Process for microbial production of an optically active 3-(3,4-dihydroxyphenyl)serine

N-acyl-DL-3-(3,4-dihydroxyphenyl)serine or a catecholic hydroxyl-protected derivative thereof is stereospecifically de-acylated by reaction with a microorganism of the genus Streptomyces or Streptoverticillium having an acylase or with an extract of said microorganism containing said acylase, to produce L-3-(3,4-dihydroxyphenyl)serine or the hydroxyl-protected derivative thereof, with the N-acyl-D-3-(3,4-dihydroxyphenyl)serine compound remaining unaltered. The L-3-(3,4-dihydroxyphenyl)serine compound so produced can be separated from the N-acyl-D-3-(3,4-dihydroxyphenyl)serine compound owing to the difference in the properties of them.

This invention relates to a process for the production of an optically 
active 3-(3,4-dihydroxyphenyl)serine which is known to be useful as a 
medicine, or an optically active 3-(3,4-dihydroxyphenyl)serine protected 
derivative in which the catecholic 3- and 4-hydroxyl groups on the phenyl 
ring have been protected and which is useful as an intermediate product 
for the production of an optically active 
3-(3,4-dihydroxyphenyl)-N-methylserine. 
It is known that L-threo-3-(3,4-dihydroxyphenyl) serine (sometime referred 
to as L-threo-DOPS) is useful as an anti-depressant agent or 
anti-hypertensive agent (see Japanese patent application unexamined first 
publication "Kokai" No. 49252/75; Japanese patent application unexamined 
first publication "Kokai" No. 20747/80; U.S. Pat. Nos. 3,920,728; 
4,319,040) and also as an anti-parkinsonian agent (see Japanese patent 
application unexamined first publication "Kokai" No. 125630/77). It is 
known that L-erythro-3-(3,4-dihydroxyphenyl)serine is useful as an 
anti-hypertensive agent (see Japanese patent application unexamined first 
publication "Kokai" No. 49252/75). Further, such an 
L-threo-3-(3,4-dihydroxyphenyl)serine O-protected derivative in which the 
catecholic hydroxyl groups present at the metha- and para-positions of the 
phenyl ring each have been protected with a known hydroxyl-protective 
group is useful as a starting material for synthesis of 
L-threo-3-(3,4-dihydroxyphenyl)-N-methylserine which is interesting as 
psychotropic agent, especially anti-depressant agent and as 
anti-parkinsonian agent (see Japanese patent application No. 221797/82; 
U.S. patent application Ser. No. 523,957 and EPC patent application 
publication No. 0112606 A1). Such an 
L-erythro-3-(3,4-dihydroxyphenyl)serine O-protected derivative in which 
the catecholic 3- and 4-hydroxyl groups on the phenyl ring have been 
protected are useful also as a starting compound for synthesis of 
L-erythro-3-(3,4-dihydroxyphenyl)-N-methylserine which is interesting as 
anti-hypertensive agent (see Japanese patent application No. 72053/83). 
For the production of an optically active, that is, L- or 
D-threo-3-(3,4-dihydroxyphenyl)serine, there are known several prior art 
methods which each comprise subjecting an optically inactive, that is, 
DL-threo-N-benzyloxycarbonyl-3-(3,4-dibenzyloxyphenyl)serine to optical 
resolution procedure using as the optically resolving agent an optically 
active ephedrine or an optically active 
threo-1-(p-nitrophenyl)-2-amino-1,3-propanediol (see Japanese patent 
application unexamined first publication "Kokai" No. 49252/75); quinine 
(see Japanese patent application unexamined first publication "Kokai" No. 
65242/77); an optically active 
threo-1-(p-methylsulfonylphenyl)-2-amino-1,3-propanediol (see Japanese 
patent application unexamined first publication "Kokai" No. 36233/79) and 
optically active S-2-amino-1,1-diphenylpropanol, 
S-2-amino-3-methyl-1,1-diphenylbutanol or 
S-2-amino-4-methyl-1,1-diphenylpentanol (see Japanese patent application 
unexamined first publication "Kokai" No. 29551/81), respectively, and then 
removing the remaining N-protecting benzyloxycarbonyl group and 
catechol-protecting benzyl group from.the optically active 
threo-N-benzyloxycarbonyl-3-(3,4-dibenzyloxyphenyl)serine so isolated. 
However, these prior art methods are commercially disadvantageous due to 
that the above-mentioned optically resolving agents are hardly available 
in commerce, that the L-3-(3,4-dihydroxyphenyl)serine of a high optical 
purity cannot be obtained without troublesome repetition of 
recrystallization and re-precipitation, and that an extra step of 
decomposing the resulting diastereomer salt as the intermediate product is 
needed. 
In this situation, we, the present inventors, have examined whether or not 
DL-threo-3-(3,4-dihydroxyphenyl) serine can be optically resolved into the 
D-form and the L-form by an enzymatic method, and we have got an idea that 
a success may be attained if an N-acyl-DL-3-(3,4-dihydroxyphenyl)serine 
compound of the general formula 
##STR1## 
wherein R.sup.1 and R.sup.2 each denote a hydrogen atom or a 
hydroxyl-protecting group for the hydroxyl groups of catecholic nature 
present at the 3- and 4-positions of the phenyl ring and R.sup.3 denotes 
an alkyl group or an aryl group which may optionally be substituted is 
prepared and is then reacted with such an acylase which is capable of 
removing hydrolytically the N-acyl group (R.sup.3 CO--) preferentially 
from the .alpha.-amino group of the L-isomer of said DL-compound of the 
formula (I) but is not capable of removing the N-acyl group from the 
.alpha.-amino group of the D-isomer of the DL-compound of the formula (I), 
so that there is produced an L-3-(3,4-dihydroxyphenyl)serine compound of 
the general formula 
##STR2## 
wherein R.sup.1 and R.sup.2 are as defined above, while the 
N-acyl-D-3-(3,4-dihydroxyphenyl)serine of the general formula (III) 
##STR3## 
wherein R.sup.1, R.sup.2 and R.sup.3 are as defined above remains 
unaltered. As a result of our experiments, we have found that all the 
known acylases which are presently available are not capable of removing 
the N-acyl group (R.sup.3 CO--) preferentially from the 
N-acyl-L-3-(3,4-dihydroxyphenyl) serine by asymmetrical hydrolysis. 
Accordingly, we have extensively studied about a variety of naturally 
occurring materials and substances and have now found that some 
microorganisms of the genus Streptomyces and some microorganisms of the 
genus Streptoverticillium are capable of removing hydrolytically the 
N-acyl group preferentially from the L-form of the 
N-acyl-3-(3,4-dihydroxyphenyl) serine which is present in the DL-compound 
of the general formula (I), and that these microorganisms of the genus 
Streptomyces and the genus Streptoverticillium have such acylase which is 
capable of hydrolysing preferetially the L-form of the threo- or 
erythro-N-acyl-3-(3,4-dihydroxyphenyl)serine to remove the N-acyl group 
therefrom. 
Thus, we have found that the threo isomer or the erythro isomer of 
DL-3-(3,4-dihydroxyphenyl)serine as prepared by a known method, or such a 
DL-3-(3,4-dihydroxyphenyl)serine O-protected derivative having the 
catecholic 3- and 4-hydroxyl groups protected may be N-acylated with an 
alkanoyl group or an aroyl group in a known manner, and the 
N-acyl-DL-3-(3,4-dihydroxyphenyl)serine or the N-acyl-O-protected 
derivative thereof so produced may then be subjected to enzymatic reaction 
with a culture broth of a microorganism of the genus Streptomyces or the 
genus Streptoverticillium having the acylase, or with a material obtained 
from a treatment of said culture broth and containing the acylase, for 
example, the cells of the microorganism separated from said culture broth, 
and an enzyme material obtained from a treatment of said cells, or with 
the acylase itself produced by and extracted from said microorganism, so 
that the N-acylated serine product is stereoselectively hydrolysed to 
remove the N-acyl group therefrom, whereby the 
L-3-(3,4-dihydroxyphenyl)serine or its O-protected derivative having the 
catecholic 3- and 4-hydroxyl groups protected is produced, while the 
N-acyl-D-3-(3,4-dihydroxyphenyl)serine or its O-protected derivative 
having the 3- and 4-catecholic hydroxyl groups protected is remaining 
unaltered without receiving the enzymatic reaction; and also that the 
former, that is, the L-3-(3,4-dihydroxyphenyl)serine compound can be 
separated from the latter that is, the 
N-acyl-D-3-(3,4-dihydroxyphenyl)serine compound by utilizing different 
solubilities of them or any difference in other physico-chemical 
properties of them. On the basis of these findings, this invention has 
been accomplished. 
According to an aspect of this invention, therefore, there is provided a 
process for producing an optically active L-3-(3,4-dihydroxyphenyl)serine 
compound of the formula (II) 
##STR4## 
wherein R.sup.1 and R.sup.2 each are a hydrogen atom or a 
hydroxyl-protecting group for the catecholic 3- and 4-hydroxyl group on 
the phenyl ring, and an optically active D-3-(3,4-dihydroxyphenyl)serine 
compound of the formula (III) 
##STR5## 
wherein R.sup.1 and R.sup.2 are as defined above and R.sup.3 is 
unsubstituted or substituted alkyl group or an unsubstituted or 
substituted aryl group, which process comprises reacting an 
N-acyl-DL-3-(3,4-dihydroxyphenyl)serine compound of the general formula 
(I) 
##STR6## 
wherein R.sup.1, R.sup.2 and R.sup.3 are as defined above, with a 
microorganism of the genus Streptomyces or the genus Streptoverticillium 
having an acylase capable of removing hydrolytically the N-acyl group 
(--CO--R.sup.3) preferentially from the L-isomer of the DL-serine compound 
of the formula (I), or with an extract of said microorganism containing 
said acylase, to remove preferentially the N-acyl group (--CO--R.sup.3) 
from the amino group of the L-isomer present in the DLserine compound of 
the formula (I) and thereby to produce the L-3-(3,4-dihydroxyphenyl)serine 
compound of the formula (II), and separating this L-serine compound (II) 
from the N-acyl-D-3-(3,4-dihydroxyphenyl)serine compound of the formula 
(III) which remains unaltered without being de-acylated. 
If required, the process of this invention may further include a step of 
removing the residual hydroxyl-protecting groups R.sup.1, R.sup.2) from 
the resulting L-3-(3,4-dihydroxyphenyl)serine O-protected derivative of 
the formula (II) in a known manner to give 
L-3-(3,4-dihydroxyphenyl)serine, and/or a step of removing the N-acyl 
group (--CO--R.sup.3) from the resulting 
N-acyl-D-3-(3,4-dihydroxyphenyl)serine compound of the formula (III), if 
necessary, followed by a further step of removing the residual 
hydroxyl-protecting groups (R.sup.1,R.sup.2) from the resultant 
D-3-(3,4-dihydroxyphenyl)serine O-protected derivative in a known manner 
to give D-3-(3,4-dihydroxyphenyl)serine. Thus, when the 
N-acyl-D-3-(3,4-dihydroxyphenyl)serine compound of the formula (III) as 
obtained in accordance with the process of this invention is hydrolyzed by 
a chemical agent in a known manner for removal of the N-acyl group 
therefrom, there is produced D-3-(3,4-dihydroxyphenyl)serine or its 
O-protected derivative having the catecholic 3- and 4-hydroxyl groups 
protected. 
According to another aspect cf this invention, there is provided a process 
for de-acylating stereo-selectively an 
N-acyl-L-3-(3,4-dihydroxyphenyl)serine compound of the formula (I') 
##STR7## 
wherein R.sup.1, R.sup.2 and R.sup.3 are as defined above, which process 
comprises reacting the N-acyl-L-3-(3,4-dihydroxyphenyl) serine compound 
(I') with a microorganism of the genus Streptomyces or the genus 
Streptoverticillium having an acylase capable of removing hydrolytically 
the N-acyl group (--CO--R.sup.3) preferentially from the N-acyl-L-serine 
compound (I'), or with an extract of said microorganism containing said 
acylase, to remove said N-acyl group from the N-acyl-L-serine compound 
(I') and thereby to produce the L-3-(3,4-dihydroxyphenyl)serine compound 
of the formula (II) 
##STR8## 
wherein R.sup.1 and R.sup.2 are as defined above. 
In accordance with the process of this invention, any optically resolving 
chemical agent is not necessary, and besides, the step of decomposing the 
intermediate diastereomer salt as formed which was necessarily involved in 
the optical resolution procedure using an optically resolving chemical 
agent is not required owing to the use of the enzyme in the present 
process, so that an optically active 3-(3,4-dihydroxyphenyl)serine or its 
catecholic hydroxyl groups-protected derivative of high optical purity can 
readily be obtained by the process of this invention. 
The process of this invention is now described in more detail. The 
microorganism which is used in accordance with this invention may be any 
strain belonging to the genus Streptomyes or the genus 
Streptoverticillium, as far as it is such microorganism which is capable 
of removing hydrolytically the N-acyl group (R.sup.3 CO--) selectively 
from the N-acyl-L-3-(3,4-dihydroxyphenyl)serine or a catecholic 
hydroxyl-protected derivative thereof which is present in the DL-serine 
compounds of the general formula (I) shown hereinbefore, or such 
microorganism which has or produces an acylase capable of removing 
hydrolytically said N-acyl group selectively from said 
N-acyl-L-3-(3,4-dihydroxyphenyl)serine or said catecholic 
hydroxyl-protected derivative thereof. Examples of such microorganism 
which may conveniently be used in the process of this invention include 
the following species: Actinomyces aureoverticillatus (IMC S-0234) (ISP 
5080) (FERM P-7216) (ATCC 19726; CBS 465.68) (IFO 12742), Actinomyces 
bicolor (IMC S-0276) (ISP 5140) (ATCC 23614; CBS 469.68) (IFO 12746), 
Streptomyces blastmyceticus (IMC S-0189) (ISP 5029) (FERM P-7217) (ATCC 
19731; CBS 470.68) (IFO 12747), Streptomyces chartreusis (IMC S-226) (ISP 
5085) (ATCC 19738; CBS 476.68) (IFO 12753), Streptomyces flavopersicus 
(IMC S-0204) (ISP 5093) (ATCC 19756; CBS 494.68)(IFO 12769), Actinomyces 
flavotricini (IMC S-0219) (ISP 5152) (ATCC 23621; CBS 495.68) (IFO 12770), 
Streptoverticillium griseocarneum (IMC S-0237) (ISP 5004) (ATCC 19763; CBS 
501.68) (IFO 12776), Streptomyces hachijoensis (IMC S-0244) (ISP 5114) 
(FERM P-7218) (ATCC 19769; CBS 507.68) (IFO 12782), Streptomyces halstedii 
(IMC S-0194) (ISP 5068) (ATCC 19770; CBS 508.68) (IFO 12783), 
Streptoverticillium hiroshimense (IMC S-0179) (ISP 5037) (FERM P-7252) 
(ATCC 19772CBS 510.68) (IFO 12785), Streptomyces tendae (IMC S-0168) (ISP 
5101) (ATCC 19812; CBS 565.68) (IFO 12822), and Streptomyces toyocaensis 
(IMC S-0163) (ISP 5030) (FERM P-7253) (ATCC 19814; CBS 567.68) (IFO 12824) 
and others. All these microorganisms mentioned above are known stored type 
strain cultures of which microbiological properties are described in the 
"International Journal of Systematic Bacteriology" Volume 18, No. 2, pages 
84-176 (April 1968). The disclosure of this book are incorporated herein 
by reference. All the above-mentioned microorganisms are publicly 
available from a Japanese depository "The Institute for Fermentation, 
Osaka (IFO) Organization" at 17- 85 Juso-honmachi 2-chome, Yodogawa-ku, 
Osaka, 532, Japan, under IFO accession numbers which are mentioned in a 
catalogue "List of Cultures" 1984, 7th Edition, issued by the IFO, or from 
the "American Type Culture Collection", Washington D.C., U.S.A. The "IMC" 
numbers given to the above-mentioned microorganisms are the applicant's 
reference numerals of the cultures stored in the applicant's laboratory 
"Institute of Microbial Chemistry", Kami-Osaki, Meguro ku, Tokyo, Japan. 
The "ISP" numbers given to these microorganisms are the standard reference 
numerals alloted by "International Streptomyces Projects". Amongst the 
above-mentioned microorganisms, the strains having FERM P-7216, FERM 
P-7217 and FERM P-7218 as the F.R.I. deposit numbers were deposited on 5th 
Sept. 1983 in the Japanese depository "Fermentation Research Institute", 
Agency of Industrial Science and Technology, Japan and now deposited there 
under the accession numbers "FERM BP-640", "FERM BP-641" and "FERM 
BP-642", respectively, since 18th Oct. 1984 under the Budapest Treaty. The 
strains having FERM P-7252 and FERM P-7253 were deposited on 20th Sept. 
1983 in the "Fermentation Research Institute" and now deposited there 
under "FERM BP-643" and "FERM BP-644", respectively, since 18th Oct. 1984 
under the Budapest Treaty. 
In the process of this invention, the starting substrate compound of the 
formula (I) may be reacted with the microorganism as used in accordance 
with this invention, by being contacted with said microorganism which is 
present in the culture broth of said microorganism. To prepare the culture 
broth of the microorganism, the microorganism is cultivated in a known 
manner using conventional culture media. The carbon sources present in the 
culture medium used may be usual ones e.g. glucose, saccharose, fructose, 
mannose, starch, molasses and the like, and the nitrogen sources may be 
usual ones e.g. organic ones such as peptone, meat extract, yeast extract, 
corn steep liquor, urea and the like, and/or inorganic nitrogen compounds 
such as aqueous ammonia, ammonium sulfate, sodium nitrate and the like. 
Such culture media further containing appropriate quantities of inorganic 
salts such as magnesium sulfate, sodium chloride, mono-potassium 
phosphate, di-potassium phosphate and the like certain compounds required 
for good growth of the microorganism, and/or additive substances for 
derivation of the enzyme are preferred. The cultivation of the 
microorganism may preferably be carried out under aerobic conditions, e.g. 
by incubation under aeration and stirring. Preferred cultivation 
temperatures may be in a range of 20.degree. to 40.degree. C. The 
cultivation duration may normally be in a range of 1 day to 10 days in 
many instances. In the process of this invention, the microorganism used 
and the culture broth of the microorganism serve as the source for the 
acylase which participates in the enzymatic de-acylation reaction involved 
in the present process. In place of using the microorganism or the culture 
broth containing the microorganism, it is also feasible to use the cells 
which have been separated from the culture broth in the intact state, 
which may be live or dead or which may also have been immobilised in a 
manner known for the techniques for immobilisation of microbial cells. The 
process of this invention may also be performed using an extract of said 
microorganism containing said acylase. This acylase-containing extract of 
the microorganism may be in the form of such filtrate of the culture broth 
of the microorganism, such material obtained from treatment of the cells, 
particularly a cell homogenate wherein the acylase is existing, or a 
solution of the acylase which has been isolated in crude or pure form. 
This solution of the acylase may be a solution of a crude enzyme (the 
acylase) which has been recovered from the culture broth or the cells of 
the microorganism by fractional precipitation method with ammonium 
sulfate, or a solution of a purified enzyme (the acylase) which has been 
purified by a gel-filtration method or other known purification method for 
enzymes. The recovery and purification of the enzyme may be achieved 
according to any known method for preparing acylases. 
The N-acyl-DL-3-(3,4-dihydroxyphenyl)serine or a catecholic 
hydroxyl-protected derivative thereof according to the general formula (I) 
employed as the starting compound in the process of this invention may be 
prepared by reacting a carboxylic acid of the general formula (IV) 
EQU R.sup.3 COOH (IV) 
wherein R.sup.3 is as defined above, or a reactive derivative (a functional 
equivalent) thereof, such as the acid chloride and acid anhydride, in the 
presence or absence of a condensation agent or catalyst, with 
DL-3-(3,4-dihydroxyphenyl)serine which may be produced by previously known 
methods (see "J. Chem. Soc." pp. 658-662 (1947); "J. Am. Chem. Soc." 76, 
pp. 1322-1326 (1954); "Chem. Ber." 87, pp. 892-901 (1954)), or with a 
catecholic hydroxyl-protected derivative of 
DL-3-(3,4-dihydroxyphenyl)serine. The N-acyl group (R.sup.3 CO--) as used 
may preferably be acetyl group, chloroacetyl group, glycolyl group, 
benzoyl group and the like, though any acyl group may be used in 
accordance with this invention as long as it is cleavable preferentially 
by the enzymatic reaction of the present process. 
The starting N-acyl-DL-3-(3,4-dihydroxyphenyl) serine or a catecholic 
hydroxyl-protected derivative thereof according to the general formula (I) 
used as the starting compound contains two asymmetrical carbon atoms in 
the molecule thereof, so that there exist two isomers of the threo form 
and the erythro form. The threo-isomer or the erythro-isomer or even a 
mixture of them may be employed as the starting substrate in the process 
of this invention. 
The starting DL-serine compound of the general formula (I) which serves as 
the substrate in the enzymatic de-acylation reaction of the present 
process may either be protected or unprotected at its catecholic 3- and 
4-hydroxyl groups on the phenyl ring, depending on the purposes for which 
the de-acylated product of the present process is utilized. Thus, for 
example, when an optically active 3-(3,4-dihydroxyphenyl)serine is to be 
obtained, the catecholic hydroxyl groups of the substrate serine compound 
(I) may be unprotected. On the other hand, when it is ultimately desired 
to obtain an optically active 3-(3,4-dihydroxyphenyl)-N-methylserine by a 
subsequent N-methylation process, the enzymatic reaction in the process of 
this invention may preferably be conducted using the starting DL-serine 
compound of the general formula (I) in the form of the hydroxyl-protected 
derivative thereof, because the catecholic hydroxyl groups of the serine 
compound must have been protected for the subsequent N-methylation 
process. When the process of this invention is carried out using a 
catecholic hydroxyl-protected derivative of the 
N-acyl-DL-3-(3,4-dihydroxylphenyl)serine, an optically active 
3-(3,4-dihydroxyphenyl) serine can, of course, be obtained readily by 
removing the hydroxyl-protecting groups from the de-acylated product by a 
conventional deprotection technique after the enzymatic reaction of the 
present process was achieved. The hydroxyl-protecting groups (R.sub.1, 
R.sub.2) available for protection of the catecholic 3- and 4-hydroxyl 
groups of the starting N-acyl-DL-serine compound (I) may be any of the 
hydroxyl-protecting groups which are conventionally employed for blocking 
the catecholic hydroxyl groups, for example, an aralkyl group such as 
benzyl, an alkoxycarbonyl group such as ethoxycarbonyl, an alkylidene 
group such as methylene and isopropylidene, or a cycloalkylidene group 
such as cyclohexylidene. A most preferred one is benzyl group. When an 
alhylidene group or a cycloalkylidene group is employed for the protection 
of the catecholic hydroxyl groups, the groups R.sup.1 and R.sup.2 taken 
together form a single alkylidene or cycloalkylidene group. 
In the process of this invention, the enzymatic reaction of reacting the 
starting N-acyl-DL-serine compound of the formula (I) with the 
microorqanism or the acylase of said microorganism may preferably be 
carried out at a pH of 5 to 9, desirably at a pH of 6 to 5 and at a 
temperature of 20.degree. to 80.degree. C., desirably 35.degree. to 
60.degree. C. The reaction medium may be water not containing or 
containing, if necessary, a proportion of an organic solvent which does 
not inactivate the enzyme, such as a lower alkanol, e.g. ethanol. If 
required or if preferred, the enzymatic reaction may be conducted in the 
presence of cation of a metal such as cobalt as the catalyst added. The 
required reaction time may vary depending on the quantity and the activity 
of the enzyme used, the reaction temperature and other various factors, 
but normally the reaction may be conducted for a time of 30 minutes to 20 
hours. A reaction time of 2 hours to 18 hours will suffice in many cases. 
The step of the enzymatic reaction may be followed by the step of 
separating the resultant L-3-(3,4-dihydroxyphenyl)serine or the catecholic 
hydroxyl-protected derivative thereof according to the general formula 
(II) from the N-acyl-D-3-(3,4-dihydroxyphenyl)serine or the catecholic 
hydroxyl-protected derivative thereof according to the general formula 
(III) which remains without receiving the enzymatic de-acylation. This 
separation may be performed by a precipitation method utilizing the fact 
that generally, the N-acyl-D-3-(3,4-dihydroxyphenyl)serine compounds are 
less soluble than the L-3-(3,4-dihydroxyphenyl)serine compounds in an 
aqueous medium at acidic pH range. Alternatively, the separation of the 
L-serine compound (II) from the N-acyl-D-serine compound (III) may be 
achieved by a transfer-dissolution method, as illustrated by Example 2 
given later, in such a manner that the enzymatic reaction solution is 
extracted with n-butanol to afford a solution of both the L-compounds of 
the formula (II) and the D-compound of the formula (III) in n-butanol, the 
n-butanol is evaporated off from said solution, the resultant residue is 
taken up into 0.05M phosphate buffered solution (pH 7.0), the resulting 
solution containing the compounds (II) and (III) is adjusted to acidic pH 
with hydrochloric acid and then extracted with ethyl acetate to transfer 
mainly the compound (III) into ethyl acetate, the ethyl acetate extract is 
in turn, extracted with alkaline water (pH 9) to transfer the compound 
(III) into the aqueous phase which is then again extracted with ethyl 
acetate under the acidic condition (pH 1), and so on. 
The final products (II) and (III) as obtained by the process of this 
invention each may further be isolated and purified by an ordinary 
chromatographic method such as silica gel column chromatography and 
ion-exchange resin chromatography. It is also possible to make isolation 
and purification of the final products in a very easy way e.g. by 
precipitation method or crystallization method with utilizing the 
difference in the solublities of these products (II), (III).

This invention is now illustrated with reference to the following Examples 
to which this invention is not limited in any way. 
EXAMPLE 1 
(a) A culture medium comprising 1% potato starch, 1% glucose, 0.75% meat 
extract, 0.75% polypeptone, 0.3% sodium chloride, 0.1% magnesium sulfate 
hepta-hydrate, 0.0007% copper sulfate penta-hydrate, 0.0001% ferrous 
sulfate hepta-hydrate, 0.0008% manganese chloride tetrahydrate and 0.0002% 
zinc sulfate hepta-hydrate was placed in 100 ml-portions into 500 
ml-conical flasks, and the culture medium was then inoculated with a 
loopful quantity of a slant culture of Streptomyces hachijoensis (IMC 
S-0244) (ISP 5114) (FERM P-7218) (FERM BP-642). The shaken incubation was 
made at 27.degree. C. for 3 days to prepare a seed culture broth. This 
seed culture (10 ml) was inoculated into the culture medium of the same 
composition as described above which was charged in 1 l-portions into 5 
l-conical flasks, and shaken incubation was made at 27.degree. C. for 4 
days. The culture broth obtained was filtered to remove the microbial 
cells, and to the resulting broth filtrate (3 l) was added ammonium 
sulfate in small portions to 80% saturation under stirring. The mixture 
was allowed to stand in a cold chamber at 5.degree. C. overnight, and then 
centrifuged at 9,000 r.p.m. under cooling to 5.degree. C. The deposited 
precipitate comprising the enzyme was collected and dissolved in 150 ml of 
0.05M phosphate buffered solution to afford a solution of a crude enzyme 
(the acylase). 
(b) Water (60 ml) was added to 1 g of 
DL-threo-N-acetyl-3-(3,4-dibenzyloxyphenyl)serine, and to the resulting 
aqueous suspension was added 30% aqueous sodium hydroxide dropwise until 
the DL-serine compound dissolved in water to give a clear solution. To 
this solution were added 20 ml of 0.5M phosphate buffered solution (pH 
7.0) and a volume of water to a total volume of 90 ml to prepare a 
solution of the substrate serine compound. This solution of the substrate 
was admixed with 10 ml of the enzyme solution prepared as above and then 
was incubated at 37.degree. C. for 18 hours for the enzymatic reaction. 
After the reaction, the reaction solution was filtered to remove the 
precipitate as formed. The filtrate obtained was adjusted to pH 1 by 
addition of hydrochloric acid and the precipitate so deposited was 
recovered by filtration to afford 150 mg of 
D-threo-N-acetyl-3-(3,4-dibenzyloxyphenyl)serine as a first crop. The 
first precipitate which was obtained by filtration of said reaction 
solution was taken up into 0.1N hydrochloric acid under heating. The 
resultant solution was allowed to stand at ambient temperature, thereby 
depositing a precipitate. This precipitate was separated from the liquid 
phase by filtration to give 240 mg of 
D-threo-N-acetyl-3-(3,4dibenzyloxyphenyl)serine as a second crop. These 
crop products showed [.alpha.].sub.D.sup.20 -20.9.degree. (c 1, ethanol). 
The filtrate which remained after the separation of the D-isomer second 
crop (240 mg) by filtration was extracted with n-butanol, the extract in 
n-butanol was distilled under reduced pressure to remove the n-butanol, 
and the residue was crystallized out of isopropanol to afford 360 mg of 
L-threo-3-(3,4-dibenzyloxyphenyl)serine hydrochloride which showed 
[.alpha.].sub.D.sup.20 -5.99.degree. (c 1, ethanol). 
(c) The D-threo-N-acetyl-3-(3,4-dibenzyloxyphenyl) serine obtained as above 
was dissolved in a liquid mixture of 1N hydrochloric acid-methanol (1:1 by 
volume) and the resulting solution was heated for 5 hours under refluxing 
to effect the removal of the N-acetyl group, affording 
D-threo-3-(3,4-dibenzyloxyphenyl)serine. While, the 
L-threo-3-(3,4-dibenzyloxyphenyl)serine hydrochloride obtained as above 
was dissolved in ethanol and then catalytically reduced (hydrogenolysis) 
under hydrogen gas at 1 atm. at ambient temperature in the presence of 10% 
palladium-on-carbon to effect the removal of the hydroxyl-protecting 
benzyl groups, thereby affording L-threo-3-(3,4-dihydroxyphenyl)serine. 
Yield 100%. 
EXAMPLE 2 
(a) The culture medium of the same composition as used in Example 1 was 
inoculated with a loopful amount of a slant culture of Actinomyces 
aureoverticillatus (IMC S-0243) (ISP 5080) (FERM P-7216) (FERM BP-640) and 
the incubation was made at 27.degree. C. for 5 days. The culture broth 
obtained was filtered to remove the microbial cells, and the resulting 
broth filtrate (100 ml) containing the acylase was mixed with 108 mg of 
DL-erythro-N-acetyl-3-(3,4dibenzyloxyphenyl)serine. The mixture was 
incubated at 37.degree. C. for 18 hours to effect the enzymatic reaction. 
(b) After the completed reaction, the reaction solution was adjusted to pH 
2 by addition of hydrochloric acid and then extracted with n-butanol. The 
extract in n-butanol was distilled under reduced pressure to remove the 
n-butanol, and the residue was taken up into 0.05M phosphate buffered 
solution (pH 7.0). The solution so obtained was again adjusted to pH 2 
with aqueous HCl and then extracted with ethyl acetate. The resulting 
organic extract containing 
D-erythro-N-acetyl-3-(3,4-dibenzyloxyphenyl)serine as transferred into 
ethyl acetate was mixed with a volume of water, and the mixture was 
adjusted to pH 9 by addition of aqueous sodium hydroxide, so that the 
D-erythro-N-acetyl-3-(3,4-dibenzyloxyphenyl)serine was transferred into 
the aqueous phase. The aqueous phase was subsequently adjusted to pH 1 
with hydrochloric acid and again extracted with ethyl acetate. This 
extract in ethyl acetate was distilled under reduced pressure to remove 
the ethyl acetate, giving 34 mg of 
D-erythro-N-acetyl-3-(3,4-dibenzyloxyphenyl)serine. [.alpha.].sub.D.sup.20 
-14.6.degree. (c 1, ethanol). 
(c) The aqueous phase which remained after the ethyl acetate extraction of 
the HCl-acidfied solution at pH 2 of the aforesaid residue as obtained in 
the above procedure (b) was extracted with n-butanol. The resultant 
extract in n-butanol was distilled under reduced pressure to remove the 
n-butanol, and the residue was mixed with methanol. The mixture was 
filtered to remove the insoluble matters therefrom, and the filtrate was 
distilled under reduced pressure to remove the methanol. Crystallization 
of the so obtained residue from isopropanol gave 26 mg of 
L-erythro-3-(3,4-dibenzyloxyphenyl)serine hydrochloride. 
[.alpha.].sub.D.sup.20 +19.5.degree. (c 1, ethanol). 
When this L-erythro-3-(3,4-dibenzyloxyphenyl)serine hydrochloride was 
subjected to catalytic hydrogenolysis in the same manner as in Example 
1(c) to effect the removal of the hydroxyl-protecting benzyl groups, 
L-erythro-3-(3,4-dihydroxyphenyl)serine was obtained in a yield of 100%. 
EXAMPLE 3 
DL-Threo-N-acetyl-3-(3,4-dibenzyloxyphenyl)serine, 
DL-threo-N-chloroacetyl-3-(3,4-dibenzyloxyphenyl)serine, 
DL-threo-N-glycolyl-3-(3,4-dibenzyloxyphenyl)serine, 
DL-threo-N-benzoyl-3-(3,4-dibenzyloxyphenyl)serine, 
DL-threo-N-acetyl-3-(3,4-methylenedioxyphenyl)serine, 
DL-threo-N-acetyl-3-(3,4-dihydroxyphenyl)serine or 
DL-erythro-N-acetyl-3-(3,4-dibenzyloxyphenyl)serine was dissolved or 
suspended at the concentration of 2 mg/ml in 0.1M phosphate buffered 
solution (pH 7.0) to prepare different solutions of the substrates (the 
starting DL-serine compounds). The solution of the substrate (0.5 ml) was 
mixed with 0.5 ml of the enzyme solution which was prepared according to 
the procedure of Example 1(a) and which was containing the acylase of the 
microorganism, Streptomyces hachijoensis. The mixture so obtained was 
incubated at 37.degree. C. for 17 hours for the enzymatic reaction to 
effect the removal of the N-acyl group (the de-acylation) from the 
substrate compound. 
After the reaction, the reaction solution was analysed by high performance 
liquid chromatography with a column of "Nucleosil" 5 C.sub.18 (4.6 
mm.times.125 mm) to determine the yield of the de-acylation product which 
was produced by the removal of the N-acyl group from the substrate 
compound through the stereoselective enzymatic reaction involved. The 
results obtained are summarized in Table 1 below. 
TABLE 1 
______________________________________ 
Yield (%) of 
Substrate De-acylation product 
______________________________________ 
DL-threo-Nacetyl-3- 
100 
(3,4-dibenzyloxyphenyl)serine 
DL-threo-Nchloroacetyl-3- 
100 
(3,4-dibenzyloxyphenyl)serine 
DL-threo-Nglycolyl-3- 
31 
(3,4-dibenzyloxyphenyl)serine 
DL-threo-Nbenzoyl-3- 
10 
(3,4-dibenzyloxyphenyl)serine 
DL-threo-Nacetyl-3- 
68 
(3,4-methylenedioxyphenyl)serine 
DL-threo-Nacetyl-3- 
20 
(3,4-dihydroxyphenyl)serine 
DL-erythro-Nacetyl-3- 
81 
(3,4-dibenzyloxyphenyl)serine 
______________________________________ 
In the above table, the "Yield (%) of Deacylation product" was calculated 
according to the following equation: 
Yield (%) of Deacylation product 
##STR9## 
EXAMPLE 4 
The culture medium of the same composition as used in Example 1 was 
inoculated with a loopful quantity of a slant culture of Actinomyces 
aureoverticillatus (IMC S-0234) (ISP 5080) (FERM P-7216) (FERM BP-640); 
Streptomyces blastmyceticus (IMC S-0189) (ISP 5029) (FERM P-7217) (FERM 
BP-641); or Streptomyces hachijoensis (IMC S 0244) (ISP 5114) (FERM 
P-7218) (FERM BP-642), and the shaken incubation was made at 27.degree. C. 
for 4 days. The culture broth obtained was filtered to remove the 
microbial cells, and the resultant broth filtrate (0.5 ml) containing the 
acylase was mixed with 0.5 ml of the substrate solution comprising a 
solution containing DL-threo-N-acetyl-3(3,4-dibenzyloxyphenyl)serine at a 
concentration of 2 mg/ml in 0.1 M phosphate buffered solution (pH 7.0). 
The mixture was incubated at 37.degree. C. for 17 hours to effect the 
enzymatic reaction. 
After the completed reaction, the reaction mixture was made acidic by 
addition of 0.1 ml of 1N hydrochloric acid and then extracted with two 1 
ml-portions of n-butanol. The combined extracts in n-butanol were 
distilled under reduced pressure to remove the n-butanol. The residue was 
dissolved in 1 ml of a mixture of 0.1M phosphoric acid and methanol (40:60 
by volume), and the resultant solution was analysed by a high performance 
liquid chromatography with the same "Nucleosil 5 C.sub.18 " column as 
used in Example 3, to determine the yield of the de-acylation product as 
formed, namely the L-threo-3-(3,4-dibenzyloxyphenyl)serine. 
The results obtained are tabulated in Table 2 below. 
TABLE 2 
______________________________________ 
Yield (%) of De-acylation 
Strain product 
______________________________________ 
Actinomyces aureoverticillatus 
75 
(ISP 5080) (FERM BP-640) 
Streptomyces blastmyceticus 
54 
(ISP 5029) (FERM BP-641) 
Streptomyces hachijoensis 
92 
(ISP 5114) (FERM BP-642) 
______________________________________ 
In the above table, the "Yield (%) of De-acylation product" was calculated 
according to the following equation: 
##EQU1## 
EXAMPLE 5 
The procedures of Example 4 were repeated but using the other strains. The 
results obtained are summarized in Table 3 below. 
TABLE 3 
______________________________________ 
Yield (%) of De-acylation 
Strain product 
______________________________________ 
Streptoverticillium hiroshimense 
&gt;30 
(ISP 5037) (FERM BP-643) 
Streptomyces toyocaensis 
&gt;30 
(ISP 5030) (FERM BP-644) 
Actinomyces bicolor 
&gt;30 
(ISP 5140) 
Streptomyces chartreusis 
&gt;30 
(ISP 5085) 
Streptomyces flavopersicus 
&gt;30 
(ISP 5093) 
Actinomyces flavotrichini 
&gt;30 
(ISP 5152) 
Streptoverticillium griseocarneum 
&gt;30 
(ISP 5004) 
Streptomyces halstedii 
&gt;30 
(ISP 5068) 
Streptomyces tendae 
&gt;30 
(ISP 5101) 
______________________________________