Note: Descriptions are shown in the official language in which they were submitted.
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Proces~ for the preparation of D-proline derivatives
using microorganisms
The invention relates to a novel enzyme with
amidinohydrolase activity, to novel microorganisms
comprising these amidinohydrolases, and to a novel
process for the preparation of D-proline derivatives
using the microorganisms or amidinohydrolases.
D-proline derivatives are important
intermediates for the preparation of pharmaceuticals
(J. Org Chem., 1994, 59, 7496-7498).
A plurality of processes are known for the
preparation of D-proline.
JP-A 92183399 describes a process for the preparation
of D-proline starting from (DL)-proline, using
microorganisms of the genus Candida or Trichospora. The
disadvantage of this process is that the conversion
time is too long and the yield of D-proline is poor.
JP-A 07127354 describes a process for the preparation
of D-proline starting from ornithine using
microorganisms of the species Proteus mitajiri. The
disadvantage of this process is that, on the one hand,
ornithine is too expensive as starting material, and,
on the other hand, that the yield of D-proline is poor.
Moreover, JP-A 07289275 describes a process for the
preparation of D-proline starting from L-proline. Here,
L-proline is racemized into (DL)-proline by
microorganisms of the genus Escherichia, which have
racemase activity, and the (DL)-proline is then grown
with L-proline-degrading microorganisms to obtain D-
proline. The disadvantage of this process is that the
resulting D-proline requires further purification,
which entails high yield losses.
It is the object of the present invention to
provide an amidinohydrolase or microorganisms which
comprise this enzyme, both of which can be employed for
an inexpensive process for the preparation of D-proline
derivatives in which D-proline is obtained in a high
yield.
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This object is achieved by the microorganisms
according to Claim 1, the amidinohydrolase according to
Claim 4 and the process according to Claim 5.
The microorganisms according to the invention
can be isolated from soil samples, sludge or waste
water employing customary microbiological methods.
According to the invention, the microorganisms are
isolated in such a way that they are grown in a medium
comprising a guanidinevaleric acid derivative of the
general formula
~I R2
H2r~ ~,O V
NH OH
in which Rl is hydrogen or hydroxyl and R2 is a halogen
atom or -NH2 as the only nitrogen source, together with
a suitable carbon source.
Examples of suitable guanidinevaleric acid derivatives
of the general formula V are: L-a-chloro-~-
guanidinevaleric acid, D-a-chloro-~-guanidinevaleric
acid, (DL)-a-chloro-~-guanidinevaleric acid, L-a-bromo-
~-guanidinevaleric acid, D-a-bromo-~-guanidinevaleric
acid, (DL)-a-bromo-~-guanidinevaleric acid, L-a-chloro-
~-hydroxy-~-guanidinevaleric acid, D-a-bromo-~-hydr
~-guanidinevaleric acid, and L-, D- or (DL)-arginine.
Then, it is expedient to select those
microorganisms from the culture obtained by growing
which utilize L-a-chloro-~-guanidinevaleric acid (Rl =
H, R2 = Cl) or L-arginine (Rl = H, R2 = NH2) as the only
nitrogen source.
Examples of growth substrates which the
microorganisms can utilize as suitable carbon source
are sugars, sugar alcohols or dicarboxylic acids.
Hexoses, such as glucose, may be used as sugar.
Glycerol is an example of a sugar alcohol which may be
used. An example of a dicarboxylic acid which can be
used is succinate. Moreover, arginine, agmatine (4-
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aminobutylguanine), glutamine or glutamic acid may be
employed as carbon source.
The selection and growth media which can be
used are those conventionally used in expert circles,
for example the medium described in Table 1. The medium
described in Table 1 is preferably used.
During growth and selection, it is expedient to
induce the effective enzymes of the microorganisms.
Enzyme inductors which can be used are, for example, a-
chloro-~-guanidinevaleric acid, L-arginine, guanidine,
guanidine hydrochloride or guanidine acetate. However,
the active enzymes may also be induced in a complete
medium such as, for example, with "nutrient yeast
broth/' (NYB), if it comprises one of the inductors.
Growth and selection are normally carried out
at a temperature of 20 to 40~C, preferably 25 to 40~C,
and at a pH of between pH 5 and pH 9.5, preferably
between pH 8 and pH 9.5.
a-chloro-~-guanidinevaleric acid-utilising
microorganisms which are preferably isolated are those
of the genera Pseudomonas, Arthrobacter, Agrobacterium
or Klebsiella, in particular microorganisms of the
species Agrobacterium radiobacter, Pseudomonas cepacia,
Klebsiella pneumoniae DSM 10593, Pseudomonas aeruginosa
DSM 10581 or Arthrobacter sp. DSM 10582, and their
functionally equivalent variants and mutants. The
strains Klebsiella pneumoniae, Arthobacter sp. and
Pseudomonas aeruginosa were deposited on 11.03.1996 in
compliance with the Budapest Treaty at the Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH,
Mascheroderweg lb, D-38124 Braunschweig.
"Functionally equivalent variants and mutants"
are to be understood as meaning microorganisms which
have essentially the same characteristics and functions
as the original microorganisms. Such variants and
mutants may be formed at random, for example by means
of W irradiation, or by mutagenic chemicals.
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T~xonomic description of Klebsiella pneumoniae (DSM
10593)
Strain characteristics
Cell shape Rods ADH
Width in ,um 1.0-1.2 LDC
Length in ~m 1.2-2.0 ODC
Motility - VP +
Gram stain reaction - Indole
Lysis by 3% KOH + H2S formation
Aminopeptidase (Cerny) + Simmons citrate +
Spores - Urease +
Oxidase - Methyl red
Catalase + Hydrolysis of
Growth, anaerobic + Gelatine
Gas from glucose + DNA
Acid from (ASA) Tween 80
Glucose +
Fructose + Abbre~iations:
Xylose + ASA: acetylsalicyclic
Erythritol - acid
Adonitol - ONPG: o-nitrophenyl-
D-Mannose + galactosidase
L-Rhamnose + ADH: alcohol dehydro-
Inositol + genase
Sorbitol + LDC: lactate decar-
a-Methyl-D-glucoside + boxylase
Cellobiose + ODC: ornithine decar-
Maltose + boxylase
Lactose + VP: Voges Proskauer
D-Arabitol +
ONPG +
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Taxonomic description of Arthrobacter sp. (DSM 10582)
Characterization Gram-positive, coryneform rods,
coccoid in older cultures, strictly
aerobic, no acid or gas formation
from glucose
Motility
Spores
Catalase +
meso-Diaminopimelic acid in cell wall: no
Peptidoglycan type: n.d.
The amidinohydrolase according to the invention
can be obtained from the microorganisms described above
and is capable of liberating urea from guanidinevaleric
acid derivatives selected from amongst compounds of the
general formula V.
The amidinohydrolase is expediently obtained
from the microorganisms of the genus Klebsiella,
Pseudomonas, Agrobacterium or Arthrobacter, in
particular from the microorganisms of the species
Agrobacterium radiobacter, Pseudomonas cepacia,
Klebsiella pneumoniae DSM 10593, Arthrobacter sp. DSM
10582 or Pseudomonas aeruginosa DSM 10581.
To obtain the amidinohydrolase according to the
invention, these microorganisms are grown (cultured) in
the customary manner in an aqueous nutrient medium
which comprises a carbon source, a nitrogen source,
mineral salts and vitamin source. The microorganisms
are expediently cultured at a temperature of from 20 to
40~C and a pH of from 5 to 8. Then, after the
microorganism cells have been ruptured, for example by
the ultrasound, French press or lysozyme method, the
amidinohydrolases can be isolated by methods of enzyme
purification which are known per se.
The amidinohydrolase expediently has the
following properties:
a) a pH optimum of pH 8.5 + 1,
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WO 98/01577 - 6 - PCT/EP97/03546
b) a temperature optimum of approx. 37~C at a pH of
between 7 and 8.
c) a KM value for the substrate L-a-chloro-~-
guanidinevaleric acid of 85.2 mM + 5 (100 mM
phosphate buffer; 37~C),
and furthermore the property that it
d) is induced by L-a-chloro-~-guanidinevaleric acid,
L-arginine, guanidine hydrochloride, guanidine
and/or guanidine acetate,
e) is inhibited by L-a-chloro-~-guanidinevaleric acid
and
f) hydrolyses the substrates arginine, L-a-chloro-~-
guanidinevaleric acid and L-a-bromo-~-
guanidinevaleric acid to the corresponding L-
aminovaleric acid derivative.
For the preparation according to the invention
of D-proline derivatives, it is expedient to convert,
in a first process step, an L-arginine derivative or a
salt thereof of the general formula
R' NH2
H2N ~ NH ~50 11
NH bH
in which Rl has the abovementioned meaning in a manner
known per se into an L-guanidinevaleric acid derivative
or a salt thereof of the general formula
R' R'
H.N ~ NH ~ O Ill
NH OH
in which Rl has the abovementioned meaning and R3 is a
halogen atom. The corresponding diazonium salt is
formed as intermediate.
L-arginine or L-~-hydroxyarginine may be used as
the L-arginine derivative.
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Salts of the L-arginine derivatives and of the
L-guanidinevaleric acid derivative which can be
employed are their hydrochlorides or hydrobromides.
The first step of the diazotization reaction is
usually carried out with a nitrite solution or with
nitric acid. A sodium nitrite or potassium nitrite
solution may be used as the nitrite solution. Nitric
acid, in particular 50 to 65% strength nitric acid, is
preferably used.
The first step of the reaction is usually
carried out in the presence of a hydrohalic acid.
Hydrochloric acid or hydrobromic acid may be used as
the hydrohalic acid; hydrochloric acid is preferably
used.
The first step of the reaction is expediently
carried out at a temperature of from -10~C to 100~C,
preferably from 0~C to 80~C.
In the second process step according to the
invention, the L-guanidinevaleric acid derivative
(formula III) is hydrolyzed using an amidinohydrolase
according to Claim 4, microorganisms according to Claim
1 and/or enzyme extracts thereof, to give an L-
aminovaleric acid derivative of the general formula
R' R3
H2~ 0 1~!
0~
in which Rl and R3 have the abovementioned meaning.
In principle, the microbiological hydrolysis
may be performed both with the amidinohydrolases which
have already been described and with the enzyme
extracts and microorganisms which have already been
described. Especially suitable for the hydrolysis are
the above-described microorganisms of the genera
Pseudomonas, Arthrobacter, Agrobacterium or Klebsiella,
in particular the microorganisms of the species
Klebsiella pneumoniae DSM 10593, Psuedomonas aeruginosa
... , . .. .. ~ _
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DSM 10581, Arthrobacter sp. DSM 10582, Agrobacterium
radiobacter or Pseudomonas cepacia, and their
functionally equivalent variants and mutants.
The microbiological hydrolysis may be carried
out using dormant cells (non-growing cells which no
longer require a carbon and energy source) - after the
microorganisms have been grown in the customary manner
- or using growing cells. The hydrolysis is preferably
carried out using dormant cells.
To perform the microbiological hydrolysis,
media which are conventionally used by those skilled in
the art may be employed, such as, for example,
phosphate and Hepes buffers of low molarity, complete
media such as, for example, "nutrient yeast broth"
(NYB), or the medium described in Table 3. The
hydrolysis is preferably carried out in a phosphate or
Hepes buffer of low molarity.
The hydrolysis is expediently carried out with
single or continuous addition of the L-guanidinevaleric
acid in such a way that the concentration does not
exceed 20% by weight, preferably 1% by weight.
In the third step, the L-aminovaleric acid
derivative (general formula IV) is cyclized to the end
product of the general formula
H
~ ~"~OH
H o
in which R1 has the abovementioned meaning.
The pH range of the medium is expediently
chosen in such a way that the second step and the third
step are carried out without isolating the L-
aminovaleric acid derivative (formula IV). In this
preferred embodiment, the pH of the medium is brought
to pH 5 to pH 13, preferably to pH 7 to pH 9.5. The L-
aminovaleric acid derivative (formula IV) spontaneously
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cyclizes to D-proline in this range. The cyclization
and the hydrolysis are expediently carried out at a
temperature of from 10 to 60~C, preferably from 30 to
40~C.
In principle, the D-proline derivatives of the
formula I may also be prepared by hydrolysing the L-
arginine derivative of the formula II or its salts
directly with the abovedescribed microorganisms, enzyme
extracts or amidinohydrolases to compounds of the
formula VI
R~ R4
H,N~"~50 ~/1
OH
in which R1 has the abovementioned meaning and R4 is
-NH2, subsequently converting these compounds into an L-
aminovaleric acid derivative of the formula IV as
described above via a diazonium salt as intermediate
and cyclizing the L-aminovaleric acid derivative of the
formula IV to the desired proline derivatives. However,
this process is less preferred.
Examples:
Example 1
Isolation of the microorganisms Klebsiella pneumoniae,
Pseudomonas cepacia, Agrobacterium radiobacter and
Arthrobacter sp.
Soil samples from garden compost or the LONZA
water treatment plant (Visp) were incubated at 37~C
with shaking in the following minimal medium.
Glucose or glycerol were provided in concentrations of
1-20 g/l as carbon source.
L-arginine, urea, (NH4)2SO4, L-a-chloro-~-guanidine-
valeric acid were employed in concentrations of 0.25 -
20 mM as nitrogen sources and/or inductors.
. .
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Yeast extract was added as supplement in concentrations
of 0.1-1 g/l.
Table 1:
A) Liquid medium
MgCl2 x 6H20 0.4 g/l
CaCl2 x 2H20 0.014 g/l
FeCl3 x 6H20 2.8 mg/l
Na2SO4 0.1 g/l
Na2HPO4 2 g/l
KH2 PO4 1 g/l
NaCl 2 g/l
Vitamin solution 1 ml/l
Trace element solution 1 ml/l
pH 6.8-8.0
B) Solid media
A) with 20 g/l agar added
Microorganisms grew on the media in question
over an incubation time of 2-30 days.
They were transferred 1-3 times to fresh media and
isolated on solid media.
Cell suspensions of the microorganisms which have been
isolated were tested for activity (cf. Example 3). The
following microorganisms were isolated: Klebsiella
pneumoniae (DSM 10293), Arthrobacter sp. (DSM 10582),
Pseudomonas cepacia and Agrobacterium radiobacter
Example 2:
Selection of spontaneous mutants
The wild type Pseudomonas aeruginosa (P.
aeruginosa) PA01 (Holloway, B.W. Bacteriol Rev., 1959,
33, 419-443) was incubated for 1-30 days at 37~C on
solid or liquid medium as described in Example l with
L-a-chloro-~-guanidinevaleric acid as the only nitrogen
source and glycerol or glucose as carbon source. After
this time, 11 identical, pale brown colonies grew on
the solid media. The single colonies were again plated
out and then grown on corresponding liquid minimal
, .
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medium. Cell suspensions of such cells were examined
for the enzyme activity in question. In this manner, a
Pseudomonas aeruginosa (DSM 10581) was isolated which
expressed a hydrolase of the desired type. After "phage
typing" and strain identification with API test (20 NE
bio Merieux SA, France), the strain was still identical
with the wild-type Pseudomonas aeruginosa PA01, it
essentially differed from the wild type by its ability
to express an L-a-chloro-~-guanidinevaleric acid
amidinohydrolase.
Example 3:
Induction and biotransformation using dormant cells of
the bacterial strains Klebsiella pneumoniae DSM 10593
and Pseudomonas aeruginosa DSM 10581
The isolated microorganisms described in
Examples 1 and 2 were grown at a temperature of 37~C on
100 ml of liquid medium A) with glycerol or glucose as
the carbon source and L-a-chloro-~-guanidinevaleric
acid as inductor and nitrogen source. After the
stationary growth phase had been reached (OD650 of
0.8-1.5), the cells were harvested by centrifugation
and taken up in the biotransformation solution at a 10-
20 times greater concentration. The biotransformation
solution was composed as follows: 10-100 mM phosphate
or Hepes buffer, 10-150 mM a-chloro-~-guanidinevaleric
acid, pH 6.8-11). Biotransformation was performed at
37~C, with gentle shaking. Samples were taken at
intervals and analyzed by thin-layer chromatography or
HPLC.
L-a-chloro-~-guanidinevaleric acid was converted into
D-proline within 5-24 hours, depending on the
concentration, the ee value of D-proline being over 96%
(according to HPLC).
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Example 4:
Induction and biotransformation using the bacterial
strain Arthrobacter sp. DSM 10582
The amidinohydrolase was induced by growing the
bacteria on the following media: NYB (Difco); NYB and
1-10 mM L-a-chloro-~-guanidinevaleric acid; medium (A)
supplemented with glycerol 25 mM and L-arginine 1-10 mM
as nitrogen source. Cells were grown over 24 hours at
37~C in 100 ml of medium. Cells were harvested as
described in Example 3 and used for biotransformation.
After the cells had been washed, the biotransformation
was performed in phosphate or Hepes buffer 10-200 mM
(pH 5-10). L-a-chloro-~-guanidine-valeric acid was
converted quantitatively into D-proline over a period
of 5-24 hours, depending on the concentration, the ee
value of D-proline being over 96% (according to HPLC).
Example 5:
Induction and biotransformation using Pseudomonas
aeruginosa DSM 10581
The strain was grown in 100 ml Erlenmeyer
flasks with baffle at 37~C with shaking, on the
following media: minimal medium (A) with the
supplements described in Table 2 below, and NYB
complete medium containing tryptone 10 g/l, "meat
extractn 5 g/l, NaCl 5 g/l). The cells were prepared
for biotransformation as described in Example 3.
Table 2
C source N source Inductor Yeast OD650 Biotrans-
mM mM mM extract 24 h formation
g/l rate %
Gluc 20 - Cl-Arg 2 0.2 - 25
Gluc 20 Arg 1 Cl-Arg 4 1.7 100
Gluc 20 Arg 1 Cl-Arg 3 0.2 - 25
Gluc 20 NH4 1 Cl-Arg 4 0.2 - 25
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Gluc 20 NH4~ 1 Cl-Arg 4 0.2 - 25
Gluc 20 Arg 2 Cl-Arg 2 0.1 1.6 30
Gluc 20 Ornit 1 Cl-Arg 1 0.1 1 10
Gluc 20Citrul 1 Cl-Arg 1 0.1 0.9 6
Gly 20 Arg 2 Cl-Arg 2 0.1 1 30
Gly 20 Ornit 1 Cl-Arg 1 0.1 0.9 20
Gly 20 Citrul 1 Cl-Arg 1 0.1 0.75 30
Gly 10 Arg 10 Cl-Arg 0.1 30
0.5
Gluc 20Guanine 5 - 0.4 23
Gluc 20 Guanine 5 Arginine 1.1 20
Gluc 20 Guanidine - 0.5 20
acetate 5
C source N source Inductor OD Biotrans-
mM mM mM 24h formation rate %
Glu 10 - Cl-Arg 5 0.6 10
Glu 20 - Cl-Arg 5 0.8 30
Gluc 20 Glu 10 Cl-Arg 5 1.2 70
Gln 10 - Cl-Arg 5 0.3 10
Gln 10 - Cl-Arg 5 0.5 10
Gluc 10 Betaine Cl-Arg 5 1.2 70
Arg 10 - Cl-Arg 5 0.5 70
Gluc 10 Arg 1 Cl-Arg 5 0.8 70
Agmatine - Cl-Arg 5 0.2 20
Gluc = glucose, Arg = arginine, Gly = glycerol, Glu =
glutamic acid, Gln = Glutamine, Cl-Arg = L-a-chloro-~-
guanidinevaleric acid, Ornit = ornithine, Citrul
citruline
L-a-chloro-~-guanidinevaleric acid was
converted quantitatively into D-proline within a period
of 5-24 hours, depending on the concentration, the ee
value of D-proline being over 96~ (according to HPLC).
.. . . . . ...
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In this manner, 13 g/l of D-proline (according to HPLC)
were formed. The best results were achieved in the
abovementioned medium supplemented with Gluc 20 mM, Arg
1 mM and Cl-Arg 4 mM (cf. Table 2).
Example 6:
Preparation of L-a-chloro-~-guanidine~aleric acid
To this end, 100 g (0.475 mol) of L-arginine
monohydrochloride were dissolved in 150 ml of
concentrated hydrochloric acid and the solution was
warmed to 65~C. 75 ml of 65% strength HNO3 were added
dropwise in the course of 30 minutes, and the addition
was accompanied from the beginning by a vigorous
evolution of gasi after a further 30 minutes at 65~C,
the evolution of gas had ceased, and the clear, pale
yellow reaction solution was concentrated in vacuo. The
residue obtained was taken up twice more in in each
case 200 ml of concentrated HCl and evaporated to
dryness in vacuo. This gave 112.2 g of a pale yellow
solid. The solid was dissolved in 750 ml of
concentrated HCl at 60~C and crystallized by cooling
the solution to 0~C. The precipitate was filtered off,
washed twice with in each case 100 ml of cold 6 N HCl
and dried in vacuo. This gave 72.9 g of a colourless,
crystalline solid, which corresponds to a yield of
66.7%.
M.p. 149~C
aD25(c=10% in H2O) = -7.87~
1H-NMR in ppm (400 MHz, in D2O)i 4.55 (dd, lH, H-1);
3.25(t, 2H, H-4); 2.15 - 1.95 (br, m, 2H); 1.8 - 1.7
(br, m, 2H)
Content 100.8%
Example 7
a) Preparation of D-proline in the 3.5 1 fermenter
Cells of the strain Pseudomonas aeruginosa (DSM
10581) were inoculated with the medium described in
Table 3 with 10% of preculture and grown for 24 hours
up to an OD600 of 13. The biomass was removed by
.. ..... _ .
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centrifugation, washed in 10 mM Hepes pH 8.5 and
resuspended in the latter. Biotransformation was
performed in a 11 Applikon fermenter with a controlled
constant pH of 8.5 at 37~C, the cell density at OD600
being 10-20. The concentration of a-chloro-~-guanidine-
valeric acid during biotransformation was always above
25 mM. 110 mM substrate were added in total. After 40
hours, 106 mM D-proline with an ee value of 98.3%
(according to HPLC) had been formed (Figure 1).
After 40 hours, the cells were removed by
centrifugation. The crude solution was purified over
Celite 535i after protein precipitation with perchloric
acid and subsequent neutralization with KOH, the
biotransformation solution was treated with active
charcoal and filtered over Celite 535. The product was
evaporated to dryness.
b) Preparation of D-proline using cell-free extract
Pseudomonas aeruginosa (DSM 10581) cells were
disrupted using a French press (3 times; 120 Mpa). 1 ml
of 50 mM L-a-chloro-~-guanidinevaleric acid in 100 mm
phosphate buffer (pH 7-8) were added to 1 ml of cell-
free extract, and the mixture was incubated at 37~C.
The entire procedure was also performed on intact cells
for comparison reasons. Samples were taken and analysed
by HPLC. It emerged that the activity of the cell-free
extract was more than twice as high as the activity of
the corresponding, undisrupted cells (Fig. 2)
Table 3
MgCl2 x 6H20 0.8 g/l
CaCl2 x 6H20 0.16 g/l
FeO4 x 7H2O 20 mg/l
Trace elements without EDTA 1 ml/l
Polypropylene glycol 2000 1 ml
Glucose 10 g/l ("glucose feed" to
30 g/l)
Na2HPO4 2 g/l
KH2PO4 1 g/l
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NaCl 2 g/l
Inductor a-chloro-~-guanidine-
valeric acid 10 mM (feed)
N-source (NH4)2SO4, 3.1 g/l
c) Derivatization of D-proline to D-Z-proline
4.6 g of D-proline which had been isolated as
above were dissolved in 20 ml of H2O. Benzyl
chloroformate (Z-Cl) was added dropwise at a constant
pH of 11.5-12 (4N NaOH). A total of 9.3 g of Z-Cl were
added dropwise. After the reaction, the mixture was
neutralized with HCl. This solution was extracted with
butyl acetate. The organic phases were discarded. The
pH was brought to 2 by adding more HCl to the aqueous
phase, and the mixture was again extracted by shaking
with butyl acetate. The organic phases were combined
and concentrated. The product crystallized from a
solution of 2.5 ml of ethyl acetate and 1.5 ml of
hexane. This gave 3.6 g of Z-D-proline.
Melting point: 68.5~C
Content: 97.86%
[aD2~](c=2 in glacial acetic acid) = +55.766~
[aS462~](c=2 in glacial acetic acid) = +66.251~
ee (according to HPLC) 95.4%
lH NMR (400 Mhz in CD30D)i ~ in ppm:
7.35 (m, 5H),
5.1 (m, 2H),
4.3 (m, lH),
3.6-3.4 (m, 2H),
2.3-2.2 (m, lH),
2.1-1.9 (m,3H)
Example 8:
Enzyme purification
Pseudomonas aeruginosa DSM 10581 cells were
grown as described in Example 1. In the late-
exponential growth phase, these cells were harvested by
centrifugation and washed with 0.85% NaCl or 30-200 mM
Hepes buffer pH 7-9. The cells were disrupted using a
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French press (3 timesi 120 Mpa). The cell extract was
removed by centrifugation for 30 minutes at 30,000 g.
The cell-free supernatant was filtered (0.4 um) and
purified over a MonoQ-FPLC column (Pharmacia). The
mobile phase used was 10 mM Hepes buffer pH 8. An Na2SO4
gradient was established to elute the enzyme from the
column. The enzyme eluted from the column at a salt
concentration of 120 mM. 25 mM L-a-chloro-~-
guanidinevaleric acid were reacted overnight with the
enriched enzyme to give D-proline.
The active fractions from the monoQ FPLC
purification were combined and further purified using a
phenyl sepharose column (Pharmacia). The mobile phase
used was 50 mM Hepes buffer pH 8, 1.7 M (NH4)2SO4. The
hydrolase was eluted from the column using 50 mM Hepes
buffer pH 8. The purified enzyme transformed 25 mM L-
a-chloro-~-guanidinevaleric acid in the course of 24
hours completely to L-a-chloro-~-guanidinevaleric acid,
which subsequently cyclized to D-proline, due to the
basicity in the medium.
Example 9:
Enzyme characterization
Enzyme characterization was performed on intact
Pseudomonas aeruginosa (DSM 10581) cells. The optical
density of the cell suspension was: OD600 = 17. The
temperature effect was determined in 100 mM phosphate
buffer (pH 7-8) at a substrate concentration of 24 mM
L-a-chloro-~-guanidinevaleric acid. It emerged, that a
40% higher activity was obtained by 37~C than at 30~C.
The pH effect was determined in the same buffer at the
same substance concentration and at a temperature of
37~C. The activity was measured at pH 7, pH 7.5, pH 8.0
and pH 8.5. It emerged that the pH optimum was pH 8.5.
By using various concentrations of L-a-chloro-~-
guanidinevaleric acid, the KM value and Vmax were
determined at a temperature of 37~C in 100 mM phosphate
buffer (pH 8.5). The KM value was 85.2 mmol/l and Vmax
was 0.38 mmol/l/min (Fig. 3).
CA 022~7~42 1998-12-08
WO 98/01577 - 18 - PCT/EP97/03546
An inhibition was detected by adding various
concentrations of L-a-chloro-~-guanidinevaleric acid
(prepared from ornithine analogously to Example 6). As
can be seen from Fig. 4, complete inhibition occurs at
a concentration of 50 mM.
