Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
13~72~
HOECHST AKTIENGESELLSCHAFT HOE 87tF 383 Dr. KH~AP
Description
A process for the enzymatic hydrolysis of ~-aminoadipinyl-
monoamino compounds
~-Glutamyltranspeptidases (~-GTP hereinafter) play an im-
portant part in amino acid metabolism and in the gluta-
thione cycLe ;n animal tissues and in m;croorganisms
~[Meth. Enzymol. 77, 237 (1981)]. They are responsible for -
the transport of various amino acids in the ~orm of their
~-glutamyl derivatives, the formation of polyglutamic acid
in Bacilli, and the breakdown of glutathione (~-glutamyl-
cysteinyl-glycine).
It has already been proposed (EP 0,275,901) to use ~-GTP
to hydrolyze adipinyl- or glutaryl-monoamino compouncls.
lt has now been found, surprisingly, that ~GTP catalyzes
the hydrolysis of ~-aminoadipinyl-monoamino compounds of
the formula I
HOOC- ~CH- (CH2)3-C-NH-R
NH2
in which
R1 denotes amino acids, dipeptides, cephems, cephams
or derivat;ves thereof.
3~ This is all the more surprising because it has hitherto
been assumed that neither C4 nor C6 side-chains are
accepted by the active center of ~ GTP (see Agric. ~iol.
Chem. 42, 1978, pages 371 - 81).
Hence the invention relates to:
1. A ~-glutamyltranspeptidase having the following
properties
:~3~72~6
-- 2
- a molecular weight of 40,000 to 80,000,
- an isoelectric point at pH 4.4 to 5.9,
- for L-~-glutamyLparanitroanilide 3s substrate a
pH optimum in the range 6.5 to 10, and a Km of 9
to 36 ~M at pH 8, and
- hydroLysis of ~-aminoadipinyl-monoamino compounds
of the formula I
HOOC-CH- (CH2)3-C-NH-Rl (I)
MH2
in which
R1 denotes amino acids, dipeptides, cephems, cephams
or derivatives thereof~
2. A process for the preparation of the ~ glutamyltrans-
peptidase having the properties mentioned in 1.,
which comprises cultivation of bacteria of the genera
Pseudomonas, Proteus, Arthrobacter and 8acillus in a
nutrient medium until the said ~-GTP accumulates in
the nutrient medium.
3. The use of the ~-glutamyltranspeptidase having the
properties mentioned in 1. for the hydrolysis of
~-aminoad;pinyl-monoamino compounds of the formula I.
The invention is described in detail hereinafter, especial-
ly in its preferred embodiments. The invention is further-
more defined in the patent claims.
The ~-glutamyltranspeptidase ( ~ GTP) catalyzes the hy-
drolys;s of ~-aminoadipinyl-monoamino compounds of the
formula I, as defined above, to give the corresponding
acid and the monoamino compound~ 7-Aminocephalosporanic
acid derivatives are preferably used as substrate.
The enzyme occurs in the periplasm of microorganisms and
extracellularly and can be characterized by a molecular
weight of 40~000 to 80,000, preferably 50,000 to 70,000,
_ 3 _ ~3~72~
especially 55,000 to 65,000, and by an isoelectric point
which is at a pH of 4.4 to 5.9, preferably 4.8 to 5.5.
The pH optimum for L- ~ glutamylparanitroan;lide as sub-
strate is in the pH range 6.5 to 10. The transpeptidase
according to the invention has a Km for the same substrate
of 9 to 36 ~M, preferably 15 to 20 uM, in particular 17.
~M, at pH 8.
The ~-GTP according to the invention is irreversibly in-
hibited in the presence of azaserine or iodoacetamide.
The enzyme shows reversible inhibition in the presence
of copper, mercury and a mixture of serine and borate,
as well as in the presence of 7-aminocephalosporanic acid.
The ~-GTP is prepared with the aid of microorganisms, as
also described in European Patent Application EP 0,275,901.
In this process, bacteria, especially of the genera
Pseudomonas, Proteus, Arthrobacter and Bacillus, are cul-
tivated in a nutrient medium until ~-GTP accumulates
in the nutrient medium. Suitable examples are: Pseudomonas
putida ATCC 17390, Pseudomonas aeruginosa NCTC 10701,
Proteus vulgaris ATCC 9634, Arthrobacter parafineus ATCC
31917 as well as Pseudomonas fragi DSM 38~1 and Bacillus
subtilis IF0 3025. The enzyme is particularly preferably
obtained from ~ac. subtilis IF0 3025. Mutants and
variants of the said microorganisms are also suitable.
The microorganisms are cultured aerobically, singly or in
mixed culture, for example submerged with shaking or
stirring in shaken flasks or ~ermenters, where appropriate
with air or oxygen being passed in. The fermentation can
take place in a temperature range from about 20 to 37C~
preferably at about 25 to 30C, in particular at 2~ to
30C. Fermentation is carried out in a pH range between
5 and ~.5, preferably between 5.5 and 8Ø Under these
conditions, the culture broth shows considerable accumu-
lation of the enzyme in general after 1 tG 3 days~ Syn-
thesis of the ~-GTP starts in the late log phase and
_ 4 _ ~ 3 ~ 7 2lI~
reaches its maximum in the stationary phase of growth.
The production of the periplasmic enzyme can be followed
with the aid of activity assays by HPLC analysis or pho-
tometry.
The nutrient solution used to produce the ~ GTP contains
0.2 to 5 %, preferably 0.5 to 2 %, organic nitrogen com-
pounds and inorganic salts. Suitable organic nitrogen
compounds are: amino acids, peptones, furthermore meat ex-
tracts, m;lled seeds, for example of corn, wheat, beans,
soybeans or the cotton plant, distillation residues
from the manufacture of alcohol, meat meals or yeast ex-
tracts. Exa~ples of inorganic salts which the nutrient
solution can conta;n are chlorides, carbonates, sulfates
or phosphates of the alkali metal or alkaline earth metals~
;ron, z;nc and manganese, but also ammonium salts and
nitrates.
The addition of assimilable carbohydrates increases the
y;eld of biomass. Carbohydrates are also added in the
abovementioned concentrations. It is possible to add as
preferred carbon source for example sugars, such as glu-
cose or sucrose, as well as carbohydrate-containing natural
products such as malt extract, to the nutrient solution.
Although the optimal fermentation conditions differ for
each microorganism, either they are already known to those
skilled in the art or they can be established in easy pre~
l;minary tests.
Purification can be carried out by classical processes
via lysozyme digestion, ammonium sulfate precipitation~
and ion exchange and gel permeation chromatography~ The
enzyme can be coupled by conventional methods (Colo~ick
and Kaplan, Meth. Enzymol., vol. XLIV).
It is possible to use for the enzymatic reaction both
~3~'72'~
-- 5
whole cells in free or immobil;zed form, with the addition
of ~-lactamase inactivators, for example clavulanic acid
or thienamycin, and the isolated enzyme ~hich can
also be carrier-bound. Examples of ~uitable materials
for the immobilization of whole cells are ch;tosan~ algin-
ate, K-carrageenan, polyacrylohydrazides and other known
substances from processes known from the literature (K.
Yenkatsubramanian, Immob. Cells (197~), ACS Symposium
Series, page 106).
The hydrolysis reaction is most suitably carried out at
about pH 6.6 to ~ and at a temperature of about 28 to 38C.
The preferred compound of the formula I is that in which
R1 denotes the radical
~ S
~N~CH2R
COOH
in which R2 ;5 hydrogen~ OH or -0-8-tH3.
The ~-GTP has industrial importance in particular for ob-
taining 7-aminocephalosporanic acid from cephalosporin C.
To date however, a yeast (Trigonopsis variabilis) has al-
~ays been used to generate- from cephalosporin C glutaryl-
7-aminocephalosporanic acid which only then could be hydro-
lyzed enzymatically, in a second reaction step, to give 7-
aminocephalosporanic acid. It is now possible with the
process according to the invention to prepare 7-amino-
cephalosporanic acid from cephalosporin C in a single step.
The invention is described in more detail in the examples
which follow. Unless otherwise indicated, percentage
clata relate to we;ght.
Exa~ple 1
The ~-GTP-producing microorganism strains are maintained
on agar slants of the follow;ng composition:
- 6 - ~3
6lucose
Casein peptone0.4 %
Meat extract 0.4 %
Yeast extract 0.05
5 Liver extract 0.05 %
NaCl 0.25 %
pH 7.2
The sLant tubes are incubated at 28C for 2 days. The
cells are then rinsed off with 10 ml of phys;ological
saliner and l ml of this suspension ;s used to inoculate
a 50 ml preculture of the following composition in an
Erlenmeyer fLask of capacity 300 ml:
Peptone 1 %
Malt extract 0.5 %
pH 7.0
The flask is incubated at 30C and 190 rpm in a rotary
shaker for 24 hours. 2.5 ml of this culture are used as
inoculum for 50 ml of ma;n culture:
Bacilli
-
Peptone 0.12 %
25 Yeast extract 0.12 %
Glucose 0.25 %
: Na lactate (60 %) 5.6 ml
NH4Cl 0.12 %
K2HPo4 0.12 %
KH2P04 0.034 %
MgS04 x 7 H20 0.025 %
NaCl 0.5 %
KCl 0~5 %
CaCl2 x 2 H20 0.0015 %
MnCl2 x 4 H20 O.Oû07 %
Fe(NH4)c;trate 0~00015 %
The culture is incubated at 28C and a shaking frequency
_ 7 _ ~ 3 ~
of 190 rpm for 24 hours and is then harvested by cent-
rifugation.
~ GTP activities of some strains are listed in the table
which follows:
Strain _ -GTP ~mU/ml culture solution)
8. subtilis IF0 3025 S0
" " IF0 3013 15
10 " " IF0 3335 25
Examp(e 2
A preculture with Pac. subtilis lF0 3025 is cultured in
analogy to Example 1. 50 ml of this culture are used as
inoculum for 2 l of main culture solution in a 5 l fer-
menter. The strain is cultured at 34C and a partial
pressure of oxygen of 70 %. The formation of the ~-GTP
;s followed by photometry, and the culture is harvested
at the maximum enzyme titer. Under the given conditions,
a ~-GTP titer of 150 mU/ml of culture solution is reached.
Exa~ple 3
9 L of culture solution are separated by means of cross-
flow filtration ~exclusion limit 300,000 dalton) into
culture filtrate and biomass. The culture filtrate ob-
tained in this way contains a ~-GTP activity of 1350 Un
The enzyme is precipitated by addition of ammonium sul
; fate to 70 % saturation and is taken up again in 1/10 of
the volume. After dialysis against 20 mM tris, pH 8~0r
the enzyme is further purified on a DEAE-cellulose column
(DE 52, Whatman). The active eluates are combined and
concentrated. A ~-GTP product obtained in this way (con-
taining about 25 U of ~-GTP/ml) is used for the conversions.
~3~72~
Example 4
The following mixture is chosen for preparative conversion
of deacetyl-CPC:
100 ~l of enzyme concentrate prepared as in Example 3, and
100 ~l of 40 mM deacetyl-CPC dissolved in 20 mM potassium
phosphate buffer, pH 7.3, are incubated at a temperature
of 33C.
Up to 16 % deacetyl-7-aminocephalosporanic acid are pro-
duced under the chosen conditions.
Exa~ple S
With incubation conditions analogous to those detailed in
Example 4, 3 % 7-aminocephalosporanic acid is liberated
from CPC.
Example 6
Determination of ~-GTP activity
a) HPLC assay
50 ~l of 80 mM deacetyl-CPC are mixed with 100 to 140
~l of 250 mM potassium phosphate buffer, pH 5.0, and
10 to 50 ~l of enzyme solution and incubated at 33C.
A 20 ~l-sample is taken every 10 minutes. The reaction
is stopped with 20 ~l of methanol. It is centrifuged
and diLuted with water in the ratio 1:10. A 10 ~l
sample is investigated by HPLC for the 7-aminocephalo-
sporanic acid content.
Stationary phase: C-18 silica gel
Mobile phase: KH2P04 50 mM in H20/MeOH ~80:20)
~ 0.001 % tetrabutylammoniu~ sulfate
b) Photometric assay
600 ~l of L-~-glutamyl-p-nitroanilide (166 ~M)
- 9 13~l ~2~
300 ~l of potassium phosphate bu~fer, pH 5.7, 5Q mM and
100 ~l of culture solution are mixed together and
incubated at 37C.
405 = 96Z0
mol . cm
