CN1900298A

CN1900298A - Method for preparing D-tyrosine by enzyme method

Info

Publication number: CN1900298A
Application number: CN 200610082015
Authority: CN
Inventors: 马云峰; 柴多里; 王组元
Original assignee: ANHUI HORAE NEW TECHNOLOGY DEVELOPMENT Co Ltd
Current assignee: ANHUI HORAE NEW TECHNOLOGY DEVELOPMENT Co Ltd
Priority date: 2006-07-17
Filing date: 2006-07-17
Publication date: 2007-01-24

Abstract

The present invention discloses preparation of chiral organic compound, and is especially process of preparing D-tyrosine from DL-tyrosine. The process includes the following steps: the first acylation reaction between DL-tyrosine and acylating reagent in the presence of organic acid to produce N-acetyl-DL-tyrosine; dissolving N-acetyl-DL-tyrosine in deionized water to compound 0.5-1.0 mol/L concentration solution; regulating pH value with alkali and filtering to obtain N-acetyl-DL-tyrosine solution; reaction between the N-acetyl-DL-tyrosine solution and D-acylating hydrolase fixed in enzyme column, eluting, decolorizing the reacted liquid with decolorizing agent, filtering, concentrating, cooling, crystallizing, rinsing with small amount of deionized water and drying to obtain D-tyrosine; and preparing L-tyrosine with the crystallized mother liquid. The process may be used in industrial production.

Description

Method for preparing D-tyrosine by enzyme method

Technical Field

The invention relates to a preparation method of a chiral organic compound, in particular to a method for preparing D-tyrosine from DL-tyrosine.

Background

Tyrosine, chemical name α -amino- β p-hydroxyphenylpropionic acid, alsoknown as p-hydroxyphenylalanine, has special physiological properties and is of great use in the food, feed and agricultural industries.

L (-) -tyrosine D (+) -tyrosine

L-tyrosine is a natural amino acid, the performance of which has long been known and is widely applied in the fields of medicine, food, feed, chemical industry and the like. For example, L-tyrosine can be used as an additive of food and feed, and can be used as a raw material of compound amino acid infusion in the pharmaceutical industry. D-tyrosine is an unnatural amino acid which is considered to be useless in the past, so that the research on the performance and the action of the D-tyrosine is only started in recent years, and particularly with the continuous deepening of scientific research, the D-tyrosine is found to have unique physiological performance and has the function which cannot be replaced by the L-tyrosine in life activities and medicine preparation. At present, D-tyrosine has been developed into a chiral source with wide application prospect, namely, the D-tyrosine can be used as a chiral precursor to synthesize a series of chiral drugs, and the chiral drugs have some special effects. For example, with D-tyrosine as a precursor, synthetic atosiban is an important tocolytic; the synthesized polypeptide has special curative effect on treating malignant tumor; the synthesized anisidine has the functions of resisting protozoa, resisting metamorphosis, resisting fungi, resisting tumors, preventing and treating plant fungi and the like; d-tyrosine can also be used for synthesizing antibacterial drugs for treating infection of mould, rhodobacter enzymaticus, yeast and parasite; in addition, D-tyrosine has wide application in synthesizing anti-inflammatory drugs with excellent curative effects on inflammations of lung, central nervous system, liver, joint, endocardium, eye, ear, skin, intestines and stomach, urinary system and the like. At present, most of the drugs are still in the clinical experimental stage of pharmacological efficacy, and if the drugs are on the market completely, the demand of D-tyrosine must be promoted to be increased sharply. Statistics show that no enterprise for producing D-tyrosine in large scale exists at home and abroad at present, and the main restriction factor is that no technology which can be applied to producing D-tyrosine in large scale and at low cost exists at present. Therefore, research and development of a D-tyrosine preparation technology are necessary.

D-tyrosine is an unnatural amino acid, and unlike L-tyrosine, D-tyrosine cannot be extracted from natural substances and can only be obtained by chiral synthesis or resolution. Chiral synthesis is one of the important methods for preparing chiral compounds, but at present, most chiral compounds still have no suitable synthetic method and reagent found, so that the resolution method is the most economically feasible chiral compound preparation method at present.

Since the knowledge of D-tyrosine is late, the research on the resolution and preparation technology of D-tyrosine is also attracting attention in recent years. Therefore, reports about the resolution preparation technology of D-tyrosine are rare at foreign countries so far, and are more rare at home.

According to the investigation, the D-acylated hydrolase extracted and purified from the microorganisms of A.dentrifians DA181 and A.faecalis among the existing D-acylated hydrolases has higher stereoselectivity and optical activity, and the enzyme has good stability and is easy to purify, so that the D-tyrosine is prepared by splitting the racemic tyrosinic acid by using the purchased D-acylated hydrolase.

Disclosure of Invention

The invention aims to provide a method for preparing D-tyrosine by an enzyme method, which has high efficiency and low cost and can be industrialized.

The method for preparing D-tyrosine by using the enzyme method disclosed by the invention comprises the following steps of:

a. firstly, feeding DL-tyrosine and an acylating reagent in a weight ratio of 1: 1-3: 1(W) under the condition of organic acid to carry out acylation reaction to prepare N-acetyl-DL-tyrosine;

b. purifying the D-acylation hydrolase, and fixing the acylation hydrolase by using an enzyme column;

c. preparing 0.5-1.0mol/L solution from N-acetyl-DL-tyrosine and deionized water, adjusting the pH value to 7-8 with alkali, and filtering to obtain an N-acetyl-DL-tyrosine solution;

d. the N-acetyl-DL-tyrosine solution passes through an enzyme column fixed with D-acylation hydrolase at the temperature of 40-60 ℃ and the reaction time is 5-8 hours;

e. eluting, decolorizing the reaction solution with decolorizing agent at 80-90 deg.C, filtering, concentrating, cooling, crystallizing, rinsing with small amount of deionized water, and drying to obtain D-tyrosine. The crystallization mother liquor can prepare L-tyrosine.

The purified acylation hydrolase is a novel D-acylation hydrolase which has high activity, strong stereoselectivity and strong inhibition resistance under the condition of industrially effective substrate concentration and only deacylates N-acetyl-D-tyrosine in N-acetyl-DL-tyrosine to synthesize D-tyrosine; the acylating agent used in the acylation process is acetic anhydride; the organic acid is acetic acid; the alkali used for adjusting the pH value of the N-acetyl-DL-tyrosine solution is sodium hydroxide solution or ammonia water; the decolorizing agent used in decolorizing process is adsorptive decolorizing agent such as activated carbon or diatomaceous earth or clay.

The invention provides a new enzymatic process for preparing D-tyrosine from DL-tyrosine, and the purified D-acylated hydrolase has the characteristics of high activity, strong stereoselectivity and strong inhibition resistance under the condition of industrially effective substrate concentration, so that the yield of the D-tyrosine reaches over 88 percent, the optical purity of a D-tyrosine product is greatly improved to over 99.5 percent ee, and the production cost is reduced. The acylating agent and the organic acid used in the acylation process, the alkali used for adjusting the pH value of the acylated DL-tyrosine solution and the decolorizing agent used in the decolorization process are all common chemical raw materials for industrial production.

In conclusion, the enzymatic novel process for preparing D-tyrosine from DL-tyrosine provided by the invention can be used for industrial production of D-tyrosine.

Detailed description of the preferred embodiments

The DL-amino acid is acylated under certain condition with acylating reagent to produce N-acetyl-DL-amino acid, which is deacetylated and hydrolyzed under the action of specific acylating hydrolase to obtain D-amino acid. In the invention, a D-tyrosine acylation hydrolase with high activity, strong inhibition resistance and high stereoselectivity to N-acetyl-D-tyrosine is purified, and the D-tyrosine is obtained by deacetylating the N-acetyl-D-tyrosine in the N-acetyl-DL-tyrosine.

Example 1

Step 1 preparation of N-acetyl-DL-tyrosine

Dissolving 36.2 gDL-tyrosine (0.2mol) and 24.0g acetic acid (0.4mol) in 200ml deionized water, adding 24.0g acetic anhydride (0.2mol), stirring and reacting at 40-60 ℃ for 3-6 hours, distilling and recovering unreacted acetic acid, cooling to room temperature, cooling in an ice water bath for 1 hour, filtering, centrifuging, washing and drying to obtain 42.6g of product with the yield of 95.5%. The obtained N-acetyl-DL-tyrosine is ready for use.

Step 2 purification and immobilization of D-acylhydrolase

The commercially available D-acylated hydrolase thallus is added into a dissolving tank (pH7.8), is partially purified by a diethylaminoethyl chromatographic column, is immobilized by an enzyme column, and is stored at low temperature for later use.

Tests show that each gram of wet thallus can respectively produce 2245 units of D-acylation hydrolase and 852 units of L-acylation hydrolase. The immobilized D-acylhydrolase contained 13500 enzyme activity units per ml of gel. The free enzyme and immobilized enzyme have a mie constant of 1.0 and 0.22, respectively, when tested using N-acetylmethionine as a substrate. The pH value in the immobilized enzyme resolution reaction is 7.5, and the temperature is 45 ℃. The free enzyme is stable in the pH range of 5.0-11.0, and the immobilized enzyme is easy to dissolve above pH 9.0. Both enzymes were stable at 40 ℃. The enzyme activity of the immobilized enzyme is still kept 90% after the immobilized enzyme is continuously resolved for 20 days at 45 ℃.

Step 3, resolving racemic tyrosine by using D-acylated hydrolase to prepare D-tyrosine

Dissolving 42.6g N-acetyl-DL-tyrosine prepared in the step 1 in 300ml of deionized water, and adjusting the pH value to 8.0 by using a sodium hydroxide solution. The reaction solution was passed through an immobilized enzyme column at 45 ℃ and then eluted with an appropriate amount of ethanol. Transferring the eluent into a three-neck flask, adding 2g of active carbon,stirring at 85 deg.C for 40 min, and filtering while it is hot. The filtrate was concentrated in vacuo at 60 ℃ to 1/3 at the original volume at which timeA large amount of crystals are separated out, the mixture is cooled and crystallized for 3 to 6 hours at the temperature of between 5 and 10 ℃, the mother liquor is centrifugally separated, the filter cake is dried, and the D-tyrosine product 32.5g is obtained, and the optical rotation of the product is detected to be [ α]]_D ²⁰The resulting product had an optical purity of 99.7% ee at +11.7 °, and an enzyme resolution yield of 94.0%. The total yield of the product is 89.8%.

Example 2

Step 1 preparation of N-acetyl-DL-tyrosine

54.3 gDL-tyrosine (0.3mol) and 36.0g acetic acid (0.6mol) are dissolved in 200ml deionized water, 24.0g acetic anhydride (0.2mol) is added, the mixture is stirred and reacted for 4 to 6 hours at the temperature of between 50 and 65 ℃, unreacted acetic acid is recovered by distillation, the mixture is cooled to room temperature and then cooled for 45 minutes by an ice water bath, and then 64.2g products are obtained by filtering, centrifuging, washing and drying, wherein the yield is 96.0%. The obtained N-acetyl-DL-tyrosine is prepared for standby.

Step 2 purification and immobilization of D-acylhydrolase

The enzyme column prepared in step 2 of example 1 was used.

Dissolving 64.2g of N-acetyl-DL-tyrosine prepared in the step1 in 300ml of deionized water, adjusting the pH value to 7.5 by using ammonia water, passing the reaction liquid through an immobilized enzyme column at 50 ℃, eluting by using a proper amount of ethanol, transferring the eluent into a three-neck flask, adding 5g of clay for decolorization, keeping the temperature and stirring for 1 hour at 90 ℃, filtering while hot, vacuum-concentrating the filtrate at 60 ℃ to 1/3 of the original volume, separating out a large amount of crystals, cooling and crystallizing for 4-5 hours at 2-5 ℃, centrifugally separating mother liquor, drying a filter cake to obtain 49.9g of D-tyrosine product, and detecting the optical rotation of the product to be [ α]]_D ²⁰The resulting product had an optical purity of 99.9% ee at +12.0 ℃ and an enzyme resolution yield of 95.8%. The total yield of the product is 92.0 percent.

Example 3

Step 1 preparation of N-acetyl-DL-tyrosine

72.4 gDL-tyrosine (0.4mol) and 42.0g acetic acid (0.7mol) are dissolved in 350ml deionized water, 24.0g acetic anhydride (0.2mol) is added, the mixture is stirred and reacted for 4 to 5 hours at the temperature of between 50 and 60 ℃, unreacted acetic acid is recovered by distillation, the mixture is cooled to room temperature and then cooled for 1.5 hours by an ice water bath, and 84.4g products are obtained by filtering, centrifuging, washing and drying, and the yield is 94.6%. The obtained N-acetyl-DL-tyrosine is ready for use.

Step 2 purification and immobilization of the D-aminoacylase

The enzyme column prepared in step 2 of example 1 was used.

Dissolving 84.4g of N-acetyl-DL-tyrosine prepared in the step 1 in 500ml of deionized water, adjusting the pH value to 7.5 by using a sodium hydroxide solution, passing the reaction liquid through an immobilized enzyme column at 45 ℃, eluting by using a proper amount of ethanol, transferring the eluent into a three-neck flask, adding 5g of activated carbon, keeping the temperature and stirring for 40 minutes at 80 ℃, filtering while hot, vacuum-concentrating the filtrate at 60 ℃ to 1/3 of the original volume, separating out a large amount of crystals, cooling and crystallizing for 5-6 hours at 2-8 ℃, centrifugally separating mother liquor, drying a filter cake, obtaining 63.4g of D-tyrosine product, and detecting the optical rotation of the product to be [ α]]_D ²⁰The resulting product had an optical purity of 99.5% ee at +11.2 ° and an enzyme resolution yield of 93.1%. The total yield of the product is 88.1 percent.

Claims

1. A method for preparing D-tyrosine by an enzyme method comprises the following steps:

2. The enzymatic process of D-tyrosine according to claim 1, characterized in that: the purified D-acylated hydrolase is a novel D-acylated hydrolase which has high activity, strong stereoselectivity and strong inhibition resistance under the condition of industrially effective substrate concentration and only deacetylates N-acetyl-D-tyrosine in an N-acetyl-DL-tyrosine solution to synthesize D-tyrosine.

3. The enzymatic process of D-tyrosine according to claim 1, characterized in that: the acylation reagent used in the acylation process is acetic anhydride, and the DL-tyrosine and the acetic anhydride are fed for acylation reaction according to the weight ratio of 1: 1-3: 1(W) under the condition of organic acid.

4. The enzymatic process of D-tyrosine according to claim 1, characterized in that: the organic acid used in the acylation process is acetic acid, and the weight ratio of DL-tyrosine to acetic acid is 1: 1.5-1: 1.7.

5. The enzymatic process of D-tyrosine according to claim 1, characterized in that: the alkali used for adjusting the pH value of the N-acetyl-DL-tyrosine solution is sodium hydroxide solution or ammonia water.

6. The enzymatic process of D-tyrosine according to claim 1, characterized in that: the decolorizing agent used in decolorizing process is adsorptive decolorizing agent, such as activated carbon or diatomaceous earth or clay, and its dosage is 0.005% by weight of DL-tyrosine.