Note: Descriptions are shown in the official language in which they were submitted.
1063Z96
The present invention relates to copolymers which are useful as
components of water-insoluble proteinic preparations in which a protein or
polypeptide is bound to the copolymer. Such proteinic preparations are used
in carrying out enzyme-catalyzed reactions. Such preparations are the subject
of our application serial number 167,343 from which this application is
divided.
The covalent bonding of substances to insoluble polymeric carriers
has gained increasingly in importance in recent years. The binding of cat-
alytically active compounds, for example enzymes, offers particular advantages
since they can, in this form, be easily separated off after completion of the
reaction and repeatedly reused.
Copolymers of maleic anhydride and vinyl compounds have already
been proposed repeatedly as carriers with suitable binding groups. However,
copolymers of maleic anhydride with ethylene and monovinyl compounds become
more or less water-soluble on reaction with aqueous enzyme solutions so that
before or during the reaction, an additional crosslinking agent, for example
a diamine, is desirably added. Enzyme preparations obtained in this way are
relatively difficult to filter and possess soluble constituents, and losses
of bound enzyme results tcompare E. Katchalski, Biochemistry 3, (1964),
pages 1905-1919).
Further, copolymers of acrylamide and maleic acid have been describ-
ed which are converted into the anhydride form by subsequent heating. These
products are relatively slightly crosslinked, swell very markedly in water
and only possess moderate mechanical stability which leads to abrasion losses
when using these resins (compare German Offenlegungsschrift 1 908 290).
Furthermore, strongly crosslinked carrier polymers have been pro-
duced by copolymerization of maleic anhydride with divinyl ethers. Because
of the alternating type of copolymerization of the monomers, these polymers
contain a very high proportion of anhydride groups - ;n each case above 50%
by weight of maleic anhydride, in the examples disclosed - which is determined
-- 1 --
1063296
by the molecular weight of the vinyl ether monomer and is therefore only
adaptable to the particular end use within relatively narrow limits (compare
German Offenlegungsschrift 2,008,996).
We have therefore attempted to find new reaction products of pro-
teins and peptides with new, strongly crosslinked, water-swellable copolymers
possessing a content of cyclic dicarboxylic acid anhydride groups capable of
great variation, and convenient process for their production. The new reaction
products of proteins and peptides with the new copolymers do not have the dis-
advantages of the previously known protein preparations or at least, have them
to a slighter extent.
There is provided according to this invention, a crosslinked copoly-
mer comprising the following copolymerized units:
tA) about 0.1 to 30 wt. %, preferably 2 to 20 wt. %, of at least
one ,~-monoolefinically unsaturated dicarboxylic acid anhydride having about
4 to 9 carbon atoms, preferably 4 to 5 carbon atoms;
(B) about 35 to 90 wt. %, preferably 50 to 85 wt. %, of at least
one di- or poly(meth)acrylate of a diol or polyol; and
(C) about 5 to 60 wt. %, preferably 10 to 50 wt. %, of at least one
hydrophilic monomer;
the hydrophilic monomer of C being comprised of units not defined under B;
the polymer having a bulk volume of about 1.4 to 30 and a specific area of 1
to 500 square meters per gram.
The crosslinked copolymer should preferably have a bulk volume of
about 2 to 20 ml/g, and a specific surface area of 1 to 400 m2/g, and contain
after saponification of the anhydride groups, 0.01 to 14, preferably 0.02 to
11, milli-equivalents of acid per gram.
There is also provided a copolymer which further comprises copoly-
merized units derived from 0.01 to 30 wt. %, based on the total monomer units
of the copolymer, of a crosslinking agent having at least 2 non-conjugated
double bonds.
There is also provided according to the invention a process for the
production of a crosslinked copolymer as defined above in which, relative to
,; - 2 -
,..
~,"~ ~
1063296
the total weight of copolymerizable monomers present:
(A) about 0.1 to 50 wt. %, preferably 2 to 25 wt. %, of the said at
least one anhydride;
~ B) about 35 to 90 wt. %, preferably 50 to 85 wt. %, of the said at
least one acrylate; and
(C) about 5 to 60 wt. %, preferably 10 to 50 wt. %, of the said at
least one hydrophilic monomer;
are copol~nerized by precipitation or bead polymerization in a diluent inert
to anhydride groups at a temperature of 20 to 200C. in the presence of a
free-radical initiator.
The copolymer thus produced can be reacted, in aqueous suspension,
with a solution of a peptide material to g~ve a water insoluble proteinic
preparation, in accordance with our application serial No. 167,343.
In this specification the term "peptide material" includes proteins,
polypeptides, oligopeptides, and amino-acids.
The starting materials required for the production of the copolymer
of the invention will first be described.
Important examples of ~,~ -monoolefinically unsaturated dicarboxylic
acid anhydrides with 4-9 carbon atoms, preferably with 4-5 carbon atoms, which
are required to provide units A are maleic, itaconic and citraconic anhydrides,
especially maleic anhydride. Mixtures of these anhydrides can also be used
for the copolymerization.
The diols and polyols from which the dimethacrylates, polymeth-
acrylates, diacrylates and polyacrylates used to provide units B in this
invention are derived comprise the following categories of compounds:
(i) Hydroxy compounds with at least two alcoholic or phenoiic, prefer-
ably alcoholic, hydroxyl groups;
(ii) the reaction products of the said hydroxy compounds (i) with alky-
lene oxides having 2 to 8, preferably 2 to 4 carbon atoms, or mixtures of
such alkylene oxides, 1 to 104, preferably 1 to 10, alkylene oxide units
1063296
being added to one mol of the hydroxy compound (i). Examples of suitable
alkylene oxides are ethylene oxide, propylene oxide, butylene oxide, tri-
methylene oxide, tetramethylene oxide, bischloromethyl-oxacyclobutane and
styrene oxide, ethylene and propylene oxides being preferred;
(iii) the reaction products of at least one of the alkylene oxides
defined in (ii) with a compound having at least two Zerewitinoff-active
hydrogen atoms which is not an alcohol or a phenol.
The di- and poly-tmeth)acrylates of diols and polyols, to be employ-
ed according to the invention, can be obtained in accordance with known
methods, for example by reaction of the diols and/or polyols with (meth)
acrylic acid chloride in the presence of about equimolar amounts, relative
to acid chloride, of tertiary amines such as triethylamine, at temperatures
below 20C. in the presence of benzene (compare German Offenlegungsschrift
1 907 666). Possible diols or polyols with at least 2 carbon atoms, prefer-
ably 2-12 carbon atoms, are, for example: ethylene glycol, 1,2-propanediol,
1,3-propanediol, butanediols, especially 1,4-butanediol, hexanediols,
decanediols, glycerine, trimethylolpropane, pantaerythritol, sorbitol, suc-
rose and their reaction products with alkylene oxides, as indicated above.
Poly-bis-chloromethyl-oxacyclobutane or polystyrene-oxide are also suitable.
Mixtures of diols and polyols can also be employed to produce the acrylates.
Preferably, diacrylates or dimethacrylates of diols with 2-4
carbon atoms and/or reaction products of one mol of these diols with 1-10
mols of alkylene oxide with 2-4 carbon atoms or trimethylolpropane trimeth-
acrylate are used.
The dimethacrylates of ethylene glycol, diethylene glycol, tri-
ethylene glycol, tetraethylene glycol or higher polyalkylene glycols with
molecular weights of up to 500, or their mixtures, are particularly advan-
tageous.
The third group of the copolymerization components, giving units C,
consists of singly unsaturated, hydrophilic monomers. As such, it is possible
~063296
to use any polymerizable singly unsaturated compound which does not possess
any functional groups which are reactive towards anhydride groups, and which
forms hydrophilic polymers.
The hydrophilic monomers C preferably possess at least one carboxyl,
aminocarbonyl, sulpho or sulphamoyl group, and the amino groups of the amino-
carbonyl or sulphamoyl radical can optionally carry as substituents alkyl
groups with 1-4 carbon atoms or alkoxymethyl groups with 1-4 carbon atoms in
the alkoxy radical.
As examples of these hydrophilic monomers, there may be mentioned
acrylic acid, methacrylic acid, maleic acid half-esters with 1-8 carbon
atoms in the alcohol radical, N-vinyl-lactams such as N-vinylpyrrolidone,
methacrylamide, N-substituted ~meth)acrylamides such as N-methyl- and N-
methoxymethyl-~meth)-acrylamide and N-acryloyldimethyltaurine.
Depending on the desired hydrophilic character and swellability of
the copolymers and on the length of the polyalkylene oxide chain or chains
of the polyfunctional ~meth) acrylic ester B, the hydrophilic monomers C are
added to the monomer mixture in amounts of 5-60% by weight.
Apart from affecting the hydrophilic character, the addition of
these monomers surprisingly also affects the structure of the polymers, which
is of very particular advantage in the production of bead polymers, since
here the choice of the diluents for the monomer mixture is greatly restricted
by the conditions that they should be insoluble in paraffin hydrocarbons and
inert towards anhydride groups.
Because of the range of variation in the composition of the monomer
mixture, the hydrophilic character, density of crosslinking, swellability and
anhydride group content of the copolymers according to the invention can be
adapted to give optimal results in the intended particular end use of the
preparation over a very broad range.
If desired, there may also be added in addition to the di- and/or
poly-(meth)acrylates to be used according to the invention, at least one
1063296
crosslinking agent having at least two non-conjugated double bonds ~e.g.
divinyl adipate, methylene-bis-acrylamide, triacrylformal, triallyl cyanurate
etc) to the monomer mixture in amounts of 0.01-30% by weight.
The polymerization can be carried out, for example, in an organic
solvent, as a precipitation polymerization, in which case the polymers start
to precipitate shortly after the start of the polymerization. Inherently,
all solvents which are inert towards anhydride groups are suitable. Particu-
larly advantageous solvents are aliphatic, cycloaliphatic and aromatic hydro-
carbons and halogen-substituted aromatic and aliphatic hydrocarbons, alkyl-
aromatic compounds and carboxylic acid esters.
As examples of suitable organic solvents there may be mentioned:
heptane, octane, isooctane, benzine fractions with boiling points of 60 to
200C., cyclohexane, benzene, toluene, the xylenes, chlorobenzene, dichloro-
benzenes, ethyl acetate, butyl acetate, and the like.
The solvent used should preferably possess a boiling point of at
least 60C. and should be easily removable from the precipitation polymer in
vacuo. For 1 part of the monomer mixture, about 2-50, preferably 5-20, parts
by weight of the solvent are used. The properties of the copolymers,
especially the bulk density and the specific surface area, are significantly
influenced by the nature and amount of the solvent.
In many cases it is advantageous to use mixtures of the above men-
tioned solvents or to start the polymerization in a solvent for the polymer
and continuously to add a precipitant for the polymer over the course of the
polymerization. The precipitant can also be added in one or more portions
at particular points in time. Furthermore, the monomer mixture can be fed,
together with a suitable initiator, as a solution or without solvent, into a
previously taken amount of solvent, so that during the polymerization a uni-
form low monomer concentration is maintained. Products with swellability,
density and specific surface area suited to the particular end use, and with
good mechanical stability, having a very wide range of properties, can be
1063Z96
produced by using (meth)acrylic monomers B of differing hydrophilic character
and varying the polymerization conditions.
The copolymers according to the invention are preferably produced
by suspension polymerization. The most customary type of bead polymerization
in which the monomers are suspended in water, optionally with the addition of
an organic solvent, meet with little success in our system since the anhydride
hydrolyzes very rapidly and the resulting dicarboxylic acid mainly passes in-
to the water phase, with only small amounts being incorporated into the poly-
mer. ~ence, the suspension polymerization is preferably carried out in an
organic medium. Paraffin hydrocarbons, such as hexane, heptane, octane and
higher homologues, cycloaliphatic compounds such as cyclohexane, and paraffin
mixtures such as benzine fractions or paraffin oil, are particularly suitable
as the continuous phase. The monomers and the initiator are generally dis-
solved in a solvent which is immiscible with paraffins and inert towards
anhydride groups, such as, for example, acetonitrile, dimethylformamide,
dimethylsulphoxide or hexamethylphosphoric acid triamide, and dispersed in
the continuous phase, in st cases with addition of at least one dispersing
agent. The volume ratio of continuous phase: monomer phase is generally 1:1
to 10:1, preferably 2:1 to 5:1.
In order to stabilize the suspension, it is possible to use, for
example, glycerine monooleate and dioleate, as well as mixtures of these
compounds, sorbitan monooleate and trioleate or monostearate and tristearate,
polyethylene glycol monoethers with stearyl alcohol or lauryl alcohol or
nonylphenol, polyethylene glycol monoesters with oleic acid, stearic acid
and other fatty acids with more than 10 carbon atoms, and the sodium salt of
sulphosuccinic acid dioctyl ester. These stabilizing substances are general-
ly employed in amounts of, preferably 0.1-10% by weight relative to the mono-
mer mixture and are generally dissolved in the hydrocarbon phase. The
particle size of the copolymeric product can be reduced either by increasing
the speed of stirring or by adding 0.01-2% by weight relative to monomer, of
1063Z96
a further surface-active substance, for example an alkylsulphonate.
Polymerization in the process of the invention is initiated by
radical initiators. Suitable ini~iators are, for example, azo compounds or
per-compounds. The most customary azo compound for initiating the polymeri-
zation is azoisobutyronitrile. Possible per-compounds are mainly diacyl
peroxides such as dibenzoyl peroxide or percarbonates such as diisopropyl
percarbonate and dicyclohexyl percarbonate, but it is also possible to use
dialkyl peroxides, hydroperoxides and redox systèms which are active in
organic solvents for the initiation.
The initiators are generally added in amounts of 0.01-10 wt. %,
preferably 0.1-3 wt. %, relative to the weight of the monomer mixture.
The polymerization is generally carried out at temperatures of
about 20-200C., preferably 50-100C., depending on the speed of decomposi-
tion of the initiators and, in most cases, below the boiling point of the
solvent, and in the case of bead polymerizations, below the miscibility
temperature of the two phases. Furthermore, it is as a rule advantageous
to carry out the polymerization in an inert atmosphere in the absence of
oxygen.
The copolymers obtained by precipitation polymerization are color-
less to pale yellow-colored powdery substances with bulk volumes of 1.5-30
ml/g, preferably 2-20 ml/g and specific surface areas of 0.1-500 m2/g, pre-
ferably 1-400 m2/g. The content of carboxyl groups determined titrimetri-
cally after saponification of the anhydride groups is about 0.01-14 m-
equiv/g, preferably about 0.02-11 m-equiv/g.
The copolymers obtained by suspension polymerization are white or
slightly colored beads which can in some cases be irregular in shape and have
a diameter of 0.03-3 mm, preferably 0.05-0.5 mm and bulk volumes of 1.4-~
ml/g, preferably 1.4-5 ml/g. Their carboxyl group content, determined after
hydrolysis of the anhydride groups, is 0.01-14 m-equiv/g. preferably 0.02-11
m-equiv/g.
~063296
The copolymers used according to the invention contain copolymer-
ized units essentially in statistical distribution, trandom copolymers).
Because of their high density of crosslinking, they are insoluble in all
solvents. Molecular weights can therefore not be determined.
The copolymers can swell in water to between 1.1 and 3 times their
bulk volume. They are outstandingly suitable for use as carrieT resins for
fixing substances which can react with the anhydride groups of the copolymers.
They also possess excellent mechanical stability and hence practically no
abrasion.
The carrier copolymers described above bind all substances which
carry a functional group which is capable of reacting with the anhydride
groups of the polymer. In the case of proteins and peptides, these are pri-
marily the terminal amino groups of lysine and the free amino groups of the
peptlde chain ends.
The following substances are examples of peptide materials which
can be bound to the copolymers of the invention:
Enzymes: Hydrolyases such as proteases, for example trypsin,
chymotrypsin, papaine and elastase; amidases, for example asparaginase,
glutaminase and urease; acyltransferases, for example penicillinacylase;
lyases, for example hyaluronidase.
Other proteins: plasma constituents and globulins (antibodies).
Oligopeptides, such as glutathione.
Polypeptides, such as kallikrein inhibitor and insulin.
Aminoacids, such as lysine or alanine.
It is however envisaged that the copolymer will primarily be used
with proteins isolated from bacteria, fungi, actinomycetes or animal
material.
After production of the copolymer, it is combined with the peptide
material. The requisite amount of polymer is introduced into a stirred
aqueous solution of the peptide material at a temperature of between 0 and
~063296
30C. The weight ratio of peptide materials to carrier resin can be varied
within wide limits and be suited to the subsequent end use. Good yields are
obtained for example with a ratio of 1 part by weight of protein to between
4 and 10 parts by weight of polymeric carrier. However, the optimum ratios
depend both on the composition and structure of the polymer and on the nature
of the protein.
In the case of numerous enzymes it is also advisable to add stabil-
izers. As such stabilizers, it is possible to use polyethylene glycols or
non-ionic wetting agents for reducing denaturation at surfaces, as well as
the known SH-reagents or metal ions in the case of special enzymes. The pH
is appropriately kept by means of a pH-stat at the optimum pH-value for the
particular bonding. This pH-value lies in a range of 2 to 9, preferably 5
to 7. When the peptide material is penicillinacylase it has proved advisable
to work at between pH 5.7 and pH 6.8. In doing so, inorganic bases (for
example caustic alkali solutions), or organic bases ~for example tertiary
~ . . .
organic amines), must be added in order to keep the pH-value constant. The
progress of the reaction is discernible from the consumption of the amount
of base required to keep the pH constant. At room temperature, about 16 hours
are required to complete the reaction and at 4C. up to 40 hours are required.
Thereafter, the copolymer is filtered off and is washed with buffers or salts,
in the concentration range of 0.2 to 1 M, in order to dissolve off a small
proportion of ionically bound protein.
The bound substance can be determined either by elementary analysis
or, in the case of enzymes or inhibitors, by the enzymatic activity or the
inhibition of the enzymatic activity. The yields of bound substance depend
on the nature of the substance and the composition of the poly~er. Thus,
for example, aminoacids and low lecular peptides were coupled practically
completely; however, even with the proteins, the yields of bound enzymatic
activity are between about 20 and over 90% of the activity introduced at
start.
- 10 -
~063Z96
According to the state of the art (German Offenlegungsschriften
1 935 711 and 2 008 990), the coupling of proteins is effected in buffer
solutions in concentrations of 0.05 M to 0.2 M. If no buffer is used, the
proportion of ionically bound protein increases (German Offenlegungsschrift
1 935 711). Surprisingly it was found that coupling with water insoluble
preparations containing the copolymer of the invention and a peptide material,
in a medium which is as free of ions as possible gives maximum yields of
enzyme bound by covalent bonds. In practice a conduction range of 0,1 - 1 m
mhos at pH 6.3 was achieved. In this procedure the pH can be kept constant
very accurately at the optimum value by means of a pH-stat.
The use of copolymer-bound enzymes is industrially of particular
importance since industrially valuable products can be manufactured with
their aid. The prior bonding of biological catalysts permits them to be
isolated completely from the reaction mixture by very simple separation
processes and makes it possible to reuse them repeatedly, which is a decisive
factor economically. Additionally, in most cases the stabili~y of the sensi-
tive and expensive proteins is decisively improved.
The following gives some examples of the use of the preparations
composed of a peptide material bound to a copolymer of the invention in
carrying out enzyme-cataly2ed conversions.
The proteases, such as trypsin, chymotrypsin, papaine and elastase,
can be used, for example, for the production of protein hydrolysis products
for microbiological processes. Furthermore, the enzymes can also be used
to remove antigenic proteins from pharmaceutically active substances.
Acylases are used industrially for the manufacture of 6-amino-
penicillanic acid from penicillins or for the separation of racemates of
acylated aminoacids. Carrier-bound amylase can be used for the hydrolytic
degradation of starch.
Special hydrolases, such as asparaginase or urease, can be employed
therapeutically, in the copolymer-bound form, in extra-corporal circulation.
- 11 -
1063296
These and numerous other possible uses have been described in the
literature. See, for example, the summaries in Chem. Eng. News of 15.2.1971,
page 86 and Rev. Pure ~ Appl. Chem. 21, 83 (1971).
A further large field of use for substances fixed to carriers is
affinity chromatography. Thus antibodies can be isolated by means of bound
antigens or haptens, and conversely antigens can be isolated with fixed
antibodies (y -globulins). Equally, enzymes can be enriched specifically
with the aid of bound inhibitors or substrate anslogues. For example, it is
known to isolate trypsin and chymotrypsin by means of bound vegetable or
animal inhibitors. Other examples are the isolation of peptidases on special
bour.d peptides or the isolation of plasmin with lysine bound to a carrier.
Summarizing works on this field of use have been published by G. Feinstein
in Naturwissenschaften 58, 389 (1971) and F. Fried in Chromatographic Reviews
4, 121, (1971).
In order to produce 6-aminopenicillanic acid (6-APA), penicillins
can be split by means of acylases from micro-organisms, for example, bacteria,
especially E. coli, Erwinia, Actinomycetes such as Streptomyces, Micromono-
spora and Nocardia; fungi such as Fusarium; and yeasts.
/
According to the process of German Patent Specification 1,111,778
for the production of 6-APA, a penicillin G solution is treated with a
bacterial sludge which contains the enzyme penicillinacylase (E.C. 3.6.1.11).
As a result of the action of the enzyme, the lateral carbonamide grouping of
the penicillin is split off without opening the ~-lactam ring.
The use of suspensions of micro-organisms has the following dis-
advantages:
(a) The suspension of micro-organisms, in addition to containing the
intra-cellular penicillinacylase, contains further proteins and enzymes as
well as constituents from the nutrient medium or their transformation products
which have been produced during the fermentation. The impurities cannot be
completely eluted from the crystalline 6-APA during working up.
1063296
tb) The suspension of micro-organisms can economically appropriately
only be employed once.
~ c) The suspension of micro-organisms contains impurities and other
enzymes which inactivate penicillin and/or 6-APA by opening the ~-lactam
ring.
(d) The suspension only contsins small amounts of penicillinacylase.
The use of more enzyme material, for example to achieve shorter reaction
times and hence better 6-APA yields with a lower content of extraneous pro-
ducts is not possible to practice.
te) The operating yields of 6-APA tepend on the varying formation of
penicillinacylase in the individual fermentation batches.
tf) The complete removal of suspended micro-organisms requires an
additional process step when working up the 6-APA batches and this causes
losses in yield. Further purification steps are necessary to remove protein-
like impurities which can cause allergic reactions ~British Patents 1,169,696,
1,078,847 and 1,114,311).
All disadvantages mentioned are avoided if instead of a suspension
of micro-organisms a penicillinacylase is used which is obtained by covalent
bonding to a water-insoluble carrier.
While attempts to produce 6-APA by enzymatic splitting of penicil-
lins with carrier-bound penicillinacylase are known, tsee on the subject,
German Offenlegungsschrift 1,917,057 and German Offenlegungsschrift 1,907,365)
it has not been possible to apply the process described on a technical scale.
The reasons for this are firstly the mechanical properties of the carrier
material used, which is very vulnerable to abrasion, while with moderate
process yields only low specific activities of carrier-bound penicillinacylase
,rere achievable.
The insoluble enzyme used in German Offenlegungsschrift 2,157,970
which was by covalent bonding of the penicillinacylase to a copolymer of
acrylamide, N,N'-methylene-bis-acrylamide and maleic anhydride, also has
1063296
disadvantages for the splitting of penicillin on an industrial scale, since
it swells strongly and is mechanically unstable. These disadvantages handi-
cap the repeated reuse of the resin, thus manufactured, on an industrial
scale. It has now been found that the disadvantages mentioned are avoided
if a preparation comprising a penicillinacylase bound to a water-insoluble
copolymer carrier of the invention is used for splitting penicillins.
The splitting of penicillins with csTrier-bound penicillinacylase
can be carried out simply and also on a large industrial scale. In a pre-
ferred form of this process, the carrier-bound insoluble enzyme is suspended
in a solution containing 75,000-150,000 IU/ml of penicillin El mg. of
potassium benzylpenicillin G corresponds to 1598 IU tInternational Units)],
for example penicillin G or penicillin V. The enzymatic splitting is carried
out at a constant pH value in the range of 6-9, particularly in the range of
the pH optimum of the particular bound penicillinacylase, for example at
pH 7.8. To neutralize the acyl radical split off, for example of phenylacetic
acid or of phenoxyacetic acid, aqueous alkali solutions, for example potassium
hydroxide solution or sodium hydroxide solution, or organic amines, prefer-
ably triethylamine, are used. The reaction velocity and the completion of
the splitting can be seen from the consumption of the base. The penicillin-
acylase catalyses both the splitting of penicillin to give 6-APA and the re-
synthesis of the penicillins from the splitting products. The equilibrium
depends on the pH value of the medium. At lower pH value the equilibrium is
displaced in favor of the starting product, penicillin. This can be utilized
for the transacylation of penicillins in the presence of other acyl radicals
or for the synthesis of penicillins from 6-APA.
The reaction temperature of the enzymatic splitting is preferably
38C. At lower temperatures, the activity of the enzyme decreases. If the
splitting is carried out, for example, at 25C., twice as much enzyme as at
38C. has to be employed if the same reaction times are to be achieved.
At a given temperature, the reaction velocity depends on the
1063Z96
specific activity and on the amount of the carrier-bound penicillinacylase.
Furthermore, the reaction velocity depends on the ratio of the amount of the
carrier-bound penicillinacylase to the concentration of the penicillin. A
splitting batch with a concentration of 100,000 IU/ml of potassium penicillin
G has been completely hydrolyzed to 6-APA and phenylacetic acid after 10
hours at pH 7.8 and 38C. if, per unit of penicillinacylase, 3.105 units of
penicillin G are employed ~one enzyme unit (U) is defined as the activity
which hydrolyzes 1 ymol of 6-nitro-3-tphenyl-acetyl)-aminobenzoic acid tNIPAB)
per minute at 25C.]. The proportion of dry enzyme resin is only 0.5-1%
of the reaction mixture. If 2 units of penicillinacylase are employed per
10 IU of penicillin G, the complete splitting only requires two hours.
Even shorter reaction times are also possible when using even more bound
penicillinacylase, when using, for example, carrier-bound penicillinacylase
from crystallized enzyme.
The carrier-bound penicillinacylase is preferably in the shape of
beads and is distinguished by high mechanical stability and a comparatively
high specific gravity. These properties allow it to be employed for pro-
longed periods of time if it is repeatedly used. These properties further-
more make it possible, in batch processes, to ~oy intensive stirring and
simple separation by centrifuging without loss through mechanical stress,
for example through abrasion. Thus, batch processes yield clear filtrates
which can, without additional filtration, be procassed further in order to
isolate the end product, 6-APA. The carrier-bound penicillinacylase also
permits rapid and simple filtration, since, as a result of the mechanical
stability, no very fine particles which block the filter surface are pro-
duced. The resin offers further advantages in the batch process because of
the comparatively high specific gravity, which causes the resin to settle
out rapidly, so that after completion of the process the supernatant solution
can easily be siphoned off. This results in a simplification of the conduct
of the process, since in the batch process the resin can remain in the
1063296
reaction vessel and be used directly for the next splitting.
The properties of the polymer furthermore permit the use of the
carrier-bound penicillinacylase not only in batch processes but also in con-
tinuous processes, for example in reaction columns, where the bead shape
permits the requisite high speed of flow through the column.
After separating off the enzyme resin, the 6-APA formed in the
enzymatic splitting can be isolated from the reaction solution in accordance
with known processes (see, for example, German Patent Specification 1,111,778)
and is crystallized at pH 4.3. In the splitting of penicillin, with the
carrier-bound penicillinacylase produced according to the invention, sub-
stantially higher yields of 6-APA are obtained than when using E. coli
sludge, but also higher yields than when using the enzyme resin according to
German Patent Application P 2 157 970.4. Thus, as is shown in Examples 14
to 17, 6-APA has been isolated in a yield of about 90% of theory. The 6-APA
thus produced does not contain any proteins as impurities. The 6-APA thus
produced also contains practically no polymers which can be produced in
other procedures. Allergic side-effects attributed to proteins or polymers
are impossible with the 6-APA produced by means of a peptide material bonded
to a copolymer of the invention.
The carrier-bound penicillinacylase can be used repeatedly over a
prolonged period of time. Even thereafter, the enzyme activity is still
retained practically completely.
The following Examples are presented to illustrate the invention
but the invention is not to be considered as limited thereto. The boiling
points in the Examples were determined at normal pressure.
EXAMPLE 1 (a)
70 g of tetraethylene glycol dimethacrylate, 20 g of methacrylic
acid, 10 g of maleic anhydride and 1 g of azoisobutyronitrile are dissolved
in 1 liter of benzene and initially polymerized for 4 hours at 60C. 1 g of
azoisobutyronitrile and 200 ml of benzine (boiling point 100-140C.) are then
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added and the polymerization is carried out for 2 hours at 70C. and 2 hours
at 80C.
The pulverulent polymer is thoroughly washed with petroleum ether
tboiling point 30-50C.) and dried in vacuo.
Yield: 96 g
Bulk volume: 8.8 ml/g
Swelling volume in water: 12.4 ml/g
Specific surface area: 8.6 m2/g
Acid content after saponification of the anhydride groups = 3.85 m-equiv/g.
EXAMPLE 1 tb)
6 g of the carrier copolymer produced according to Example 1 (a)
are suspended in a solution of 610 U of penicillinacylase in 150 ml of water.
The pH value is kept at 6.3 by adding 1 N NaOH, using a pH-stat, and the
suspension is stirred for 20 hours at 25C.
The copolymer is then filtered off on a G 3 glass frit and washed
with 300 ml of 0.05 M phosphate buffer of pH 7.5, containing 1 M sodium
chloride, and with 300 ml of the same buffer without sodium chloride. No
further activity can be eluted by further washing. The supernatant liquid
and the wash solutions are combined and their enzymatic activity is deter-
mined. The enzymatic activity of the moist copolymer is measured in aliquot
amount.
Result:
Enzymatic activities (NIPAB test):
Starting solution 610 U
Supernatant liquid + wash solutions 112 U
Carrier copolymer after the reaction 561 U
(i.e. 92% of the starting activity)
The enzymatic activity of the penicillinacylase is measured
colorimetrically or titrimetrically with 0.002 M of 6-nitro-3-tN-phenyl-
acetyl)-aminobenzoic acid (NIPAB) as the substrate at pH 7.5 and 25C. The
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molar extinction coefficient of the resulting 6-nitro-3-aminobenzoic acid
is E405 nm = 9~090 1 unit ~U) corresponds to the conversion of 1 ~mol of
substrate per minute.
EXAMPLE 1 (c)
400 mg of the carrier copolymer produced according to Example 1 ta)
are suspended in a solution of 40 ml of urease in 32 ml of water. The pH
value is kept constant at pH 6.0 by adding 1 N sodium hydroxide solution,
while constantly stirring at room temperature. After 16 hours, the reaction
is complete and the copolymer is filtered off and washed with buffer as in
Example 1 ~).
Result:
Enzymatic activity:
Starting solution 168 U
Supernatant liquid + wash solutions 64.5 U
Carrier copolymer after the reaction 87 U
~i.e. 52% of the starting activity)
The enzymatic activity of the urease is determined titrimetrically
with 0.17 M urea as the substrate, at 25C. and pH 6.1. 1 unit (U) corre-
sponds to the amount of enzyme which consumes 1 ~mol of urea, that is to say
2 ~mol of hydrochloric acid, per minute.
EXAMPLE 1 (d)
500 mg of the carrier copolymer produced according to Example 1 (a)
are suspended in a solution of 100 mg of trypsin in 32 ml of 0.02 M calcium
chloride. The pH value is kept constant at pH 6.3 by addition of 1 N sodium
hydroxide solution while constantly stirring at 4C. After 16 hours the
copolymer is filtered off and washed with buffer as in Example 1 ~b).
Result:
Enzymatic activity:
Starting solution 110 U
Supernatant liquid + wash solutions 15.6 U
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Carrier copolymer after the reaction 64 U
(i.e. 58% of the starting activity)
The enzymatic activity was determined colorimetrically according
to Tuppy. Z. Physiol. Chem. 329 (1962? 278, with benzoyl-arginine-p-nitro-
anilide (BAPNA) as the substrate. 1 unit (U) corresponds to the splitting
of 1 ~mol of substrate per minute at 25C. and pH 7.8.
EXAMPLE 2 (a)
A solution of 80 g of tetraethylene glycol dimethacrylate, 10 g of
methacrylic acid, 10 g of maleic anhydride and 1 g of azoisobutyronitrile in
1 liter of benzene is polymerized for 4 hours at 60C., 2 hours at 70C. and
1 hour at 80C., while stirring slowly. The polymer is filtered off, stirred
three times in benzene, washed with petroleum ether and dried in vacuo.
Yield: 70 g
Bulk volume: 7.0 ml/g
Swelling volume in water: 8.2 ml/g
Specific surface area: 5.3 m2/g
Acid content after saponification of the anhydride groups = 2.55 m-equiv/g.
EXAMPLE 2 (b)
1 g of the polymer produced according to Example 2 (a) is reacted,
analogously to Example 1 (b), with penicillinacylase in 33 ml of water at
pH 6.3.
Result:
Enzymatic activities (NIPAB test):
Starting solution 118 U
Supernatant li~uid + wash solutions 21 U
Carrier copolymer after the reaction 82 U
(i.e. 69% of the starting activity)
EXAMPLE 3 (a)
60 g of tetraethylene glycol dimethacrylate, 30 g of methacrylic
acid, 10 g of maleic anhydride and 1 g of azoisobutyronitrile are dissolved
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in 300 ml of acetonitrile. This solution is suspended in 1 liter of benzine
(boiling point 100-140C.) which contains 5 g of a mixture of glycerine
monooleate and glycerine dioleate and is polymerized for 22 hours at 60C.
The polymer beads are filtered off, suspended three times in ben-
zene and subsequently twice in petroleum ether tboiling point 30-50C.) and
dried in vacuo.
Yield: 94 g of white spheres
Bulk volume: 4.4 ml/g
Swelling volume in water: 5.5 ml/g
Specific surface area: 6.6 m2/g
Mean particle diameter: ~ 200,u
Acid content after saponification of the anhydride groups = 4.3 m-equiv/g.
EXAMPLE 3 tb)
1 g of the polymer produced according to Example 3 ~a) is reacted
analogously to Example 1 (b) with penicillinacylase in 33 ml of water at
pH 6.3.
Result:
Enzymatic activity (NIPAB test):
Starting solution 118 U
Supernatant liquid + wash solutions 43 U
Carrier copolymer after the reaction 96 U
(i.e. 81% of the starting activity)
EXAMPLE 4 (a)
A solution of 2,100 g of tetraethylene glycol dimethacrylate,
600 g of methacrylic acid, 300 g of maleic anhydride and 30 g of azoiso-
butyronitrile in 7.5 liters of acetonitrile is suspended in 21 liters of
benzine (boiling point 100-140C.) in which 150 g of a mixture of glycerine
monooleate and glycerine dioleate have been dissolved, and is polymerized
for 1 hour at 50C. and 20 hours at 60C.
The bead polymer is filtered off, twice stirred with toluene and
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once with petroleum ether (boiling point 30-50C.) and dried at 60C.
Yield: 2.95 kg
Bulk volume: 4.7 ml/g
Swelling volume in water: 5.4 ml/g
Mean particle diameter: approx. 0.3 mm
Acid content after saponification of the anhydride groups = 4.0 m-equiv/g.
EXAMPLE 4 tb)
1 g of the polymer produced according to Example 4 (a) is reacted
as described in Example 1 (b) with penicillinacylase in 33 ml of water at
pH 6.3
Result:
Enzymatic activity (NIPAB test):
In the starting solution 107 U
Supernatant liquid and wash solutions 27 U
Carrier copolymer after the reaction 67 U
(i.e. 63% of the starting activity)
EXAMPLE 4 (c)
20 g of the polymer produced according to Example 4 (a) are intro-
duced into 600 ml of a solution of 1.0 g of nonspecific elastase from pig
pancreas at room temperature, while stirring. At the same time the pH is
kept constant at 5.8. After a reaction time of 16 hours, the copolymer is
filtered off and worked-up in the manner described in Example 1 (b).
Result:
Enzymatic activity (casein test):
Starting solution 2,180 U
Supernatant liquid + wash solutions993 U
Carrier copolymer after the reaction 1,225 U
(i.e. 56% of the starting activity)
The enzymatic activity of the nonspecific elastase is determined
30titrimetrically with casein as the substrate (concentration 11.9 mg/ml) at
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pH 8.0 at 25C. 1 unit (U) corresponds to a consumption of 1 ~mol of
potassium hydroxide solution per minute.
EXAMPLE 5 ~a)
A solution of 80 g of tetraethylene glycol dimethacrylate, 10 g of
methacrylic acid, 10 g of maleic anhydride and 1 g of azoisobutyronitrile in
300 ml of acetonitrile is polymerized as described in Example 3 (a).
Yield: 92 g of white, egg-shaped particles
Bulk volume: 2.5 ml/g
Swelling volume in water: 3.2 ml/g
Specific surface area: 1.7 m2/g
Mean particle diameter: 125 ,u
Acid content after saponification of the anhydride groups = 3.5 m-equiv/g.
EXAMPLE 5 (b)
l g of the polymer produced according to Example 5 (a) is reacted
as described in Example 1 (b) with penicillinacylase in 33 ml of water at
pH 6.3.
Result:
Enzymatic activity (NIPAB test):
Starting solution 118 U
Supernatant liquid + wash solutions34 U
Carrier copolymer after the reaction61 U
(i.e. 52% of the starting activity)
EXAMPLE 6 (a)
A solution of 50 g of ethylene glycol dimethacrylate, 40 g of
methacrylic acid, 10 g of maleic anhydride and 1 g of azoisobutyronitrile
in 300 ml of acetonitrile is suspended in 1 liter of benzine ~oiling point
100-140C.) in which 5 g of a mixture of glycerine monooleate and glycerine
dioleate are dissolved, and is polymerized for 20 hours at 60C. The bead
polymer is filtered off, suspended three times in benzene and twice in
petroleum ether (boiling point 30-50C.) and dried in vacuo at 50C.
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Yield: 87 g
Bulk volume 4.8 ml/g
Swelling volume in water: 5.6 ml/g
Specific surface area: 13.2 m2/g
Acid content after saponification of the anhydride groups = 4.2 m-equiv/g.
EXAMPLE 6 (b)
1 g of the polymer produced according to Example 6 (a) is reacted
as described in Example 1 (b) with penicillinacylase in 33 ml of water at
pH 6.3.
Result:
Enzymatic activity (NIPAB test):
Starting solution 118 U
Supern~tant liquid and wash solutions 29 U
Carrier copolymer after the reaction55 U
~i.e. 47% of the starting activity)
EXAMPLE 7 (a)
A solution of 50 g of trimethylolpropane trimethacrylate, 30 g of
methacrylic acid, 20 g of maleic anhydride and 1 g of azoisobutyronitrile in
300 ml of acetonitrile is polymerized as described in Example 6 ~a).
Yield: 86 g
Bulk volume: 2.0 ml/g
Swelling volume in water: 2.2 mltg
Mean particle diameter: _ 30 y
Acid content after saponification of the anhydride groups = 4.1 m-equiv/g.
EXAMPLE 7 (b)
1 g of the copolymer produced according to Example 7 ~a) is added
to a solution of 100 mg of lysine in 32 ml of water at room temperature,
with constant stirring. The pH value is kept constant at 6.3. After 16
hours, the copolymer is filtered off, suspended in 50 ml of 1 M sodium
chloride solution, filtered off and rinsed with 100 ml of water. The residue
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is dried in vacuo at 100C. and the nitrogen content is determined by
Kjeldahl's method.
Result:
Dry weight: 860 mg
Nitrogen content: 1.5~, corresponding to a content of 65 mg of the lysine
in the copolymer. This represents 65% of the amount of lysine employed.
EXAMPLE 8
The following were polymerized as described in Example 6 ta)
(variation of the amount of the maleic anhydride):
Experi-
ment No. a b c d e f g
TGDM 75 70 65 60 55 50 45
(g)
MAA 20 20 20 20 20 20 20
(g)
MA 5 10 15 20 25 30 35
(g)
AIBN
(g)
Aceto-
nitrile 250 250 250 250 250 250 250
(ml)
Benz-
inel,000 1,000 1,000 1,000 1,000 1,000 1,000
(ml)
Emuls-
ifier
(g) 5 2.5 5 5 5 5 5
Yi~l~95 93 86 88 80 79 70
Vs(ml/g) 3.4 3.4 3.6 2.6 3.0 3.2 2.5
VQ~ml/g) 4.4 6.0 4.5 3.5 4.0 4.3 3.4
Acid
(meq./g) 3.1 4.2 3.6 3.8 3.9 4.7 4.3
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TGDM = Tetraethylene glycol dimethacrylate
MAA = Methacrylic acid
MA = Maleic anhydride
AIBN = Azoisobutyronitrile
Emulsifier = Mixture of glycerine monooleate and glycerine dioleate
VS = Bulk volume
VQ = Swelling volume in water
Acid = Acid content after saponification of the anhydride groups.
EXAMPLE 9 (a)
A solution of 70 g of tetraethylene glycol dimethacrylate, 20 g of
acrylic acid, 10 g of maleic anhydride and 1 g of azoisobutyronitrile in 1
liter of benzene is warmed for 4 hours at 60C., 2 hours at 70 and 2 hours
at 80C., with exclusion of air and while stirring slowly. The finely
divided precipitation polymer is suspended three times in benzene and twice
in acetonitrile, filtered off and dried in vacuo.
Yield: 94 g
Bulk volume: 6.8 ml/g
Swelling volume in water: 7.6 ml/g
Acid content after saponification of the anhydride groups = 1.64 m-equiv/g
EXAMPLE 9 (b)
1 g of the polymer produced according to Example 9 (a) is reacted
as described in Example 1 (b), with penicillinacylase in 32 ml of water at
pH 6.3.
Result:
Enzymatic activity (NIPAB test):
Starting solution 107 U
Supernatant liquid + wash solution 26 g
Carrier copolymer after the reaction 38 U
(i.e. 36% of the starting activity)
EXAMPLE 10 (a)
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A solution of 50 g of tetraethylene glycol dimethacrylate, 40 g of
N-vinylpyrrolidone, 10 g of maleic anhydride and 1 g of dicyclohexyl percar-
bonate in 200 ml of acetonitrile is suspended in l liter of benzine (boiling
point 100-140C.) which contains 5 g of a mixture of glycerine monooleate
and glycerine dioleate and is initially warmed to 50C. for 18 hours, while
stirring. A further 1 g of the initiator is then added and the mixture is
stirred for a further 8 hours at 60C. The polymer spheres are thoroughly
washed with benzene and petroleum ether and dried in vacuo.
Yield: 54 g of clear beads
Bulk volume: 1.6 ml/g
Swelling volume in water: 2.4 ml/g
Mean particle diameter: _ 0.3 mm
Acid content after saponification of the anhydride groups = 3.3 m-equiv/g
Nitrogen content: 3.4% ^ 27% of N-vinylpyrrolidone
EXAMPLE 10 (b)
1 g of the polymer produced according to Example 10 (a) is reacted
as described in Example 1 (b) with penicillinacylase in 33 ml of water at
p~ 6.3.
Result:
Enzymatic activity (NIPAB test):
Starting solution 107 U
Supernatant liquid ~ wash solutions 26 U
Carrier copolymer after the reaction82 U
(i.e. 77% of the starting activity)
EXAMPLE 11 (a)
A solution of 70 g of tetraethylene glycol dimethacrylate, 20 g of
methacrylamide, 10 g of maleic anhydride, 1 g of azoisobutyronitrile and 200
ml of acetonitrile is suspended in benzine and polymerized as described in
Example 5 (a).
Yield: 93 g
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~o63296
Bulk volume: 2.0 ml/g
Swelling volume in water: 3.3 ml/g
Acid content after saponification of the anhydride groups: 1.7 m-equiv/g
EXAMPLE 11 (b)
1 g of the polymer produced as described in Example 11 ~a) is
reacted as described in Example 1 tb) with penicillinacylase in 32 ml of
water at pH 6.3.
Result:
Enzymatic activity tNIPAB test):
Starting solution 107 U
Supernatant liquid and wash solutions 42 U
Carrier copolymer after the reaction 33 U
(i.e. 31% of the starting activity)
EXAMPLE 12 ~a)
A solution of 60 g of tetraethylsne glycol dimethacrylate, 30 g of
N-methoxymethyl-acrylamide, 10 g of maleic anhydride and 1 g of azoiso-
butyronitrile in 200 ml of acetonitrile is suspended in 1 liter of benzine
in which 5 g of a mixture of glycerine monooleate and glycerine dioleate
are dissolved, and is polymerized for 20 hours at 60C.
The bead polymer is filtered off, suspended three times in ethyl
acetate and dried in vacuo at 50C.
Yield: 97 g
Bulk volume: 1.6 ml/g
Swelling volume in water: 3.1 ml/g
Mean particle diameter:~_- 0.3 mm
EXAMPLE 12 tb)
1 g of the polymer produced according to Example 12 (a) is reacted
as described in Example 1 ~b) with penicillinacylase in 32 ml of water at
pH 6.3.
Result:
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Enzymatic acti~ity (NIPAB test):
Starting solution 107 U
Supernatant liquid + wash solutions 49 U
Carrier copolymer after the reaction 25 U
ti-e- 23% of the starting activity)
EXAMPLE 13 (a)
If in batch 12 ~a) 30 g of N-methoxymethylmeth-acrylamide are
employed instead of N-methoxymethyl-acrylamide, the following result is
obtained:
Yield: 94 g
Bulk volume: 2.4 ml/g
Swelling volume in water: 3.9 ml/g
Mean particle diameter: ~ 0.6 mm
EXAMPLE 13 (b)
1 g of the p~lymer produced according to Example 13 ~a) is reacted
as described in Examples 1 (b) with penicillinacylase in 32 ml of water at
pH 6.3.
Result:
Enzymatic activity (NIPAB test):
Starting solution 107 U
Supernatant liquid and wash solutions 60 U
Carrier copolymer after the reaction 24 U
~i.e. 22% of the starting activity)
EXAMPLE 14
76.1 kg (moist weight) of carrier-bound penicillinacylase produced
according to Example 4 ~b) having an activity of 737,000 U (NIPAB test) and
125 kg of potassium penicillin G (purity 98%) are successively added to
2,000 liters of water and the mixture is stirred at 38C. The pH value of
the reaction batch is kept constant at 7.8 by continuous addition of tri-
ethylamine. After 8 hours, no further triethylamine is taken up. The
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carrier-bound penicillinacylase in centrifuged off, rinsed with 60 liters of
water and 80 liters of 0.2 M phosphate buffer of pH 6.5, and reemployed for
further splitting batches. The filtrate, including the wash water, is con-
centrated to 300 liters in vacuo. The 6-APA is precipitated by addition of
half-concentrated hydrochloric acid at the isoelectric point at pH 4.3 in
the presence of 200 liters of methyl isobutyl ketone. After one hour the
product is filtered off and rinsed with 200 liters of water and then with
200 liters of acetone. It is dried in vacuo at 40C.; melting point 208C;
yield 66.7 kg, representing 91.9% of theory; purity 98%.
The carrier-bound penicillinacylase was successively employed for
a total of 30 batches. Even after 30 splitting reactions, the carrier-
bound penicillinacylase has not been consumed. The reaction time does not
change. Working up took place as described above. The yields of 6-APA
achieved are listed below:
1st splitting reaction 91.9% of theory
2nd splitting reaction 91.4% of theory
3rd splitting reaction 91.6% of theory
4th splitting reaction 91.0% of theory
5th splitting reaction 91.2% of theory
206th splitting reaction 90.3% of theory
7th splitting reaction 91.5% of theory
8th splitting reaction 90.9% of theory
9th splitting reaction 91.9% of theory
10th splitting reaction 90.7% of theory
11th splitting reaction 91.2% of theory
12th splitting reaction 90.1% of theory
13th splitting reaction 90.9% of theory
14th splitting reaction 90.7% of theory
15th splitting reaction 91.0% of theory
3016th splitting reaction 90.2% of theory
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17th splitting reaction 90.3% of theory
18th splitting reaction 89.9% of theory
19th splitting reaction 89.8% of theory
20th splitting reaction 89.1% of theory
21st splitting reaction 91.7% of theory
22nd splitting reaction 91.9% of theory
23rd splitting reaction 91.6% of theory
24th splitting reaction 91.1% of theory
25th splitting reaction 91.8% of theory
26th splitting reaction 90.7% of theory
27th splitting reaction 91.7% of theory
28th splitting reaction 91.4% of theory
29th splitting reaction 90.8% of theory
30th splitting reaction 90.3% of theory
= 90.95% of theory
EXAMPLE 15
330 g of carrier-bound penicillinacylase produced according to
Example 1, having an enzymatic activity of 3,410 U (NIPAB test) and 129 g
of potassium penicillin G are successively added to 2,000 ml of water and
stirred at 38C. and pH 7.8 as described in Example 14. The penicillin G
is completely split to 6-APA and phenylacetic acid over the course of 2
hours. The 6-APA is isolated as described in Example 14. The carrier-bound
penicillinacylase is employed twenty times in succession. Even after the
twentieth splitting, the reaction time does not have to be extended to
achieve complete splitting.
Yields of 6-APA:
1st splitting reaction 67.5 g (90.1% of theory)
2nd splitting reaction 68.4 g (91.1% of theory)
3rd splitting reaction 68.7 g (91.9% of theory)
4th splitting reaction 68.5 g (91.5% of theory)
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5th splitting reaction 68.4 g ~91.0% of theory)
6th splitting reaction 68.1 g (90.8% of theory)
7th splitting reaction 6R.7 g (91.6% of theory)
8th splitting reaction 68.4 g (91.2% of theory)
9th splitting reaction 68.5 g (91.5% of theory)
10th splitting reaction 68.1 g ~90.9% of theory)
11th splitting reaction 68.5 g ~91.3~ of theory)
12th splitting reaction 68.6 g ~91.7% of theory)
13th splitting reaction 68.5 g ~91.3~ of theory)
1014th splitting reaction 68.1 g (90.8% of theory)
15th splitting reaction 68.3 g (91.1% of theory)
16th splitting reaction 67.8 g (90.5% of theory)
17th splitting reaction 68.0 g (90.6% of theory)
18th splitting reaction 67.4 g ~89.9% of theory)
l9th splitting reaction 67.5 g ~90.0% of theory)
20th splitting reaction 66.8 g ~89.1% of theory)
= 90.89% of theory
EXAMPLE 16
120 g of carrier-bound penicillinacylase (moist weight) according
to Example 4, with an activity of 1,160 U ~NIPAB test) and 120 g of potassium
penicillin G, are introduced into 1,300 ml of water and stirred for 9 hours
at 38C. and pH 7.8, as described in Example 14. The mixture is worked up
as described in Example 14. The carrier-bound penicillinacylase is employed
five times in succession for the enzymatic splitting reaction. The yields
of 6-APA achieved are listed below:
1st splitting reaction 61.2 g ~87.6% of theory)
2nd splitting reaction 62.0 g (88.9% of theory)
3rd splitting reaction 63.3 g (90.6% of theory)
4th splitting reaction 62.7 g (89.9% of theory)
305th splitting reaction 63.1 g (90.4% of theory)
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EXAMPLE 17
290 g of carrier-bound penicillinacylase according to Example 4,
having an enzymatic activity of 2,803 U (NIPAB test) and 138 g of potassium
penicillin V are added to 2,000 ml of water and stirrea for 9 hours at 38C.
The pH value is kept constant at 7.8 by continuous addition of triethylamine.
The mixture is worked up as described in Example 14. Yields of 6-APA 64.3 g
t86.1~ of theory); purity 97.4%.
The invention has been described herein with reference to certain
preferred embodiments. However, as variations thereon will become obvious
to those skilled in the art, the invention is not to be considered as limited
thereto~
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