Process for preparing L-threonine

Disclosed is a process for preparing L-threonine which comprises causing D-threonine-aldolase, or D-threonine-aldolase and L-allothreonine-aldolase to act on a solution containing at least DL-threonine thereby obtaining L-threonine from a mixture containing at least DL-threonine.

BACKGROUND AND DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to a process for preparing L-threonine which 
comprises causing D-threonine-aldolase, or D-threonine-aldolase and 
L-allothreonine-aldolase to act on a solution containing at least 
DL-threonine. 
More particularly, the present invention relates to a process for preparing 
L-threonine which comprises causing D-threonine-aldolase to act on 
DL-threonine or causing D-threoninealdolase and L-allothreonine-aldolase 
to act on a mixture of DL-threonine and DL-allothreonine thereby obtaining 
L-threonine from DL-threonine or the mixture of DL-threonine and 
DL-allothreonine by decomposing D-threonine, D-allothreonine and 
L-allothreonine asymmetrically into glycine and acetaldehyde. 
L-threonine is one of the essential amino acids for human and animals, and 
because of the relatively small content thereof in various animal and 
plant proteins, the potential demand for L-threonine as one of the 
additives into foods and feeds is large enough. Hitherto, L-threonine has 
been produced by a method of extraction from natural materials or a method 
of fermentation of natural materials, however, due to the relatively high 
cost of L-threonine produced by such a method, a process for preparing 
thereof at a lower cost has been required. 
On the other hand, although L-threonine is easily synthesizable while using 
glycine, etc., D-threonine is by-produced in the same amount as that of 
L-threonine with the simultaneous formation of D-allothreonine and 
L-allothreonine as their DL-isomer. Accordingly, the steps of isolating 
and pruifying L-threonine from the reaction products are extremely 
complicated. The yield of L-threonine is low and the price of L-threonine 
produced by synthetic process is very high. For instance, as an optical 
resolution method of DL-threonine, the methods disclosed in 
Bull.Soc.Chim., Vol. 20, page 903(1953) and ibid., Vol. 23, page 447(1956) 
have been known, however, these methods for optical resolution of 
DL-threonine are extremely troublesome and only give L-threonine in a low 
yield. In addition, for removing allothreonines, a very troublesome and 
inefficient method is inevitably used such as the method disclosed in 
Japanese Patent Publication No. 36-19562(1961) wherein 
bis(acetaldehyde)threonine copper is used, or the method disclosed in U.S. 
Pat. No. 2,461,847 wherein allothreonine and threonine are converted into 
their sodium salts in ethanol by using sodium ethylate, and the salts are 
separated by utilizing the difference between their solubilities. 
Besides, there have been demerits in the synthetic process that there are 
scarcely any demand for D-threonine and allothreonine in the market, and 
that the racemization of D-threonine into L-threonine is not so easily 
effected. 
As a result of the present inventors' efforts in studying for developing a 
process for preparing L-threonine at a low cost by utilizing enzymatic 
reactions while dissolving the technical problems, the present inventors 
have found a novel enzyme which catalyzes D-threonine and also 
D-allothreonine to convert them into glycine and acetaldehyde, and termed 
the enzyme D-threonine-aldolase. Namely, the present inventors have 
further found that in the case where D-threonine-aldolase is brought into 
action on the product of the synthesis, i.e., DL-threonine, or in the case 
where D-threonine-aldolase in combination with L-allothreonine-aldolase is 
brought into action on the more complicated products of synthesis, i.e., a 
mixture of DL-threonine and DL-allothreonine, L-threonine together with 
the useful decomposition-product, i.e., glycine and acetaldehyde are 
obtained. As the result, the preparation of L-threonine is easily carried 
out and the by-products are utilizable while dissolving all the problems 
shown above. 
In an aspect of the present invention, there is provided a process for 
preparing L-threonine which comprises causing D-threonine-aldolase or 
D-threonine-aldolase and L-allothreonine-aldolase to act on a solution 
containing at least DL-threonine. More particularly, there is provided a 
process for preparing L-threonine which comprises causing 
D-threonine-aldolase to act on DL-threonine or causing 
D-threonine-aldolase and L-allothreonine-aldolase to act on a mixture of 
DL-threonine and DL-allothreonine thereby obtaining L-threonine from 
DL-threonine or the mixture of DL-threonine and DL-allothreonine. 
Namely, the present invention relates to a process for obtaining 
L-threonine from DL-threonine or a mixture of DL-threonine and 
DL-allothreonine, comprising the step of catalyzing DL-threonine contained 
in an aqueous solution with D-threonine-aldolase or catalyzing a mixture 
of DL-threonine and DL-allothreonine contained in an aqueous solution with 
a mixture of the D-threonine-aldolase and L-allothreonine-aldolase. 
The D-threonine-aldolase according to the present invention is a novel 
enzyme which decomposes D-threonine into glycine and acetaldehyde and also 
catalyzes D-allothreonine to decompose thereof into glycine and 
acetaldehyde. An enzyme produced by a strain of Alcaligenes faecalis, IFO 
12669 (deposited in Institute for Fermentation Osaka, Japan), an enzyme 
produced by a strain of Pseudomonas DK-2, deposited in Fermentation 
Research Institute, Agency of Industrial Science and Technology, Ministry 
of International Trade and Industry, Japan under a deposite number of 
FERM-P No. 6200, and an enzyme produced by a strain of Arthrobacter DK-19, 
also deposited in the latter Institute under a deposite number of FERM-P 
No. 6201 respectively possess an activity of deocmposing D-threonine and 
D-allothreonine and accordingly, each of them can be used according to the 
present invention. 
The bacteriological properties of the strain of Pseudomonas DK-2 (FERM-P 
No. 6200) and the strain of Arthrobacter DK-19 (FERM-P No. 6201) are shown 
below. 
______________________________________ 
(a) Morphological properties: 
Item Pseudomonas DK-2 
Arthrobacter DK-19 
______________________________________ 
Shape of cells 
rod-shaped rod-shaped 
Size of cells (.mu.) 
1.5 .times. 0.8 
2.5 .times. 0.8 
Pleiomorphism 
none Mixture of rods of 
ordinary curved line- 
like ones, V-shaped 
ones and stick-like 
ones 
Mobility positive positive: 
monotrichous very active 
peritrichous 
Spore none none 
Gram-staining 
negative having gram-positive 
granules within the 
gram-negative cells 
Acid-fastness 
none none 
______________________________________ 
______________________________________ 
(b) Growth state in various culture media: 
Item Pseudomonas DK-2 
Arthrobacter DK-19 
______________________________________ 
Plate culture 
semi-transparent 
semi-transparent 
medium of agar 
circular colonies 
cream-coloured circu- 
with bouillon 
with convex-circu- 
lar colonies with 
lar protuberance,. 
convex-circular pro- 
lustrous tuberance, lustrous 
Slunt culture of 
growth moderate, 
growth moderate, semi- 
agar with semi-transparent 
transparent thread- 
bouillon and lustrous like colonies with 
cream-like colour, 
lustrous 
Liquid culture 
growth moderate 
growth favorable 
medium with flocculent 
bouillon 
Stab culture with 
growth favorable 
growth favorable on 
gelatin and 
on the surface of 
the surface of the 
bouillon the culture medium 
culture medium as 
filiform 
Litmus-milk 
change to alkaline 
discoloration without 
color liquefying 
______________________________________ 
______________________________________ 
(c) Physiological properties: 
Item Pseudomonas DK-2 
Arthrobacter DK-19 
______________________________________ 
Reduction of nitrate 
+ - 
Denitrification 
+ + 
MR test - - 
VP test - - 
Production of indole 
- - 
Production of H.sub.2 S 
+(weak) +(weak) 
Hydrolysis of starch 
- - 
Utilization of citric 
acid in 
Koser's medium 
- + 
Christensen's 
- + 
medium 
Utilization of inorg. 
nitrogen source 
nitrate - - 
ammonium salt 
- - 
Production of dye 
- - 
Activity as urease 
- - 
Activity as oxidase 
+ - 
Activity as catalase 
+ + 
Range of growth 
pH 6 to 9.5 4.5 to 9.5, 
preferably 
8 to 8.5 
temperature (.degree.C.) 
5 to 50 15 to 38, 
preferably 
28 to 30 
Aerobism yes yes 
O-F test (method of 
oxidative oxidative 
Hush Leifson) 
Production of acid 
acid gas acid gas 
and gas from sugar 
L-arabinose - - - - 
D-xylose - - - - 
D-glucose - - - - 
D-mannose - - - - 
D-fructose - - - - 
D-galactose - - - - 
Maltose - - - - 
Sacrose - - - - 
Lactose - - - - 
Trehalose - - - - 
D-solbitol - - - - 
D-mannitol - - - - 
Inositol - - - - 
Glycerol +(weak) - - - 
Starch - - - - 
Raffinose - - - - 
Inulin - - - - 
D-ribose - - - - 
Dulcitol - - - - 
Sorbose - - - - 
Carboxymethyl- 
- - - - 
cellulose 
Halotolerance in 
5% by weight of 
does grow does grow 
aqeuous solution 
of sodium chloride 
10% by weight of 
does not grow does grow slightly 
aqueous solution 
of sodium chloride 
Decompositive 
-- -- 
activity to gelatine 
Activity as -- -- 
DNA-ase 
Essential vitamines 
thiamine and pantothenic acid 
folic acid and nicotinic acid 
Source of isolation 
soil soil 
______________________________________ 
On classifying these two strains on the ground of the bacteriological 
properties while referring to "Manual of Determinative Bacteriology, 8th 
Ed. (1974)" by Burgey, the strain DK-2 was identified to belong to the 
genus Pseudomonas, because it is a gram-negative rod which is 
monotrichous, positive in oxidase activity and positive in 
denitrification. 
On the other hand, the strain DK-19 was identified to belong to the genus 
Arthrobacter, because it is a weakly grampositive rod having a 
pleiomorphism and is peritrichous and impossible to utilize saccharide. 
The enzyme having both the activity of D-threonine-aldolase and the 
activity of D-allothreonine-aldolase according to the present invention 
can be produced by culturing, for instance, one of the strains in a 
nutrient culture medium which may be the same as those for culturing 
ordinarily any strain of bacterial containing saccharide such as glucose, 
glycerol, molasses and the like or organic carboxylic acid such as acetic 
acid, malic acid and the like as a carbon source, ammonium sulfate, 
ammonium chloride, urea and the like as a nitrogen source, yeast extract, 
pepton, meat extract, corn-steep liquor, etc. As an organic nutrient and 
magnesium, iron, manganese, potassium, phosphate, etc. as inorganic ion. 
The cultivation may be effected under the conventional conditions, that 
is, at a pH of 4 to 10 of the culture medium, at a temperature of 
20.degree. to 60.degree. C. for 1 to 3 days of aerobic cultivation after 
being inoculated. 
By culturing one of the strain under the conditions, the enzyme having both 
the activity of D-threonine-aldolase and the activity of 
D-allothreonine-aldolase is produced in the bacterial bodies and 
accumulated therewithin. In order to isolate the enzyme in a purer state 
from the cultured medium thereof, the proliferated bodies of the strain of 
microorganism (hereinafter referred to as "the bacterial cells") are 
destroyed by a known method such as a mechanical method, a treatment with 
an enzyme and an autolysing method to obtain a crude extract of the enzyme 
and then the crude extract was subjected to purification by a suitable 
combination of precipitation with ammonium sulfate or an organic solvent 
such as acetone or methanol, and chromatography while using an 
ion-exchanger such as diethylaminoethyl (hereinafter referred to as 
"DEAE")cephalose, DEAE-cephadex and calcium phosphate gel, etc. or an 
adsorbent, etc. In order to obtain the manifastation the enzymic activity 
thereof, the presence of a coenzyme, pyridoxal-5'-phosphate, is necessary 
in its reaction in an ordinary amount of 10.sup.-5 to 10.sup.-3 M. 
Physico-chemical properties of the novel enzyme according to the present 
invention are explained as follows. 
(1) Activity and substrate-specificity: 
The novel enzyme according to the present invention decomposes both 
D-threonine and D-allothreonine into glycine and acetaldehyde, and on the 
other hand, does not act at all on L-threonine and L-allothreonine. 
(2) Optimum pH: 
From the result of determination of aldehyde produced by the novel enzyme 
from D-threonine as a substrate at 30.degree. C. for 10 min. at one of a 
series of pH, it is found that the optimum pH of the novel enzyme was in a 
range of 7 to 9. The respective buffer solutions used are a 0.1M phosphate 
buffer for a range of pH of 4 to 7.5, a 0.1M tris-HCl buffer for a range 
of pH of 7 to 9 and a 0.1M sodium carbonate buffer for a range of 9 to 11. 
(3) pH range in which the novel enzyme is stable: 
From the result of determination of the remaining activity of the novel 
enzyme after heating a solution of the enzyme for one hour at 30.degree. 
C. at one of a series of pH, the pH range in which the novel enzyme can 
exist in a stable state is 6 to 9. The respective buffer solution used in 
the calturing are a 1.0M phosphate buffer for a range of pH of 4 to 7.5, a 
0.1M tris-HCl buffer for a range of pH of 7 to 9 and a 0.1M sodium 
carbonate buffer for a range of 9 to 11. 
(4) Method for determination of the enzymic activity: 
The amount of acetaldehyde formed when 0.1 ml of a liquid containing the 
novel enzyme is added to 0.4 ml of a 0.1M tris-HCl buffer solution 
containing 100 micromols of D-threonine at pH of 8.0 and the mixture is 
heated at 30.degree. C. for 10 min is determined by the method of Paz 
(refer to Arch.Biochem.Biophys., Vol. 109, page 548(1965)), and the 
enzymic activity on decomposing 1 micromol of D-threonine at 30.degree. C. 
is taken as a standard, i.e., one unit (U). 
(5) Range of the optimum temperature for the activity: 
From the determination of the amount of acetaldehyde produced by the novel 
enzyme under the conditions of the optimum pH (8.0) at one of a series of 
temperatures for 10 min. while using a 0.1M tris-HCl buffer solution, it 
is found that the range of the optimum temperature for the enzyme was 
40.degree. to 50.degree. C. 
(6) Heat-stability of the novel enzyme: 
From the determination of the remaining activity after heating a solution 
of the novel enzyme in a 0.1M tris-HCl buffer solution at pH of 8.0 for 
one hour at one of a series of temperatures, it is found that the 
temperature at which the enzyme is stable was below 40.degree. C. 
(7) Conditions of in-activation of the novel enzyme: 
The novel enzyme according to the present invention is in-activated 
completely at a pH below 5 and over 11, and also completely in-activated 
after heating for one hour at a temperature over 70.degree. C. 
(8) Agents inhibiting, activating or stabilizing the activity: 
The novel enzyme is activated and stabilized by mercaptoethanol, sodium 
sulfite, sodium hydrogen sulfite, dithiothreitol, and Mn.sup.2+, 
Co.sup.2+, Fe.sup.2+ or Mg.sup.2+, and on the other hand, the activity 
thereof is inhibited by monovalent Ag.sup.+, Cu.sup.2+, Hg.sup.2+, 
Zn.sup.2+, Pd.sup.2+, hydroxylamine and p-chloromercuribenzoate. 
(9) Coenzyme: 
The coenzyme of the enzyme is pyridoxal-5'-phosphate. 
(10) Molecular weight: 
The molecular weight of the enzyme is in a range of 100,000-150,000 as a 
result of gel-filtration by Cephadex.RTM. G-200. 
(11) Elementary analytical composition: 
50.7-52.7% of carbon, 
6.8-8.8% of hydrogen and 
14.7-16.7% of nitrogen 
Since the known threonine-aldolase and allothreonine-aldolase decompose 
only L-threonine and L-allothreonine respectively, and those decomposing 
the D-isomer have never been known, each enzyme found in the strains 
respectively is a novel enzyme having a new activity. 
Namely, any anzyme have D-threonine-aldolase activity and can be used in 
the process of the present invention as far as the enzyme can decompose 
both D-threonine and D-allothreonine to convert them into glycine and 
acetaldehyde. 
L-allothreonine-aldolase used in the process according to the present 
invention is an enzyme catalyzing L-allothreonine to convert thereof into 
glycine and acetaldehyde, and the presence thereof has been known in the 
sheep liver and corn seed in germination, however, the microbiological 
production thereof has never been known. 
However, the present inventors have found out that some bacteria 
respectively belonging to the genera Bacillus, Pseudomonas, Arthrobacter 
and Alcaligenes produce the enzyme, and have found the method for 
producing the enzyme industrially in a large scale. As the examples of the 
microorganism having the productivity of L-allothreonine-aldolase, a 
strain of Bacillus DK-315 (deposited in Fermentation Research Institute, 
Agency of Industrial Science and Technology, Ministry of International 
Trade and Industry, Japan under a deposite number FERM-P No. 6202) 
isolated from a soil, a strain of Arthrobacter DK-19 (FERM-P No. 6201), 
the strain of Pseudomonas DK-2 (FERM-P No. 6200) and a strain of 
Alcaligenes faecalis (deposited in Institute for Fermentation Osaka, Japan 
under a deposite number of IFO-12669) can be used. 
The bacteriological properties of the strain of Bacillus DK-315 (FERM-P No. 
6202) are as follows. 
(a) Morphological properties: 
(1) Shape and size of the cells: rod-shaped of 0.8=2.0 micrometers, 
(2) Pleiomorphism: none, 
(3) Mobility: positive, multitrichous, 
(4) Spore: ellipsoidal in shape existing in the position out from the 
center, 
(5) Gram-staining: positive, 
(6) Acid-fastness: none. 
(b) Growth state in various culture media: 
(1) Plate culture medium of agar with bouillon: favorable with circular 
colonies, 
(2) Slunt culture of agar with bouillon: favorable, semitransparent with 
luster, 
(3) Liquid culture medium with bouillon: favorable, 
(4) Stab culture with gelatin and bouillon: favorable filiform in the 
surface of the culture medium, 
(5) Litmus-milk: decolorated and liquefied. 
______________________________________ 
(c) Physiological properties: 
______________________________________ 
(1) Reduction of nitrate: 
+ 
(2) Denitrification: + 
(3) MR test: + (weak) 
(4) VP test: - 
(5) Production of indol: 
- 
(6) Production of H.sub.2 S: 
- 
(7) Hydrolysis of starch: 
+ 
(8) Utilization of citric 
acid 
in Koser's medium: 
- 
in Christensen's - 
medium: 
(9) Utilization of 
inorganic nitrogen 
nitrate: - 
ammonium salt: + 
(10) Production of dye: 
- 
(11) Activity as urease: 
- 
(12) Activity as oxidase: 
+ 
(13) Activity as catalase: 
+ (weak) 
(14) Range of growth 
pH: 5 to 12 
temperature: 5 to 40.degree. C. 
(15) Aerobism: yes 
(16) O-F test (Hush F 
Leifson's method): 
(17) Production of acid 
(acid) (gas) 
and gas from sugars: 
L-arabinose - - 
D-xylose - - 
D-glucose + + 
D-mannose + + 
D-fructose + + 
D-galactose + + 
Maltose + - 
Sucrose - - 
Lactose - - 
Trehalose + - 
D-sorbitol - - 
D-mannitol + - 
Inositol - - 
Glycerol + + 
Starch + - 
(18) Halotrelance in an 
does not 
aqueous 5% sodium 
grow 
chloride solution: 
(19) Decompositive + 
activity to gelatine: 
(20) Activity as + 
DNA-ase: 
(21) Essential vitamins: 
none 
______________________________________ 
From the above-mentioned bacteriological properties, while referring to 
"Manual of Determinative Bacteriology, 8th Ed(1974) by Burgey, the strain 
DK-315 was identified to belong to the genus Bacillus because of moving by 
peritricha and of gram-positive rod having a capacity of forming spores. 
L-allothreonine-aldolase can be produced by culturing each of the 
above-mentioned strains in a nutrient culture medium which is usually used 
for culturing a bacterial strain containing a saccharide such as glucose, 
glycerol and molasses or an organic acid such as acetic acid, malic acid 
and the like as a carbon source, ammonium sulfate, ammonium chloride, urea 
and the like as a nitrogen source, and an inorganic ion such as ammonium 
sulfate, ammonium chloride and urea, as an organic nutrient source of 
yeast-extract, peptone, meat-extract, corn-steep liquor and the like and a 
metal salt such as magnesium, iron, manganese, potassium and phosphates 
usually according to the conventional method of cultivating bacteria at a 
pH of 4 to 10 for one to three days at 20.degree. to 60.degree. C. 
aerobically at the pH of the culture medium of 4.0 to 10.0. 
Thus, L-allothreonine-aldolase is produced and accumulates in the bacterial 
bodies, and accordingly, in the case of isolating the enzyme from the 
cultured medium, the bacterial cells are broken by a known method such as 
a mechanical means, an enzymic means or a autolysing method and then the 
crude extract of the enzyme is prepared. The crude extract was treated by 
a suitable combination of precipitation with ammonium sulfate or a solvent 
such as acetone or ethanol and chromatography while using ion-exchangers 
such as DEAE-cepharose, DEAE-cephadex, gel of calcium phosphate or 
adsorbents to be the enzyme product of a high quality. The followings are 
the simple physico-chemical properties of the thus purely obtained 
L-allothreonine-aldolase. 
(1) Optimum pH; 8 to 9, 
(2) Optimum temperature: 60.degree. to 70.degree. C., 
(3) Conditions for inactivation: inactivated within one hour at 30.degree. 
C. and pH of 5 to 11 or in one hour at a temperature of higher than 
50.degree. C. and pH of 8, 
(4) Inhibitants: Cu.sup.2+, Hg.sup.2+ and Ag.sup.1+, 
(5) Stabilizers: mercaptoethanol, dithiothreitol and sodium sulfite, 
(6) Coenzyme: pyridoxal-5'-phosphate. 
Since L-allothreonine-aldolase requires pyridoxal-5'-phosphate as a 
coenzyme for exhibiting its activity, usually 10.sup.-3 to 10.sup.-5 M of 
pyridoxal-5'-phosphate is made to coexist with the enzyme whenever it is 
reacted. 
(7) Molecular weight: 100,000 to 150,000 according to the determination of 
gel-filtration by Cephadex.RTM.G-200. 
(8) Elementary analytical data: 
C: 51.4-53.4% 
H: 6.5-8.5% and 
N: 14.2-16.2%. 
L-allothreonine-aldolase used in the present invention is enough for the 
purpose if it decomposes L-allothreonine into glycine and acetaldehyde, 
and it goes without saying that the enzyme is not restricted to that 
derived from microorganism. 
The enzymes used in the present invention, that is, D-threonine-aldolase 
and L-allothreonine-aldolase, are respectively enough for the purpose in 
the case where they are respectively under the condition of capable of 
exhibiting the enzymatic activity thereof, and they are never restricted 
under isolated condition, and accordingly, half-purified products, crude 
extract, moreover, the cultured medium, living cells, freeze-dried cells, 
dried cells by acetone, ground cells, ground sheep liver and the like may 
be used as it is. The immobilized enzyme or the immobilized cells by a 
known means can be used. As a method of immobilization, a method of 
combining with a carrier, a method of cross-linking, a method of 
entrapping, a method of agglutination and the like are broadly usable. 
DL-threonine or a mixture of DL-threonine and DL-allothreonine may be the 
product obtained by any known method, and for instance, a method wherein 
an acetoacetate is used as the starting material for obtaining an ester of 
alpha-amino-beta-hydroxybutyric acid and the amino-hydroxybutyrate is 
reacted with thionyl chloride to form an oxazoline-ester, and then the 
ester is hydrolyzed into the product by heating (refer to J.Am.Chem.Soc., 
71, 1101(1949)) and a method wherein vinyl acetate as the starting 
material is subjected to hydroformylation by the oxo process to give 
alpha-acetoxypropionaldehyde and the aldehyde is reacted with hydrogen 
cyanide and ammonia to be alpha-amino-beta-hydroxybutyronitrile which is 
in turn reacted with phosgen to be a derivative of oxazolidone and then 
the derivative is subjected to hydrolysis to be the product (refer to 
Japanese Patent Publication No. 40-11608(1965)) are utilizable for the 
purpose. However, since in the process according to the present invention, 
the object product is L-threonine and by-product consists of glycine and 
acetoaldehyde, a synthetic method which produces the allo-forms in a 
smaller amount and requires glycine and acetaldehyde as the starting 
compounds is preferable. In this connection, a method wherein a metal salt 
is reacted with glycine to be a metal complex of glycine and then 
acetaldehyde is condensed with the metal complex of glycine (refer to 
Japanese Patent Publications Nos. 36-19562(1961) and 47-39093(1972)) is 
the suitable method for producing a mixture of DL-threonine and 
DL-allothreonine. The solution containing the mixture may be an aqueous 
solution which is obtained by dissolving the once-obtained product as 
crystals into water, however, it may be the liquid obtained by the 
synthesis, or may be any intermediate liquid in the course of obtaining 
the crystals of the mixture. In short, the liquid containing DL-threonine 
and DL-allothreonine used in the process according to the present 
invention may be a solution containing DL-threonine and DL-allothreonine, 
and the ratio of D-isomer to L-isomer in the solution and the ratio of 
threonines to allothreonines in the solution are out of the question. 
Further, the solution may contain any other impurities in addition to 
DL-threonine and DL-allothreonine. Any impurities inhibiting the enzyme 
reaction, if any, should be removed in advance. For instance, in the case 
where a metal ion used in the synthesis is deleterious in the enzyme 
reaction, it should be removed by a cation-exchanging resin, etc. in 
advance of the enzyme reaction. 
There is no particular difficulty in treating (catalyzing) a solution 
containing DL-threonine with D-threonine-aldolase or treating (catalyzing) 
a solution containing both DL-threonine and DL-allothreonine with the 
mixture of D-threonine-aldolase and L-allothreonine-aldolase. In short, 
the indicated enzyme may be made present in the aqueous solution of the 
indicated substance. The concentration of DL-threonine and/or 
DL-allothreonine in the solution may be an extent which does not 
remarkably inhibit the enzymatic activity, and it is preferably 0.1 and 2 
mol/liter. The solvent of the enzyme reaction system is, in principle, 
water, however, an organic solvent may be contained if it does not inhibit 
the enzyme reaction. Although the pH of the reaction system depends on the 
enzyme, it is preferably around pH 7 to 10 in the cases where the enzyme 
is obtained in Examples described. Although the reaction temperature 
depends on the enzyme, it is preferably 30.degree. to 45.degree. C. in the 
case of using the enzyme prepared in Examples described. In addition, in 
the case of using the enzyme prepared in Example, the enzyme reaction can 
be accelerated by bringing 10.sup.-3 to 10.sup.-5 molar amount of 
pyridoxal-5'-phosphate coexist in the system as a coenzyme. Furthermore, a 
surfactant may be added to the reaction system for various purposes. The 
enzyme reaction can be carried out by a batch system or in a continuous 
system. D-threonine-aldolase and L-allothreonine-aldolase may be added 
together with or separately. 
The reaction time can be selected optionally according to the purpose of 
L-threonine for use. For example, in the case where L-threonine in which 
the existence of undecomposed isomer is allowable is to be obtained, the 
reaction may be stopped before the completion of enzymatic decomposition 
of the isomer. At any rate, a reaction time of 5 to 100 hours is 
sufficient for every enzyme. 
After the enzyme reaction finishes, the suspending matters in the reaction 
mixture are removed by centrifugation or filtration, if necessary, and the 
obtained reaction mixture is purified by treatment of ion-exchanging resin 
and crystallization, and after decolorizing the reaction solution by 
activated carbon, etc., the decolorized solution is condensed to obtain 
the crystals of L-threonine in a pure state. The reaction product other 
than L-threonine comprises glycine and acetaldehyde in the case of using 
D-threonine-aldolase and also in the case of using D-threonine-aldolase 
and L-allothreonine-aldolase, and glycine can be separated and isolated by 
chromatography, for example, using ion-exchanging resin. Because of the 
non-enzymatic condensation of acetaldehyde with glycine during the enzyme 
reaction or of the re-combination with glycine by each enzyme, it may be 
better to recover acetaldehyde during the enzyme reaction by distillation, 
etc. 
According to the process of the present invention, the removal of 
D-threonine or DL-allothreonine from threonine mixture which has been 
difficult can be carried out by one convenient and simple step. 
Accordingly, the present invention has dissolved the large problem which 
has hindered the separation of the isomer and has provided with the method 
for producing L-threonine at a low price from threonine which is 
synthetically produced. Particularly, when combined with the synthetic 
method for producing threonine from glycine and acetaldehyde, it is more 
preferable, because the by-products of the enzyme reaction can be re-used 
as the starting materials.

The present invention will be more precisely explained while referring to 
Examples as follows. 
However, the present invention is not restricted to Examples under 
mentioned. From the foregoing description, one skilled in the art can 
easily ascertain the essential characteristics of this invention, and 
without departing from the spirit and scope thereof, can make various 
changes and modifications of the invention to adapt it to various usage 
and conditions. 
In addition, all the percentage are % by weight. The activity of 
D-allothreonine-aldolase and that of L-allothreonine-aldolase were 
measured as in the case of measuring the activity of D-threonine-aldolase 
except for changing the substrate corresponding to each enzyme. The titer 
of the activity was designated that the activity to decompose 1 micromol 
of allothreonine, the substrate, for one min is one unit (1 U). 
PREATION EXAMPLE 1 
Production of an enzyme having the activity of D-threoninealdolase and the 
activity of D-allothreonine-aldolase 
A culture medium adjusted to pH 7.5 comprising 0.5% of polypeptone, 0.2% of 
yeast extract, 0.1% of potassium dihydrogen sulfate, 0.05% of magnesium 
sulfate, 0.1% of L-glutamic acid and 0.5% of D-threonine was prepared, and 
in a 5-liter culturing vessel, 3 liters of the culture medium was 
introduced, and then the medium was sterilized at 120.degree. C. for 10 
min. In the thus sterilized culture medium, a strain of Arthrobacter DK-19 
(FERM-P No. 6201) was inoculated and cultured for 20 hours at 30.degree. 
C. while aerating and agitating. 
After the culturing finished, the cells were collected from the culture 
liquid by centrifugation, and after washing the cells with aqueous 0.9% 
sodium chloride solution, the wet cells were suspended in 100 ml of 0.1M 
phosphate buffer solution containing 10 mM of mercaptoethanol and 0.1 mM 
pyridoxal-5'-phosphate. After treating the thus obtained cell suspension 
with an ultrasonics of 20 kHz for each 5 min. in total 5 times, the 
suspension was centrifugated to collect the supernatant liquid. After 
adding protamin sulfate to the supernatant liquid to remove nucleic acid, 
the liquid was fractioned by ammonium sulfate to collect a fraction 
containing an enzyme having the activity of D-threonine-aldolase. 
After subjecting the fraction having activity of D-threonine-aldolase into 
dialysis against a 0.01M phosphate buffer containing 10 mM of 
mercaptoethanol and 0.1 mM pyridoxal-5'-phosphate at pH 7.5, the dialyzate 
was passed through a column filled with 100 ml of DEAE-Cephadex.RTM.A-50 
and then the adsorbed fraction on the column was subjected to elution 
fractionally by a method of concentration-gradient of potassium chloride 
to collect the fraction containing the enzyme having the activity of 
D-threonine-aldolase. By condensing the fraction while using a method of 
ultrafiltration, 10 ml of a solution of the purified D-threonine-aldolase 
was obtained. The activity of D-threonine-aldolase and the activity of 
D-allothreonine-aldolase of the thus obtained solution were 5 U/ml and 
12.5 U/ml, respectively. 
PREATION EXAMPLE 2 
Production of enzymes having the activity of D-threoninealdolase and the 
activity of D-allothreonine-aldolase 
A strain of Pseudomonas DK-2 (FERM-P No. 6200) and a strain of Alcaligenes 
faecalis IFO 12669 were respectively cultured in the same kind of culture 
medium by the same procedures as in Preparation Example 1, and from the 
thus cultured medium, each 10 ml of a solution of purified 
D-threonine-aldolase was obtained by the same procedures in Preparation 
Example 1. The activity of D-threonine-aldolase and the activity of 
D-allothreonine-aldolase of the enzyme obtained by culturing the strain of 
Pseudomonas DK-2 were 4.5 U/ml and 10.8 U/ml, respectively, and those 
obtained by culturing the strain of Alcaligenes faecalis were 4.2 U/ml and 
9.5 U/ml, respectively. 
PREATION EXAMPLE 3 
Production of L-allothreonine-aldolase 
A strain of Bacillus DK-315 (FERM-P No. 6202) was cultured in the same 
culture medium and in the same manner as in Preparation Example 1, and 10 
ml of aqueous solution of purified L-allothreonine-aldolase was obtained 
by the same procedures of isolation and purification as Preparation 
Example 1. The activity of L-allothreonine-aldolase of the thus obtained 
solution was 4.5 U/ml, and the solution showed no activity at all on 
D-allothreonine and D-threonine. 
SYNTHESIS EXAMPLE 1 
Synthesis of DL-threonine 
Into 5 liters of hot water, 75 g of glycine was dissolved, and 100 g of 
basic copper carbonate was slowly added to the solution and the mixture 
was reacted by heating. After the reaction finished, an excess of basic 
copper carbonate was filtered off from the hot solution, and after 
condensing the filtrate under a reduced pressure, the concentrated 
filtrate was cooled to obtain 110 g of crystalline copper-glycine complex. 
Into one liter of water, 58 g of copper salt of glycine, 40 of an 
anion-exchange resin SA 21A (HCO.sub.3 -type) and 45 ml of acetaldehyde 
were added, and the mixture was brought into reaction by heating to 
40.degree. C. for 2 hours under agitation. After the reaction was over, 
the reaction mixture was left as it is at 5.degree. C. for 24 hours to 
obtain crystals of bisacetaldehydethreonine copper-complex separated out. 
After collecting the crystals together with the catalyst by filtration, 
the solid matters were suspended in 0.6 liter of an aqueous 3% ammonia 
solution and the suspension was filtered. The filtrate was passed through 
a column filled with 1.2 liters of a chelate resin, Dowex.RTM.A-1 
(NH.sub.4 -type), to remove copper from the filtrate, and the fraction 
reacting positively to ninhydrin was collected and condensed under a 
reduced pressure. By adding methanol to the condensate, crystals were 
obtained and recrystallized from aqueous methanolic solution to be 41 g of 
DL-threonine not containing allothreonine at all. 
SYNTHESIS EXAMPLE 2 
Synthesis of a mixture of DL-threonine and DL-allothreonine 
Into 5 liters of hot water, 75 g of glycine was dissolved, and 100 g of 
basic copper carbonate was slowly added to the solution and the mixture 
was reacted by heating. After the reaction finished, an excess of basic 
copper carbonate was filtered off from the hot solution, and after 
condensing the filtrate under a reduced pressure, the concentrated 
filtrate was cooled to obtain 110 g of crystalline copper-glycine complex. 
Into 0.8 liter of methanol containing 6 g of potassium hydroxide, 58 g of 
copper-glycine complex and 50 g of acetaldehyde were added to react at 
50.degree. to 60.degree. C. for 1.5 hours under agitation. After the 
reaction was over, the reaction mixture was filtered to remove a small 
amount of undissolved material, and after adding 6.4 g of acetic acid to 
the filtrate, it was subjected to distillation under a reduced pressure to 
remove the solvent. After sufficiently washing the crude copperthreonine 
compound which remained as a distillation residue with water, the 
crystalline compound was disolved in 0.5 liter of 6N aqueous ammonia 
solution, and the solution was passed through a column filled with 2 
liters of Dowex.RTM. A-1 (NH.sub.4 -type), a chelating resin, to remove 
copper from the solution and collect the fraction containing threonine. 
The fraction was condensed under a reduced pressure to 100 ml, and 400 ml 
of methanol was added to the condensate, and then the mixture was cooled 
to obtain crystals which were recrystallized from aqueous methanolic 
solution to be 38 g of crystalline DL-threonine containing 10% of 
DL-allothreonine. 
EXAMPLE 1 
Into 20 ml of an aqueous solution containing 10.sup.-6 mol of 
pyridoxal-5'-phosphate, 10.sup.-4 mol of mercaptoethanol, 10.sup.-3 mol of 
manganese chloride and 2.38 g of the crystals of DL-threonine which was 
obtained in Synthesis Example 2 and contained 10% of DL-allothreonine, 10 
ml of the solution containing the purified enzyme which was obtained in 
Preparation Example 1 and had both the activity of D-threonine-aldolase 
and the activity of D-allothreonine-aldolase and 10 ml of the solution of 
purified D-allothreonine-aldolase which was obtained in Preparation 
Example 3 were added, and while subjecting the mixture to distillation 
under a reduced pressure to remove and recover acetaldehyde formed in the 
enzyme reaction, the mixture was brought into reaction at 30.degree. C. 
for 24 hours. The pH of the reaction mixture during reaction was kept at 
7.5 to 8.5 by adding 0.1N sodium hydroxide solution. 
After the reaction finished, the reaction mixture was neutralized with 
dilute hydrochloric acid, and the neutralizate was passed through a column 
filled with 100 ml of Dowex.RTM. 50 WX-8 of H.sup.+ -type. The column was 
washed with water, and threonine, glycine etc. which had been adsorbed 
onto the column were eluted with aqueous 0.2N ammonia solution. After 
condensing the eluate to dryness under a reduced pressure, the residual 
solid matter was dissolved in 20 ml of water, and after adjusting the pH 
of the solution with dilute hydrochloric acid to 3, the solution was again 
passed through a column filled with 100 ml of Dowex.RTM.50 WX-8 of H.sup.+ 
-type. After washing the column with water, aqueous 0.1N ammonia solution 
was passed through the column to elute a fraction containing threonine and 
the eluate was dried to solid under a reduced pressure. The solid matter 
was dissolved in 4 ml of water and 10 ml of ethanol was slowly added to 
the solution to obtain crystals which were isolated. The dry weight of the 
crystals was 0.9 g and it was confirmed by paper-chromatography that the 
crystals were pure threonine. The specific rotation of the crystal was 
[.alpha.].sub. D.sup.27 =-28.7.degree.(in water) which was coincided with 
that of the authentic specimen of pure L-threonine. 
The aqueous 0.1N ammonia was further passed through the remaining column to 
elute a fraction containing glycine. The eluate was dried to solid under a 
reduced pressure, and the solid matter was dissolved in 1 ml of water. By 
adding 4 ml of ethanol slowly to the solution, crystals appeared in the 
solution, which were isolated and dried to be 0.65 g of glycine after 
confirming by paper-chromatography. 
On the other hand, acetaldehyde recovered during the reaction under a 
reduced pressure was 0.34 g. 
Example 2 
In the same manner as in Example 1 except for using 10 ml of the solution 
of purified D-threonine-aldolase obtained from Pseudomonas DK-2 FERM-P 
6200 in Preparation Example 2 instead of the solution of purified 
D-threonine-aldolase obtained in Preparation Example 1, decomposition of 
DL-threonine containing 10% of DL-allothreonine was carried out to obtain 
0.91 g of crystals of threonine not containing allothreonine at all, 0.66 
g of crystalline glycine and 0.34 g of acetaldehyde. The specific rotation 
of the crystalline threonine was [.alpha.].sub.D.sup.27 =-28.7.degree.(in 
water), namely showing that the crystals consisting only of L-threonine. 
Example 3 
In the same manner as in Example 1 except for using 10 ml of the solution 
of purified D-threonine-aldolase obtained from Alcaligenes faecalis IFO 
12669 in Preparation Example 2 instead of the solution of 
D-threonine-aldolase obtained in Preparation Example 1, decomposition of 
DL-threonine containing 10% of DL-allothreonine was carried out followed 
by the operation of isolation. Thus, 0.91 g of crystalline threonine not 
containing allothreonine at all, 0.66 g of crystalline glycine and 0.34 g 
of acetaldehyde were obtained. The specific rotation of the crystalline 
threonine was [.alpha.].sub.D.sup.27 =-28.7.degree.(in water), showing 
that the crystals consisting only of L-threonine. 
Example 4 
Into 30 ml of an aqueous solution containing 2.38 g of DL-threonine 
obtained in Synthesis Example 1, 10.sup.-6 mol of pyridoxal-5'-phosphate, 
10.sup.-4 mol of mercaptoethanol, 10.sup.-3 mol of manganese chloride, 10 
ml of the solution of purified D-threonine-aldolase obtained in 
Preparation Example 1 was added and while removing and recovering 
acetaldehyde formed during the enzymatic reaction, the mixture was brought 
into reaction at 30.degree. C. for 24 hours. The pH of the reaction system 
was kept at 7.5 to 8.5 by adding aqueous 0.1N sodium hydroxide solution. 
After the reaction finished, the reaction mixture was neutralized with 
dilute hydrochloric acid and passed through a column filled with 100 ml of 
Dowex.RTM.50 WX-8 (H.sup.+ -type). After washing the column with water, 
threonine and glycine adsorbed onto the column were eluted with aqueous 
0.2N ammonia solution. After drying the eluate to solid under a reduced 
pressure, the solid matter was dissolved in 20 ml of water, and the 
solution was adjusted to pH 3 by addition of dilute hydrochloric acid and 
then passed through the column filled with 100 ml of Dowex.RTM.50 WX-8 
(H.sup.+ -type). After washing the column with water, aqueous 0.1N ammonia 
solution was passed through the washed column to elute the fraction 
containing threonine. The eluate was dried to solid under a reduced 
pressure, and then the solid matter was dissolved in 4 ml of water. By 
adding 10 ml of ethanol slowly to the solution, crystals appeared in the 
solution, which were isolated and dried. The thus obtained crystals 
weighing 0.95 g were confirmed to be pure threonine having a specific 
rotation of [.alpha.].sub.D.sup.27 =-28.7.degree. (in water) coinciding 
with that of the authentic specimen of L-threonine. 
The solution of 0.1N ammonia was further passed through the above-mentioned 
column to elute the fraction containing glycine, and the eluate was 
condensed to dryness under a reduced pressure. After dissolving the solid 
matter in 1 ml of water, 4 ml of ethanol was slowly added to the solution 
to obtain crystals separating out. After drying, the crystals were weighed 
to be 0.6 g which consisted solely of glycine according to the 
paper-chromatography. On the other hand, the recovered acetaldehyde during 
the reaction under a reduced pressure was 0.3 g. 
Example 5 
The same procedures of the enzymatic reaction and separation of the 
products were carried out as in Example 1 except for not carrying out the 
distillation under a reduced pressure during the enzymatic reaction. As a 
result, 0.8 g of crystalline threonine containing 10% of allothreonine, 
0.3 g of crystalline glycine and 0.05 g of acetaldehyde were obtained. 
Example 6 
The strain of Pseudomonas DK-2 (FERM-P No. 6200) was cultured in the same 
conditions as in Preparation Example 1 and the thus obtained fraction 
amounting to 10 ml having the activity of D-threonine-aldolase and the 
thus obtained fraction amounting to 10 ml having the activity of 
L-allothreonine-aldolase were respectively condensed by ultrafiltration to 
obtain two kinds of solutions, respectively containing 
D-threonine-aldolase and L-allothreonine-aldolase. 
By adding the thus obtained two kinds of solutions into a mixture of 2 g of 
DL-threonine and 1 g of DL-allothreonine in the same conditions as in 
Example 1 except for a temperature of 35.degree. C. instead of 30.degree. 
C. in Example 1, 0.85 g of crystalline L-threonine not containing 
D-threonine nor DL-allothreonine, 1.01 g of glycine and 0.51 g of 
acetaldehyde were obtained. The specific rotation of the thus obtained 
L-threonine was [.alpha.].sub.D.sup.27 =-28.6.degree.(in water).