FI59265C - FOERFARANDE FOER FRAMSTAELLNING AV 6-AMINOPENICILLANSYRA - Google Patents

Description

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Pttant- och iwgirtMvtyralMn 'Amaktn uthgd och utUknfun puUkmd 31.03.8l (32) (33) (31) · «/« TuoJkeu * —B «ftrd prloritat 13 · 08.7 * + 26.OU.75 United Kingdom-Storl» ritannien (GB) 35556M, 17W75 (71) Beecham Group Limited, Beecham House, Great West Road, Brentford,

Middlesex, England-England (GB) (72) Richard Anthony Godwin Smith, Wellington, Surrey, England-England (GB) (7 * 0 Berggren Oy Ab (5 ^) Method for the preparation of β-aminopenicillanic acid - For the preparation of 6-aminopenicillanic acid (62) Separated from application 752275 - Avdelad frän ansokan 752275

The present invention relates to a process for the preparation of 6-aminopenicillanic acid and to a possible re-use of the enzyme used in the reaction.

Enzymatically catalyzed reactions are important steps in some chemical processes, e.g. benzylpenicillin or phenoxymethylpenicillic acid, using penicillin acylase.

Enzymatic reactions are generally performed with the enzyme dissolved in a medium containing a substrate. (In this specification, the term "substrate" refers to the substance that is modified by the enzyme to obtain the desired end product). Because the enzyme is dissolved in the reaction medium, it is often very difficult to separate the enzyme from the substrate or final product at the end of the reaction. Once 2,59265 products have been separated from the reaction mixture, this separation method generally causes inactivation of the enzyme. This, of course, means that the enzyme cannot be reused.

To avoid this separation difficulty and to provide an enzyme system that can be reused, it is known to attach the enzyme to an insoluble solid support either by adsorption (see e.g. British Patent Publication No. 1,264,147) or by covalent bonds either directly or indirectly by bridging groups. (See, e.g., British Patent Publication Nos. 1,349,498, 1,387,460 and 1,365,886). However, such insoluble preparations have a number of disadvantages. First, because they are solids, they are susceptible to mechanical damage and potentially decompose. Second, the attachment of the enzyme to the surface of the solid support and thus the specific activity of the preparation is often low, and third, the entry of the substrate into the active site of the enzyme is often prevented. Attempts to improve the specific activity of such insoluble enzyme polymer preparations by increasing the surface area of the solid require a reduction in the particle size of the solid preparation which makes handling and especially separation by filtration more difficult, and increasing the internal surface area by making the particles more porous.

Also disclosed are water-soluble enzyme-polymer complexes to be prepared (see, e.g., British Patent Publication No. 1,284,925 and Finnish Patent No. 50882) in which the enzyme is bound either directly or indirectly by a bridging group to a water-soluble polymeric carrier. These enzyme-polymer complexes can be recovered from the aqueous reaction medium by ultrafiltration. However, ultrafiltration is a difficult and expensive method, especially on a large scale, and as a result is ill-suited for industrial use.

The utilization of hydrophobic groups in chromatography, e.g. in protein separation, is discussed by Er-el et al. in Biochem. Biophys. Res. Comm., 49 (1972) p. 383 and hofstree, ibid 50 (1973) p. 751 and Bartling et al. In Nature, 243 (1973) p. 342.

3,59265

Er-el et al. and Hofstree study the purification of enzymes using hydrophobic (affinity) chromatography in which the enzyme is attached to a support (sepharose) via alkyl chains. The alkyl chains of the carrier thus lose their hydrophobicity when bound to the enzyme. This binding does not involve a chemical reaction but is based solely on the hydrophobic effect. Bartling et al. describe the binding of an enzyme to a copolymer of styrene and 4-vinylbenzoic acid. Attachment of the enzyme to the polymer occurs via an amide bond formed by the amino group of the enzyme and the carboxyl group of the polymer. The coupling reaction is performed in a water-soluble polar, organic solvent, dimethylformamide.

Another method for separating enzymes from reaction media is microencapsulation (see, e.g., "Immobilized Enzymes", O.R. Zaborsky, CRC Press 1973, pages 93-101). This method is based on the formation of a physical barrier or semipermeable film around a water droplet containing the enzyme. The barrier is sufficiently permeable to allow the substrate to enter the microcapsule so that it can react with the enzyme and to release the product formed, while instead allowing the enzyme to escape.

Permanent microcapsules can be prepared, for example, by forming a semipermeable film of a polymeric material around a water droplet containing the enzyme. The remaining microcapsules are dispersed in an aqueous medium containing a substrate and then removed by filtration.

Volatile microcapsules can be prepared by enclosing an enzyme-containing water droplet through an organic liquid inside a surfactant film. The surfactant stabilizes the liquid film, which surfactant accumulates at the interface between the entrapped water droplet and the organic liquid. In use, an organic liquid dispersed with enzyme-containing water droplets is in turn dispersed in an aqueous medium to form droplets of organic liquid, which in turn contain droplets of the enzyme-containing aqueous medium. The liquid is selected so that the substrate can penetrate the membrane and react with the entrapped enzyme, after which any product formed can be removed while the removal of the enzyme is prevented. The organic droplets 4,59265 are allowed to coalesce and then the enzyme can be separated by removing the organic phase that carries the entrapped enzyme with it.

It has now been found that certain enzymes can be attached to non-polar groups to make a preparation separable from an aqueous medium based on its affinity for water-immiscible liquids.

According to the present invention there is provided a process for the preparation of 6-amino-penicillanic acid comprising contacting penicillin with an enzyme preparation in an aqueous medium consisting of penicillin acylase bound directly or alternatively to a dialdehyde containing 3 to 6 carbon atoms and a diamine containing 1 to 6 carbon atoms. the copolymer of epichlorohydrin or methyl vinyl ether and maleic anhydride, the recovery of 6-aminopenicillanic acid from an aqueous medium and possibly the reuse of the enzyme by contacting it with a freshly bound and unsubstituted polymer, and the invention is essentially characterized in that the enzyme when contacted with an inert water-immiscible solution, such as an alkane or alcohol containing 7 to 10 carbon atoms, to an insoluble solution and is separable from an aqueous medium containing 6-aminopenicillanic acid.

In this context, "inert, water-immiscible liquid" means one that does not react with the enzyme preparation to substantially degrade the activity of the enzyme.

The term "transferred" as used above means any interaction between the non-polar groups of an enzyme preparation and the molecules of a water-immiscible liquid that is sufficiently stable to effect separation of the enzyme preparation from the aqueous medium.

5,59265

When some enzymes are used, the attachment of a certain number of non-polar groups directly to the enzyme molecule causes a deformation and a change in the activity of the enzyme. Due to this and in a few cases the structure of the enzyme, it is not always possible to attach enough non-polar groups to the enzyme to make the preparation separable from the aqueous medium without adversely reducing the activity of the enzyme.

A suitable class of water-soluble copolymers are methyl vinyl ether / maleic anhydride copolymers sold under the trademark "Gantrez AN" (manufactured by GAF Corporation, New York, USA).

The enzyme may be attached directly to a polymer containing a non-polar group, or alternatively, bridge-forming groups to which the enzyme may be attached may be attached to the polymeric carrier. The bridging group may be daldehyde having 3 to 6 carbon atoms or di-Amine having 1 to 6 carbon atoms. The amino or aldehyde function of the bridging group is attached to the polymer and the other is free to attach to the enzyme. The use of such bifunctional bridging groups also provides some degree of crosslinking between enzyme and polymer units. Bridging groups can be used between the enzyme and the carrier, between the enzyme and the non-polar group (in the presence of the carrier), or between the carrier and the non-polar group.

The amount of these non-polar groups attached to the compositions of the invention depends on several factors. Attachment of non-polar groups to the polymer and / or enzyme provides a hydrophobic environment in the vicinity of the enzyme. When a few enzymes are used, too high a degree of hydrophobicity results in inactivation of the enzyme due to a concomitant structural change.

A balance is required between the attachment of the minimum number of non-polar groups necessary to allow separation of the aqueous medium and the maximum number allowed by the stability of the enzyme. This balance is determined on the basis of standard tests and corrected errors. The factors which must be used to determine this balance are as follows: 6 59265 (1) Molecular weight of the polymeric carrier.

(2) Molecular weight of the monomer unit.

(3) Percentage of monomer units in the polymer substituted with non-polar groups.

(4) Size of non-polar groups.

(5) Quality of the enzyme.

(6) Degree of polymer substitution by enzyme.

For example, when the carrier is Gantrez AN 119 (a polymer having a molecular weight of 230,000, a molecular weight of 156 monomer units), wherein 1 mole of Gantrez AN 119 is substituted with 1 mole of penicillin acylase, the necessary minimal substitution occurs to achieve efficient separation from the aqueous medium when about 25% the monomer units are substituted with n-decyl groups, and the maximum substitution that retains enzymatic activity occurs when about 50% of the monomer units are substituted with n-octadecyl groups.

*

The enzyme preparation used in the process of the invention can be prepared by contacting a compound of formula (I): R to X (I) wherein R is a non-polar group and X is a functional group with a penicillan acylase attached to an active group capable of reacting with a group. With X.

The group X may be a dialdehyde having 3 to 6 carbon atoms, such as Gly-oxal or glyceraldehyde, or a diamine having 1 to 6 carbon atoms, such as 1,6-hexamethylenediamine or ethylenediamine.

The active group attached to the enzyme and capable of reacting with the group X may be a group present in the enzyme, either originally present or provided by modification, such as a carboxyl, amino, thiol or phenol hydroxy group or anhydride bond, or a polymeric carrier material, containing such an active group, or a reactive coupling group, such as in the case of the above-mentioned group X.

7 59265

The reaction between the compound of formula (I) and the enzyme is most preferably carried out at a substantially neutral pH, suitably in the pH range of 4-9, and in the temperature range of -4 to + 40 ° C depending on the enzyme. Preferably, the pH is about 7.0 and the temperature is in the range of 0-5 ° C.

It is preferred that the support be first modified to add a non-polar group and, if necessary, to add bridging groups before attaching it to the enzyme. In this embodiment, the group X in the above formula (I) means a polymeric material bonded to a non-polar group R and a functional group.

Enzyme / Modified Polymer Preparations can then be prepared by any known method for incorporating enzymes into polymers. Such coupling methods are described, for example, in British Patent No. 1,325,912 and involve the coupling of an enzyme to a polymer using reagents such as halogen cyanides, especially bromocyanide, s-triazines, especially 2-amino-4,6-dichloro-s-triazine, acylazides, diazonium compounds, e.g. 2-hydroxy-3- (p-diazophenyl) propyl ether, organic cyanates such as phenyl cyanate, and carbodiimides. Often, such coupling agents are first reacted with the polymer to form reactive groups, which groups are then reacted with the enzyme. In a few cases, the enzymes may react directly with the polymer, e.g. if it contains or is modified to contain, active groups such as anhydride bonds or acid azide groups.

As a result, it should be noted that the reaction method and conditions used to prepare the modified polymers and bind them to the enzymes vary depending on the nature of the enzyme and the polymeric material.

For most enzyme / polymer preparations, a suitable method is shown in Scheme I. The first step is to form non-polar groups in the polymer (a) preferably via a primary or secondary amine R * NH 2 or R 2 * NH, and (b) bridging groups such as a, The incorporation of,-diamines into the enzyme via dialdehyde groups. These reactions are conveniently carried out in aqueous solution or suspension at an alkaline pH (suitably pH 8-12) and at room temperature. The enzyme containing dialdehyde groups can then be coupled to the modified polymer in an aqueous medium at a substantially neutral pH.

<· 59265 8

GRAPH I

---- -P_ nh. R

Polymer -_y Polymer y_ --RNH9 or IUNH -k.

+2 2 ^ NH (CH2) xNK2 nh2 (ch2) xnh2

. CH0 (CHo) CHO Enzyme “N = CH (CH2) CHO

Enzyme__L2L___) y y_

___ NH. R

Polymer

--- J— NH. (CH?) -N

-Ί V

Enzyme _ N = CH- (CH2) V-CH

1 / wherein R is a non-polar group as defined above; ΝΗ2 (0Η2) χΝΗ2 is an α, ω-diamine; and CHO (CH2) yCHO is a dialdehyde.

It is often necessary to activate the groups in the polymer, preferably with cyanogen bromide, before coupling with non-polar groups is possible.

On the other hand some polymers are such groups that can be use to provide a connection with a non-polar groups and / or enzyme, if desired, without sillanmuodostusryh-groups. The enzyme preparations according to the invention prepared in this way do not have the cross-linking caused by the use of bridging groups and often have the above-mentioned advantageous properties.

For example, in polymers based on a maleic anhydride monomer, such as Gantrez materials, backbone anhydride groups can be used to attach both non-polar groups and enzymes. The polymer is suitably modified by first attaching non-polar groups to it, and the remaining anhide groups in the polymer are then used directly to effect coupling with the enzyme. This method is shown in Scheme II: 959265

t CHART II

OCH, I 3 CHp-CH-CH-CH-- + m RNh2 -ycc ΑΛ 0 0 0 _ _n (j) CH3 och5 --CH2-CH-CH-CH ----- CHo-CH-CH-CH - M li C0, HC = 0 pn nhr NHK 000 - —_ __n-m x enz-nh2 och, och, och, I> I 3 I 3? --CHp-CH-CH-CH --- CHP-CH-CH-CH-CH --- Chg-CFF-CH

h, and „„ 1 1 M II

COpH C = 0 CO2H <j? = 0 CO2M COpH

NHR _ NH

L. et al L enz_x [_ n- (m + x) wherein R is the aforementioned non-polar group; ENZ-NHp is an enzyme with an amino group.

Gantrez polymers, e.g. Gantrez AM 119 and Gantrez AN 1 9 9, have the further advantage that they are soluble without reacting with certain aprotic, non-aqueous solvents, such as dimethylformamide, which are also good solvents for the amines RNHp and RpNH. A homogeneous reaction mixture is possible, and as a result, such solvents provide preferred reaction media for attaching non-polar groups to these polymers in the preparation of the enzyme preparations of the invention.

Thus, the enzyme-polymer preparations used in the process of the invention can be prepared by reacting a polymeric material having anhydride groups in the backbone with an amine of the formula RNH2 or R2NH in an aprotic, non-aqueous, polar solvent, wherein R is non-aqueous. polar group and then attaching the modified polymer to the enzyme.

It is preferred that the amine be reacted with the polymer in the presence of a tertiary base such as pyridine. The term "tertiary base" as used herein means a tertiary amine or heterocyclic aromatic amine.

The non-aqueous aprotic solvent is preferably N, N-dimethylformamide or the tertiary base itself. The temperature at which the reaction is carried out depends on the nature of the polymeric support and the amine. However, the reaction is generally carried out at a temperature between room temperature and 100 ° C.

The modified polymeric carrier can be separated from the medium in which it is prepared by precipitation as a less polar solvent, preferably diethyl ether, or by adding the solution to water to cause swelling and hydrolysis of the anhydride and separating the hydrolyzed modified polymer from the aqueous medium using water-immiscible.

The enzyme is then attached to the above-mentioned separated, modified support by contacting the modified support with the enzyme in an aqueous solution, optionally using a coupling agent, e.g., a substituted carbodiimide. Alternatively, a non-hydrolyzed reaction mixture or a non-hydrolyzed modified polymer dissolved in a small volume of a polar, non-aqueous solvent can be added directly to an aqueous solution of the enzyme.

Alternatively, the coupling can be accomplished by first providing the aforementioned non-hydrolyzed support and treating it with hydrazine hydrate in dimethylformamide solution. This treated support, which is then hydrolyzed and separated from the aqueous medium by centrifugation, can be coupled with the enzyme in an aqueous medium using sodium nitrite as an activator.

11 59265

Alkanes or alcohols having 7 to 10 carbon atoms are used to separate the enzyme preparation from the aqueous medium. Suitable alkanes include, for example, heptane, octane, nonane, decane, hexadecane, and suitable alcohols include C 1 -C 4 aliphatic alcohols such as n-decanol.

After contact with such a liquid, the enzyme preparation forms a surface layer in the contact area between the two. If it is desired to maximize this area of agitation, the contacting step usually comprises mixing, e.g. with a mixing device or by shaking, to break up the water-immiscible liquid into small droplets. Each drop is then surrounded by an enzyme preparation which adheres to its surface.

How the enzyme preparation relates to the water-immiscible liquid varies depending on the polymeric material and / or the non-polar groups contained in the enzyme preparation. For example, when enzyme / polymer preparations with significant crosslinking are used, solid aggregates are formed and these particles adhere to the droplets of the water-immiscible liquid. However, when non-crosslinked preparations such as "Gantrez" carriers (and non-polymer preparations) are used, there is generally no transverse attachment of solids to the droplets and a simple layer of enzyme preparation is formed on the droplets. This is very preferred when the enzyme reaction is carried out by first contacting the enzyme preparation with a water-immiscible liquid to provide such a system.

Whatever the nature of the attachment, contact between the enzyme preparation and a water-immiscible liquid (usually in the form of droplets) allows the enzyme preparation to be separated from the aqueous medium. The density of the droplets coated with the enzyme preparation is approximately the same as the density of the water-immiscible liquid. When the droplets are allowed to stand, they either rise to the surface of the aqueous medium (in the case of liquids that are lighter than water) or settle to the bottom (in the case of heavier liquids). Thus, a separate layer is provided containing a water-immiscible liquid together with an enzyme preparation which can be easily recovered from the aqueous medium.

In the case of liquids which do not float on the surface of the aqueous medium or settle to the bottom quickly enough, other separation methods can be used, such as centrifugation or passing the mixture through a water-repellent filter, after which the aqueous medium passes through it and a water-immiscible liquid (together with enzyme preparation). ) remains on the filter and is separated from it. In the process of the invention, 6-aminopenicillanic acid is prepared from penicillin using a preparation based on the enzyme penicillin acylase. The acylase enzyme is preferably obtained from bacteria, such as the Escherichia coli strain, such as that used to cleave benzylpenicillin, or a fungus of the genus Actincmycetes can be used to treat fencinethylpenicillin. Such enzymes are well known. They are used in the preparation of 6-APA in the pH range 6.0-9.0, preferably 7.0-8.5. Since deacylation of penicillin causes the separation of the free acid from the penicillin side chain, it is necessary to maintain the above pH range during the process of the invention by adding, if necessary, an alkali such as sodium or ammonium hydroxide or triethylamine solution.

The continuous enzymatic reaction process shown in the examples can be carried out, for example, in an apparatus as shown in the accompanying drawing 1, which schematically shows a suitable reactor.

As shown in Figure 1, it has a reactor vessel 1 with a mixing device 2, supply lines 3, 4 and 5 for substrate, alkali and enzymease, respectively, and an outlet line 6. The vessel 1 also has a pH measuring device 7 controlled by a valve 8, which is connected to the alkali supply line 4. The discharge line 6 leads through the pump 9 to the separation tank 10 via the inlet line 11. The separation tank has an upper discharge line 12 connected to the reactor vessel supply line 5, a lower discharge line 13 for discharging the reaction product through the pump 14, and a glass sinter filter 15 arranged above the lower discharge line 13. The substrate supply line 3 has a pump 16, and the pumps 14 and 16 are connected so that they operate at the same speed.

During the treatment, the substrate is introduced into the reaction vessel 1 via line 3 in an aqueous medium together with the enzyme preparation according to the invention and the water-immiscible liquid. The mixture is dispersed by means of a mixer 2 to form an emulsion. The tank 1 can be kept at a constant temperature, e.g. by means of a water jacket (not shown) which surrounds the tank. The alkali is added to the emulsion in the tank via line 4, and such an addition rate is controlled by means of a valve 8, which in turn is controlled by a sampling point 7 so that the pH of the reaction mixture is kept within the range required for optimal reaction. During the reaction, the pump 9 operates and the mixture is discharged from the discharge line 6 and further to the separation tank 10 via the line 11. In the tank 10, the water-immiscible liquid lighter than water forms a separate upper layer 17, which also contains the enzyme preparation. The lower aqueous layer 18 containing the reaction product is removed through a filter 15 and then through a line 13 by means of a pump 14. The upper layer 17 containing the 59265 enzyme preparation together with the water-immiscible liquid is allowed to flow through the outlet line 12 and is returned to the reaction vessel 1 via the line 5. After the product has been removed via line 13, more substrate solution is added to the tank 1 via supply line 3 by means of a pump 16. The pumps 14 and 16 are adjusted so that the substrate feed rate is equal to the product discharge rate.

Flow rates, residence time in the reactor vessel, and other reaction parameters depend on the enzyme preparation-substrate system used. Preliminary results indicate that using a 1000 kg enzyme preparation based on penicillin acylase attached to a Gantrez AN 149 support in a 10,000 liter reactor can treat about 2000 kg of penicillin G per day at a concentration of 5% w / v at 22 ° C and pH. 7.8.

The following examples illustrate the preparation and properties of some of the penicillin acylase polymer complexes used in the invention.

The following abbreviations are used in the examples to define polymers and modified polymers, enzyme, linking groups, non-polar groups, and coupling agents.

Enzyme: PA: Penicillin acylase

Carriers: F: Ficoll (copolymer of sucrose and epichlorohydrin, manufactured by Pharmacia AB, Uppsala, Sweden) GAN: Gantrez (copolymer of vinyl methyl ether and maleic anhydride, manufactured by GAF Corporation, New York, USA)

Enzyme / carrier linker: HD: 1,6-diaminohexane

Non-polar groups: D: n-decylamino DD: n-dodecylamino OD: n-octadecylamino 5 5 92 65

Crosslinking Agents and Activating Agents and Activating Groups: CMC: 1-Cyclohexyl-3- (2-morpholinoethyl) carbodiimide methano-p-toluenesulfonate G: Glutaraldehyde A: Hydrazide

The integer after the entry indicates the batch number.

For example, the enzyme preparation of Example 1 is labeled PAFHDDG. The enzyme is then penicillin acylase (PA). The carrier is Ficoll (I · ') with hexamethylenediamine groups (HD) to effect attachment to the enzyme and also decyl groups (D) as non-polar groups. Coupling between the free amino group and the enzyme of the hexamethylenediamine mixture is accomplished using glutaraldehyde (G).

GAN 1U9 HDOD is also a modified carrier prepared from Gantrez 1 ^ 9 modified with 1,6-diaminohexane and octadecylamine, and PAGAN 1 * 19 HDOD-G is such a modified carrier mentioned above to which penicillin acylase is attached to glutaraldehyde. acid.

Example 1 (a) n-Decylamino, (6-aminohexylamino) -Ftcoll (FHDD-1)

Ficoll (5 g) was dissolved in water (300 ml) and adjusted to pH 11.0. Cyanogen bromide (Ig) was added and the pH was maintained at the correct level during the reaction with 2N NaOH. The reaction was complete after 20 minutes at room temperature and n-decyl Arni itu (Ig) and 1,6-diamino-k-cane (Ig). .5 g) dissolved in methanol (10 ml) was then added. The pH was then adjusted to 9.5 with 2N HCl and the mixture was stirred for 16 hours at 1 ° C. The resulting colloidal solution was centrifuged at 22,000 g / l for 1 hour to give a white cake (wet weight 35 g) which was collected and the cloudy liquid above the precipitate was discarded.

(b) Penicillin acylase / n-decylamino, (6-aminohexylamino) -Ficoll / (PAFHDDG-1), FHDD-1 (21.6 g, wet weight) was mixed with 0.1 M sodium phosphate buffer, pH 6.0 ( 20 mL), and adjusted to pH 6.2 with 2N HCl.

A solution of E. coli penicillin acylase (50 ml, 3.10 mg protein, partially purified) was adjusted to a pp of 6.2 at 0 ° C and 16,59265 tert-glutaraldehyde (5 ml, 25% w / v aqueous solution) was added. . After stirring at 0 ° C for 30 min, the FHDD-1 suspension was added. The mixture was stirred at 0 ° C for 3 hours and at * 0 ° 0 * 1 hours and then cooled (~ 20 ° C) for 16 hours. After thawing, the suspension was centrifuged (20,000 g / 4 ° C / 45 min) to give a brown gel cake. This gel was resuspended in 0.1 M sodium phosphate buffer, pH 7.0 (30 mL), and recentrifuged (20 ° C / g / min / min). The liquids above the precipitate were combined and the brown cake (wet weight about 6.3 g) was suspended in 0.1 M sodium phosphate buffer, pH 7.0, and stored at 10 ° C. The cake suspension mixed with at least its own weight of n-decanol floats with decanol on the surface of aqueous solutions, leaving an enzyme-free solution. The enzyme activities of the cake, the solution above the precipitate and the basic enzyme are shown in Table 1. Thus, the% origin-. enzyme activity is recovered virtually entirely in the conjugate. The activity of the complex can be recovered substantially from penicillin solutions by either flotation or conventional centrifugation.

Table 1 Penicillin acylase activity of PAFHDDG-1

Test method: Penicillin G (sodium salt) is dissolved in a suitable concentration in 20 ml of 0.02 M sodium phosphate buffer, pH 7.8, at 37 ° C and an enzyme sample is added. The addition of 0.1 or 0.5 NaOH maintains the desired pH and activity is shown based on the initial rate of preparation of C-aminopenicillanic acid (6-ΛΡΛ). In Control Experiment 1., 2 ml of n-decanol is added to the stirred reaction mixture, and after the order, the suspension is allowed to separate into two layers. The upper layer is removed by suction and reused. In treatment 2, the final suspension (2000 g / 15 min) is centrifuged and the cake is reused.

17 59265,. Initial preparation of 6-APA- M. . ,. ........% (w / v) flow rate moles /

Material Amount of penicillin min _____ G in test x io ~ ^ ·

Basic enzyme 1 ml 5 0 PAPHDDG-1 supernatant 1 ml 5 0 008 PAPHDDG-1 0,5 g 5 1,500 PAPHDDG-1 specific tests Treatment 1. 1. 0,5 g 5 0,860 '2 · °> 5 g 5 0,550 5 · S 5 0,500 Processing 2. 1. 0,5 g 5 1,500 2 · 0,5 g 5 1,210 3 · 0,5 g 5 1,110

Jl · °> 5 g δ 1,030

Example 2 (a)> (6-Aminohexylamino) -Little 1 (FODKD-1) Ficoll (5 g) was dissolved in water (300 ml) and the solution was treated with cyanogen bromide 11a (1 g) at pH 11.0 as described above.

After 20 minutes, the solution was adjusted to pH 10.0 with 2N HCl and a solution of n-octadecylamine (5 g) and 1,6-diaminohexane (0.5 g) in methanol (20 mL) was added. The pH was then readjusted to 10.0 with 2N HCl and the mixture was stirred at H ° C for 72 hours. The product was centrifuged (20,000 gM ° C / 5 min). What remained was a loose white cake (25 ml) and a clear liquid above the precipitate with foam on the surface (octadecylamine).

(b) Penicillin acylase / n-octadecylamino, (6-aminohexylamino) -Ficoll.7 (PAFODHDG-i)

Penicillin acylase (25 ml, 170 mg protein) was adjusted to pH 6.2 at room temperature and glutaraldbhyde (2.5 ml, 25% w / v) was added. The mixture was stirred at room temperature for 15 min, FODHD-1 (10 mL) was added and the suspension was adjusted to pH 6.2 with 2N HCl. After stirring at 1 ° C for 3 hours, the centrifuged material (20,000 g / l | ° C / 30 min) was centrifuged. The pink cake was resuspended in 0.3 M sodium phosphate buffer, pH 7.0, and stored at 4 ° C. The suspension was floated with n-decanol and its activity is shown in Table 2. The recovery of activity was about 60%.

18 59265

Table 2 PAFQDHDG-1 activity

Test method: same as in Table 1. 5% w / v was used in all regulations. penicillin G.

f 6-APA original preparation-Material_Quantity_ speed moles / min x 10_

Basic enzyme 1 ml 0.885 PAFODHDG-1 0.5 g 0.940

Control experiments (method 2) 1. 0.5 g 0.800 2. 0.5 g 0.585

Example 3 (a) GAN149HDOD-1

N-Octadecylamine (1 g) and 1,6-diaminohexane (0.2 g) were suspended in methanol (10 ml) and the suspension was added to 0.2 M sodium phosphate buffer, pH 8.0 (200 ml) containing methanol ( 40 ml). Gantrez 149 (2 g) was added in small amounts with vigorous stirring. The mixture was stirred for 16 hours at room temperature and then centrifuged (12,000 g / 1 h) at room temperature. The foam and supernatant were discarded and the cake was resuspended in 0.1 M sodium phosphate buffer, pH 7.0 (100 mL), and centrifuged again as described above. The gel cake (80 g) was stored at 4 ° C.

(b) PAGAN149HDOD-1-G

_7

A penicillin acylase solution (20 mL, 1.36 x 10 units, 136 mg protein) was adjusted to pH 6.2 and glutaraldehyde (2 mL; 25% w / v in water) was added. The mixture was stirred at room temperature and a suspension of GAN149HDOD-1 (10 g) in 0.1 M sodium phosphate buffer, pH 6.0 (12 ml) was added. The pH was maintained at 6.2 Stirring was continued at room temperature for 2 hours. The suspension was centrifuged for 1 hour (20,000 g / 4 ° C) and the cake (14 g) was collected. The cake was resuspended in 0.1 M sodium phosphate buffer, pH 7.0 (10 mL), and centrifugation was repeated. The final cake was stored at 4 ° C.

i9 5 9 2 6 5

Specific activity: 3 x 10 ^ moles of 6-APA min ^ (5% peninylin G, pH 7.8 22 ° C)

Recovered activity: 21%. Can be floated with n-decanol or n-decane.

Example 4 (a) GÄN149HDOD-2

N-Octadecylamine (5 g) was dissolved in ethanol (50 ml) and added to 0.1 M sodium phosphate buffer, pH 9.0 (1 liter) containing ethanol (200 ml). Gantrez 149 (5 g) was added in small amounts over 3/4 hours with stirring. The pH was maintained between 8 and 9 with 2N NaOH. After the addition of Gantrez, 1,6-diaminohexane (5 g) was added and the mixture was stirred at room temperature for 6 hours. The pH was then lowered to 3.0 with concentrated HCl and the suspension was centrifuged (12,000 g / 1 h) at room temperature. The supernatant was discarded and the cake was resuspended in water (550 mL) and recentrifuged to give the final white cake (150 g) which was stored at 4 ° C.

(b) PAGAN149HDOD-2-G

A solution of penicillin acylase (100 mL, 9.8 x 10 6 units, 88 mg protein) was mixed with GAN149HDOD-2 (15 g), 0.1 M sodium phosphate buffer, pH 6.0 (20 mL), and 2% weight / vol. sodium azide (1 ml) and homogenized briefly at 0 °. The mixture was adjusted to pH 6.8 at 0 ° C for 4 1/2 hours and then centrifuged (20,000 g / 4 ° C / 1 h). The cake was resuspended in 0.1 M sodium phosphate buffer, pH 7.0, and centrifuged again. These treatments were then repeated to obtain a final cake, weighing 11.2 g, which was stored at 4 ° C.

Specific activity: 1.8 x 10 5 moles of 6-APA min-^ g-1.

(Conditions are the same as above)

Activity recovery: 55%. It could be floated with n-decanol or n-decane.

Example 5

Penicillin acylase (n-octadecylamino Gantrez AN 119) (PAGAN1190D) Gantrez AN 119 (0.5 g) was dissolved in dimethylformamide (2.5 ml) and heated to 80 ° C with a water bath. Octadecylamine (0.28 g) was also dissolved in dimethylformamide (2.5 ml) at 80 ° C and added rapidly to the Gantrez solution while hot. The mixture was stirred and pyridine (0.1 ml) was added. After cooling to room temperature over 1 hour, the whole solution was added to penicillin acylase (175 mg) in water (30 ml), pH 7.0, and briefly homogenized at 0 ° C.

The pink mixture was then stirred at 0 ° C for 3 hours, the pH was maintained at 4 ° C for 16 hours, sodium chloride (5.8 g) was dissolved in the suspension and the mixture was centrifuged (25,000 g / 0 ° C / 1 h). The resulting pink cake weighed 4.0 g and was resuspended in 0.1 M sodium phosphate buffer, pH 7.0 (20 mL).

When using 5% w / v. penicillin G in 0.02 M sodium phosphate drop buffer, pH 7.8 (20 mL) at 25 ° C, had a specific activity of 4.0 x 10 μmol / min / ml in this suspension. This corresponds to approximately 70% recovery of activity in the conjugate. The above suspension (4.0 mL) was mixed with 0.02 M sodium phosphate buffer (20 mL) and n-decanol (5 mL) and homogenized (4000 rpm, 5 min) at 0 ° C. The emulsion was centrifuged (100 g) at room temperature for 20 min and separated into an upper organic emulsion and a lower aqueous layer. The lower layer was retained and the upper layer was resuspended in 0.02 M sodium phosphate buffer (20 mL) and centrifuged again. The total activity of the original lower layer was 1.25 x 10 ~ mol / min and the activity of the washed upper layer was 9.5 x 10 ~ 5 mol / min. The ratio of specific activities (calculated by volume) is 21.6 (upper / lower). After determining the activity by the above method, the organic layer was carefully recycled by centrifugation and removing the aqueous layer as shown in Table 5.

Table 5 Percentage recovery of initial activity of PAGAN 1190D-n-decanol phase in successive flotation reuse cycles

Period No. Activity% Period No. Activity% 1 80 7 46 2 86 8 46 3 93 9 44 4 73 10 42 5 64 11 45 6 47 12 43 2i 5 9265

Example 6

Penicillin acylase (n-dodecylamino Gantrez AN 119) (PAGAN119DD) In dimethylformamide (1 ml) was dissolved n-dodecylamine-Gantrez AN 119 (0.25 g) and added to a solution of penicillin acylase (about 100 mg in 14 ml 0.1 M sodium phosphate buffer, pH 7.0).

After a short homogenization at 0 ° C, the suspension was stirred for 16 hours at 4 ° C. Ammonium sulfate (7.8 g) was then added to a homogeneous solution at 4 ° C and the pH was maintained at 7.0 with 1 M sodium carbonate solution. After dissolution of the ammonium sulfate, a cloudy suspension formed. This material was centrifuged (25,000 g / 0 ° C / l h). The cake was resuspended in a solution of ammonium sulfate (7.8 g) in water (30 mL), pH 7.0, and centrifuged again. The final cake weighed 1.36 g and was redissolved in water (10 mL) to give a viscous solution. The activity of the cake (above experiment) was 1.62 x 10 ~ 4 moles / min / g wet weight, corresponding to a recovery of 80% activity. The immobilization of the water-soluble conjugate on n-decanol droplets is illustrated by the following example: 1 ml of the above solution was dissolved in 0.02 M sodium phosphate buffer (20 ml) and homogenized with n-decanol (10 ml) at 4000 rpm for 3 min at 0 ° C. . The emulsion was separated into layers by centrifugation (300 mg) for 10 min. The total activity of the upper layer was 1.14 x 10 μmol / min and its activity in successive recirculations is shown in Table 6.

In another experiment, 2 ml of enzyme conjugate solution was homogenized under the same conditions with n-decanol (2.5 ml) and 0.02 M sodium phosphate buffer (10 ml). After centrifugation and washing with -5 ml of the same buffer, the activity of the upper layer was 1.8 x 10 mol / min and the activity of the lower layer was 8.0 x 10 mol / min. The ratio of specific activities (top layer / bottom layer by volume) was about 7.5.

Table 6 Percentage of initial activity recovery of PAGAN119DD-n-decanol phase in successive flotation reuse cycles

Section No. Activity% 1 96 2 66 3 66 4 53 2 2 59265

Example 7 (a) Hydrolysed n-octadecylamino Gantrez AN 119 hydrazide (GAN1190DH)

Gantrez AN 119 (5 g) was dissolved in dimethylformamide (20 ml) and heated to 60 ° C in a water bath. A solution of n-octadecylamine (2.8 g) in hot (80 ° C) dimethylformamide (30 ml) was then added and the mixture was maintained at about 60 ° C for 1 hour. After cooling to room temperature, a solution was added to a mixture of hydrazine hydrate (10 ml) and dimethylformamide (50 ml) at room temperature. A slightly brownish precipitate formed and the mixture was stirred for 10 min and water (500 ml) was added. A soapy green solution was obtained and after stirring for 15 min the pH was adjusted to 7.0 with concentrated HCl and sodium chloride (50 g) was added. Flocculation occurred and the suspension was centrifuged (10,000 g / 30 min / 4 ° C). The pellet was resuspended in water (400 ml) and mixed with 16 g at 4 ° C and then centrifuged as described above to give a blue gray cake (wet weight 39 g).

(b) PAGAN1190DH coupled with penicillin acylase (n-octadecylamino Gantrez AN 119) hydrazide

GAN1190DH (5 g wet weight) was suspended in 2N HCl (50 mL) and stirred at 0 ° C for 15 min. A solution of sodium nitrite (0.5 g) in water (5 ml) was added and the mixture was stirred at 0 ° C for 10 min, then centrifuged (25,000 g / 30 min / 0 ° C) and the pellet resuspended in 0.1 M sodium phosphate buffer. pH

8.0 (50 mL), at 0 ° C. This suspension was added to a penicillin acylase solution (100 ml containing 55 mg of protein) and adjusted to pH 8.0. The suspension was stirred at 4 ° C for 72 hours and then centrifuged (25,000 g / 1 h / 0 ° C), resuspended in 0.1 M sodium phosphate buffer, pH 7.0, and centrifuged again. The final weight of the pellet was 3.74 g. Specific activity (experiment performed as described above): 5.2 x 10 mol / min / g. Activity recovery: 32.5%.

A suspension of the above conjugate in 0.1 M sodium phosphate buffer, pH 7.0 (0.29 g / ml, 2.5 mL) was mixed with 0.02 M sodium phosphate buffer, pH 7.8 (20 mL). and homogenized with n-decanol (7.5 mL) at 0 ° C for 2 min, and centrifugation at 5000 rpm (100 g, 10 min) gave two layers.

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The total activity of the lower layer was 1.8 x 10 mol / min and the activity of the upper layer after washing with the above buffer (20 ml) was 1.9 x 10 mol / min. The ratio of specific activities (upper / lower by volume) was about 30. Recirculation results using the upper phase are shown in Table 7.

Table 7 Percentage recovery of initial activity of PAGAN1990DH-n-decanol phase using successive flotation reuse cycles

Section No. Activity% 1 90 2 84 3 74

Example 8

Enzymatic cleavage of benzylpenicillin to prepare 6-aminopenicillanic acid 1.0 g of Gantrez AN 119 was dissolved in 10 ml of dimethylformamide and 0.56 g of octadecylamine was added. The mixture was stirred overnight at room temperature.

48 ml of the partially purified penicillin acylase preparation was adjusted to pH 9.0 with 1 M sodium carbonate solution. A solution of Gantrez resin was then added in two equal portions while homogenizing and the pH was adjusted between the two additions. The mixture was then stirred for 3 hours, maintaining the pH at 9.0 by the addition of 1 M sodium hydroxide solution.

The enzyme resin was then recovered by centrifugation, resuspended in 120 ml of 0.1 M phosphate buffer, pH 7.0, and homogenized for 30 sec. The enzyme resin was recovered by centrifugation and washed.

20 g of enzyme gel was homogenized with 80 ml of n-decane and 20 ml of 0.02 M phosphate buffer, pH 7.8, for 1 min. The homogenate was added to 250 ml of distilled water. The mixture was heated to 37 °, adjusted to pH 7.8 and potassium benzylpenicillin (21.8 g) was added.

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The mixture was stirred for 5 hours and the pH was maintained at 7.8 by the addition of 4% (w / v) sodium hydroxide solution. At the end of the reaction, the mixture was added to a separatory funnel and the enzyme was allowed to separate from the aqueous phase. This took 30 min. The aqueous layer was then removed and 6-amino-penicillanic acid was extracted as follows.

The solution was concentrated to 1/4 volume, cooled to 5 °, and an equal volume of methyl isobutyl ketone was added with stirring. The pH was lowered to 4.3 by the addition of concentrated nitric acid, after which 6-aminopenicillanic acid was precipitated. The solid was collected by filtration, washed with water and acetone and dried overnight at 40 °.

7.65 g of 6-aminopenicillanic acid were obtained.