BE832366A - ENZYME PREPARATION - Google Patents

Description

       

  Enzyme preparation.

  
 <EMI ID = 1.1>

  
enzyme and more particularly relates to preparations for carrying out chemical transformations, which can be recovered from the reaction medium for reuse.

  
enzyme-catalyzed transformations or conversions are important stages in a large number of chemical processes, for example in the saponification of lipids.

  
 <EMI ID = 2.1>

  
protein preparation using a proteolytic enzyme such as trypsin; production of steroids using dehydrogenase; the production of glucose syrups from starch using a hydrolase; and the production of 6-amino-penicillanic acid from penicillin such as benzylpenicillin or phenoxymethylpenicillin using penicillin acylase.

  
In general, the enzymatic reactions are carried out while the enzyme is in solution in the medium containing the

  
 <EMI ID = 3.1>

  
substance which the enzyme transforms or converts to give the product_7. Since the enzyme is dissolved in the reaction medium, it is frequently very difficult to separate the en-

  
 <EMI ID = 4.1>

  
when the reaction is complete. Generally, when the product is isolated from the reaction mixture, the separation process causes deactivation of the enzyme. This of course makes the enzyme unrecoverable.

  
In order to overcome this difficulty of separation, and to provide an enzyme system which can be reused, it is known to fix the enzyme on an insoluble solid support by adsorption (see for example British Patent No. [deg.] 1,264. 147) or by covalent bonds, directly or indirectly by bridging groups (see for example British Patents Nos. 1,349,498, 1,387,460 and 1,365,886.). However, such insoluble preparations have a number of drawbacks. First of all, because they are solid, they are subject to mechanical degradation and possibly to rupture or dissociation. Moreover, the incorporated

  
 <EMI ID = 5.1> <EMI ID = 6.1>

  
third, access of the substrate to the active center of the enzyme is often inhibited or hampered. Attempts to improve the specific activity of such insoluble enzyme and polymer preparations by increasing the outer surface area of the

  
Solids require a decrease in the particle size of the solid preparation making handling and in particular separation by filtration more difficult, and those increasing the interior surface area by making the particles much more porous result in particles having lower mechanical strength.

  
In addition, certain water-soluble enzyme and polymer complexes have been described (see e.g.

  
 <EMI ID = 7.1>

  
N [deg.] 53822/72 and. 44542 /? 3); in this case the enzyme is attached directly or indirectly by a bridging group to a water soluble polymer support. These enzyme and polymer complexes are recoverable from the aqueous reaction medium by ultrafiltration. However, ultrafiltration is a difficult and expensive technique, particularly on a large scale, and therefore undesirable for industrial applications.

  
The research which led to the invention has shown that certain enzymes can be attached to nonpolar groups to make the preparation separable from aqueous media by affinity for water immiscible liquids.

  
The invention is therefore embodied in an enzyme preparation containing an enzyme attached to non-polar groups in sufficient number so that, by contacting the preparation in aqueous media with an inert liquid immiscible with water, the preparation becomes associated with the inert liquid immiscible with water and can therefore be separated from the aqueous medium.

  
By the expression "inert water immiscible liquid" is meant herein a liquid which does not react with the enzyme preparation in a manner capable of substantially modifying the enzymatic activity.

  
The term "associated with" used above relates to any form of interaction between the nonpolar groups of the enzyme preparation and the water-immiscible liquid molecules. sufficiently stable to provide a means <EMI ID = 8.1> production process suitable for incorporation into the preparations according to the invention, and there may be mentioned, as enzymes of this type, Amylase, Asparaginase, Neutral Protease or Alca -

  
 <EMI ID = 9.1>

  
Trypsin.

  
Preferably, the enzyme is Lipase, Penicillin Acylase or Trypsin.

  
As suitable nonpolar groups which can be attached to the enzyme, mention may be made of hydrocarbyl groups having at least 6 carbon atoms, and suitably the groups.

  
 <EMI ID = 10.1>

  
heptyl, n-octyl, n-nonyl, n-decyl, n-dodecyl, n-octadecyl,

  
 <EMI ID = 11.1>

  
 <EMI ID = 12.1>

  
 <EMI ID = 13.1>

  
aralkyl, for example benzyl, 2-phenylethyl, 4-phenylbutyl, 2-tolylethyl.

  
The enzyme can be attached directly to the non-polar group, or else indirectly via a bridging group.

  
With some enzymes, the attachment of a number of nonpolar groups directly to an enzyme molecule produces a conformational change and a change in the activity of the enzyme. Because of this fact and in some cases because of the structure of the enzyme, it is sometimes not possible to attach nonpolar groups in sufficient number to an enzyme so that the preparation can be separated from aqueous media without inadmissibly reduce the activity of the enzyme.

  
To overcome this difficulty, it is often preferable to produce an enzyme preparation in which the enzyme is linked to a polymeric carrier or. adsorbed on such a support, which can then carry nonpolar groups instead

  
 <EMI ID = 14.1> <EMI ID = 15.1>

  
the relative proportions of nonpolar groups and enzyme in the preparation. It is also possible, of course, to attach an enzyme bearing sufficient nonpolar groups to render it separable from aqueous media on a polymeric support which may or may not bear other nonpolar groups.

  
According to a second aspect, the invention consequently relates to an enzyme preparation as defined above in which the enzyme is attached to a polymer support.

  
Binding of the enzyme to a polymer as used herein includes both covalent bonding with and adsorption to the polymer support, provided that the adsorption is strong enough to retain the enzyme. on the support when the preparation is combined with the liquid not

  
 <EMI ID = 16.1>

  
The carrier suitable for use in the above preparations may be soluble or insoluble in water.

  
When a water-insoluble polymeric material is used in the preparations according to the invention, it is preferably in a finely divided state, so as to have a large surface area for the binding of the enzyme, and thus to impart a high specific activity to the preparation.

  
Suitable water insoluble materials include powdered cellulose and cellulose derivatives.

  
 <EMI ID = 17.1>

  
ions, nylon, high molecular weight polysaccharides,

  
 <EMI ID = 18.1>

  
modified, with modifying agents such as epichlorohydrin, or modified to contain carboxymethyl or aminoethyl groups; polyacrylates and polymethacrylates

  
 <EMI ID = 19.1>

  
and macro-crosslinking crosslinked polyacrylate and polymethacrylate type resins.

  
As suitable water-soluble materials, mention may be made of polymers of natural origin, for example

  
 <EMI ID = 20.1>

  
long and the original, natural polymers which have been modified, sum partially degraded starch or cellulose; the

  
 <EMI ID = 21.1>

  
 <EMI ID = 22.1>

  
Among the saccharides and oligosaccharides mentioned modified above, it has been found that copolymers of epichlorohydrin and lactose, dextrose or sucrose

  
are particularly suitable. A preferred polymer is

  
 <EMI ID = 23.1>

  
a molecular weight of the order of 400,000. The term "Ficoll" is a trade name.

  
As well as polymers of natural origin, it is also possible to use synthetic water-soluble polymers, for example polymers of polyvinyl alcohol and copolymers of maleic or acrylic anhydride with ethylene, styrene, methyl vinyl ether, divinyl ether or., vinyl acetate. Such polymers are described for example in German patent applications

  
n [deg.] 1,948,177 and 1,948,298, and in British patents

  
N [deg.] 1.290.701 and 1.223.28 <1>.

  
A suitable class of water-soluble copolymers is formed by the copolymers of ethyl vinyl ether and maleic anhydride known under the name "Gantrez".

  
 <EMI ID = 24.1>

  
The enzyme can be attached directly to a polymer containing the non-polar group, or alternatively it can be attached to the bridging buttocks of the polymer support, to which the enzyme can be attached itself. bridging group is an aliphatic alpha, omega-diamine in

  
 <EMI ID = 25.1>

  
amino function of the bridging group is attached to the polymer and the other is free for coupling to the enzyme, for example through a water soluble dialdehyde such as glyoxal or glutaraldehyde. Using groups in

  
 <EMI ID = 26.1>

  
between the enzyme and polymer units. Bridging groups can be used between the enzyme and the support; the enzyme and the non-polar group (with or without the additional presence of a support); or the support and the non-polar group.

  
The number of nonpolar groups incorporated in the preparations according to the invention depends on a number of factors. The attachment of non-polar groups to the polymer and <EMI ID = 27.1>

  
me. With some enzymes, too much hydrophobicity results in deactivation of the enzyme as a result of a concomitant conformational change.

  
 <EMI ID = 28.1>

  
minimum of nonpolar groups necessary to allow separation from aqueous media and that of the maximum number compatible with the stability of the enzyme. Determining a balanced cat must, of necessity, remain a matter of determination by experience and classical error. The criteria by which this balance can be determined are as follows:

  
1 - molecular weight of the polymer support,

  
2 - molecular weight of the monomer unit,

  
3 - percentage of monomer units in the substituted polymer

  
by non-polar groups,

  
4 - size of non-polar groups,

  
5 - identity of the enzyme,

  
6 - degree of substitution of the polymer by the enzyme.

  
For example, when the support is Gantrez AN 119
(polymer molecular weight 230,000, monomer unit molecular weight 156) in which one mole of Gantrez AN 119 is substituted with one mole of penicillin acylase, the minimum substitution necessary to effect separation by

  
 <EMI ID = 29.1>

  
monomer units are substituted with n-decyl groups; and maximum substitution to maintain enzyme activity

  
 <EMI ID = 30.1>

  
replaced by n-octadecyl groups.

  
The invention also relates to a process for the production of an enzyme preparation as defined above, said process comprising contacting a compound of formula.

  
 <EMI ID = 31.1>

R - X (I)

  
wherein R is a non-polar group and X is a functional group with an enzyme attached to an active group capable of reacting with the group X.

  
The group. X can be, for example, an amino, carboxyl, aldehyde, hydroxy, thiol or azo group; or reactive Linking groups derived, for example, from dialdehydes such as.

  
glyoxal or glutaraldehyde, and (or) such as

  
 <EMI ID = 32.1>

  
polymeric support containing any such functional group. Preferably, X is an amino group or else a mole fraction bearing an amino group.

  
The active group attached to the enzyme and capable of reacting with the X group can be a group inherently present on the enzyme, or by modification, for example a group.

  
 <EMI ID = 33.1>

  
anhydride bran; or a polymeric support material containing such an active group; or a reactive linking group such as those indicated for group X above.

  
The reaction between the compound of formula (I) and the enzyme is most advantageously carried out at a substantially neutral pH, suitably in a pH range from 4 to

  
 <EMI ID = 34.1>

  
 <EMI ID = 35.1>

  
between 0 and 5 [deg.] C.

  
When the enzyme preparation is formed essentially by the enzyme and the nonpolar groups alone, it is preferable that the X group is derived from a dialdehyde and (or) from an alpha, omega-diamine, that is that is, the non-polar groups are attached to the enzyme by such bridging groups.

  
When producing an enzyme preparation according to the invention also including a polymeric support, it is first preferable to modify the support so as to incorporate the non-polar group and, when necessary, the group. bridging, before binding to the enzyme

  
 <EMI ID = 36.1>

  
Feels like a polymeric material which is linked to the non-polar group R and to a functional group.

  
Enzyme and modified polymer preparations can then be obtained by any known method for linking enzymes to polymers. Such binding methods are described, for example, in the British patent

  
 <EMI ID = 37.1> .the polymer by the use of reagents such as halogenu. cyanogen res, for example cyanogen bromide; the <EMI ID = 38.1>

  
acyl azothhydrides, diazonium compounds, for example

  
 <EMI ID = 39.1>

  
organic compounds such as phenyl cyanate; and carbodiimides. Often, such linkers are used first for reaction with the polymer, and they provide reactive groups thereon, and then these groups are caused to react with the enzyme. In some cases, enzymes can react directly with the polymer, for example if the polymer contains or has been modified to contain active groups such as anhydride linkages or azothhydride groups.

  
It will therefore be understood that the reaction method

  
 <EMI ID = 40.1>

  
modified mothers and to bind them to enzymes will vary depending on the nature of the enzyme and that of the polymeric material.

  
For most enzyme and polymer preparations, a judicious process is shown by Procedure I. The first step is to attach to the polymer (a) the nonpolar groups, preferably via

  
 <EMI ID = 41.1>

  
bridging groups such as alpha, omega-diamines, for subsequent attachment to the enzyme via dialdehyde groups. These reactions are conveniently carried out in an aqueous solution or suspension at pH

  
 <EMI ID = 42.1>

  
you. The enzyme bearing dialdehyde groups can then be coupled with the modified polymer in aqueous media at substantially neutral pH.

  
 <EMI ID = 43.1>
 <EMI ID = 44.1>
 in which R is a nonpolar group as designated above:

  
 <EMI ID = 45.1>

  
It is often necessary to activate the groups carried by the polymer, preferably with cyanogen bromide, before attachment to the nonpolar groups if this is possible.

  
Further, some polymers include groups which can be used for coupling with nonpolar groups and (or) the enzyme, if desired without an intermediate bridging group. Enzyme preparations according to the invention prepared in this way then do not exhibit the crosslinking associated with the use of bridging groups and often possess advantageous properties as indicated below.

  
For example, in polymers based on the monomer formed by maleic anhydride, such as "Gantrez" type materials, the anhydride groups located along the backbone can be used for attachment of both

  
 <EMI ID = 46.1>

  
is first modified by attachment of the nonpolar groups, and the remaining anhydride groups of the polymer are then used- <EMI ID = 47.1>

  
is illustrated by operating diagram II.

OPERATING DIAGRAM II.

  

 <EMI ID = 48.1>


  
'R represents the nonpolar group mentioned above; ENZ-NH2 represents the enzyme carrying an amino group.

  
Polymers of the Gantrez type, for example Gantrez AN 119 and. the Gantrez AN 149, have yet 'another advantage in that they dissolve without reaction in certain solvents

  
 <EMI ID = 49.1>

  
 <EMI ID = 50.1>

  
R2NH. A homogeneous reaction mixture is possible and therefore such solvents constitute advantageous reaction media for the attachment of nonpolar groups to these polymers during the production of the enzyme preparations according to the invention.

  
Consequently, according to a pre- <EMI ID = 51.1>

  
tion are prepared by reacting a compre- polymeric material. nant anhydride groups on the dorsal skeleton with a

  
 <EMI ID = 52.1>

  
than non-aqueous in which R is a non-polar group, and then by attaching the modified polymer to an enzyme.

  
It is preferable to react the amine with the polymer in the presence of a tertiary base, for example pyridine.

  
The expression “tertiary base” used herein denotes a tertiary amine or else an aromatic heterocyclic amine.

  
Preferably, the non-aqueous aprotic solvent is

  
 <EMI ID = 53.1>

  
temperature at which the reaction is carried out depends on the identity of the polymeric support and the amine. However, in general, the reaction is carried out at a temperature between

  
 <EMI ID = 54.1>

  
The modified polymer support can be isolated from the medium in which it is prepared by precipitation with a less polar solvent, most suitably with diethyl ether, or by adding the solution to water when swelling occurs. and hydrolysis of the anhydride, with isolation of the hydrolyzed modified polymer from the aqueous medium, using a water immiscible liquid, or by centrifugation.

  
The enzyme is then attached to the aforementioned isolated modified support by contact of the modified support with the enzyme in aqueous solution, optionally with a coupling agent, for example with a substituted carbodiimide. Alternatively, the unhydrolyzed reaction mixture or the unhydrolyzed modified polymer dissolved in a small volume of a polar, nonaqueous solvent can be added directly to an aqueous solution of the enzyme.

  
Alternatively, the coupling can be provided by first adapting the aforementioned non-hydrolyzed support by treating it with hydrazine hydrate in solution in dimethylformamide. This suitable carrier, which is then hydrolyzed and isolated from the aqueous medium by centrifugation, can be coupled with the enzyme in aqueous media using

  
 <EMI ID = 55.1>

  
In another aspect, the invention relates to a process - <EMI ID = 56.1>

  
both in bringing into contact, in an aqueous medium, an enzyme preparation as defined above with a substrate of the enzyme, then in separating the enzyme preparation from the aqueous reaction mixture by contact with a liquid immiscible in water, to recover the product of the reaction from the aqueous phase, and optionally to reuse the separated enzyme preparation by contacting it with a new substrate.

  
A wide variety of water-immiscible liquids can be used to separate the enzyme preparation from the aqueous medium. Suitable liquids include alkanes such as heptane, octane, - nonane, decane, hexadecane, aromatic hydrocarbons, higher aliphatic esters

  
 <EMI ID = 57.1>

  
Upon contact with such a liquid, the enzyme preparation forms a surface layer in the contact zone between the two. In order to. to make this contact zone maximum, the contacting stage usually involves agitation, for example by shaking or normal agitation, to dissociate the water immiscible liquid into small droplets. Each droplet is then surrounded by the enzyme preparation which adheres to its surface.

  
This contact of the enzyme preparation with the water-immiscible liquid can be carried out before, during or after the enzymatic reaction. Preferably, it is ensured before the enzymatic dispersion reaction, by stirring the enzyme preparation with a liquid immiscible with water in an aqueous medium containing the substrate. Thus, during the enzymatic reaction, the preparation is associated with droplets of water immiscible liquid, which is present as an emulsion in the aqueous medium, and the dispersion is maintained during the reaction by stirring. When separation of the enzyme preparation is required, stirring is discontinued and the phases merge.

  
Alternatively, the enzyme preparation may be present in the aqueous reaction mixture as a solution or a suspension and may be contacted with

  
 <EMI ID = 58.1>

  
 <EMI ID = 59.1>

  
 <EMI ID = 60.1>

  
zyme and the water-immiscible liquid will vary depending on the polymeric material and (or) the non-polar groups incorporated in the enzyme preparation. For example, when the enzyme and polymer preparations exhibit significant crosslinking, solid aggregates form and these particles adhere to the water-immiscible liquid droplets. However, in the case of uncrosslinked preparations, among which may be mentioned by way of example the materials fixed on supports of the type "Gantrez" as indicated above (and the preparations not containing a polymer), there is no Normally there is significant adhesion of the solid to the droplets, and a single layer of enzyme preparation forms over these droplets.

  
This is particularly advantageous when the enzymatic reaction is carried out after first bringing the enzyme preparation into contact with the water-immiscible liquid to produce such a system.

  
Whatever the nature of the association, the contact between the enzyme preparation and the water immiscible liquid
(usually in the form of droplets) allows the separation

  
 <EMI ID = 61.1>

  
 <EMI ID = 62.1>

  
ximply the same density as that of water immiscible liquids. When allowed to stand, the droplets either float to the surface of the aqueous medium (for liquids less dense than water) or settle to the bottom (for denser liquids). Thus, a separate layer is produced which contains the water-immiscible liquid associated with the enzyme preparation, which is easily recovered from the aqueous medium.

  
For liquids which do not float on aqueous media or which do not settle there sufficiently rapidly, other means of separation can be used, for example centrifugation, or one can act by passage of the mixture through a water-repellent filter, so that the aqueous medium passes through this filter and the water-immiscible liquid (as well as the enzyme preparation) remains behind and is therefore separated., An enzymatic process Preferred according to the invention is the preparation of 6-aminopeni: illanic acid from a penicillin using a preparation

  
 <EMI ID = 63.1> <EMI ID = 64.1>

  
such as strains of Escherichia coli when used for dissociating benzylpenicillin, or fungi of the actinomycete type when used for dissociating phenoxymethylpenicillin. Such enzymes are generally well known. They are used for the production of 6-APA in a pH range from 6.0 to 9.0 and from

  
 <EMI ID = 65.1>

  
tion of a penicillin results in the release of a free acid from the side chain of penicillin, 3 is necessary to maintain the above pH range during the process of preparing 6-APA by addition, depending on the needs, an alkali such as a sodium hydroxide solution, ammonium hydroxide, or triethylamine.

  
A particular advantage resulting from the use of enzyme preparations according to the invention is the ease of application to a continuous method of enzymatic reaction, which can be carried out in an apparatus such as that shown in FIG. 3 of the accompanying drawing, which is a schematic representation of a suitable reactor.

  
Referring to Fig. 3, it can be seen that a reactor vessel 1 is equipped with stirring means in the form of a rotary stirrer 2, inlets 3, 4 and 5 for the substrate, the alkali and the phase formed by the enzyme respectively, and of an .output 6. the tank 1 also contains a probe 7 for detecting the pH, which controls a valve 8 connected to the inlet of the alkaline 4. The outlet 6 of the tank feeds through a 9 pumps a separator vessel 10, via an inlet 11. The separator has an upper outlet 12 connected to the inlet 5 of the reactor vessel, a lower outlet 13 for discharging the product of the reaction through the pump 14, and a filter in the form of sintered glass 15 disposed above the lower outlet 13.

   The substrate inlet 3 is supplied from a pump 16 and the pumps 14 and 16 are interconnected to work at the same flow rates.

  
In service, the reactor vessel 1 receives, via the inlet 3, the substrate in a medium

  
 <EMI ID = 66.1> <EMI ID = 67.1>

  
tank 1 can be maintained at a constant temperature, for example by means of a water jacket (not shown) surrounding this tank. The alkaline is added to the emulsion which is in the tank, via the inlet 4, and the speed of addition is regulated by the valve 8 controlled by the probe 7, so that the pH of the reaction mixture is kept within the range required for optimum reaction. During the reaction, the pump 9 works and the mixture is taken

  
at outlet 6 and transferred to separator 10 through inlet 11.

  
In the separator. 10, the water-immiscible liquid (in this case this liquid is less dense than water) forms a top layer.

  
 <EMI ID = 68.1>
-zyme. The lower aqueous layer 18 containing the product of the reaction is taken off by the filter 15 and consequently by the outlet 13 by means of the pump 14. The upper layer 17 containing the enzyme preparation associated with the water immiscible liquid. can flow. through the outlet 12 and is returned to the reactor vessel 1 through the inlet 5. When the product is withdrawn through the outlet 13, a new quantity of substrate solution is added to the vessel 1 through the inlet 3, by means of of the pump 16. The pumps 14 and 16 are coordinated such that the inlet flow rate of the substrate is equal to the outlet flow rate of the. product.

  
Flow rates, reactor residence time, and other reaction parameters will depend on the particular enzyme and substrate preparation system considered. It can be noted from preliminary results that a reactor with a capacity of 10,000 liters, using
1,000 kg of an enzyme preparation based on penicil acylase <EMI ID = 69.1>

  
about 2,000 kg of penicillin G per day according to a concentration

  
 <EMI ID = 70.1>

  
have improved with enzyme preparations having higher specific activity.

  
The examples given below without limitation illustrate the production and the properties of certain compleces of polymer and enzyme according to the invention.

  
In the examples, the following abbreviations are used to denote polymers and modified polymers, - <EMI ID = 71.1>. And coupling agents.

  
Enzymes:

  
L: Lipase

  
PA: Penicillin Acylase

T: Trypsin '

  
Supports:

  
 <EMI ID = 72.1>

GAN: Gantrez

  
SR: Sepharose

  
T 2000: Dextran T.2000

  
Enzyme / Support Linkage Group:

  
 <EMI ID = 73.1>

  
Non-polar groups:

  
D: n-decylamino

  
DD: n-dodecylamino

  
OD: n-octadecylamino

  
Liaison and Activation Agents and Activated Groups:

  
CMC: Methane-p-toluenesulfonate of 1-cyclohexyl-3- (2-

  
 <EMI ID = 74.1>

  
A: Hydrazide.

  
The integer number following the notation represents the load number:

  
For example, the enzyme preparation of Example 1 is

  
 <EMI ID = 75.1>

  
 <EMI ID = 76.1>

  
hexamethylenediamine (HD) for attachment to the enzyme, and also decyl groups (D) as nonpolar groups.

  
 <EMI ID = 77.1>

  
ene-diamipe and the enzyme is made using glutaraldehyde (G).

  
Moreover, GAN149HDOD is the modified support prepared from GANTREZ 149 modified with 1,6-diaminohexane and

  
 <EMI ID = 78.1>

  
above to which penicillin acylase is attached in the presence of glutaraldehyde.

  
EXAMPLE 1 <EMI ID = 79.1>

  
Dissolve Ficoll (5 g) in water <EMI ID = 80.1>

  
gene (1 g) and the pH is maintained during the reaction with

  
2N NaOH. The reaction ceased after 20 minutes at room temperature and n-decylamine (4 g) and 1,6-diaminohexane (0.5 g) dissolved in methanol (10 ml) were added. We adjust

  
 <EMI ID = 81.1>

  
 <EMI ID = 82.1>

  
g / hour, resulting in a white pellet (wet weight 35 g) which is retained, and a cloudy liquor which floats and is discarded.

  
(b) Penicillin acylase &#65533; n-decylamino, (6-aminohexylamino) -Ficoll_7 (PAFHDDG - 1)

  
FHDD - 1 (wet weight 21.6 g) is mixed with a

  
 <EMI ID = 83.1>

  
 <EMI ID = 84.1>

  
E-coli penicillin (50 ml, 340 mg protein, partial

  
 <EMI ID = 85.1>

  
 <EMI ID = 86.1>

  
Within 30 minutes, the suspension of FHDD-1 is added. We shake the

  
 <EMI ID = 87.1>

  
frozen (-20 [deg.] C) for 16 hours. After thawing, we

  
 <EMI ID = 88.1>

  
a brown dumpling = - similar to a gel. We put it back

  
 <EMI ID = 89.1>

  
nutes. The liquors which supernate are combined and the brown pellet (wet weight approximately 6.3 g) is suspended in a

  
 <EMI ID = 90.1>

  
 <EMI ID = 91.1>

  
n-decanol, the suspension containing the pellet floats with the decanol on the surface of aqueous solutions, leaving an underlying mass not containing the enzyme in the form of particles. The enzymatic activities of the pellet, of the liquor which floats and of the starting enzyme are indicated in the ta-

  
 <EMI ID = 92.1>

  
the enzyme, virtually all of which is in the complex. The activity of the complex can be recovered substantially from the penicillin solutions by flotation or by centrifugation, in a conventional manner.

  
 <EMI ID = 93.1>

  
PAFHDDG-1 penicillin acylase activity Titration process. Penicillin G (sodium salt) is dissolved in the appropriate concentration in

  
 <EMI ID = 94.1>

  
 <EMI ID = 95.1>

  
Award-winning initial rate of 6-aminopenicillanic acid (6-APA) production. In confirmatory titration procedure 1, 2 ml of n-decanol is added to the stirred reaction mixture, and after the determination the suspension is allowed to separate into two layers. The top layer is removed with a syringe and reused. In process 2, the final suspension is centrifuged at 2,000 x g for 15 minutes and the formed pellet is reused.

  

 <EMI ID = 96.1>

EXAMPLE 2

  
(a) n-Octadecylamirio; (6-aminohexylamino) -Ficoll (FODHD-1)

  
Ficoll (5g) is dissolved in water (300ml) and the solution treated with cyanogen bromide (1g) at

  
 <EMI ID = 97.1>

  
 <EMI ID = 98.1> <EMI ID = 99.1>

  
methanol (20 ml). The pH is readjusted to 10.0 with 2N HCl and

  
 <EMI ID = 100.1>

  
loose white color (25 ml, retained) and a clear supernatant liquor, with a foam on the surface (octadecylamine).

  
 <EMI ID = 101.1>

  
Penicillin acylase (25 ml,

  
 <EMI ID = 102.1>

  
 <EMI ID = 103.1>

  
at room temperature for 15 minutes, add

  
 <EMI ID = 104.1>

  
After stirring at 4 [deg.] C for 3 hours, the material is centrifuged at 20,000 g / 4 [deg.] C / 30 min. The pink pellet is resuspended in sodium phosphate buffer

  
 <EMI ID = 105.1>

  
effective flotation under the effect of n-decanol, its activity being indicated in ... Table 2. Recovery of the activity

  
 <EMI ID = 106.1>

TABLE 2

  
PAFODHDG-1 activity

  
Titration procedure: as for Table 1. Penicillin G at 5% w / v is used for the whole.

  

 <EMI ID = 107.1>


  
EXAMPLE 3 <EMI ID = 108.1> cyanogen ripe (2.5 g) as previously described. After 20 <EMI ID = 109.1>

  
at 4 [deg.] C for 16 hours then centrifugation is carried out at <EMI ID = 110.1> <EMI ID = 111.1>

  
1 hour). The second ball is kept (wet weight approximately 50 g).

  
 <EMI ID = 112.1>

  
T2000HDD-1 (wet weight 10 g) is homogenized in sodium phosphate buffer solution

  
 <EMI ID = 113.1>

  
penicillin acylase (50 ml - 340 mg protein) at pH

  
 <EMI ID = 114.1>

  
store at -20 [deg.] C for 16 hours. After thawing, the suspension is stirred at room temperature for 1 hour.

  
 <EMI ID = 115.1>

  
fraction which floats and the pellet is resuspended in

  
 <EMI ID = 116.1>

  
performs homogenization. Centrifuge as before then resuspend and centrifuge again to give a brown pellet (wet weight 8.0 g). This undergoes effective flotation with n-decanol and less effectively with n-heptane. The activity of this preparation

  
 <EMI ID = 117.1>

  
tial of the enzyme are present in the complex.

TABLE 3 ..

  
 <EMI ID = 118.1>. Titration process: as before, we use <EMI ID = 119.1>
 <EMI ID = 120.1>
 EXAMPLE 4
(a) n-Decylamino, (6-aminohexylamino) Sepharose 4B (SRHDD-1)

  
Sepharose 4B (dry weight 2 g) activated by cyanogen bromide is swelled in 50 ml of 10-3N HCl.

  
 <EMI ID = 121.1>

  
two times 100 ml of 10-3N HCl. Add the gel to a solution

  
 <EMI ID = 122.1>

  
decylamine (0.5 g) and 1,6-diaminohexane (0.1 g). The mixture is slowly rotated at 4 [deg.] C for 16 hours, then filtered through glass, sintered. The gel is washed under suction

  
 <EMI ID = 123.1>

  
times 50 ml of water, then resuspended in water and

  
 <EMI ID = 124.1> <EMI ID = 125.1> <EMI ID = 126.1>

  
SRHDD-1 in the form of a suction-dried gel (1.5 g) is suspended in a phosphate solution of

  
 <EMI ID = 127.1>

  
(0.5 ml, 25% w / v). The mixture is stirred for 2 hours at room temperature and dried by suction over sintered glass. The gel is then added to a mixture of pear acylase.

  
 <EMI ID = 128.1>

  
0.1M pH 6.0 (3 mL). This mixture is stirred for 24 hours at

  
 <EMI ID = 129.1>

  
with 5 times 20 ml of sodium phosphate buffer solution

  
 <EMI ID = 130.1>

  
The resulting beads float on the addition of n-deca-

  
 <EMI ID = 131.1>

  
from aqueous suspensions containing n-decanol. The active- <EMI ID = 132.1>

  
approximately of the initial activity are present in the complex.

  
 <EMI ID = 133.1>

  
 <EMI ID = 134.1>

  
Titration process: as before, constantly using 5% w / v penicillin G.

  

 <EMI ID = 135.1>


  
EXAMPLE 5

  
Penicillin n-Decylaminoglutaraldehyde acylase (PADDG-1)

  
Penicillin acylase (40 ml,
272 mg) at pH 6.2 and glutaraldehyde (4 ml, 25%

  
 <EMI ID = 136.1>

  
decanol (6 ml) and n-decylamine (0.6 ml) and a

  
 <EMI ID = 137.1>

  
room temperature for 1 hour. Centrifugation of the

  
 <EMI ID = 138.1>

  
brown in color that is removed and stored suspended in

  
 <EMI ID = 139.1>

  
This floats spontaneously on the surface of aqueous suspensions, but it can only be dispersed somewhat less easily than polymer complexes, and it tends to force large aggregates. 1 ml of the suspension, titrated as in Example 1, exhibits an activity of

  
 <EMI ID = 140.1>

  
 <EMI ID = 141.1>

  
EXAMPLE 6

  
 <EMI ID = 142.1>

  
(TFODHDG-1)

  
Trypsin (50 mg, no salt) is dissolved in 0.1 M sodium phosphate buffer solution at pH 6.0.

  
 <EMI ID = 143.1> <EMI ID = 144.1>

  
 <EMI ID = 145.1>

  
centrifugation is carried out at 20,000 g / 4 [deg.] C / 30 min. The pellet is resuspended in 20 ml of water and filtered using Whatman IPS phase separation papers. The residue is resuspended in HCl <1> 0- <3> M (20 mL) and filtered again. Resuspension (] here 10-3M) and centrifugation (10,000 g / 14 [deg.] C / 20 min) are then carried out twice, which gives 2.65 g of a brown gel. This material can be made

  
 <EMI ID = 146.1>

  
tee of the complex is shown in Fig. 1, on which the incubation times in minutes have been plotted on the abscissa and the activities on the ordinate. The titration process is as follows:

  
 <EMI ID = 147.1>

  
resulting in a 2 x 10-314 solution of the chromogenic substrate. Gold

  
 <EMI ID = 148.1>

  
ge at 22 [deg.] 0. The enzyme sample is added and 5 ml aliquots of the mixture are taken at intervals. When allowed to stand briefly or when subjected to gentle centrifugation, the heptane layer separates (causing

  
 <EMI ID = 149.1>

  
 <EMI ID = 150.1>

  
ment of p-nitroanilide. Heptane droplets give a significant "zero" absorption value. In some cases,

  
 <EMI ID = 151.1>

  
sorption. After reading, both the hepta layer and the aqueous layer are returned to the stirred reaction mixture. The activity

  
 <EMI ID = 152.1>

  
initial amidolytic activity.

  
EXAMPLE 7

  
 <EMI ID = 153.1>

  
Candida Cylindracea lipase is dissolved

  
 <EMI ID = 154.1>

  
 <EMI ID = 155.1>

  
 <EMI ID = 156.1> <EMI ID = 157.1>

  
proud again the precipitate in the water. Homogenization of 10 g of the resulting gel is carried out in sodium phosphate

  
 <EMI ID = 158.1>

  
glutaraldehyde. The pH is adjusted to 6.2 and the mixture is stirred at 4 [deg.] C for 16 hours. The suspension is centrifuged

  
 <EMI ID = 159.1>

  
 <EMI ID = 160.1>

  
 <EMI ID = 161.1>

  
suspends the final dumpling (wet weight 8.4 g) in

  
 <EMI ID = 162.1>

  
be made floating with both n-decanol and n-heptane. The activity of the complex is shown in Fig. 2, on which the incubation times in minutes on the abscissa and the activities on the ordinate have been plotted here again. The titration process is as follows:

  
P-nitrophenyl laurate is stirred rapidly
(10- <2> M in n-heptane), 5 ml, with sodium phosphate

  
 <EMI ID = 163.1>

  
However, non-enzymatic ester hydrolysis cannot be detected in an hour or more. After addition of the enzyme, 5 ml aliquots are taken at certain intervals and

  
 <EMI ID = 164.1>

  
 <EMI ID = 165.1>

  
minutes after taking the first aliquot. The aqueous layers and heptane are then returned to the stirred mixture. (Since the substrate is in the nonaqueous phase during this experiment, the rate of stirring, ie the degree of dispersion, should be kept constant throughout the experiment). About 1% of the initial lipase activity is present in the complex.

EXAMPLE

  
 <EMI ID = 166.1>

  
nohexane (0.2 g) suspended in methanol (10 ml) and

  
 <EMI ID = 167.1>

  
 <EMI ID = 168.1>

  
 <EMI ID = 169.1>

  
The mixture is stirred for 16 hours at room temperature.

  
1 <EMI ID = 170.1>

  
Room temperature. The foam and the floating fraction are discarded, and the pellet is resuspended in a solution.

  
 <EMI ID = 171.1>

  
centrifuge again as before. The gel-shaped pellet (80 g) is stored at 4 [deg.] C.

  
 <EMI ID = 172.1>

  
A penicillin acylase solution is adjusted

  
 <EMI ID = 173.1>

  
 <EMI ID = 174.1>

  
the water). The mixture is stirred at room temperature and a suspension of GAN149HDOD-1 (10 g) in water buffer is added.

  
 <EMI ID = 175.1>

  
to 6.2 and stirring is continued at room temperature for 2 hours. The suspension is centrifuged for 1 hour at
20,000 g at 4 [deg.] C and keep the meatball (14 g). This pellet is resuspended in sodium phosphate buffer

  
 <EMI ID = 176.1>

  
ve the final dumpling at 4 [deg.] C.

  
 <EMI ID = 177.1>

  
5% cillin G, pH 7.8, 22 [deg.] C)

  
 <EMI ID = 178.1>

  
n-decane.

  
 <EMI ID = 179.1>
(a) GAN149HDOD-2

  
N-Octadecylamine (5g) is dissolved in ethanol (50ml) and added to a phosphate buffer solution.

  
 <EMI ID = 180.1>

  
 <EMI ID = 181.1>

  
 <EMI ID = 182.1>

  
 <EMI ID = 183.1>

  
for 6 hours. The pH is lowered to 3.0 with concentrated HCl and the suspension centrifuged at 12,000 xg for 1 hour at room temperature. Discard the supernatant fraction and resuspend the pellet in water (550 ml) and centrifuge again, which gives a final white pellet.

  
 <EMI ID = 184.1> <EMI ID = 185.1>

  
A solution of penicillin acylase is mixed

  
 <EMI ID = 186.1>

  
HDOD-2 (15 g), sodium phosphate buffer solution at pH

  
 <EMI ID = 187.1>

  
 <EMI ID = 188.1>

  
 <EMI ID = 189.1>

  
trifuge again. These operations are then repeated, which gives a final ball weighing 11.2 g, which we keep

  
 <EMI ID = 190.1>. Specific activity: 1.8 x 10-5 mole of 6-APA min
(Conditions as before).

  
 <EMI ID = 191.1>

  
decanol or n-decane.

EXAMPLE 10

  
Trypsin (n-decylamino-Gantrez AN119) (TGAN119D)

  
 <EMI ID = 192.1>

  
methylformamide (DMF) (5 mL) and stirred vigorously. N-decylamine (0.3 g) in DMF (1 ml) is added and the solution becomes more viscous. After stirring for 10 minutes, the solution is added dropwise to a solution of bovine trypsin (300 ml) in 0.1M sodium phosphate buffer solution pH 8.0 (60 ml), at 22. [deg.] C. The pH is maintained with 2N sodium hydroxide and when no other variation occurs, sodium chloride (4 g) is added. The resulting solution is poured into a sodium phosphate buffer solution.

  
 <EMI ID = 193.1>

  
ammonium fate (50 g) and the mixture is stirred at 4 [deg.] C for

  
 <EMI ID = 194.1>

  
re-homogenize the gel-like pellet in 0.1 M sodium phosphate buffer solution at

  
 <EMI ID = 195.1>

  
two more rounds of homogenization / centrifugation, resulting in a final loose gel (7.5 g). The preparation is titled by

  
 <EMI ID = 196.1>

  
Nitroanilide (BANA), 2mM, in 0.1M veronal buffer at pH

  
 <EMI ID = 197.1> 0.001 per minute at 400 nm. Since the gel causes appreciable scattering of light, it is necessary to <EMI ID = 198.1>

  
attempted...

  
Gel activity: 168 units / mg wet weight, <EMI ID = 199.1>

  
 <EMI ID = 200.1>

  
The immobilization of the enzyme complex on the solvent droplets is illustrated by the following experiment:

  
 <EMI ID = 201.1>

  
in a small tissue mill with n-decane (2.5 ml) and a

  
 <EMI ID = 202.1>
4.000 revolutions / minute and 0 [deg.] C, for 5 minutes. The milky slurry is separated to form an upper layer comprising tightly packed decane droplets and a <EMI ID = 203.1> layer

  
layers using, the same spectrophotometric method. Again, the cuvette is shaken at regular intervals when the top layer is titrated and the average rate of increase in optical density is measured.

  
Phase activity, upper: 2,280 units / ml

  
Activity of the lower phase: 57 units / ml.

  
Since the apparent activity of the upper phase is probably significantly lower than the actual activity as a result of the method used, this result indicates that at

  
 <EMI ID = 204.1>

  
your solvent.

  
 <EMI ID = 205.1>

  
Penicillin acylase (n-octadecylamino Gantrez AN119)
(PAGAN1190D).

  
Gantrez AN119 (0.5 g) is dissolved in the di-

  
 <EMI ID = 206.1>

  
also dissolves octadecylamine (0.28 g) in the

  
 <EMI ID = 207.1>

  
Gantrez solution while it is still hot. The mixture is stirred and pyridine (0.1 ml) is added. After cooling to room temperature for 1 hour, the whole acylase solution of penicillin (175 mg) in water (30 ml) at pH 7.0 is added and homogenized briefly.

  
 <EMI ID = 208.1>

  
 <EMI ID = 209.1>

  
 <EMI ID = 210.1> <EMI ID = 211.1>

  
 <EMI ID = 212.1>

  
0.1 M sodium phosphate buffer pH 7.0 (20 ml).

  
 <EMI ID = 213.1>

  
 <EMI ID = 214.1>

  
 <EMI ID = 215.1>

  
 <EMI ID = 216.1>

  
in the complex. The above suspension (4.0 ml) is mixed with 0.02M sodium phosphate buffer (20 ml) and ndecanol (5 ml) and homogenization is carried out at 4000 rpm.

  
 <EMI ID = 217.1>

  
at 100 g at room temperature for 20 minutes and separated into an upper organic emulsion and a lower aqueous layer. The lower layer is retained and the upper layer is resuspended in 0.02M sodium phosphate buffer (20 mL) and centrifuged again. The lower layer

  
 <EMI ID = 218.1>

  
 <EMI ID = 219.1>

  
x 10-5 mole / minute. The relationship between specific activities
(on a volumetric basis) is equal to 21.6 (above / below). After determining the activity by the above method, the organic layer is recycled by gentle centrifugation and removal of the aqueous layer as indicated in the table.
5.

  
TABLE 5

  
 <EMI ID = 220.1>

  

 <EMI ID = 221.1>


  
 <EMI ID = 222.1>

  
 <EMI ID = 223.1>

  
(0.25 g) in dimethylformamide (1 ml) and add at <EMI ID = 224.1>

  
 <EMI ID = 225.1>

  
suspension-for 16 hours at 4 [deg.] C. Am- sulfate is added

  
 <EMI ID = 226.1>

  
 <EMI ID = 227.1>

  
After dissolution of ammonium sulphate, a cloudy suspension forms. This material is centrifuged at 25,000 g / 0 [deg.] C / 1 hour. The pellet is resuspended in a sulphate solution.

  
 <EMI ID = 228.1>

  
centrifugal again. The final pellet weighs 1.36 g and is dissolved again in water (10 ml), resulting in a viscous solution. The activity of the dumpling (titration as above

  
 <EMI ID = 229.1>

  
represents an activity recovery of 80%. the immobilization) of the water-soluble complex on the droplets of n-decanol is illustrated by the experiment below:

  
1 ml of the above solution is dissolved in 0.02M sodium phosphate buffer (20 ml) and homogenized.

  
 <EMI ID = 230.1>

  
 <EMI ID = 231.1>

  
lion at 300 g for 10 minutes. The top layer has an ac-

  
 <EMI ID = 232.1>

  
of successive recycling is shown in table 6.

  
In a second experiment, homogenization of an enzyme complex solution is carried out under the same conditions with n-decanol (2.5 ml) and 0.02M sodium phosphate buffer (10 ml). ). After centrifugation and washing with
20 ml of the same buffer, the top layer has an activity of

  
 <EMI ID = 233.1>

  
 <EMI ID = 234.1>

  
specific (upper / lower, volumetric base) is 3 'approximately 7.5.

  
TABLE 6

  
initial activity as a percentage of phase PAGAN119DD-n-deca-

  
 <EMI ID = 235.1>

  

 <EMI ID = 236.1>
 

EXAMPLE 13

  
Trypsin (n-octadecylamino-Gantrez AN 149 hydrolyzed) TGAN
14-9DCMC.

  
 <EMI ID = 237.1>

  
 <EMI ID = 238.1>

  
 <EMI ID = 239.1>

  
1-cyclohexyl- metha-p-toluenesulfonate is added.

  
 <EMI ID = 240.1>

  
 <EMI ID = 241.1>

  
biante, bovine trypsin (75 mg in buffer

  
 <EMI ID = 242.1>

  
mixture at 5.9 and this pH is maintained for the next hour with 2N sodium hydroxide. After 3 hours at room temperature, the gel is centrifuged 25,000 g / 30 min / 4 [deg.] C. The pellet is resuspended in 0.1M sodium phosphate buffer at pH.

  
 <EMI ID = 243.1>

  
final with the aforementioned buffer (15 ml) and n-decane

  
(10 ml) and briefly homogenized. This process gives incomplete dispersion and the mixture is treated at 20 KH in successive charges of 5 over 5 minutes, with intermittent cooling in ice. The final emulsion is centrifuged at
100 g / 15 min. The top layer was washed three times with the phosphate buffer (25 ml). The final volume of the top layer is 11ml and the layer has activity (titrated as

  
 <EMI ID = 244.1>

  
 <EMI ID = 245.1>

  
 <EMI ID = 246.1>
(a) n-Octadecylamino-Gantrez AN 119-hydrazide hydrolyzed
(GAN1190DH) <EMI ID = 247.1> add a solution of n-octadecylamïne (2.8 g) in dimethylformamide (30 ml) hot (80 [deg.] C) and the mixture is kept at

  
 <EMI ID = 248.1>

  
ambient temperature, the solution is added to a hydrate mixture

  
 <EMI ID = 249.1>

  
ambient erasure. A slightly brown precipitate forms and

  
 <EMI ID = 250.1>

  
This resulted in a green soapy solution and after stirring - <EMI ID = 251.1> <EMI ID = 252.1>

  
flocculation and the suspension is centrifuged at 10,000 g / 30 min /

  
 <EMI ID = 253.1>

  
stirred for 16 hours at 4 [deg.] C, then centrifuged as before, which gives a blue-gray pellet (wet weight

  
39 g).

  
(b) Penicillin acylase (n-octadecylamino-Gantrez AN 119) - <EMI ID = 254.1>

  
 <EMI ID = 255.1>

  
 <EMI ID = 256.1>

  
a solution of sodium nitrite (0.5 g) in water (5 ml) and the mixture is stirred at 0 [deg.] C for 10 min, then centrifuged at
25,000 g / 30mn / 0 [deg.] C and the pellet is resuspended in a

  
 <EMI ID = 257.1>

  
add this suspension to a solution of penicillin acylase (100 ml containing 55 mg of protein) and adjust to pH

  
 <EMI ID = 258.1>

  
 <EMI ID = 259.1>

  
 <EMI ID = 260.1>

  
 <EMI ID = 261.1>

  
that (titration as above): 5.2 x 10-5 mol / min / g. Activity recovery: 32.5%.

  
A suspension of the above complex is mixed in 0.1M sodium phosphate buffer at pH 7.0 (0.29 g / ml, 2.5 ml) with 0.02M sodium phosphate buffer at pH?, 8 (20 ml) and

  
 <EMI ID = 262.1>

  
then centrifuged at 5,000 revolutions / minute (100 g, 10 min), which gives two layers. The lower layer has full activity

  
 <EMI ID = 263.1>

  
 <EMI ID = 264.1>

  
on / min. The ratio between specific activities (upper / lower, volumetric basis) is approximately 30. The results of recycling with the upper phase are shown in Table 7.

  
TABLE 7

  
 <EMI ID = 265.1>

  
iecanol during successive cycles of flotation and reuse.

  

 <EMI ID = 266.1>


  
EXAMPLE 15

  
Enzymatic dissociation of benzyl-penicillin to produce 6-aminopenicillanic acid.

  
1.0 g of Gantrez AN119 is dissolved in 10 ml of dimethylformamide and 0.56 g of octadecylamine is added. The whole is stirred overnight at room temperature.

  
48 ml of a partially purified preparation of penicillin acylase to pH 9.0 are adjusted with 1M sodium carbonate solution. The Gantrez resin solution is then added in two aliquots with homogenization and pH adjustment between the two additions. The mixture is then stirred for 3 hours, the pH being maintained at 990 by the addition of a 1M sodium hydroxide solution.

  
The enzyme-resin complex is then recovered by cen. trifugation, resuspended in 120 ml of a buffer

  
 <EMI ID = 267.1>

  
The enzyme-resin is recovered by centrifugation and the washing is repeated.

  
20 g of the enzyme gel is homogenized with 80 ml of n-decane and 20 ml of 0.02M phosphate buffer at

  
 <EMI ID = 268.1>

  
250 ml of distilled water. We heat the whole to 370C, we adjust

  
 <EMI ID = 269.1>

  
(21.8 g). The mixture is stirred for 5 hours and the pH is maintained at 7.8 by the addition of a 4% (w / v) sodium hydroxide solution. At the end of the reaction, the mixture is poured into a funnel

  
separator and the enzyme is allowed to separate from the aqueous phase. This took 30 minutes. The aqueous layer is then removed

  
 <EMI ID = 270.1>

  
boasts:

  
Concentrate the liquor to a quarter of its volume,

  
 <EMI ID = 271.1>

  
ketone while shaking. The pH is reduced to 4.3 by adding concentrated nitric acid, after which the 6-amino acid is precipitated <EMI ID = 272.1> <EMI ID = 273.1> <EMI ID = 274.1>

  
Changes can be made to the modes of

  
implementation described, in the field of technical equivalences, without departing from the invention.

  
 <EMI ID = 275.1>

  
1. - Enzyme preparation, characterized in that it contains an enzyme attached to non-polar groups in sufficient quantity so that, when the preparation is brought into contact in aqueous media with an inert liquid immiscible with water, said preparation combines with the inert liquid immiscible with water and can then separate from the aqueous medium.


    

Claims (1)

2. - Enzyme preparation according to claim 1, characterized in that the enzyme is attached to a polymer support. 3. - Enzyme preparation according to claim 2, characterized in.:.ce that the polymer support is a polysaccharide-based material. <EMI ID = 276.1> characterized in that the polymeric support is dextran or Sepharose. 5. - Enzyme preparation according to claim 2, characterized in that the polymer support is a copolymer of an oligosaccharide and of epichlorohydrin. 6. - Enzyme preparation according to claim 5, characterized in that the polymer support is a copolymer of sucrose and epichlorohydrin. 7. - Enzyme preparation according to claim 2, characterized in that the polymer support comprises anhydride groups attached to the backbone of the polymer. <EMI ID = 277.1> characterized in that the polymeric support is a copolymer of methyl vinyl ether and maleic anhydride. <EMI ID = 278.1> claims 1 to 8, characterized in that the enzyme is amylase, asparaginasé, neutral or alkaline protease, chymotrypsin, cellulase, dextranase, lipase, oxynitrilase , pepsin, penicillin acylase or trypsin. 10. - Enzyme preparation according to claim 9, characterized in that the enzyme is penicillin acylase. 11. - Preparation of enzyme according to any one of <EMI ID = 279.1> <EMI ID = 280.1> [deg.] 6- [deg.] 20 'cycloalkyl, substituted aryl or aralkyl. 12. The enzyme preparation according to claim 11, <EMI ID = 281.1> <EMI ID = 282.1> <EMI ID = 283.1> n-decyl, n-dodecyl or n-octadecyl. 14. - Enzyme preparation according to any one of <EMI ID = 284.1> binds the enzyme to the polymer support; and (or) the enzyme to the non-polar group; and (or) the polymeric support to the non-polar group. 15. - Enzyme preparation according to claim 14, characterized in that the bridging group is derived from an alpha, <EMI ID = 285.1> luble in water. 16. - Enzyme preparation according to any one of claims 2 to 13, characterized in that the enzyme is linked directly to the polymer support. 17. - Process for the production of an enzyme preparation according to any one of claims 1 to 16, characterized in that one.met in contact with a compound of formula (I): <EMI ID = 286.1> wherein R is a nonpolar group and X is a functional group with an enzyme attached to an active group capable of reacting with the group X. <EMI ID = 287.1> in that X is an amino group. 19. - The method of claim 17 or 18, characterized in that X represents a polymeric material bearing at least one functional group. 20. - Process according to any one of the claims <EMI ID = 288.1> lution or, in aqueous suspension, 21. - Method according to claim 20, characterized in that it is carried out at a pH between 4 and 9. 22. - The method of claim 19, characterized in that reacts a polymeric material comprising anhydride groups on the backbone of the polymer with a <EMI ID = 289.1> aqueous aprotic formula wherein R is a nonpolar group, and the resulting modified polymer is subsequently attached to an enzyme. <EMI ID = 290.1> in that the non-aqueous aprotic solvent is N, N-dimethylformamide. 24. - Process for carrying out an enzymatic reaction, characterized in that one brings into contact, in an aqueous medium, an enzyme preparation according to any one of claims 1 to 16 with a substrate of the enzyme, then one separates the enzyme preparation from the aqueous reaction mixture by contact with a water-immiscible liquid, the reaction product is recovered from the aqueous phase, and optionally the separated enzyme preparation is reused by contact with a water phase. new amount of substrate. 25. - Method according to claim 24, characterized <EMI ID = 291.1> aliphatic hydrocarbon, a higher aliphatic ester or a <EMI ID = 292.1> 26. - Process according to claim 24 or 25, characterized in that the contacting between the enzyme preparation and the water-immiscible liquid is provided by stirring. <EMI ID = 293.1> in that the contacting between the enzyme and the water immiscible liquid is ensured before carrying out the enzymatic reaction. 28. - Process according to any one of claims 24 to 2 ?, characterized in that the enzyme preparation is based on penicillin acylase. 29. - Process according to claim 28, characterized <EMI ID = 294.1> 30. - Process for the preparation of 6-aminopenicillanic acid from 'benzylpenicillin, characterized in that <EMI ID = 295.1> aqueous, with an inert water-immiscible liquid and an enzyme preparation comprising penicillin acylase attached to non-polar groups in sufficient quantity for the preparation to be combined with the inert water-immiscible liquid , then the aqueous and water-immiscible layers are allowed to separate, thereby separating the enzyme preparation from the reaction mixture. aqueous as a result of its association with. <EMI ID = 296.1> aminopenicillanic acid from the aqueous phase, and optionally: The separate enzyme preparation is reused by contacting with a further quantity of benzylpenicillin. <EMI ID = 297.1> <EMI ID = 298.1>