a variety of important biochemical reactions in the human body (such as purine and thymine synthesis, etc.). L-5-methyltetrahydrofolate can be used as medicine and food additives, vitamin preparations, prevention of neural tube defects, treatment of depression, treatment of megaloblastic anemia, etc. Studies have shown that L-5-methyltetrahydrofolate is The only folic acid drug that can penetrate the blood-brain barrier has the effect of preventing and treating Alzheimer's disease (senile dementia), so i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D475/04C12P17/18
CPCC07D475/04C12P17/182
Inventor 庞正查张仲景陈天松梁超刘露王程鹏
Owner ZHEJIANG SHENGDA BIO PHARM
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