DE2143062A1 - Water-insoluble enzyme preparation and process for its production - Google Patents

Description

"Water-insoluble enzyme preparation and process for its production"

Priority: August 27, 1970 Great Britain registration no. 41262/70 k. August 1971 Great Britain registration no. 36601/71

The invention relates to water-insoluble enzyme preparations which contain at least one deacylase with cleavage activity in relation to the Contain amide bond of penicillin bodies.

Accordingly, it is an object of the present invention to provide water-insoluble enzyme preparations which can be isolated from the reaction medium in question for reuse and which are also very suitable for storing or transporting the enzyme in question in this form. An advantage of the enzyme preparations according to the invention is, moreover, that the when used; 6-Amiiiopenicillanäure available has a higher degree of purity than if a water-soluble Pr-ni oillin deacylase is used, since when using the inventive enzyme preparations in the conversion product generally no more traces of enzyme.

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can be found.

In addition, the enzyme preparations according to the invention can also be used for protecting groups from N-ayl-DL-amino acids to be separated enzymatically and thus recovered in pure form.

The water-insoluble enzyme preparations are characterized by that they consist of at least one deacylase with cleavage activity

exist, with regard to the amide bond of Penieil3inkö-p vn / whereby this deacylase is bound to a solid, water-insoluble absorbent by means of a water-soluble dialdehyde.

The deacylase in question is preferably obtained from bacteria, for example from Escherichia coil, if the enzyme preparation is to be used for splitting benzylpenicillin and for obtaining 6-aminopenicillanic acid. Palis to undergo cleavage by means of the preparation phenoxymethylpenicillin, the deacylase is obtained from appropriate w PiIzarten and actinomycetes.

The water-soluble dialdehyde is preferably glyoxal or glutaraldehyde.

As a solid, water-insoluble, absorbent substance, one can use Any water-insoluble absorbent material can be used, but finely divided polymeric materials are preferred because a

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such enzyme preparation has a greater activity. The water-insoluble absorbent material must be selected in such a way that at least 25 g of the finished enzyme preparation in the form of a pack in a column of 30.8 cm × 2.5 ^ cm with an effectiveness of at least 90 S? during a period of at least 12 hours at 37 ° C, 5 πΊ .1e hour of a 0.5piOzentigen (weight / volume) aqueous solution of DL-phenylacetamido-hydroxyphenylglycine (hereinafter abbreviated as PAPG) to the DP-hydroxyphenylglycine, in addition, at least 50 g of the enzyme preparations according to the invention at least 70 % of the potassium salt of benzylpenicillin contained in 100 ml of a 1 percent (weight / volume) aqueous solution at a pH of 7 * 8 in a stirred tank after 6 hours and at a temperature of 37 C. convert into 6-aminopenicillanic acid.

Well-suited water-insoluble, absorbent, solid substances are cellulose powder and the numerous known cellulose derivatives, also ion exchange resins, silica gel, colloidal silica and polymeric substances.

A particularly suitable polymeric material is nylon, preferably in finely divided form. Particularly useful here is nylon of the 6.6 type with a low molecular weight and a part size such that the powder is passed through an AST! ^{# 35 1} 'fine sieve (500 micron sieve), this nylon being said to have a high content of amino end groups, for example see

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200 to 500 gram equivalent of such groups, 10 grams each can also partially hydrolyze the nylon, for example

Number of means of diluted mineral acid in order to / for the deposition of the Enzyme to increase the available free amino groups with the help of the dialdehyde.

The invention also relates to a process for the preparation of these enzyme preparations, which is characterized 'by bringing the ψ individual components in an aqueous medium at a pH of preferably 5 to 9 contact each other and the enzyme complex formed is then separated. The water-absorbing material can first be brought into contact with the deacylase or first with the dialdehyde, or contact can be made with these two components at the same time.

If the absorbent is first mixed with the "" enzyme in water Brings solution into contact, a residence time of about 12 to 24 hours is very useful in order to achieve good absorption. Then the loaded absorbent is then usually for about 1 to 24 hours at a temperature of 0 to 50 ° C brought into contact with the dialdehyde. Even if the absorbent is treated first with the dialdehyde and then with the enzyme or if the contact is made with both components at the same time, treatment times are the specified duration very welcome. The solid enzyme preparation can then be washed with water to remove unbound protein and unreacted To remove dialdehyde.

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If the water-insoluble absorbent substance does not have any chemical groups which can react with the aldehyde, so the absorbed enzyme molecules are cross-linked by the action of the dialdehyde. If, on the other hand, the absorbent Contains reactive groups which can react with the dialdehyde, so occurs in addition to a covalent crosslinking of the enzyme molecules with the absorbent substance, with the help of the dialdehyde, there is also a crosslinking of the enzyme molecules with one another on.

Examples 1 and 2 below illustrate the preparation of a penicillin deacylase. The remaining examples explain the Production of the water-insoluble enzyme preparations according to the invention.

example 1

A partially purified penicillin deacylase is prepared starting from a strain NC IB 87 ^ 3 of Eseherichia coli ,, which produces penicillin deacylase. Enz: / m is made from the cells of this bacterium in a homogenizer mechanical means released, then centrifuged, the supernatant solution treated until 40 percent saturation with Ammonium sulfate, discards the precipitate, continues to add ammonium sulfate to 75 percent saturation, then filters it precipitate formed, dissolve it in water, dialyzed "" ·

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against water and finally subjects the freeze-dry

Example 2

A partially purified "penicillin saeylase" is made from a Mutant strain of Escherichia coll. NC IB 8743 "produced by the enzyme is released from the cells of the bacterium, then adjusted to pH 4.5 with acetic acid and finally the cell debris is filtered off or removed by centrifugation.

Example 3

25 g of a commercially available diethylaminoethyl cellulose are suspended in 100 ml of a 0.1 M sodium borate buffer at a pH of 8.0 and then 2.5 g of a partially purified penicillin deacylase are added. This mixture is gently stirred for 24 hours at room temperature, then 5 % (weight / volume) glutaraldehyde is added and stirring is continued for an additional hour.

Then the pest is filtered off with a small one Amount of buffer solution washed out and then carefully first with Im sodium chloride solution, then with O, Im sodium hydroxide solution and finally with an Im ammonium chloride buffer solution washed out to a pH of 8.6. The cellulose is left in contact with the buffer solution overnight and then removed

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from it and finally washes it with 1m sodium chloride solution and distilled water. This water-insoluble enzyme preparation has such an activity that it is 100 percent effective for 20 hours at 5 ml / hour of a 0.5 percent (weight / Volume) PAPG solution into the corresponding D-p-hydroxyphenylglyoin convicted.

Example 4

The procedure of Example 3 is followed using a commercially available cellulose fiber powder as the absorbent
repeated. This enzyme preparation has such an activity that it contains 5 ml / hour of 0.5 percent (weight / volume) PAPG solution for 18 hours with 100 percent effectiveness
enzymatically cleaves, with 10 g of this preparation 18 hours
Split 32 to 3 ml of a 5 percent (weight / volume) substrate for a long time with 100 percent effectiveness.

Example 5

The procedure of Example 3 is carried out using commercially available carboxymethyl cellulose as the absorbent material
repeated except that only 2 g of the enzyme are used.
This enzyme preparation has an activity such that it enzymatically cleaves 5 ml / hour of a 0.5 pΐΌ-cent (weight / volume) PAPG solution for 19 hours with 100 percent effectiveness.

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Example 6

The procedure of Example 3 is followed using 25 g commercially available epichlorohydrin-triethanolamine celluloses as an absorbent repeatedly. But the buffer solution has one pH value of 8.75 This enzyme preparation has an activity such as that it is 5 ml / hour for 19 hours with 100 percent effectiveness a 0.5 percent (weight / volume) PAPG solution.

Example- 7

5 g of diethylaminoethyl Sephadex A5O are allowed to swell for 3 days in an 0.1m sodium borate buffer solution at a pH of 8.0. The excess buffer solution is then decanted off and 1 g of enzyme is added. The mixture is stirred for 24 hours at room temperature. Then 5 % (weight / volume) glutaraldehyde is added and stirring is continued for a further hour. Finally, the enzyme preparation is carefully washed out according to the procedure of Example J>. This enzyme preparation has an activity such that it enzymatically cleaves 5 ml / hour of a 0.5 percent (weight / volume) PAPG solution for 20 hours with 100% effectiveness.

Example 8

25 g of the ion exchange resin Amberlite IR-45 (OH) become suspended in 100 ml of a 0.1 m phosphate buffer at pH 7> 0. Then 4 g of partially purified penicillin deacylase are used

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added. The further treatment with glutaraldehyde and the following Careful washing is carried out according to the procedure of Example 3. With this enzyme preparation, the following Results achieved;

1) 25 g of the preparation have an activity such that

it for 500 hours with 100 percent effectiveness 70 ml / hour a 5 percent (weight / volume) PAPG solution enzymatically columns.

2) 40 g of this enzyme preparation, on the other hand, become a series

of three enzymatic treatments of a 4 percent (weight / ' Volume) solution of potassium benzylpenicillin at 37 ° C and one. pH value of 7 * 8 used, with 400 ml of solution being treated. The enzyme is filtered off after each enzymatic treatment. In all three cases the benzylpenicillin is within Converted to 6-aminopenicillanic acid for 6 hours.

B e ispie l 9

According to the procedure of Example 2, a clarified E. coli homogenate is prepared which, by adding 16.4 g pinodium hydrogen phosphate and 1.1 g sodium dihydrogen phosphate is buffered to a pH of 7. 1 liter of the hornogenate is used for this. 200 g are then suspended in it the ion exchange resin Amberlite 1R-45 (OH). This suspension is stirred for 24 hours at room temperature.

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Then 5 % (weight / volume) glutaraldehyde is added and the mixture is left to stand for two hours. Eventually the feast. The substance is filtered off, washed with a 5 percent solution (weight / volume) of sodium bisulfite and then with a 0, Im sodium chloride solution and with water. This enzyme preparation has such an activity that it cleaves 80 to 100 ml / hour of a 5% (weight / volume) PAPG solution for 300 hours with 100% effectiveness, with practically no loss of activity being observed at the end of the experiment.

Example 10

According to the procedure of Example 2, a clarified cell homogenate from E.coil is produced and the pH is adjusted to 5 * 0. 200 g of the ion exchange resin Amberlite IR-45 (OH) are then suspended in the homogenate and the mixture is stirred for 24 hours at room temperature. The pH is then adjusted to 8.0 and 1 % (weight / volume) glutaraldehyde is added. The further work-up is carried out according to the procedure of Example 9. This enzyme preparation has such an activity that it enzymatically cleaves 35 ml / hour of a 5% weight / volume PAPG solution for 300 hours with 100% effectiveness. In addition, 20 g of the enzyme preparation are used to produce 50 ml of a 2 percent (weight / volume) solution of

-Potassium salt
Benzylpenicillin / at a pH of 7 * 8 and a temperature

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enzymatically to cleave at 37 ° C. In both attempts, within A ßO percent conversion to 6-aminopenicillanic acid within 3 hours achieved.

Example U

The procedure of Example 3 is followed using silica gel as an absorbent and from J5 $ (weight / volume) glutaraldehyde repeated. This enzyme preparation has such an activity that it lasts 20 hours with 100% effectiveness 5 ml / hour of a 0.5 percent (weight / volume) PAPG solution enzymatically cleaves.

Example 12

25 g of a commercially available aluminum oxide (grade II) are suspended in 100 ml of distilled water and then 1.5 g of enzyme are added. The pH is adjusted to 5 * 0 and then stirred for 24 hours at room temperature. Then 2 % (weight / volume) glutaraldehyde is added and the pH is adjusted to 8.0. After stirring for a further hour, the solid is filtered off and carefully washed out according to the procedure of Example 3. This enzyme preparation has an activity such that it enzymatically cleaves 5 ml / hour of a 0.5 percent (weight / volume) PAPG solution for 15 hours with 100 percent effectiveness. In addition, 21 g of this preparation are used to enzymatically ©

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To carry out deacylation of a 1 percent (weight / volume) solution of the potassium salt of benzylpenicillin at a pH value of "J, and a temperature of J57 ° C. in a stirred reactor with a capacity of 400 ml. In both experiments, over 90 % of the benzylpenicillin salt in 6 -Converted aminopenicillanic acid.

Example 13

10 g of a commercially available colloidal silica are in ' 50 ml of a 0.1 im phosphate buffer solution with a pH of 7 * 0 suspended, which also contains 0.1 g / m of a partially purified penicillin deacylase. Shake gently for 24 hours long.

Then 5 % (weight / volume) glutaraldehyde are added and the mixture is shaken for a further hour. Finally, the solid is filtered off and carefully washed out according to the procedure of Example J). This enzyme preparation has an activity such that it enzymatically cleaves 5 ml / hour of a 0.5% PAPG solution for 20 hours with a 100% effectiveness.

Example 14

7.6 g of acrylamide, 0.4 g of Ν, Ν'-methylene-bisacrylamide, 18 mg of ammonium persulfate and 20 μΐ of β, β-dimethylaminopropiononitrile are added to 32 ml of distilled water. This mixture will

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carefully deaerated, and a stiff gel then forms, which is passed through a 30-mesh sieve.

20 g of this polyacrylamide gel and 2 g of a partially purified penicillin deacylase are introduced into 50 ml of a 0.1 lm sodium borate buffer with a pH of 8.0 and this mixture is agitated for 24 hours at room temperature. Then 5 % (weight / volume) glutaraldehyde is added and the mixture is left to react for a further hour. The enzyme preparation is then carefully washed out according to the procedure of Example J>. The finished preparation has such an activity that it enzymatically cleaves 5 ml / hour of a 0.5 percent (weight / volume) PAPG solution for 20 hours with 100 percent effectiveness.

Example 15

The procedure of Example 8 is repeated using the ion exchange resin Amberlite IR-45 (OH), but the glutaraldehyde is replaced by 5 % (weight / volume) glyoxal. This enzyme preparation has such an activity that it enzymatically cleaves 5 ml / hour of a 0.5 percent (weight / volume) PAPG solution for 20 hours with 100 percent effectiveness.

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Example l6

10 g of the ion exchange resin Amberlite XAD 7 (a macro network tes acrylic acid ester resin) are stirred into 100 ml of a phosphate buffer with a pH of 5.0, which contains penicillin deacylase. The mixture is stirred for 16 hours at room temperature. The pH is then raised to 8.0, and 10 ml of 50 percent (volume / volume) glutaraldehyde solution is added and the mixture is stirred for a further 2 hours at room temperature. The pesticide is then filtered off and washed with a 5 percent (weight / volume) solution of sodium bisulfite, then with 0.5m phosphate buffer of pH 7.8 and finally with water. This preparation is used to enzymatically break down 160 ml of a 6 percent (weight / volume) solution of the potassium salt of benzylpenicillin. In three successive experiments, over 80 % of the benzylpenicillin is converted into 6-aminopenicillanic acid within 4 to 5 hours using the same enzyme preparation. -

Example 17

25 g of commercially available diethylaminoethyl cellulose are suspended in 100 ml of an 0.1 lm phosphate buffer solution with a pH of 5.0 and then 2.5 g of enzyme are added. This mixture is stirred for 20 hours at room temperature. The pH is then raised to 8.0 and 5 % (weight / volume) glutaraldehyde is added and the mixture is stirred for a further 1 hour. The pesticide (wet weight: 40 g) is filtered off and then according to the procedure

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from Example 9 carefully washed out.

20 g of this enzyme preparation are used for the enzymatic cleavage of 100 ml of a 2 percent (weight / volume) solution of the potassium salt of benzylpenicillin, working at 37 ° C. and a pH of 7.8. This takes 2 hours. In five subsequent experiments, 95 % of the benzylpenicillin salt is converted into 6-aminopenicillanic acid.

At s ρ i e 1 l8

25 g of a commercially available diethylaminoethyl cellulose are suspended in 100 ml of a phosphate buffer with a pH of 8.0. Then 5 % (weight / volume) glutaraldehyde is added and the mixture is stirred for a further 30 minutes. Finally, 2.5 g of the partially purified penicillin deacylase are added and this mixture is stirred for a further 2 hours. The solid is then filtered off and carefully washed according to the procedure of Example 9.

20 g of this enzyme preparation are used to enzymatically cleave 100 ml of a 2 percent (weight / volume) solution of the potassium salt of benzylpenicillin within 2 hours at a pH of 7.8 and a temperature of 37.degree. In two successive experiments, 95 % of the benzylpenicillin salt is converted into 6-aminopenicillanic acid.

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Ex iel 19

The procedure of Example 17 is followed using a commercially available aminoethyl cellulose as a solid absorbent Fabric repeated.

20 g of this enzyme preparation are used for the enzymatic cleavage of 100 ml of a 1 percent (weight / volume) solution of the potassium salt of benzylpenicillin at a pH of 7 * 8 and a temperature of 37 ° C, the treatment lasting 2 hours. In two successive experiments, 95 % of the benzylpenicillin salt is converted into 6-aminopenicillanic acid.

Example 20

2 g nylon (particle size: 53 to 75 microns; content of free Amino groups: 472 gram equivalents per 10 grams) are with

aqueous solution of 200 ml of enzyme solution and 2 ml of a 25 percent / glutaraldehyde Stirred at 3 ° C for 16 hours. The enzyme solution is prepared by 240hig of the enzyme obtained according to Example 1 stirred into 200 ml of a 0.05m sodium phosphate buffer solution with a pH of 7 * 5. After 16 hours one centrifuges and thus wins the solid, loaded absorbent, which is washed out well with water.

The activity of the solid enzyme preparation is such that 1? 950 JuMol phenylacetamide per hour and per gram enzymatically

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be split.

In addition, 4 g of the enzyme preparation are used to split 750 ml of a 15m solution of benzylpenicillin in 0.05m sodium phosphate buffer solution at a pH of 8 and a temperature of 37.degree. After just 17 minutes, 70 % of the benzylpenicillin has been converted into 6-aminopenicillanic acid. The degree of conversion is 90 % after 25 minutes and 95% after 33 minutes.

Example 21

10 g of nylon (particle size: .53 to 75 microns; content of free amino groups: 4-72 gram equivalents per 10 grams) are stirred for 1 hour at 3 ° C. with 50 ml of a 25 percent aqueous solution of glutaraldehyde. The pesticide is then separated off and used to remove excess glutaraldehyde. Water washed. Is then added 100 ml of an enzyme solution are added and stirring is continued for l6 hours at 3 ⁰ C. This enzyme solution was obtained by mixing 240 mg of the prepared in Example 1 enzyme preparation in 100 ml of a O, 05m-sodium phosphate buffer solution with pH of 8, 0 resolves. After 16 hours the pest is centrifuged off and washed out well with water. The activity of the enzyme preparation is such that 1 260 ^ u-MoI phenylacetamide per hour and per gram are enzymatically decomposed.

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Claims (9)

Claims 1. Water-insoluble enzyme preparation, consisting of at least one deacylase with cleavage activity in relation to the amide bond of penicillin bodies, which are attached to a solid, water-insoluble, absorbent substance by means of a water-soluble dialdehyde is bound. 2. Water-insoluble enzyme preparation according to claim 1, characterized in that it is a deacylase produced by bacteria contains. 3. Water-insoluble enzyme preparation according to claim 2, characterized characterized as producing one of Escherichi'a coli Contains deacylase. 4. Water-insoluble enzyme preparation according to Claims 1 to J5 * characterized in that it is a solid ^ water-insoluble. material absorbent / cellulose powder, a cellulose derivative, an ion exchange resin, Contains silica gel, colloidal silica and / or a polymer. 5 water-insoluble enzyme preparation according to claims 1 to 4, characterized in that it contains a nyloh type polymer as the absorbent material. 209810/1705 6. Water-insoluble enzyme preparation according to claim 1 to 5 * characterized in that it contains the solid, water-insoluble absorbent material in finely divided form. 7. Water-insoluble enzyme preparation according to claim 1 to 6, characterized in that it is used as a water-soluble dialdehyde Contains glutaraldehyde or glyoxal. 8. Process for the production of a water-insoluble enzyme preparation according to claim 1, characterized in that one brings the individual components into contact with one another in an aqueous medium at a pH of preferably 5 to 9 and the enzyme complex formed is then separated off. 9. Use of an enzyme preparation according to claim 1 for the production of 6-aminopenicillanic acid from benzylpenicillin or a salt of this penicillin. 209810/1705 * ORIGINAL INSPECTED