Process for producing D-amino acids with composite immobilized enzyme preparation

A process for the efficient production of a D-amino acid from the corresponding DL-5-substituted hydantoin by one-step reaction which comprises using a composite immobilized enzyme at a pH about neutrality, said composite immobilized enzyme being obtained by immobilizing a hydantoinase having its optimal pH within an alkaline range and a D-N-carbamyl-.alpha.-amino acid amidohydrolase having its optimal pH about neutrality in a coexisting state on an immobilizing support, simultaneously, is disclosed.

FIELD OF THE INVENTION 
The present invention relates to a composite immobilized enzyme preparation 
and a process for producing D-amino acids using the preparation. In 
particular, the composite immobilized enzyme preparation of the present 
invention is useful for the production of D-.alpha.-amino acids which are 
intermediate compounds for the production of antibiotics, such as 
D-(p-hydroxyphenyl)glycine to be used for the production of the 
antibiotic, amoxycillin and the like. 
BACKGROUND OF THE INVENTION 
Optically-active D-amino acids are important compounds as intermediate 
compounds for drugs and it has been known that they can be produced 
efficiently by combining an asymmetric hydrolysis reaction of 
5-substituted hydantoins into the corresponding D-N-carbamyl-.alpha.-amino 
acids with the enzymes, hydantoinases (hereinafter sometimes abbreviated 
as "Hase") (JP-B 62-30785), and an conversion reaction of the resultant 
D-N-carbamyl-a-amino acids into the corresponding D-.alpha.-amino acids 
with the enzymes, D-N-carbamyl-.alpha.-amino acid .alpha.-midohydrolases 
(hereinafter sometimes abbreviated as "decarbamylase" or "DCase") 
(PCT/JP91/01696: WO 92/10579). 
In addition, JP-A 63-185382, WO 92/00739 and the like disclose that the 
respective reactions are carried out more efficiently by using these 
enzymes in the form of so- called immobilized enzymes wherein they are 
immobilized on supports such as ion exchange resins and the like. 
However, a two-step reaction has been employed for carrying out these 
reactions because the optimal and stable pH's of both enzymes are 
considerably different from each other. Therefore, both immobilized 
enzymes should be prepared separately and complicated reaction operations 
are required. 
OBJECTS OF THE INVENTION 
The present invention relates to a technique for producing D-.alpha.-amino 
acids efficiently by using an immobilized enzyme resin obtained by 
immobilizing both hydantoinase and decarbamylase on one immobilizing resin 
support in a coexisting state of the enzymes (hereinafter referred to as a 
"composite enzyme"), simultaneously. 
In these two enzymatic reactions for converting 5-substituted hydantoins 
into D-.alpha.-amino acids, in general, the optimal pH of the hydantoinase 
reaction is pH 8 to 9 and the solubility of the substrate is increased as 
increase in pH. In addition, the racemic reaction of the hydantoin ring is 
promoted in an alkaline range. Therefore, it is desired to carry out the 
hydantoinase reaction in the pH ranging from 7 to 10, preferably in an 
alkaline range. On the other hand, in general, the optimal pH of the 
decarbamylase reaction is pH 6.5 to 9.0 but the hindrance of the reaction 
by ammonia formed is remarkably increased as increase in pH. Therefore, it 
is desired to carry out the decarbamylase reaction at pH about neutrality. 
If so-called one-step reaction can be employed for carrying out these 
reactions, i.e., these two enzymatic reactions can be carried out in one 
reaction vessel simultaneously, in comparison with so-called two-step 
reaction wherein two different enzymes react with the substrates 
separately, reaction operations become simple and the overall reaction 
time can be shortened. In addition, by combining the hydantoinase reaction 
which is a reversible reaction and the decarbamylase reaction which is a 
irreversible reaction, the conversion yield of hydantoin can be improved 
as well as the subsequent purification of D-amino acids can be simplified. 
Thus, it is expected to significantly reduce the production cost. However, 
when the respective enzymes are immobilized on different immobilizing 
supports and they are mixed upon using them, the reactivities become 
inferior because of the difference in optimal pH and there is a problem in 
the stability of the immobilized enzymes. 
The present inventors have intensively studied to produce a composite 
immobilized enzyme preparation wherein both enzymes are immobilized on one 
immobilizing support simultaneously. If these two enzymes are immobilized 
simultaneously, two enzymatic reactions occur successively in micro-spaces 
of resins. Therefore, movement of the substrate for a decarbamylase, a 
D-N-carbamyl-.alpha.-amino acid, from immobilized Hase to immobilized 
DCase by diffusion is not required and pH variation in the micro-spaces 
can be minimized. Thus, it is expected that, in addition to increase in 
reactivities of the respective enzymes and relief of the product hindrance 
due to ammonia, stability of enzymes upon using repeatedly is improved. 
SUMMARY OF THE INVENTION 
The present invention provides a process for the efficient production of a 
D-amino acid from the corresponding DL-5-substituted hydantoin by one-step 
reaction which comprises using a composite immobilized enzyme at a pH 
about neutrality, said composite immobilized enzyme being obtained by 
immobilizing a hydantoinase having its optimal pH within an alkaline range 
and a D-N-carbamyl-.alpha.-amino acid amidohydrolase having its optimal pH 
about neutrality in a coexisting state on an immobilizing support of an 
anionic ion exchange resin, simultaneously (hereinafter referred to as 
composite enzyme).

DETAILED EXPLANATION OF THE INVENTION 
As for the hydantoinase used in the present invention, the enzymes from 
animals, plants and microorganisms can be used. The hydantoinases from 
microorganisms are suitable for industrial production. As such a 
microorganism, those disclosed in JP-B 62-30758 are exemplified. The 
bacteria include Achromobacter, Aerobacter, Aeromonas, Aqrobacterium, 
Alcaliqenes, Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, 
Enterobacter, Erwinia, Escherichia, Klebsiella, Microbacterium, 
Micrococcus, Protaminobacter, Proteus, Pseudomonas, Sarcina, Serratia, 
Xanthomonas and the like. The Actinomycetes include Actinomyces, 
Mycobacterium, Nocardia, Streptomyces, Actinoplanes and the like. The 
filamentous fungi include Asperqillus, Paecilomyces, and Penicillium and 
the like. The yeasts include Candida, Pichia, Rhodotorula, Torulopsis and 
the like. 
Among the above microorganisms, examples of strains which have relatively 
high hydantoinase activities and are excellent in practical use include 
Aerobacter cloacae IAM 1221, Agrobacterium rhizogenes IFO 13259, 
Brevibacterium incertum IFO 12145, Corynebacterium sepedonicum IFO 3306, 
Microbacterium flavum ATCC 10340, Micrococcus roseus IFO 3764, Pseudomonas 
striata IFO 12996, Mycobacterium smecmatis ATCC 607, Nocardia corallina 
IFO 3338, Streptomyces flaveolus IFO 3241, Bacillus sp. KNK108 (FERM 
P-6056), Bacillus sp. KNK245 (FERM BP-4863) and the like. 
In addition, the enzymes produced by artificial microorganisms in which 
hydantoinase productivity is imparted or increased by gene recombinant 
technique, for example, E. coli HB101pTH104 (FERM BP-4864), or the 
dihydropyrimidinase having similar activity as disclosed in JP-A 53-136583 
can be used. 
As for the decarbamylase used in the present invention, the origins thereof 
is not specifically limited and those derived from animals, plants and 
microorganism can be used. However, for industrial production, the enzymes 
from microorganisms are suitable. Examples of such microorganisms include 
naturally occurring microorganisms such as Agrobacterium Pseudomonas, 
Arthrobacter, Alcaligenes, Achromobacter, Moraxella, Paracoccus, 
Aerobacter, Aeromonas, Brevibacterium, Bacillus, Flavobacterium and 
Serratia disclosed in JP-B 57-18793, JP-B 63-20520 and JP-B 1-48758, or 
the artificial microorganisms described in WO 91/01696 in which 
decarbamylase productivity is imparted or increased by gene recombinant 
technique. Representative examples of such microorganisms include 
Agrobacterium sp. KNK712 (FERM BP-1900), Pseudomonas sp. KNK003A (FERM 
BP-3181), Pseudomonas sp. KNK505 (FERM BP-3182), Escherichia coli 
JM109pAD108 (FERM BP-3184), E. coli JM109pPD304 (FERM BP-3183) and the 
like. 
When a stabilized decarbamylase in which the amino acid responsible for 
heat resistance of the decarbamylase is replaced by another one is used, 
the following transformants disclosed in WO 94/03613 can be used. For 
example, the transformants include E. coli JM109pAD402 (FERM BP-3912), E. 
coli JM109pAD404 (FERM BP-3913), E. coli JM109pAD406 (FERM BP-3914), E. 
coli JM109pAD416 (FERM BP-3915), E. coli JM109pAD428, E. coli JM109pAD429 
(FERM BP-4035), E. coli JM109pAD431, E. coli JM109pAD434, E. coli 
JM109pAD435, E. coli JM109pAD439, E. coli JM109pAD441, E. coli 
JM109pAD445, E. coli JM109pAD447, E. coli JM109pAD448, E. coli 
JM109pAD450, E. coli JM109pAD421, E. coli JM109pAD422, E. coli 
JM109pAD423, E. coli JM109pAD424 (FERM BP-4034), E. coli JM109pAD425, E. 
coli JM109pAD426, E. coli JM109pAD427, E. coli JM109pAD451, E. coli 
JM109pAD452, E. coli JM109pAD453, E. coli JM109pAD461, E. coli 
JM109pAD454, E. coli JM109pAD455 (FERM BP-4036), E. coli JM109pAD456, E. 
coli JM109pAD468, E. coli JM109pAD469, E. coli JM109pAD470, E. coli 
HB101pNT4553 (FERM BP-4368) or the like. When such stabilized enzymes are 
used, better results can be obtained in repeated use of the composite 
immobilized enzyme preparation of the present invention. 
The enzymes used in the present invention can be produced simultaneously by 
using a microorganism which is capable of producing both enzymes. 
Alternatively, the enzymes can be produced separately or simultaneously 
using microorganisms which are capable of producing respective enzymes 
alone. As the microorganism which is capable of producing both enzymes, a 
microorganism isolated from a natural source such as an Aqrobacterium 
disclosed in JP-B 63-250520 and the like can be used. In addition, a 
recombinant microorganism prepared by isolating genes of both enzymes from 
the microorganisms isolated from a natural source and introducing them 
into a host, for example, the microorganisms disclosed in WO 94/00577, can 
be used. Alternatively, genes of both enzymes can be isolated from the 
same or different microorganisms and inserted into the same vector in the 
expressible form of both genes, or inserted into different vectors having 
different replication modes, for example, pUC19 and pACYC184. Then, the 
recipient host such as E. coli can be transformed with the above vector or 
vectors so that a single microorganism can produce both enzymes. In these 
cases, by selecting the kinds of promoters having various capabilities and 
the plasmids having different copy numbers, the ratio of production of 
each enzyme can be varied according to a particular purpose. However, it 
is preferred to adjust the ratio so as to obtain almost equal amounts of 
enzyme proteins. In the case that microorganisms which produce different 
enzymes separately are used, strains isolated from natural sources or 
recombinant microorganisms prepared by using gene recombinant technique 
can also be used. In such case, the production of the enzymes can be 
carried out either by cultivating the producer microorganisms separately, 
or cultivating in a mixed culture at various ratios, preferably, at such a 
ratio that almost equal amounts of the enzymes can be produced. 
The cultivation can be carried out aerobically, for example, by shaking 
culture using flasks or by spinner culture with aeration. As for culture 
medium used in the cultivation, normally, an nutrient medium which 
contains generally used natural nutrients such as meat extract and 
polypeptone and the like can be used. When hydantoinase is produced 
separately or simultaneously with decarbamylase, a good result can be 
obtained by carrying out the cultivation with addition of manganese ion in 
an amount of, for example, as manganese chloride, 1 to 100 mg/liter, 
preferably about 20 mg/liter. 
In the present invention, the enzymes can exist in the immobilized enzyme 
preparation in the purified, partially purified or crude form, or in some 
cases, in the form of microbial cells per se. And, in so far as the 
enzymes are under the conditions that the enzymatic activities can be 
exhibited the enzymes can exist in any form, and can be accompanied by any 
substance. 
In the preparation or use of the composite immobilized enzyme preparation 
of the present invention, better results can be obtained by adding an 
antioxidant for a repeated use. As such an antioxidant, there can be used 
dithiothreitol, 2-mercaptoethanol, L-cystein hydrochloride, cysteamine 
hydrochloride, dithioerythritol, a mixture of dithiothreitol and 
dithioerythritol, reduced glutathione and the like. 
An immobilized support used can be varied according to particular use 
conditions of the immobilized enzymes. When a crude enzyme solution such 
as a cell-free extract, or a partially purified enzyme solution treated by 
ammonium sulfate precipitation or the like are subjected to 
immobilization, polymer supports having ion exchanging groups or covalent 
bonding groups can be used. 
As for a polymer support having an ion exchanging group, for example, 
Duolite A (registered trade mark) series, or Amberlite IRA (registered 
trade mark) series, the exchanging groups of which are primary, secondary, 
tertiary and quaternary amines; or a polystyrene resin having a diethanol 
type functional group, for example, Diaion EX can be used. 
As for a support with a covalent bonding group, a substituted 
polymethacrylate polymer having an aldehyde as a bonding group, a high 
density alumina covered with a complex of polyethyleneimine/glutaraldehyde 
and the like can be used. 
To immobilize whole microbial cells such as live cells or dried cells, a 
polymer such as polyacrylamide, polyurethane or calcium alginate, or a 
porous material such as alumina can be used. 
The composite immobilized enzyme preparation of the present invention is 
produced as follows. A solution of the crude composite enzyme in which 
activities of Hase and DCase are appropriately adjusted is contacted with 
a support to adsorb the respective enzymes and treated with a 
cross-linking agent for stabilization. To prepare a solution of the crude 
composite enzyme, firstly, each enzyme is produced by cultivating 
microbial cells. In this case, a cell-free extract can be prepared by 
collecting the cells and disrupting them with, for example, sonication, 
mechanical disruption (for example, homogenizer) or enzyme treatment, when 
a microorganism capable of producing the enzymes simultaneously is used. 
When a solution of the composite enzyme is prepared by using different 
microorganisms, it can be prepared by cultivating the microorganisms 
separately, collecting the cells, preparing respective cell-free extracts 
and mixing them. Or, it can be prepared by cultivating respective producer 
microorganisms together and disrupting the cells at the same time to 
prepare a cell-free extract in which both two enzymes are mixed. When both 
enzymes are produced by different microorganisms, the productivities vary 
to a large extent according to the kinds of the microorganisms and 
difference in the cultivation methods. However, in general, regarding 
hydantoinase, a normal producer microorganism produces hydantoinase at 
about 0.1 to about 10 units/ml (one unit of the enzyme used herein is 
defined as an amount required to convert the substrate 
5-(p-hydroxyphenyl)hydantoin into D-N-carbamyl-p-hydroxy-phenylglycine at 
pH 8.7, 40.degree. C., for 1 min) and a recombinant artificial producer 
microorganism produces it at about 5 to about 150 units/ml. Regarding 
decarbamylase, a normal producer microorganism produces decarbamylase at 
about 0.01 to about 2 units/ml (one unit of the enzyme used herein is 
defined as an amount required to convert the substrate 
D-N-carbamyl-p-hydroxyphenylglycine into D-p-hydroxyphenylglycine at pH 
7.0, 40.degree. C., for 1 min) and a recombinant artificial producer 
microorganism produces it at about 0.1 to about 20 units/ml. The ratios of 
Hase and DCase activities in a solution of the crude composite enzyme are 
adjusted so that the reaction producing D-.alpha.-amino acid from 
5-substituted hydantoin can proceed most efficiently. As described 
hereinafter, this reaction is carried out at a pH of neutrality which is 
near the optimal pH for DCase and therefore the conditions are different 
from those for exhibiting maximal activity of Hase. Then, it is desired 
that both enzymatic activities are adjusted so that almost equal 
activities are exhibited at the reaction pH. For example, in the case of 
carrying out the reaction at pH 7.5, the desired composite immobilized 
enzyme preparation can be produced by carrying out the immobilization 
reaction using an enzyme solution in which there are almost equal amounts 
of proteins and enzymatic activities so that hydantoinase is about five 
times as much as the DCase in units. To maintain such activities after 
immobilization, it is desired that the ratio of the enzyme activities 
(Hase: DCase) in a solution of the crude composite enzyme should be in a 
range of 1 to 10:1. Therefore, after disruption of the microbial cells and 
before adsorption, the activities of the crude composite enzyme are 
adjusted to such levels. 
In addition, to adsorb appropriate amounts of enzymes on the support, it is 
preferred to adjust the concentration of decarbamylase in the solution of 
the crude composite enzyme to 10 to 300 units/ml. The activity adsorbed is 
about 20 to about 90% of the activity added and no substantial difference 
can be seen between both enzymes. 
The support is used after activation of its exchanging group with, for 
example, aqueous solution of sodium chloride and equilibrating, for 
example, in a buffer solution. It is preferred that the ratio of the 
solution of the crude composite enzyme and the support is adjusted so that 
the total amount of the protein in the crude enzyme solution are almost 
the same as the maximum adsorption capacity of the support. If necessary, 
1 to 10 mM of an antioxidant and/or 0.5 to 20 mM of manganese ion can be 
present. The mixture is stirred at 4 to 30.degree. C., preferably at 
15.degree. C., and the support is collected by filtration after the amount 
of enzyme adsorbed reaches the given amount (normally 8 to 48 hours, 
preferably this is carried out under the atmosphere of inert gas, such as 
nitrogen). Then, the support is washed and insolubilized by treating with 
a cross-linking agent to stabilize it. As for a cross-linking agent, a 
known agent such as at less than 1%, preferably 0.1 to 0.2% glutaraldehyde 
can be used. The cross-linked composite enzyme immobilized preparation is 
washed with distilled water and a buffer solution (preferably containing 
0.1 to 20 mM, normally 1 to 5 mM antioxidant as described above) and 
stored in wet state in a sealed vessel at a low temperature (4.degree. 
C.). In general, the activities of the composite immobilized enzyme 
preparation thus obtained are 5 to 80 units/g support with respect to 
decarbamylase. For hydantoinase, its activity is 1 to 10 times as much as 
the decarbamylase activity according to the adsorption conditions. 
The process for producing D-a-amino acids from 5-substituted hydantoins by 
using the composite immobilized enzyme preparation of the present 
invention will be described hereinafter. 
The reaction is carried out by reacting the substrate, 5-substituted 
hydantoin, with the composite immobilized enzyme preparation, if 
necessary, in the presence of an antioxidant agent and/or manganese ion. 
Normally, it is preferred that the reaction is carried out in the presence 
of 0.1 to 20 mM of an antioxidant agent and manganese ion. The reaction 
proceeds according to the following reaction scheme: 
##STR1## 
wherein R represents phenyl group, phenyl group substituted with hydroxy 
group, alkyl group, substituted alkyl group, aralkyl group or thienyl 
group. 
The concentration of the substrate 5-substituted hydantoin to be used is 
0.1 to 30% (w/v), preferably 1 to 5% (w/v). It is preferred that the 
amount of the composite immobilized enzyme preparation to be used is about 
10 to about 20 units/g substrate as decarbamylase activity. The reaction 
temperature varies according to particular enzymes but, in general, it is 
30 to 60.degree. C. Enzymes having heat resistance be used at much higher 
temperature. 
The reaction pH is suitably selected from the range of 6.5 to 8.0. The pH 
range is almost optimal for DCase. However, it is considerably far from 
the optimal pH for Hase and the Hase activity decreases several times as 
low as the activity at the optimal pH. The rate of racemization also 
decreases. However, ultimately, the production of D-.alpha.-amino acid is 
governed by the enzyme of the last step, DCase, it is preferred to set the 
amount of the enzyme and the reaction conditions based on the DCase (i.e., 
the optimal conditions for DCase) and to combine Hase activity in 
proportion to that DCase activity. In general, the ratio of the enzymes to 
be adsorbed to the support is adjusted so that almost equal activities can 
be expressed, although this varies according to particular reaction 
conditions. The "almost equal activities" mean the situation where the 
ratio of the hydantoinase activity measured at pH 7.5 (represented in 
"units (pH 7.5)"), and the decarbamylase activity as described above is 
about 0.5 to 1.5:1. The reaction is carried out with adjusting a reaction 
pH to the pH range thus selected, normally to pH 7.5. By these operations, 
the unfavorable conditions for Hase are overcome and the reaction 
equilibrium is declined toward the production of D-N-carbamyl amino acid. 
Thus, the reaction can be carried out more efficiently than expected and 
the conversion of the substrate, a 5-substituted hydantoin, can be 
improved. When the reaction is carried out at an alkaline pH range near 
the optimal pH of hydantoinase, the decarbamylase activity decreases to 
below the half of the activity at the optimal pH, though the production of 
a D-N-carbamyl amino acid can proceed successfully. In addition, it has 
been observed that the whole yield decreases and that the stability of 
decarbamylase itself is lowered because the reaction is largely inhibited 
by ammonia produced by the reaction. For these reasons, the reaction using 
the composite immobilized enzyme preparation can be carried out 
efficiently when the composite immobilized enzyme preparation having such 
immobilization ratio that the decarbamylase reaction is slightly 
rate-determining is used under conditions near the optimal reaction 
conditions and the substrate concentration of decarbamylase. In addition, 
it is advantageous that the whole activities of the composite immobilized 
enzyme preparation is easily controlled because the productivity of Hase 
is high and the DCase catalyzes the irreversible reaction. 
The reaction is carried out normally by column method or by suspending the 
composite immobilized enzyme preparation in a reaction vessel. In the 
latter case, a batch reaction is usually carried out and the reaction time 
is about 6 to about 48 hours per batch. By using the composite immobilized 
enzyme preparation of the present invention, the corresponding D-amino 
acid can be produced from a 5-substituted hydantoin at a high yield in a 
one-step reaction. The following examples further illustrate the present 
invention in detail but are not to be construed to limit the scope 
thereof. 
EXAMPLE 1 
Firstly, for preparing an enzyme solution of hydantoinase, Bacillus sp. 
KNK108 (FERM P-6056) was inoculated to a 250 ml of seed culture medium 
(meat extract 1.0%, polypeptone 1.0%, yeast extract 0.5% (pH 7.0)) and 
cultivated at 33.degree. C. for about 25 hours. This seed culture was 
inoculated to 2.5 liter of a culture medium (meat extract 1.0%, 
polypeptone 1.0%, yeast extract 0.5%, uracil 0.1%, MnCl.sub.2 20 ppm (pH 
7.5)) and cultivated at 33.degree. C. for about 16 hours. The microbial 
cells were collected by centrifugation and suspended in 50 ml of 20 mM 
MnSO.sub.4 aqueous solution. After adjusting the pH to 8.5, the cells were 
disrupted by sonication and the residue was removed to obtain a crude 
enzyme solution of hydantoinase (107 units/ml). 
In addition, to prepare an enzyme solution of decarbamylase, E. coli 
HB101pNT4553 (FERM BP-4368) was cultivated in 20 ml of 2YT medium (Bacto 
peptone 1.6%, Bacto yeast extract 1.0%, NaCl 0.5%) supplemented with 50 
.mu.g/ml ampicillin at 37.degree. C. for about 16 hours. This culture was 
inoculated to 1.4 liter of 2YT medium in an amount of 1%, and cultivated 
at 37.degree. C. for about 28 hours. The cells were collected by 
centrifugation and suspended to 140 ml of 5 mM dithiothreitol solution. 
After adjusting the pH to 7.0, the cells were disrupted by sonication and 
the residue was removed to obtain the supernatant as a crude enzyme 
solution of decarbamylase (36 units/ml). 
EXAMPLE 2 
By using the crude enzyme solutions obtained in Example 1, immobilization 
of the enzymes was carried out. Duolite A-568 (Rohm & Haas) as a 
immobilization support was washed with firstly 1M NaCl and deionized water 
and then put into the deionized water and the pH was adjusted to 7.5. To 
8.4 g of this resin were added 20 ml of the crude enzyme solution of 
hydantoinase and 11.5 ml of the crude enzyme solution of decarbamylase the 
pH of both of which had been adjusted to 7.5 and the mixture was stirred 
at 15.degree. C. for about 20 hours under nitrogen atmosphere. After 
washing this resin twice with 0.5 mM MnSO.sub.4 solution, the resin was 
suspended in five time volumes of deionized water. After the pH was 
adjusted to 7.5, the suspension was stirred for 35 minutes with addition 
of 544 .mu.l of 2.5% glutaraldehyde with portions. This suspension was 
treated with 50 mM Tris-HCl (pH 7.5), 5 mM DTT and 1 mM MnSO.sub.4 
overnight and then the composite immobilized enzyme preparation was 
collected by filtration (hydantoinase activity 8.2 units (pH 7.5) and 
decarbamylase activity 9.9 units per 1 g resin). 
Then, as controls, resins to which two enzymes were separately immobilized 
were prepared. 
Each of 20 ml of the crude enzyme solution of hydantoinase and 60 ml of the 
crude enzyme solution of decarbamylase was mixed with each of 4.2 g and 
22.0 g of the above immobilization support and the same operations as 
described above was carried out to obtain each immobilized enzyme resin in 
which each enzyme was separately immobilized (hydantoinase immobilized 
enzyme: 24 units/g resin, 2.7 units/g resin (pH7.5), and decarbamylase 
immobilized enzyme: 37 units/g resin). 
EXAMPLE 3 
By using the composite immobilized enzyme preparation and control 
immobilized enzymes containing respective single enzymes obtained in 
Example 2, the reactions to produce the corresponding D-.alpha.-amino acid 
from a 5-substituted hydantoin were carried out. 
After addition of 1g of 5-(p-hydroxyphenyl)hydantoin as the substrate to 
100 ml of 0.1 M KPB (pH 7.5), 1 MM MnSO.sub.4 and 5 mM DTT, nitrogen gas 
was sufficiently bubbled in and the reaction conditions were adjusted to 
40.degree. C. and pH 7.5. In order to obtain the same enzymatic activities 
of both enzymes, 0.97 g of the composite immobilized enzyme preparation or 
3.0 g of the hydantoinase immobilized enzyme and 0.26 g of the 
dacarbamylase immobilized enzyme were added. The reaction was carried out 
with bubbling of nitrogen gas and controlling the reaction pH to 7.5 with 
2N H.sub.2 SO.sub.4 or 6N NaOH. The reaction was carried out for 29 hours 
with periodical samplings. The amount of p-hydroxyphenylglycine produced 
at each sampling point was determined by high performance liquid 
chromatography (Nippon Bunko, Finepack SIL C-18 column). 
The results are shown in FIG. 1. 
As is seen from FIG. 1, it is found that the reaction can be carried out 
more efficiently when two enzymes were immobilized simultaneously on the 
same resin. 
EXAMPLE 4 
The stability of the enzyme activities was investigated by carrying out the 
reaction repeatedly with the composite immobilized enzyme preparation. Two 
grams of 5-(p-hydroxyphenyl)hydantoin was added to 100 ml of 1 mM 
MnSO.sub.4 and 5 mM DTT and pH was adjusted to 7.5. To the mixture was 
added 8.9 g of the composite immobilized enzyme preparation obtained in 
Example 2 was added and the reaction was carried out at 40.degree. C. for 
23 hours with bubbling of nitrogen gas and controlling the pH to 7.5. 
After filtering the reaction mixture with suction, another mixture was 
added to the composite immobilized enzyme according to the same manner as 
described above and the reaction was carried out. This operation was 
repeated five times and both enzyme activities were determined at the end 
of each reaction. 
The relative activities to those in the first reaction are shown in FIG. 2. 
The decrease in the activities were scarcely observed in these five 
reactions. 
EXAMPLE 5 
For preparing an enzyme solution of hydantoinase, E. coli HB101pTH104 (FERM 
BP-4864) containing a hydantoin gene from Bacillus sp. KNK245 (FERM 
BP-4863) was cultivated in 20 ml of 2YT medium at 37.degree. C. for about 
16 hours. This culture was transferred to 1.2 liter of 2YT medium 
supplemented with 50 .mu.g/ml of ampicillin and 400 ppm of 
MnCl.sub.2.4H.sub.2 O and cultivated for 26 hours at 37.degree. C. The 
cells were collected by centrifugation and suspended in 80 ml of 1 mM 
MnSO.sub.4 aqueous solution. After adjusting the pH to 8.5 with ammonia 
water, the cells were disrupted by sonication and the residue was removed 
by centrifugation. After adjusting the pH of the supernatant to 8.5, heat 
treatment at 60.degree. C. was carried out for 20 min. The denatured 
proteins were removed by centrifugation to obtain a crude enzyme solution 
of hydantoinase (1,100 units/ml). 
By using this crude enzyme solution and a crude enzyme solution of 
decarbamylase prepared according to the same manner as described in 
Example 1 (240 units/ml), the composite immobilized enzyme preparation was 
prepared according to the same manner as described in Example 2. By using 
30 ml of the crude enzyme solution of decarbamylase and 13 ml of the crude 
enzyme solution of hydantoinase, the enzymes were immobilized on 29 g of 
the resin according to the same manner as described in Example 2 
(decarbamylase: 45 units, and hydantoinase: 119 units, 44 units (pH 7.5) 
per 1 g of resin) 
As controls, resins on which two enzymes were separately immobilized were 
used. The decarbamylase immobilized enzyme (43 units/g-resin) was prepared 
as described in Example 2. The hydantoinase immobilized enzyme was 
prepared according to the method described in Example 2 by mixing 21.8 g 
of the resin for immobilization with 60 ml of the crude enzyme solution 
(177 units/g-resin, 51 units/g (pH 7.5)) 
EXAMPLE 6 
By using 5 g of the composite immobilized enzyme preparation obtained in 
Example 5, and as immobilized enzymes prepared by immobilizing respective 
enzymes on different resins, a mixture of 5.6 g of the hydantoinase 
immobilized enzyme and 5.2 g of the decarbamylase immobilized enzyme 
obtained in Example 5 (the decarbamylase activity (pH 7.5) was equal to 
the composite immobilized enzyme preparation and the hydantoinase activity 
(pH 8.7) was twice as much as the composite immobilized enzyme 
preparation), the reaction was carried out with 3% substrate according to 
the method described in Example 3. 
The results are shown in FIG. 3. 
As shown in FIG. 3, the reactivity of the composite immobilized enzyme 
preparation was similar to the reactivity of the separately immobilized 
enzymes in spite that the hydantoinase activity was one half of the 
separately immobilized enzyme. Thus, it has been found that the reaction 
by the composite immobilized enzyme preparation is more efficient and that 
the amount of the expensive resin for immobilization can be largely 
reduced. 
EXAMPLE 7 
By using each 5 g of the composite immobilized enzyme obtained in Example 
5, the effect of the reaction pH was investigated. In three pH levels of 
7.0, 7.25 and 7.5, the reactions with 3% substrate were carried out. The 
times required to convert 99% of the substrate was measured. The times 
were 5.5, 5.4 and 6.75 hours, respectively. Thus, it have been found that 
pH 7.0 and 7.25 are advantageous to the reaction. 
As described hereinabove, according to the present invention, by using of 
the composite immobilized enzyme preparation produced by immobilizing the 
hydantoinase and the decarbamylase simultaneously on the same resin in the 
production of the corresponding D-.alpha.-amino acids from 5-substituted 
hydantoins, it is possible to carry out the one-step reaction with much 
simpler operations than two-step reaction and more efficiently than the 
reaction by the mixture of the two immobilized enzymes obtained by 
immobilizing these enzymes separately to the different resins.