Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
106395Z
Th~ l)res~nt lnvcntion relates to cll~ymic cata-
lys~s contail~ing p~l~icillin~yl~se (E~C. 3.5.1.11), to a
process for tl~ir ~roduc~ioll, alld to t~e us~ o sucll cata-
lysts in the production of 6-amino-penicill~nic acid.
Penlcillinacyl~se is an enzyme used in large-
scale indus~rial processcs for th~ production of 6-amino-
penicillanic ~cid ~G-~P~). 6-APA is an intermedia~e pro-
duct, i.e. ~he pelliclllln nucleus, ~hich is val~ahle or
~he preparation of a wide varie~y o semi-synthe~ic peni-
cilllns by subse~uent reaction oE the 6-APA nucleus with
the desired side chain.
; According to the process o German Patent Speci-
fication 1,111,778 for the preparation of 6-APA, a solution
of benzylpenicillin (Pen. G) is ~reated with a bacterial ~-
f~
`~ slurry which contains penicillinacylase. As a result o ¦
the catalytic action of the enzyme, the side chain carbon-
amide grouping of the penicillin is split off without open-
ing of the ~-lsctam ring.
Xowever, this process has great disadvantages.
Thus, the 6-AP~ prepared according to this process contains
many impurities which originate, inter alia, from the
notrient medium, the fermentation liquor and the bacteria.
; In addition, the enzymatic activity of the bacterial sus-
pension is virtually spent af er a single use, so that it
cannot be re-used.
To avoid these disadvantages, water-insoluble
~ !
`: ' - 1 - ~ I
.` , . I
.,. ~
.
:
lG~3952
enzymic catalysts containing penicillinacylases have been
uset instead of a suspension of bacteria.
Various methods by which such catalysts can be
prepared have already been disclosed:
1. By covalent bonding to water-insoluble carriers
[G.J.H. ~elrose, Rev. Pure and Appl. Chem. 21, 83 (1971)1.
2. By inclusion in the lattice of a porous gel
tK. Mosbach, R. Mosbach, Acta Chem. Scand. 1966, 20, 2807].
3. By micro-encapsulation 1T.M.S. Chang, Nature
229, 117 (1971)].
4. By inclusion of enzymes in the fibrous struc-
ture lGerm~n Offenlegungsschrift (German Published Specifi-
cation) 1,932,426].
Such water-insoluble enzymic catalysts are hetero-
geneous catalysts and can be separated off snd re-used after
each reaction. On the other hand, however, they suffer
from several disadvantages: in fact, processe~ described in
German Offenlegungsschriften (German Published Specifications)
1,917,057 and 1,907,365 for the preparation of 6-APA with
penicilli~acylase covalently bound to a carrier cannot be
tran~ferred to an industrial scale. The reasons for thi3
are, firstly, the mechanical properties of the carrier
; material u8ed, which is excessively suscept~ble to abrasion,
and, secondly, that the process yields are derate and only
low 8pecific activities of carrier-bound penicillinacylases
can be achieved.
~ ~ ~ 39 5 Z
The insoluble enzymic catalyst used according
to German Offenlegungsschrift (German Published Specifica-
tion) 2,143,062 also has disadvantages in industrial use.
It employs penicillinacylase which is bound to solit, water-
insoluble, adsorbing substances, for example, nylon, by
means of a water-soluble dialdehyde. Here, however, in
addition to a crosslinking of the enzyme molecules with one
another~ covalent crosslinking with the carrier can also
occur if the latter contains active groups which are able
to react with the dialdehyde. Insolubilized penicillin-
acylase produced in this way can split off soluble prote$n
as a result of hydrolysis. There are therefore limitations
on the re-use of the insoluble catalyst produced in this
way.
In the known processes for enclosing enzymes in
porous polymers, conditions under which enzymes are easily
denatured are used. Accordingly, high losses in yield must
be accepted in the preparation of such insoluble catalysts.
In addition, free enzyme can diffuse through the pores of
the polymer into the reaction medium. Thi~ results in a
continuous deterioration of the catalytic activity of the
catalysts.
In the process described in German Offenlegungs-
schrift (German Published Specification) 1,932,426, enzymes
are incorporated into an easily produced fibrous structure.
The enzyme is included in separate cavities, which prevent
.. ~ .
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". :~
10~3952
the enzyme from diffusing out. However, it is a great di~-
advantage of this process that after the incorporation of
the enzyme into the fiber a high proportion of the orig~n-
ally present enzyme ac~ivity is lost. Furthermore, while
on the one hand the pore sizes of the fibers must be 80
small that no enzyme can diffu~e out, on the other hand with
such small pore sizes the diffusion of the substrate through
the fiber structure to the enzyme and the rediffusion of
ehe reaction products to the reaction medium are greatly
restricted. Thus, the incorporated enzyme is not utilized
to the optimum extent. Furthermore, the incorporated en-
zyme is in an unbound form and free enzymes are denatured
re rapidly than enzymes covalently bound to carriers
[B.D. Orth and W. Br~mmer, Angewandte Chemie, 84, pages
319-368 (1972)1. Accordingly, enzymes incorporated in
polymers are for these reasons inferior to enzymes covalent-
ly bound to carriers.
It has furthermore already been disclosed ~hat
enzymes can be bound to waeer-soluble carr~ers. Thus,
according to U.S. Patent No. 3,625,827, enzymes are coupled
to a water-soluble polymer of ethylene and maleic anhydride~
~owever, this process is not suitable for the preparaticn
of high molecular enzyme derivatives since the carrier
used becomes insoluble as the molecular weight increases.
Furthermore, the enzyme can also react several t~me~, with
different carrier molecules. This cause~ crosslinking, as
,
106395Z
a result of which the enz~ne-polymer derivative becomes
insoluble.
More particularly, the present invention is con-
cerned with enzymic catalysts comprising penicillinacylase
covalently bound to a water-soluble polymeric carrier. The
enzymic catalysts may be either a water-soluble catalyst or
a water-insoluble catalyst. In the latter case, the water-
soluble catalyst above described is incorporated in or en-
capsulated in a water-insoluble polymer. Both the water-
soluble and the water-insoluble catalysts of the present
invention are useful as above noted in the production of
6-APA from penicillins by the enzymic splitting of the 6-
position side chain of the penic~llin leaving the 6-APA
nucleus for isolation and recovery.
In the case of the water-soluble enzymic catalyst,
the preferred water-soluble carriers are polysaccharides,
especially dextran, starch, levan and carboxymethylcellulose.
The water-soluble catalyst is produced by reacting an acti-
vated derivative of the carrier in aqueous solution with
the penicillinacylase. Preferably a carrier containing
vicinal hydroxyl groups is selected such as a polysaccharide,
and this is reacted with a cyanogen halide to produce the
activated derivative of the carrier. Cyanogen chloride,
bromide and iodide are all useful but cyanogen bromide is
preferred. The water-soluble catalyst can be obtained in
almost quantitative yield, as appears from the enzymic
.. ...
_ 5 _
~0~3952
activities of the starting materials and the resulting
products.
The water-insoluble catalyst of the present in-
vention is produced as described above by incorporating the
water-soluble catalyst in or encapsulating the water-soluble
catalyst in a water-insoluble polymer.
The water-insoluble polymer may be a polymeric
matrix in which the water-soluble catalyst is dispersed or
a fibrous polymer in the interstices of which the water-soluble
cata~yst is trapped.
A preferred polymeric matrix in which the water-
soluble catalyst may be incorporated is the copolymer of
acrylamide and N,N'-methylene-bis-acrylamide, but many others,
such as the ethylene-maleic anhydride polymers known in the
art, may be used. This water-insoluble catalyst may, accord-
ing to the invention, be produced by preparing the water-
" insoluble polymer by polymerization in the presence of the
water-soluble catalyst of the invention.
The water-insolùble catalyst of the invention in
which the polymeric carrier is fibrous can, according to the `
present invention, be prepared by spinning the polymeric
carrier in the presence of the water-soluble catalyst. Suit-
able water-insoluble polymeric carriers for this purpose are
known in the art (see, for example, German Published Speci-
fication No. 1,932,426). --
As mentioned above, the present invention also
includes an improved process for the~produc~on of 6-APA
,. ; . . . .
10ti3952
which comprises contacting either a water-soluble catalyst
of the present invention or a water-insoluble catalyst of
the present invention with a penicillin. The water-soluble
catalyst of the present invention is used in aqueous solu-
tion and may be recovered after the reaction from the aqueous
reaction mixture by ultrafiltration, The water-insoluble
catalyst of the present invention can be recovered after the
reaction by simple filtration. Both the enzymic catalysts
oP the present invention can be re-used repeatedly without
substantial loss of activity.
As compared to the unbound enzyme, the enzymic
catalyst according to the present invention has the great
advantage of being more stable even at higher penicillin
concentrations. Thus, the use of the enzymic catalyst of
the present invention represents an advance of significant
value in the preparation of 6-APA.
If a polysaccharide is first reacted with a cyano-
gen halide, to produce an àctivated derivative, which is then
reacted with penicillinacylase to produce the water-soluble
catalyst of the invention ~compare German Offenlegungsschrift
1,768,5121, the course of the reaction can be represented by
the following equation:
~c=~ ~2~-Protoln ~c--e--~--Protel~l
~,.
~ nt
~0 ~ 39 5Z
wherein X ls NH or an oxygen atom.
In preparing the water-soluble catalyst of the
present invent~on, water is generally used as the diluent
or solvent. The reaction temperatures are generaLly between
0C and 50C and the reaction can be carried out both under
normal pressure and under elevated pressure.
During the preparation of the activated derivative,
the reaction medium is generally kept in the pH range of 8
to 13, preferably at pH 11.0, by adding a base, for example,
sodium hydroxide solution. The reaction of the cyanogen
halide with the polysaccharide is usually complete after 10
to 20 minutes. No cyanogen halide should remain in the
solution when the activated derivative is reacted with peni-
cillinacylase in the next step. Also, the next step must be
carried out as soon as possible since the activated deriva-
tive can easily be inactivated by hydrolysis. Before the
` reaction with penicillinacylase, the pH value of the solution
containing the activated derivative is preferably adjusted
to 8.5. After addit~on of the penicillinacylase, the mixture
is stirred at 0C - 50C, preferably 5C - 10C. The co~-
" tent of unreacted enzyme can be determined if ~extran of
molecular weight of 500,000 is used. For example, the high
molecular water-soluble catalyst can be separated from the
unbound enzyme by ultrafiltration using membranes impermeable
to molecules of molecular weight exceeding 100,000.
The ratio of polysaccharide, cyanogen halide and
~ , .
.. .
- 8 -
: ' ' :
1~395Z
penicillinacylase can be varied within wide limitations.
Complete conversion of the enzyme is reliably achieved if
at least 5 parts by weight of polysaccharide aré used per
part by weight of enzymic protein.
It has furthermore been found that the ratio of
the amounts of cyanogen halide and polysaccharide has an
extraordinary influence on ~he keeping qualities of the water-
soluble catalyst. Thus, natural penicillinacylase in aqueous
solution of pH 7.8 and 45C is inactivated to the extent of
up to 95% within 24 hours (see Figure 1, curve a). Under
identical conditions, the water-soluble catalyst of the in-
vention containing, as polymeric carrier, dextran of molecu-
lar weight 500,000, only loses 47% of its activity in 24
hours, if ~7 mg of cyanogen bromide are employed per gram
of dextran (see Figure 1, curve b). However, if 80 mg of
cyanogen bromide are used per gram of dextran, the enzyme
activity only decreases by 5% in 24 hours (see Figure 1,
curve c). It is surprising that, at constant coupling yield,
'` the stability o the water-soluble catalyst can be increased
~` 20 extraordinarily by increasing the proportion of cyanogen
` halide. Possibly, more covalent bonds are formed between
the carrier molecule and the enzyme molecule as a result of
increasing the amountof cyanogen halide. This could lead
` to the tertiary structure of the enzyme being stabilized in-
.,
creasingly as the number of covalent bonds produced between
the carrier and the enzyme increase. The amount of cyanogen
-.
,
~ O ~ 39 5Z
halide employed is, however, limited because sparingly
soluble and/or insoluble enzyme derivatives can be produced
as a result of crosslinking. Thus, it is not advisable to
employ more than 150 mg of cyanogen bromide per gram if dextran
of molecular weight about S00~000 is used.
If dextran of lower molecular weight, for example
20,000, is used, there are no problems in using 300 mg of
- cyanogen bromide per gram of dextran for the reaction. How-
ever, the use of carriers of lower molecular weights reduces
the stability of the enzyme. Figure 2 shows the variation
with time of the residual activities of water-soluble peni-
cillinacylase-dextran catalysts according to the invention
after pre-incubation at 45C and pH 7.8.
In this Figure, the curves represent the following:
Curve a: unbound penicillinacylase.
Curve b: penicillinacylase covalently bound to dextran
of approximate molecular weight 20,000 (80 mg
of cyanogen bromide per gram of dextran).
Curve c: like b, but 160 mg of cyanogen bromide per gram
o dextran.
Curve d: penicillinacylase covalently bound to dextran of
approx~mate molecular weight 60,000 (80 mg of
cyanogen bromide per gram of dextran).
80 mg of cyanogen bromide were used per gram of
dextran to obtain curves (b) and (d). It was possible to
improve perceptibly the stability of the catalyst by using
'` ' ` "'............................ """`'"" :
- 10 -
1063952
twice a~ much cyanogen bromide as in experiment (d) -
see curve (c).
With starch as the water-soluble carrier, extreme-
ly stable water-soluble catalysts according to the invention
can be produced. The stability of the catalyst also depends
on the amount of cyanogen halide employed. Preferably, at
least 150 mg of cyanogen bromide are used for the reaction
per gram of starch. The distinct stabilization of the peni-
cillinacylase by covalent bonding to starch, in accordance
with the invention, is illustrated by Figure 3. The curves
show the residual activities after pre-incubation at 45C
and pH 7.8.
The curves represent the following:
;` Curve a: unbound penicillinacylase.
Curve b: penicillinacylase bound to starch (80 mg of
cyanogen bromide per gram of starch).
~
Curve c: penicillinacylase bound to starch (160 mg of
cyanogen bromide per gram of starch).
.
As stated above, the water-soluble catalysts
prepared in this way can, according to the invention, be
encapsulated or incorporated in water-insoluble polymers,
to provide water-insoluble catalysts. It is particularly
, ~ . .
; advantageous to encapsulate them in fibers by spinning
lcompare German Offenlegungsschrift 1,932,426] or to in-
~ corporate them in a polymerization mixture. Thus, for
! e~ample, it was possible to incorporate, by polymerization,
~,~
~ .. ,
,~ .
- 11 -
, :
, . .
~;3952
a penicillinacylase-dextran water-soluble catalyst accord-
ing to the invention (dextran of molecular weight 500,000)
in yields of 60% of the original enzyme activity, using
methods known from the literature [H. Nilsson, R. Mosbach
and K. Mosbach, Biochim. BiophysO Acta 268 (1972) 253-256].
In a control experiment with natural unbound penicillin-
acylase, only 10% of the originally present enzyme activity
was obtained ;.fter incorporation by polymerization, under
identical experimental conditions. The water-insoluble
catalyst proved to be enzymically extremely stable even after
being employed repeatedly for the preparation of 6-APA. The
water-insoluble catalyst can be separated rapidly and simply
from the reaction medium, for example by f~ltration, since
the polymer can be produced in particle sizes of more than
" 1 mm in diameter.
The new water-soluble and water-insoluble catalysts
display a strong enzymatic activity ~hich makes them highly
suitable for the preparation of 6-APA. Usually, the enzymatic
splitting of penicillins to produce 6-APA is carried out in
dilute solutions in order not to inactivate the enzyme. Prior
to isolating the 6-APA, about 80% of the water must be eva-
porated off in order to achieve good yields of 6-APA.
:~ Since the catalysts according to the invention
` allow the splitting to be carried out also at higher con-
`~ centrations, less water has to be evaporated, owing to the
increased amount of penicillin present. Hen~e, the pro-
duction of 6-APA using the penicillinacylase~atalysts
....
- 12 -
~ 3952
according to the invention is more economical than corres-
ponding processes using unbound penicillins.
Examples of penicillins that may according to
the invention be used in conjunction with the catalysts of
the invention to produce 6-APA are benzylpenicillin (Pen.G)
and its salts, potassium penicillin G. The reaction is
generally carried out in aqueous solution with the continu-
ous or repeated addition of base (especially triethylamine)
to neutralize the acid formed in the reaction; it is pre-
ferred to keep the pH between 7 and 8, especially at about
7.8. After the reaction is completed, the solution con-
tains unconverted penicillin, phenylacetic acid, and 6-APA;
the catalyst can be separated by filtration or ultrafiltra-
tion according to its particle size.
Commercially available membrane filters which are
impermeable to molecules of molecular weights about 100,000
are particularly suitable for separating the water-soluble
catalyst of the invention.
The 6-APA formed can be isolated according to
known methods [see, for example, German Patent Specification
1,111~778] after removal of the catalyst, and~ can be cry-
stallized, preferably at pH 4.3.
In the enzymatic splitting of penicillin with the
catalysts according to the invention, substantially higher
yields of 6-APA are obtained than when E. coli slurry is
used. Thus, we have isolated 6-APA in yields of about
.
10~;3952
85 - 90%, after use of both the water-soluble and the
water-insoluble catalysts according to the invention.
The penicillinacylase catalysts according to the
present invention can be employed repeatedly over a long
period, because o their high stability. Even after a long
period, their en~ymic activity is still retained practi-
cally completely. For the economical use of the water-
soluble carrier-bound penicillinacylase, this stability
after repeated re-use is of great importance.
The enzyme activities (U) quoted in the examples
which follow are defined as the activity which hy`drolyzes
1~ mol of penicillin G to 6-APA and phenylacetic acid, per
minute at 37~C and pH 7.8.
The penicillinacylase used was prepared according
to Canadian Application 153,721, filed October 12, 1972.
The following non-limitative e~amples more parti-
cularly illustrate the present invention.
Example 1
` (a) 5 g of dextran 500, of approximate molecular ~;
weight 500,000, are dissolved in 165 ml of water and the
solution is ad~usted to pH 11.0 with 2 N sodium hydroxide
solution. 85 mg of cyanogen bromide are added at a tempera-
ture of 20C while stirring and the pH value of the solution
is kept at 11.0 with 2 N NaOH. 10 Minutes after addi$ion of
the cyanogen bromide, the solution is adJusted to pH 8.5
- 14 -
~ !
~06395Z
with 2 N hydrochloric acid and mixed with 170 ml of an
aqueous solution of 550 ng of penicillinacylase (enzymatic
activity 7.3 U/?~g). The solution is stirred for 16 hours
in a refrigerator at 4C. After ultrafiltration, no
enzyme activity is detectable in the filtrate. It m?ay
therefore be assumed that coupling with the carrier has
taken place completely. The yield of enzymatic activity is
`` 98% relative to the initial ac~ivity. After freeze-drying,the enzymic activity remains preserved com~pletely.
A solution of the water-soluble catalyst thus
produced was stored at 45C and pH 7.8 to test its stability.
The residual activity determined after various times are
. shown in Figure 1, curve b. Curve a was obtained corres-
pondingly with natural, unbound penicillinacylase.
(b) 5 g of dextran 500, of approxim te molecular
weight 500,000,are dissolved in 165 ml of water and adjusted
to pH 11.0 with 2 N sodium hydroxide solution. 400 mg of
cyanogen bromide are added at a temperature of 20C, while
stirring, and the pH value of the solution is kept at 11.0
by means of 2 N sodium hydroxide solution. 20 Minutes after
addition of the cyanogen bromide, the solution is ad3usted
to pH 8.5 with 2 N hydrochloric acid and mixed with 170 ml
of ~n aqueous solution of 550 mg of penicillinacylase
(enzymatic activity 7.3 U/mg). The solution is stirred
for 16 hours in a refrigerator at 4C. Under these condi-
tions, the enzyme is bound completely to the carrier. The
r?
- 15 -
~O 6 39 52
yield of enzymatic activity is 96% relative to the initial
activity. The keeping quality of the water-soluble cata-
lyst thus produced - determined as in Example l(a) - is
shown graphically in Figure 1, curve c.
Example 2
(a) 5 g of dextran 20 of approximate molecular weight
20,000 are reacted, as indicated in Example 1 (b), with r
400 mg of cyanogen bromide and then with 550 mg o peni-
cillinacylase. After the reaction, the yield of enzymatic
activity is 96%, relative to the initial activity. The
keeping quality of this water-soluble catalyst - determined
as in Example 1 (a) - is shown graphlcally in Figure 2, curve
b.
Cb) 5 g of dextran 20 are reacted, as indicated in
Example 1 (b), with 800 mg of cyanogen bromide and then with ;
550 mg of penicillinacylase. The yield of enzymatic acti-
vlty after the reaction is 87%, relative to the in~tial
activity. The keeping quality of this water-soluble cata- `
lyst - determined as in Example 1 (a) - is shown graphically
in Figure 2, curve c.
~c) 5 g of dextran 60 of approximate molecular weight
60,000 are reacted, as indicated in Example 1 (b), with 400
mg of cyanogen bromide and then with 550 mg of penicillin-
acylase. The yield of enzymatic activity after the reaction
i8 99%, relative to the initial activity. The keeping
s
nt
- 16 -
~ .
.
.
.~ . ,
3952
quality of this water-soluble catalyst - determined as in
Example 1 (~) - is shown graphically in Figure 2~ curve d.
Example 3
~a) 5 g of soluble starch, prepared according to
Zulkowsky, are dissolved in 165 ml of water and the solution
is ad~usted to pH 11.0 wlth 2 N sodium hydroxide solution.
400 mg of cyaA~ogen bromide are added at a temperature of
20C, while stirring, and the pH value of the solution is
kept at 11.0 by means of 2 N NaOH. 20 Minutes after addi-
`` 10 tion of the cyanogen bromide, the solution is ad~usted to
pH 8.5 with 2 N hydrochloric acid and mixed with 170 ml of
an aqueous solution of 550 mg of penicillinacylase (enzymatic
activity 7.3 U/mg). The solution is stirred for 16 hours
in a refrigerator at 4C. After the reaction, the yield
of enzyme activity is 98%, relative to the ini~ial activity.
The wate--soluble catalyst thus produced, which contains
~ penicillinacylase bound to starch by covalent bonds, can
`~ be freeze-dried without loss of activity. Yield 6.2 g, with
~ .
an enzymatic activity of 0.65 U/mg. The stability of the
water-soluble catalyst was tested, as indicated in Example
1 (a), in aqueous solution at 45C and pH 7.8 (see graphi-
cal representation, Figure 3, curve b).
(b) 5 g of soluble starch prepared according to
Zulkowsky are reacted, as indicated in Example 3 (a), with
800 mg of cyanogen bromide and then with 550 mg of penicillin-
, ....
!- - 17 -
106395Z
acylase~ After the reaction, the yield of enzyme activity
is 98%, relative to the initial activity. The stability of
the water-soluble cataly3t thus produced was tested in aqueous
solution at 45C and pH 7.8 (see graphical representation,
Figure 3, curve c). Yield after freeze-drying, 6.1 g, of
enzymatic activity 0.65 U/mg.
Example 4
210 ml of a solution of water-soluble penicillin-
acyla~e-dextran catalyst, prepared according to Example 1 (b)
and having an activity of 9.6 U/ml, are added to a solution
of 63 g of poeassium penicillin G in 800 ml of water and the
mixture is stlrred at 38C. The pH value of the reaction
mixture is kept constant at 7.8 by continuous addition of `~
~riethylamine. After 6 hours, no further triethylamine i8 . ~ .
taken up. The solution is filtered through an ultrafiIter
down to a residual volume of 100 ml, 250 ml of water are
. .~
; added to the residual solution and the mixture is again
filtered down to 100 ml. The filtrate, including the wash
water, is concentrated to 150 ml in vacuo. The 6-APA is
precipitated at the isoelectric point at pH 4.3, in the pre-
sence of lQ0 ml of methyl isobutyl ketone, by addition of
6 N hydrochloric acid. After one hour, the 6-APA is filtered
and rinsed with 100 ml of water and 100 ml of acetone. It
is dried in vacuo at 40C; melting point 208C; yield 31.9 g
of 6-APA, representing 87.2% of theory.
The water-soluble catalyst separated off by ultra-
- 18 -
~0~3952
filtration call be employed for furthcr splitting batches.
After r~peating the splitting five times, no enzyme acti-
vity has been consumed, so that the reaction time does not
have ~o be lengthened.
2.9 g of water-soluble penicillinacylase-starch
catalyst, having an activity of 650 U/g and prepared accord-
ing to ExRmple 3 (b), are dissolved in 600 ml o 0.05 M
triethanolamine/hydrochloric acid buffer of pH 7Ø 85.5 g
o~ acrylamide, 4.5 g of N,N'-methylene-bis-acrylamide and
2.5 g of ammonium peroxydisulphate dissolved in 5 ml of the
above buffer and 5 ml of N,N,N',N'-tetramethylethylenediamine
are added to the solution tgiving solution A).
2,900 ml of toluene, 1,100 ml of chloroform, 5 ml
of N,N,N^,N'-tetramethylethylenediamine and 10 ml of emul-
` sifier 1736 (Bayer AG - the reaction product of oleyl alcohol
and ethylene oxide) are introduced into a three-necked flask
equipped with a stirrer, cooled to 4C and stirred at a
` speed of 250 revolutions/minutes. Solution A is added
d~opwise from a dropping funnel, under nitrogen as a pro-
tective gas, the mixture is stirred for 30 minutes and the
polymer is filtered off. It is rinsed twice with 1 liter of
toluene and 2 liters of 0.5 M sodium chloride solution. The
polymer con~ains 63% of the original penicillinacylase acti-
` vity, and is a water-insoluble catalyst according to the
invention.
~, .
. - - 19 - ' I .
., , !
. I
, . .
1~ ~ 39 5 2
The water-insoluble catalyst is added to a solu-
tion of 31.5 g of potassium penicillin G in 500 ml of water
and the mixture is stirred at 38C. The pH value of the
reaction mixture is kept constant at 7~8 by addi ion of
triethylamine. After 6 hours, no further triethylEmine is
consumed. The water-insoluble catalyst is filtered off and
washed with a little water. The fil~rate, including the
wash water, is concentrated to 80 ml in ~acuo. The 6-APA
is precipitated at the iso-electric point at pH 4.3, in the
presence of 80 ml of methyl isobutyl ketone, by adding 6 N
hydrochloric acid. After one hour, the 6-APA is filtered
off and rinsed with 75 ml of water and 75 ml of acetone.
It is dried in vacuo at 40C. Melting point 208C, yield
16.7 g (91% of theory).
. . .
The water-insoluble catalyst can be used, without
perceptible loss of enzymic activity, for at least 20
successive batches.
~ .
Example 6
210 ml of a solution of the water-soluble peni-
cillinacylase-dextran catalyst, of activity 9.6 U/ml, pre-
pared according to Example 1 (b), are added to 63 g of
potassium penicillin G and 290 ml of water and the mixture
~ .
is stirred constant at 38C. The pH value of the reaction
mixture is kept constant at 7.8 by continuous addition of
triethylamine. After 12 hours, no further triethylamine is
taken up. The reaction batch is worked up analogously to
. ~ .
I - 20 -
..
, .
1063952
Example 4. The enzymic solution separated off by ultra-
filtration contains 92% residual activity. Yield of 6-APA
30.8 g (84% of theory).
In a parallel experiment with an equal amount of
natural unbound penicillinacylase, unreacted penicillin was
still present after a splitting time of 12 hours in a re-
action mixture which was treated analogously. The residual
penicillinacylase activity still present was 67Z of theory.
The water-soluble catalyst of the invention thus shows,
under the same conditions, a better degree of conversion,
and better retention of its activity, than unbound peni-
` cillinacylase.
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