Note: Descriptions are shown in the official language in which they were submitted.
:~.05 4~
The present invention is concerned -with a formed
body with an enzymatically-active surface ~Ld with the
production thereof.
En~ymes f`ixed to a solid, insoluble carrier, i.e.
so-called i~mobil.ised enzymes, are of increasing importance
in many different ~ields of use, such a~ research, industry
and medicine. Depending upon the method of immobilisation
employed, the immobilised enzymes are divided into the
classes of bound, included and cross-linked enzymes. ~ound
enzymes can be obtained by covalent bonding to active
carriers, heteropolar bonding and/or van der Waal's exchange
action on ion exchangers, as well as on adsorbents.
Included enzymes are enzymes which are mechanically
immobilised in cross~ ed pol~mers and microcapsules, as
well as in regenerated cellulQse derivatives. Finally,
enzymes can also be cross-linked with bifunctional low mole-
cular weight reagents and thus made insoluble. `~
A disadvantage of the immobilised enzymes is that thecarriers suitable for fixing frequently have inadequate
mechanical properties and either cannot be worked up to
give formed bodies with enzymatically-active surfaces or
can only be so worked up with great difficulty. In principle~
o~ly those carriers based on synthetic resins, biopolymers
or inorganic substances c~n be used in which reactive groups -
are present or can be produced and which, if possible, con-
tain hydrophilic groups or centres. On the other hand,
however, for many fields of use for immobilised enzymes,
mecha~Lically ~table formed bodies are necessary. Thus, for
example, membranes, films, tubes and other formed bodies
with enzymatically-active surfaces are much more suitable
r~
~5~079 ABSl'RACT OF l~E DISCLOSURE MAY 8 1979
Enzyme containing bodies are provided useful in re-
search, industry and medicine, the bodies comprise formed bodies
with an enzymatically-active surface, wherein the surface of a
formed body is at least partially coated with adhesive and on the
adhe~ive layer there are present particles which contain at least
one enzyme im~obilised on a solid carrier, suitable bodies
include synthetic re~in, metal, glas~ or a synthatic ox natural
polymer.
~ .
, , ~,
11
V'`~
The pr~sent invention is concerned with a formed
body with ~n enzymatically-active sur~ace and with the
production thereof.
Enzymes fi~ed to a solid, insoluble carrier, i.e.
~o-oalled immobilised enzymes, are of increa~ing importance
ln many different fields of use, such as research, industry
and medicine. Depending upon the method of immobilisation
employsd, the immobilised enzymes are divided into the
classes of bound, included and cros~-linked enzymes. ~ound
enzyme~ can be obtained by covalent bonding to active
carriers, heteropolar bonding and/or van der Waal's exchange
action on ion exchangers, as well as on adsorbents.
Included enzymes are enzymes which are mechanically
immobilised in cross-linked polymers and microcapsules, as
well as in regenerated cellulose derivat:ives. ~inally,
enzymes oan also be cross~linked with bifunotional low mole-
oular weight reagents and thus made insoluble.
A disadvantage of the immobilised enzymes is that the
carriers suitable for fixing frequently have inadequate
mech~nical properties and either cannot be worked up to ;~
give formed bodies with enzymatically-active surfaces or
can only be so worked up with great difficulty. In p~inciple
only those carriers based on synthetic resins, biopolymers
or inorganic substances cQn be used in which reactive groups
are present or can be produced and which, if possible, con-
tain hydrophilic groups or centres. On the other hand,
however, for many fields of use for i~mobilised enzymes,
mechanically-~stable formed bodies are necessary. ~hus, for
example, membranes, films, tubes and other formed bodies
with enzymatically-active surfaces are ~uch more suitable
,;, ~ '
40 r~ ~3
tubes, pipes, rods, foils, ~iller bodies or in any other
desired for~ for which an enzymatically~active surface is
desired, ~or example~ in the case of reaction vessels.
'rhe surEace of -the formed body according -to -the pre-
sent invention is wholly or partially coated with an ad-
hesive layer. IJsually, the adhesive layer is presen-t on
those parts of the surface on which an enzymatic activit~
is desired. Any known and conventional -type o~ adhesive
can be used ~hese include, for example~ water-insoluble
vegetable adhe~ ves, such as adhesives made from rubber,
natural rssins and cellulose derivatives which are soluble
in organic solvents, as well as synthetic adhesives 7 such
as cellulose esters, butadiene-styrene and butadiene-
acrylonitrile co-polymers, pol~chlorobutadiene, phenoplasts~
such as phenol-~ormaldehyde condensation products and
resorcinol-formaldehyd2 condensation products, aminoplasts 9
such as urea-formaldehyde and mela~i~e-~ormaldeb~ co-nden-
sation products~ polyes-ters, polyisocyanates, epoxy resins,
silicones, polyvinyl adhes~ves, for example, polyvinyl
.~ . ,.
chloride, pol~isobutylene, po]yst~rene, polgvinyl alcoholS -
polyvinyl acetate, polyvinyl ethers and polyacrylic and
polymethacrglic acid esters, as well as mixtures thereof.
On the adhesive layer, there are present particles -
which consist of a solid carrier on which are fixed the
desired enzyme or enzymes, According to the present inven-
tion, these include not only natural carriers, i.e~ micro~
organisms, cells and cell fragments, which carr~ or co~tain
the enzyme, but also conventional enz~mes immobilised on
solid carriers and especially enz~mes covalently fixed on
to solid carriers. Immo~ilised enz~mes in which the carrier~
consists preponderantly of acrylamide ~nits are preferred,
~: 4
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and the yields obtained are ex-tremely lot,r. ~'hese known
processes also scarcely permit the simultaneous fixing of
several enzymes. ~ince an acti~ated carrier must always
be present, there is -the further feature that charged
sub3tratcs or reactants are adsorbed, -the pH optimum of
the enzyme is displaced and the Michealis constants are
changed and, in the case of cross~ ked enzymes, high
losses of a~tivity due to irreversible denaturing must be
taken into account. Furthermore 9 in some cases, fixlng
only takes place by adsorption so that the activity rapidly
'bleeds a~Jay".
It is, therefore, an object of the present invention
to overcome these disadvantages and to provide a formed
body with an enæymatically-active surface which can be used
as widely as possible in analytical and preparative
chemi~try and, on the other hand, can be produced in a
simple manner. -~
Thus, according to the present invention, there is
provided a ~ormed body with an enzymatically-active sur~ace,
wherein the surface of the formed body is at least partially
coated with an adhesive and on the adhesive layer there are
present particles which contain at least one enzyrne
immobilised on a solid carrier.
'~he formed bodies according to the present invention
can consist of any desired organic or inorganic material ~ ; `;
and can be of any desired shape. Thus, the formed bodies --
can be made of inorganic materials, such as glass, metal,
corundum or the like, or of organic materials, such as ` ;~
, : .
natural or synthetic polymers, synthetic resins and the ~`~
.
like. They can be in the forrn of, for example~ spheroids7
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tubes, pip~s, rods5 foils, filler bodies or in any other
desired ~orm for wllich an enzyma-ticall~active surface is
desired, for example, in the case of reaction vessels.
~rhe surface of the formed body according to -the pre-
sent invention is wholl~ or partially coated with an ad-
hesive la~er. IJsually~ the adhesive layer is present on ?
those parts of the surface on which an enzymatic activitg
is desired. Any known and conventional -type of adhesive
can be used. ~hese lnclude, for example~ wa-ter-insoluble
vegetable adhesives, such as adhesives made from rubber,
natural reSin~ and cellulose derivatives which are soluble
in organic solvents, as well as sgnthetic adhesives, such
as cellulose esters, butadiene-stgrene and butadiene-
acrylonitrile co-pol~mers, pol~chlorobutadiene, phenoplasts~
such as phenol-formaldehyde condensation products and
resorcinol-formaldehyde condensation products, aminoplasts,
such as urea-formaldehyde and mela~ine-formaldeh~ conden-
sation products~ polyesters, pol~isocganates, epoxy resins,
silicones, polgvinyl adhesiYes, for example, pol~vinyl ~-
chloride, polgisobutglene, pol~styrene, polyvingl alcohol,
pol~vin~l acetate, polyvinyl ethers and pol~acr~lic and
.
polgmethacr~lic acid esters, as well as mixtures thereof.
On the adhesive layer, there are present particles ~ -~
..
which consist o~ a solid carrier on which are fixed the
desired enzyme or enzymes, According to the present inven~
tion9 these include not onlg natural carriers, i.e. micro- ;
organisms, cellæ and cell fragments, which carr~ or contain
-the en~yme, but also convantional enz~mes immobilised on
solid carriers and especiallg enzgmes cova~entlg fixed on
to solid carriers. Immo~ilised enz~mes in which the carrier~
consists preponderantl~ of acrglamide ~nits are preferred,
,~
.- . . .... , .. - . , .... - - ... . - " .. ..
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be used formed bodies, the surfaces of which can be con-
verted n situ, by a chemical or physical treatment, into
an adhesiveO This i~ possible, for example, in the case
of cer-tain synthetic resins, such a~ polyvinyl chloride
(PVC) by treatment with an appropriate organic solvent
and/or pla~ticiser or modification a~ent.
~'he particles of the carrier-bound immobilised
e"~f7/~yed
enzyme ~k~e~ preferably have a size of from about 0.0001
to about 1 mm.~ the range of from 0.001 to 0.1 mm. being
especially preferred. The particle size ~hich is best
suited for a particular purpose depends upon the fixed
enzyme, the carrier in question and ~lso upon the adhesive
and especially upon the organic solvent used in the
adhesive. In the case of especially sensitive enzymes,
use is preferably made of a particle size in the upper
half of the above-stated range of particle sizes. ~he
more stable is the immobilised enzyme, the smaller can be ;
the particle size.
An important advantage of the present invention is
~hat enzymatically active formed bodies can be obtained,
the physical properties of which are practically independent
of the physical properties of the "primary carrier", i.e.
of the particulate material employed for the immobilisation
of the enzyme. The actual formed body does nob need to be
converted into an active form ~nd, in principle, carrier~
bound enzymes produced by all known methods of immobilis-
ation can ~e employed for the enzymatic activation of the
surfaces of the formed bodyO Apart from co-valently bound
enzymes and preferably enzymes co-valently bound by protein
co-polymerisation, there can ad~antageously also be employed,
~ ~ .
``~ '
.
10540 ~9
according to the present invention, enzyme~ immobilised by
inclusion.
The formed bodies according to the present invention
can be used, as already mentioned, for a large number of
different purposes. For example, they can be used in the
form of tubes, membranes and of other types of formed bodies
in apparatus used for enzymatic analysis. ~hus, for example,
it is possible, by the use of tubular material with enzymatically-
active inner qurEaces, to carry out enzymatic analyses with
10 the use of conventional photometers which operate with flow-
through cuvettes~ Another possibility of use is in automatic
analysis devices which utilise dialysis membranes, us~ thereby
being made of enzymatically-activated membranes covered by .
dialysis membranes. For simple detection reactions, enzymati- ;~
cally-active test tubes and object carriers can be used. In
the case of preparative chemistry, formed bodies according to ~ ;
the present invention are especially useful which are in the
form of reaction vessels, tubes, pipes and column fillings.
The invention is further described with reference
20 to the accompanying drawings in which:
Figure 1 illustrates graphically the enzyme ~ .
activity of a formed body of the
invention according to Example 1,
Figure 2 illustrates schematically an arrangement
for analysis of the enzyme activity of
a formed body of the invention according
to Example 1,
Figure 3 is a cross~section of one embodiment
of a formed body of the invention, and : :.
Figure 4 is a cross-section of another formed
body of the invention.
- 8 - :
~ ~ .
1 ()5~L~)r~
The following Examples are given for the purpose
of illustrating the present invention:-
Example 1
.
Enzy~el qlucose oxidase/catalase on tubesStarting materials:
tube made of polyvinyl acetate-polyethylene co-
polymer, internal diameter 1.5 mm.
5 ml. adhesive based on synthetic rubber (Uhu- :
Kontakt 2000 )
10 ml. methylene chloride
100 mg. of a glucose oxidase (GOD)-ca~alase tcab)
'~ ''.
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~40~g :~
mixed enzyme covalently bound to cross-linked
polyacrylamide carrier ~Enzygel GOD/Cat),
finely powdered
~ethod: ,
The adhesive ~olution, diluted with methylene
chloride, is introduced with a syringe into the vertically
suspended tube (length ~ 70 cm.). As soon as the
solution hai~ run -through completely (after 1 - 2 minutes),
the lower end of a spheroidially-shaped pipette tip, open at ;
both ends, is introduced into the tube. ~he pipette
,~ ~ . . . :;
synthetic resin sphere contains the enzyme powder. On the
other part of the sphere~ with an opening of about 8 -
12 mm., there is attached a pressure tube. Subsequently, `~
the enzyme powder is blown with about 1 - 3 ats. compressed
air or nitrogen into the adhesive-coated tube. Exoess
enzyme powder can be collected again at the lower end o~
the tube and used again. -
The tube coated with the enzyme powder is, after ' ~ ;~
drying (about 1 hour), rinsed with a buffer solution until
, . . .
no more enzyme particles come away. The ~inished enzyme ~
tube lS then used for the automatic analysis of glucose. ~ ;
A length of tube o~ about 48 cm~ shows 9 in the case of a
si~mple ~requency of 24/hour and a ratio of sample to wash
of 1:4, the activity indicated in Fig.1 of the accompanying
drawing3. The sample frequency can be increased to about
40/hour.
~ he analysis can be carried out with the i~rrangement
schematically illustrated in Fig.2 of the accompanying
drawings~
The details of the apparatus iand measurement device ; ~ -
are as follows~
-10-
lOS~O~
Photometer: Eppendor~ 1101 M
Flowthrough cuvette: ~ellma No. 178, lcm. layer depth
Pump: Ismatic MP 13 G ~-10 :
Connector (air inlet): DO, H, Technicon No. 116-0203-00
Coil: 14 windings, Technicon , inner diameter 2 mm~
Air separator: C-5, Technicon No. 116-0202-05
Tubes: 1. air, inner diameter 1 mm.
2. sample, inner diameter 2.5 mm.
3. reagent, inner diameter 2.5 mm. ~ ~
4. enzyme tube, inner diameter 1.5 mm., length 48 cm. - -
Sample solution: glucose 0.25 mg./lOO ml. - 200 mg./100 ml.
in 0.LM phosphate buffer (pH 7.0)
Reagent: 1.1 g. 2,2'-azino-di-(3-ethyl-benzthiazolin)-6-
sulphonate (ABTS) ~ 0.6 ml. peroxidase (Boehringer,
POD-l) in 1000 ml. 0.1 M phosphate buffer pH 7.0
Measurement radiation: 405 nm, temperature 25C.
Analogously as in the case of the Technicon Auto-
Analyzer , not only~ the sample but also the reagent solution ~ ;
are segmented in the tube by air bubbles and the substrate
concentration measured co~inuously as well as discontinuously
by comparision with a standard solution. ;
As Fig. 2 of the accompanying drawings shows, the -
sample solution (2) is segmented, before entry into the
enzyme reactor, with air bubbles (1) and subsequently reacted
in the enzyme tube (or by an enzyme foil present in a bifilar
milled spiral). By the addition of reagent solution (3), a
colour reaction takes place (or some other indicator reaction)
which, after emergence of the air bubbles (immediately before
.. . .
the flowthrough cuvette) can be measured photometrically. ~ -
- li - . .
*trademark
: " ',: ' '~ '": "
The readings registered with a recorder are directly
proportional to the substrate concentration~
Example 2
Glucose oxidase/catalase on foil ',
Starting materials: ' ,
polyamide-polyester random fibre fleece tViledon '
fleece material, H 3003)
5 mlO adhesive as in Example 1
7 ml. methylene chloride
500 mg. of the same enzyme particle preparation as
in Example 1
Method:
Tpe adhesive is diluted with methylene chloride
and thinly applied to the foil. After some time, the finely-
divided enzyme powder is applied to the still moist and
sticky foil, with the use of a sieve. After slow drying,
the foil is slowly stirred in a O.lM phosphate buffer
solution (pH 7.0) and, in this way, non-bound particles
are removed. The foil is subsequently transferred into a ,!
bifilar milled spiral and employed'for substrate dete~mination
in a similar way to the enzyme tube.
1 cm of the foil produced by the above-described ~ -
process weighs, in a dry,state, 23.3 mg. and has an
enzymatic activity of 16.1 U/g., corresponding to
0.375 U/cm .
Coatinq of a glass tip
Starting materials:
1 ml. adhesive as in Example 1
1.5 ml. methylene chloride
50 mg. enzyme powder as in Example 1.
- 12 -
*trademark
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The ylass tip is dipped once or twice into the
diluted adhesive solution and, after a few seconds, dipped
into the enzyme powder. Af-ter slow drying, it is trans-
ferred to a 0.1M buffer solution (pH 7.0) and tested after
about 12 hours. In the case of a tip of 1 - 2 mm. diameter
and with a length of 2 - 3 mm., there is obtained an
average activity of 0.5 U/tip.
Example 4
A round polystyrenerod of 2.4 mm. diameter is coated -
with an adhesive which was obtained from 6 g. of a PVC
adhesive (Tangit of the firm Henkel) and 2 ml. tetrahydro-
furan. The rod is then dipped into a finely powdered
enzyme preparation which consists of GOD (230 U/g.) and
Cat (3600 U/g.~ fixed via acryloyl chloride by the process
of enzyme co-polymerisation. The rod is then allowed to
dry and the activity determined. It is found to be
0.49 U GOD/mm surface area. ;
Example 5
The process of Example 4 is repeated with the use
of an alkaline phosphatase with an activlty of 200 U/g. ~ ;
bound to the same carrier. The polystyrene rod is, aft0r
dipping into the adhesive,left to dry for 15 to 30 seconds
in the air, whereafter the enzyme dust is sprinkled on, the
adhesive is left to dry and the rod is washed with a
buffer. The activity is found to be 0.23 U/mm surface area. --
Example 6
The process of Example 5 is repeated w1th the use
of an enzyme powder which contains 5 U/g. trypsin on a maleic
- 13
; *trademark
~ ` ~
- .: , .
~054~) ~9
anhydride-acrylamide carrier (according to German Patent
Specification No. 1,908,290). The activity of the formed
body is subsequently found to be 0.003 U/mm surface area.
Example 7
Exàmple 5 is repeated with the use of a two-
component epoxy resin adhesive (Uhuplus ) and the GOD/Cat
powder of Example 4. The measured activity of the formed body
is found to be 0.5 U/mm2 surface area.
Example 8
The process of Example 5 is repeated with the use
of a contact adhesive based on polychlorobutadiene, with the
addition of resins and organic solvents (Pattex of the firm
Henkel), as well as of the enzyme powder of Example 4. The
measured activity of the formed body is found to be 0.45 U/mm2.
Example 9
The process of Example 8 is repeated with the use
of the alkaline phosphatase enzyme powder according to
Example 5. The measured activity of the formed body is
found to be 0.021 U~mm .
ExamPle 10
Example 5 is repeated with the use of a commercially
available adhesive based on polyvinyl resin with acetone
; and ethyl acetate as solvents (Uhu Alleskleber ) and of the
enzyme powder of Example 4. The enzymatic activity
measured on the formed body is 0.5 U/mm2.
Example 11
.
Example 10 is repeated with the use of the enzyme
powder of Example 6. The measured activity is found to be
; 0.0026 U/mm . ' ;
.
*trademark
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.
~35~ ri~
Example 12
A round rod of PVC o~ 3 mm. diameter is superficially ;`
dissolved with cyclohexane until the surf`ace thereof
becomes sticky. The enzyme powder of Example 4 is then
~prinkled on, the rod is left to dry and thereafter it is
washed with a buffer solution. '~he enzymatic activity of
the formed body is 0.38 U/mm2.
xample 1~.
Ex~mple 12 is repeated with the use of the alkaline
phosphatase enzyme po~der according to Example 5. ~he ~-
enzymatic acti~ity of the formed body is 0.117 U/mm2.
xample 14. `
The process of ~xample 12 is repeated with the use ~ ;
of the trypsin-containing enzyme powder of ~xample 6. ~The
enzymatic activity of the formed body is 0.0042 U/mm2.
E_am~le 15.
A glass rod of 2.4 mm. diameter is dipped into the ;~
adhesive of Example 8, thereafter allowed to dry in the -
air for 15 to 30 seconds and sprinkled with the enzyme
p~wder of Example 4. After drying the rod9 it is washed
with buffer. The enzymatic activity of the finished rod `
is 0.66 U/mm .
a ~ . ` ;~
A steel wire of 1 mm. diameter is dipped into the
adhesive of ~xample 8, left to dry in the air for 15 to
30 seconds and then sprinkled with the enzyme powder of
Example 4. The enzymatic activity of the finished formed
body is found to be 0.80 U/mm2.
Example 17.
The process of Egample 16 is repeated, with the use
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of the all~aline pho~ hatase enz~me powder accorcling to
Exam~le 5. r~le enzymatic activit~ of the formed bod~ is
found to be 0.003 U/mm2.
~æample 18~
Example 4 i9 repeat~d but, ins-tead of the poly-
styrene rocl, there is used a '~eflon-coated magnetic
s-tirrer rod with the dimensions 6.5 x 10.9 m~, ~he
enz~matic a~-tivitg of the formea body is found to be
0.15 U/m~n2.
~ .
Example 5 is repeated but, instead of -the polg-
st~rene rod, there was used a ~eflon-coated magnetic
stirrer rod as described in Example 18. '~he enzymatic
activitg of the formed body is 0.135 U/mm2,
E ~
~ wo foils were coated with adhesive J 6610-21
(50~ polyacrylic ester) of the firm Henkel. The la~er
thickness was 25 ~ and 50~, respectivel~ ~
cells were applied to 50 cm2 pieces of the foils.
~he foils were vigorously s-tirred overnight in a phosphate
buffer (pX 7.0) in order -to remove non-~ixed cells. The
amount of ~ niger stuck on was determined by ~ -
weighing the dried foils. n the 25~ foill there was
ascertained 0,18 mg, ~ ~ cells per cm and
on the 50 ~ foil, 0;20 mg. ~ Q ~ ce~ls per cm .
~he activit~ o~ the micro-organisms on the ~oils was
0~027 U/cm2 in the case of the 25~ foil and 0~0~2 U/cm2
in the case of the 50~4 foil, ~he la~t runnings amounted
to 15~, Since the ~ ~ ells used had an
activity of 200 U/g., referred to GODI the calculated
activit~ ~ield was 75~ in the case of the 25~ ~oil and
80~G in the case o~ the 50~ foll.
-16~
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.. : , :
i~540 ï9
; ', ':
Nocardia ~ micro-organism cells were
stuck to the same foils as were used in 3xample 20~ The
Nocardia ~ cells used had a chole,sterol oxidase
activitg of 16lJ/gD '~he 25~ foil was ~ound to ha~e 0.2 mg~
of ce]ls per cn2 with an activitvv of 0.002U/cm2, which
corresponcls to an activity yield of 62.5'~, In the case o~
the 50~ foil, 0.~8 m~. o~ cells per cm2 were found with
an activlty of 0.005 U/cm29 which corresponds to a gield
o~ 64~ here were no last runnings~
3xample 22,
On the same foîls as wers used in Example 20, there
were stuck GOD/Cat commonly bound to a polymeric carrier
and having an activitv of 300 U/g, ~he 25~ foil was found
to have 0,2 mg. enzygel/cm2 with an activity of 0.047 U/
cm2, which corresponds -to an activitv yield of 78.3~ In
the case of the 50t~ foil, 0.2 mg. enz~el/cm2 were found~
with an activi ty of 0.049 U/cm2, which corresponds -to an
activit~ yield of 81.6~. ~here were no las~ runnings~
Figs. 3 and 4 of the accompan~ing drawings illust- -
ra-te two embodiments of the present invention. Fig, 3
shows a cross section through a trlangular-shaped rod
coated on the surface with an adhesive lavver and with
enzvvme-con-taining parti&les and Figo 4 shows a cross-
section through a tube, the inner sur~ace of which is
coated with an adhesive to which has been applied
enz~me-containing particles.
'
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~54~
The enzymes particularly referred to in this
disclosure have the following enzyme numbers:
glucose oxidase 1.1.3.4
catalase 1.11.1.6
alkaline phosphatase 3.1.3.1
trypsin 3.4.21.4 ~
~ . .
.
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- 18 -
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