CN1057518C

CN1057518C - Process for preparation of optically active amino-acid by hot- hydrolysis of nitrogen-ammonia formyl-amino acid

Info

Publication number: CN1057518C
Application number: CN95116833A
Authority: CN
Inventors: 江宁; 贺鹏; 孙万儒; 卢大军; 强亚静; 杨柳
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 1995-09-29
Filing date: 1995-09-29
Publication date: 2000-10-18
Anticipated expiration: 2015-09-29
Also published as: CN1146447A

Abstract

The present invention relates to a method for preparing optically active amino acid, which is characterized in that D type amino acid or L type amino acid of aliphatic series and aromatic series with optical activity is prepared by the pyrohydrolysis reaction of N-carbamyl amino acid; the concentration of reactant is from 0.1 to 45%(W/V), and the reaction temperature is from 80 to 140 DEG C; the reaction process is always kept within the range of weak acid with the pH of 3.0 to 6.0 or within the range of weak alkali with the pH of 9.6 to 10.8, and the reaction time is from 4 to 72 hours. The reactant is concentrated, and the pressure is reduced under the pressure of 10 to 600 mmHg after the reaction is finished; the volume of reactant is reduced to one twentieth to one half of initial volume; the ph value of concentrated liquid is adjusted to the isoelectric point of the amino acid by 30% of NaOH or 3NHCl; the concentrated liquid is cooled and crystallized at the room temperature or in a cold water bath, etc.; the crystallization temperature is from -2 to 30 DEG C, and the amino acid described by the present invention can be obtained. The preparation method has high efficiency, low cost and no pollution.

Description

The method of nitrogen-preparation of optically active amino-acid by hot-hydrolysis of nitrogen-ammonia formyl-amino acid

The present invention relates to amino acid whose preparation method, relate in particular to a kind of chiral compounds-optical activity D type or the amino acid whose preparation method of L type.

As is generally known, prepare the D type in recent years or the amino acid whose development work of L type has suitable progress.As hydantoin derivative from 5 replacements; generate nitrogen-carbamyl-D (or L)-amino acid through single-minded glycolylurea enzyme open loop; hydrolysis obtains D (or L)-amino acid again; be the new way [syldatk of a preparation optically active amino acids of nearly more than ten years exploitation; C.et al, in Advances inBiochem.Engin./Biotechnol.Fiechter, A; ed.VOl.41,291-316 (1990)]:

Racemic hydantoin derivative can synthesize [Bucherer, H.T., Steiner, W., J.Prakt.Chem., 140,291-316 (1934)] with cheap raw material aldehyde.Reaction (1) can spontaneously be carried out.Reaction (2) used glycolylurea enzyme claims dihydropyrimidinase (EC 3.5.2.2.) again, the substrate stereospecificity of D or L, discovery [Waltach, D., Grisoloia are all arranged in animal and plant and microorganism, S.J., Biol.Chem., 226,227-288 (1957)], at present obtained producing the higher microorganism [Sun Wanru of enzyme activity, the microorganism journal, 23 (2), 133-142 (1983); Jiangning etc., microorganism journal, 35 (5) .347-350 (1995)].Reaction (3) can pass through chemical method [Tankahashi, S.et al., J.Ferment.Technol., 4,328-332 (1979)] or enzyme process [Olivieri, R.et al, Enzyme Microb.Technol., 1,201-204 (1979)] carries out.Its reaction is as follows:

These two synthetic routes are longer for producing non-natural D type amino acid or pathways metabolism, have important use with the not high L type amino acid of fermentative Production rate and are worth.

But there is following problem in these two lines in practical application:

Carcinogens nitrous acid has been used in reaction (3-1), can bring serious environmental to pollute.The enzyme that reaction (3-2) is used, because microbial metabolism itself, its vigor has only 1/100 to 1/10 of glycolylurea enzyme, causes reaction (3) and reaction (2) to be difficult to coupling, has limited the practical application of this route.

The objective of the invention is to a non-environmental-pollution, effectively reaction replaces the chemical reaction of (3-1) or enzyme reaction (3-2) again.This reaction is exactly nitrogen-carbamyl amino acid thermal hydrolysis reaction (the amino acid whose concentration of reactant nitrogen-carbamyl is the 0.1-45% weight/volume) that we find:

When temperature surpassed 80 ℃, reaction (3-3) can be carried out, and reaches balance very soon, but as long as maintenance system under weak acid or weak base condition, constantly adds 3NHCl in reaction process, the PH of maintenance system makes CO between 3.0-6.0 ₂Constantly discharge: Or constantly adding 30%NaOH, the PH that keeps reaction system makes NH between 9.6-10.8 ₃Gas is constantly discharged:

Just can disequilibrate, reaction is carried out fully.

Above-mentioned two reactions (3-3-1) and temperature (3-3-2) are high more, speed of response is fast more, but consider the withstand voltage and molecular balance of equipment, temperature is also unsuitable too high, be suitable generally with 80 ℃-140 ℃, 95 ℃-120 ℃ more suitable, is suitable with 4-72hr. generally in this kind following reaction times of condition, and 16-48hr. is more suitable.Another key of this reaction is control PH, and strong excessively as acidity, nitrogen-carbamyl amino acid becomes hydantoin derivative with closed loop, promptly reacts the reversed reaction of (2).Strong excessively as alkalescence, the amino acid that obtains loses optical activity with racemization.

Reaction generally under 10-600mmHg pressure, better is evaporated to 1/20 to 1/2 of original volume after finishing under 10-200mmHg pressure, concentrating degree can be determined according to the starting point concentration of reactant.Transfer PH to this amino acid whose iso-electric point (can find or record) with 3NHCl or 30%NaOH again from relevant handbook with experiment, then, crystallisation by cooling in room temperature or cooling bath or ice-water bath or cryosel bath, Tc is-2 ℃-30 ℃, can obtain optically active amino acid after the filtration.

Resulting like this optical activity D type or L type amino acid, its structure is:

In the formula, the R-base is CH ₃-, Or

The invention solves the pollution problem of chemical method, except the soda acid of transferring PH to use, do not need any chemical reagent.Its efficient has increased significantly than enzyme process again.

For technology contents of the present invention further is described in further detail, for following embodiment:

Embodiment 1

In the 250ml round-bottomed flask, add 4.0g nitrogen-carbamyl-D-(-)-phenylglycine, add 100ml distilled water again, thorough mixing is transferred liquid PH to 10.0 with 30%NaOH.

Above-mentioned flask is loaded onto reflux, and after being heated to 100 ℃ of boilings on the electric mantle, reheat back flow reaction 35hr. constantly adds 30%NaOH therebetween and keeps PH between 10.0-10.6, and reaction conversion ratio is 65% when stopping.

Above-mentioned reaction solution is concentrated under 80mmHg pressure reduced pressure, stop when being concentrated into 30ml, change in the beaker, add 3NHCl accent PH to 5.0 down in stirring, have a large amount of D-(-)-phenylglycine crystal to separate out, beaker is cooled off in ice-water bath, suction filtration gets D-(-)-phenylglycine crystal after liquid is crossed in 4 ℃ of placement crystallizations, get crystal 1.78g, polarimetry purity 80%, yield 56% after the drying.

Embodiment 2

In the 250ml round-bottomed flask, add 4.0g nitrogen-carbamyl-D-(-)-phenylglycine; add 100ml distilled water again; behind the thorough mixing; transfer PH=3.0 with 3NHCl; on electric mantle, be heated to 100 ℃ of boiling 30hr.; constantly add 3NHCl therebetween and keep PH between 3.0-5.0, reaction records transformation efficiency when stopping be 90%.

Above-mentioned reaction solution is concentrated into about 30ml under 80mmHg pressure reduced pressure, change in the beaker, it is molten to add 30%NaOH while stirring, transfers PH=5.0, has a large amount of D-(-)-phenylglycine crystal to separate out, beaker is cooled off in ice-water bath, after 4 ℃ of placement crystallizations were spent the night, suction filtration got D-(-)-phenylglycine crystal, got crystal 2 .40g after the drying, polarimetry purity 97%, yield 75%.

Embodiment 3

In the 250ml beaker, add 4.0g racemization benzene glycolylurea, and add 50ml0.05MNa ₂HPO ₄The aqueous solution, thorough mixing.

The microorganism cells 50ml0.05MNa that will have D-glycolylurea enzyme activity ₂PO ₄The aqueous solution suspends, and this bacterium suspends and benzene glycolylurea solution merges, and at 35 ℃ of following stirring reactions, in 8.5, transformation efficiency reaches more than 90% stopped reaction after the 8hr. with the constant reaction solution PH of NaOH.

Transfer reaction solution PH=5.0 with HCl, boil, take advantage of heat filtering, must contain the solution of 3.70g nitrogen-carbamyl-D-(-)-phenylglycine.

Transfer this solution PH=3.0 with NHCl, change in the 250ml round-bottomed flask, be heated to 100 ℃ of boiling 30hr. on electric mantle, constantly add 3NHCl therebetween and keep PH between 3.0-5.0, reaction conversion ratio is 90%.

Above-mentioned reaction solution is concentrated into about 30ml under 80mmHg pressure reduced pressure, change in the beaker, transfer PH=5.0 in stirring down with 30%NaOH, have a large amount of D-(-)-phenylglycine crystal to separate out, beaker is cooled off in ice-water bath, place 4 ℃ of crystallizations to spend the night, suction filtration after the crystal drying, gets D-(-)-phenylglycine crystal 2 .17g, polarimetry purity 94%, yield 60%.

Embodiment 4

In the 100ml round-bottomed flask, add 0.4g nitrogen-carbamyl-L-(+)-phenylalanine; add 20ml distilled water again; thorough mixing; transfer PH3.0 with NHCl; on electric mantle, be heated to 100 ℃ of boiling 30hr.; constantly add 3NHCl therebetween and keep PH between 3.0-5.0, recording reaction conversion ratio when stopping is 88%.

Above-mentioned reaction solution is concentrated into about 30ml under 80mmHg pressure reduced pressure, change in the beaker, add 3NHCl while stirring and transfer PH=5.5, have a large amount of L-(-)-phenylalanine crystal to separate out, beaker is cooled off in ice-water bath, after placing 4 ℃ of crystallizations to spend the night, suction filtration after the crystal drying, gets L-(-)-phenylalanine 0.24g, polarimetry purity 97%, yield 73%.

Claims

1. optical activity D type or the amino acid whose preparation method of L type; it is characterized in that by the amino acid whose thermal hydrolysis reaction of nitrogen-carbamyl; to have optically active nitrogen-carbamyl amino acid to slough carbamyl, optically active D type or L type amino acid are arranged accordingly, this method comprises:

1) the amino acid whose concentration of reactant nitrogen-carbamyl is the 0.1-45% weight/volume;

2) temperature of reaction is 80-140 ℃;

3) with the PH of 3NHCl or 30%NaOH conditioned reaction system, in reaction process, PH is remained in the scope of weak acid PH3.0-6.0 or weak base PH9.6-10.8;

4) reaction times is 4-72 hour;

5) after reaction finishes, under 10-600mmHg pressure, be evaporated to the 1/20-1/2 of original volume;

6) concentrated solution transfers PH to this amino acid whose iso-electric point with 30%NaOH or 3NHCl;

7) crystallisation by cooling in room temperature or cooling bath or ice-water bath or cryosel are bathed, Tc be-2-30 ℃, promptly obtain optical activity D type or L type amino acid;

Described D type or the amino acid whose structure of L type are:

Wherein, the R-base is CH ₃-, Or

2. optical activity D type according to claim 1 or the amino acid whose preparation method of L type is characterized in that described temperature of reaction is 95-120 ℃.

3. optical activity D type according to claim 1 or the amino acid whose preparation method of L type is characterized in that the described reaction times is 16-48 hour.

4. optical activity D type according to claim 1 or the amino acid whose preparation method of L type is characterized in that described pressure is 10-200mmHg.