FI58941B - FOERFARANDE FOER AKTIVERING AV LEVANDE IMMOBILISERADE MICROORGANISMER SOM ANVAENDES SOM TRANSFORMERARE I 1-DEHYDROGENERINGS- ELLER 11 BETA-HYDROXYLERINGSREAKTIONER AV STEROIDER OCH I TRANSFORMATIONSREAKTIONER AV BENZY - Google Patents

Description

Γοΐ nn ANNOUNCEMENT cqQA1 TOfik lBJ (11) UTLÄGGNI NGSSKRIFT 3 ÖV 4 1 vS & W C / 4 «Patent ay; r .. '.: tty 11 05 1« JC1 ”Patent ineddelat (51) K» .ik.3 / mt. a.3 C 12 N 11/08 (21) l * Uanttihak «mut - PK« ntant6knlnf T72012 (22) Haktmiipilvl - Amttknlngfdig 28.06.77 (FI) (23) Alkuplivft —GIMfhetadai 28.06.77 (41) Tullut julklMkal - Bllvlt offwKlif 07-01.78

National Board of Patents and Registration (44) Nihttviksipunon] · kuLLuiuiiui «<n pvm.—

Patent and registration authorities Ansekan utiagd och utUkriften pubiicand 30.01.81 (32) (33) (31) Pretty atuotkus — B «flrd prloritvt 06.07-76

Sweden-Sweden (SE) 7607698-3 (71) Aktiebolaget Fermenta, Fack, S-152 00 Strängnäs, Sweden-Sweden (SE) (72) Per-01of Larsson, Lund, Klaus Hermann Mosbach, Furulunds Station,

Sten Albert Ohlson, Lund, Sweden-Sverige (SE) (7 * 0 Berggren Oy Ab (5 * 0 Method for the activation of immobilized living micro-organisms used as transformers in Δΐ-dehydrogenation or ιιβ-hydroxylation and benzylpenicillin conversion reactions of steroids) This invention relates to a method for activating immobilized microorganisms, and more particularly to this method is directed to a method of activating immobilized microorganisms, in particular to a method for activating immobilized microorganisms. to activate organisms used to ΔΙ-dehydrogenate or 113-hydroxylate steroids and convert benzylpenicillin.

Immobilized microorganisms have gained increasing interest as catalysts in recent years (Biotechnol. Bioeng. 17, 1797-1804 (1975); J. Appl. Chem. Biotechnol. 25, 115-141 (1975); Biotechnol. Bioeng. 12, 19 They have the same functional advantages as immobilized enzymes (FEBS Lett. 62. (supplement) E 80 to E 90 (1976)), are reusable, are well suited for continuous use under controlled conditions and are also relatively Immobilized microorganisms offer the added advantage of avoiding cumbersome and costly separation of the enzyme, the enzyme is usually stable due to its "natural environment" and usually no micronutrient factor is required. organisms are therefore very promising catalysts, provided that competing reactions can be eliminated. repeatedly, however, batch activity degrades fairly rapidly with the use of immobilized microorganisms. To date, this problem has not been satisfactorily solved with immobilized living microorganisms.

In particular, the present invention contemplates a unique property characteristic of immobilized living whole cell catalysts, namely, the ability to activate enzymatic activity in situ.

Suitable modification substrates with which the activation according to the invention can be applied are 1) steroids, in particular corticosteroids, e.g. cortisol

2) antibiotics such as penicillin G

Activation can be applied to several systems such as the conversion of corticosteroids to cortisol N 2 -dehydrogenase ^ prednisolone. The reaction can be catalyzed by Coryne bacterium simplex (Arthrobacter simplex) encapsulated in polyacrylamide.

Suitable transformations for the activation according to the invention include 1) N -dehydrogenation. Addition of a double bond to the 1,2-position of the steroid molecule. Example:

A1_dehydrogenation of cortisol and cortisol derivatives, e.g. cortisol to prednisolone.

2) IgG hydroxylation. Attachment of a hydroxy group to the IgI position of the steroid molecule. Example:

IIIs hydroxylation of cortexolone and its derivatives, e.g. cortexolone to cortisol.

58941 3) Conversion of penicillin G. Conversion of benzylpenicillin (penicillin G) to 6-aminopenicillanic acid.

Suitable organisms useful in the context of the invention are 1) Arthrobacter simplex (also called Corynebacterium simplex, e.g. ATCC 6946). This organism can be used for Δ 2 -dehydrogenation.

2) Curvularia Lunata, e.g., ATCC 12017. This organism can be used for 110-hydroxylation.

3) Escherichia coli, for example ATCC 9637. This organism can be used to prepare 6-aminopenicillanic acid from penicillin G.

The microorganisms are immobilized on a suitable carrier such as polyacrylamide, agar (2.5-15% w / v) or calcium alginate (1-5%).

The object of the present invention is to provide a method for preventing the deterioration of the activity of immobilized living microorganisms and for increasing the activity in repeated or continuous use. It is a particular object of the present invention to provide a method for activating immobilized living microorganisms used for ΔΙ-dehydrogenation or Ιΐβ-hydroxylation of steroids and for the conversion of benzylpenicillin using immobilized microorganisms, and the invention is mainly known to immobilize a microorganism containing organisms and the substrate to be converted, peptone, glucose or a mixture of peptone and glucose is added, preferably in an amount of 0.1-1.0% (w / v). Peptone should preferably be used at 0.1-1.0% (w / v). Good results are also obtained with 0.1% (w / v) peptone together with 0.2% (w / v) glucose. The process temperature should be 20-30 ° C and the process should be performed under aerobic conditions.

The invention is described in more detail below by means of examples.

58941

Example 1 In this experiment, a corticosteroid conversion system to cortisol N 2 -dehydrogenase ^ prednisolone was studied. This reaction was catalyzed by Arthrobacter simplex (also called Corynebacterium simplex) encapsulated in polyacrylamide.

A. simplex cells were grown in 0.25% yeast extract medium. N 2 -dehydrogenase activity was initiated by adding cortisol to the culture 12 hours before repair, which was performed by continuous centrifugation at 10,000 x g. The cells (5 g wet weight) were suspended in 20 ml of ice-cold 0.1 M Tris-HCl buffer, pH 7.5 and mixed with 25 ml of an ice-cold aqueous monomer solution containing 7.13 g of acrylamide and 0.38 g of g of N, N-methylene-bis-acrylamide. This mixture was poured into a laminated polymerization vessel (made of two 20 x 20 x 0.2 cm glass plates separated by a piece of latex tube 2 mm apart) and the catalysts, potassium persulfate (50 mg) and tetramethylethylenediamine (100 mg) were added. in water (1 ml). Nitrogen gas was bubbled through the suspension and the polymerization was started within 2 minutes. The polyacrylamide gel plate was partitioned in a blender and the gel granules (average size 0.2 mm) were washed thoroughly with Tris buffer and then stored at -20 ° C, at which temperature the preparation was stable for several months.

The 3-ketosteroid N-dehydrogenase activity of the immobilized A. simplex * was analyzed spectrophotometrically and the only product, prednisolone, was identified by thin layer chromatography. Approximately 40% of the dehydrogenase activity was retained during immobilization (all added bacteria became immobile and no bacterial release was observed during the incubations). However, initial experiments showed that the activity decreased relatively rapidly when large amounts of cortisol were repeatedly converted in batches and this was only compensated to a limited extent by the addition of artificial electron-accepting 2-methyl-1,4-naphthoquinone (menadione). Instead, the stabilizing effect of various nutrients and salts was studied. The results are shown in Tables I and II. In media containing water or buffer, activity decreased. In media containing peptone and glucose, on the other hand, the activity not only remained but also increased considerably several times higher than the original activity of 5,58941. 0.5% peptone medium and 0.1% peptone + 0.2% glucose medium were selected for further study and the experiment was performed with repeated batch transformations. Both media were approximately equally effective and Table III shows the results obtained with 0.5% peptone medium. As can be seen, the conversion capacity increased significantly in each run, thus 100% conversion was achieved in the first run only after 18 hours while the last batch was completed in less than 2 hours. The conversion capacity at the end of the experiment was about 0.5 g steroid / day / g gel (wet weight).

Example 2

Preliminary experiments have shown that the so-called the pseudo-crystal fermentation technique is also applicable to closed A. simplex *. Cortisol was added in an amount (3.6 g / L) that significantly exceeded its solubility in the medium and was completely converted at approximately the same rate as in the experiments with dissolved cortisol. The precipitated prednisolone product could be separated simply by filtration after the relatively dense gel granules had first allowed to settle. This technique made it possible to reduce the volume of the medium in terms of size and also of nutrients.

5 g of acrylamide gel containing 10% of A. simplex * was incubated in 20 ml of medium on a shaking table. The medium contained 181 mg of cortisol, 5% methanol, and either water or 0.5% peptone + 0.2% glucose. Samples were taken at regular intervals and analyzed by high performance liquid chromatography (HPLC). After 24 hours, the gels were collected and incubated with fresh medium. The results are shown in Table I.

Table I

Activating effect of nutrients on the pseudo-crystal fermentation of cortisol to predhisolone using A. simplex immobilized on polyacrylamide.

Medium Conversion rate (=% prednisolone formed in 5 hours) in incubation No. 1 2 3 4 5 0.5% Peptone 38 50 67 77 77 0.2% Glucose H 2 O 32 21 20 15 13 58941

Table II

Activating effect of nutrients, buffers and salts on the immobilized A.-simplex 3-ketosteroid-N-dehydrogenase activity of polymrylamide cl) b} Medium3 · Initial rate of cell conversion0 (%) 0 2 6 10 after 16 days

Peptone 0.5% 100 460 500 650 530

Glucose 0.2% 100 210 170 110 90 K2HPO4, 0.1 IM, pH 7.0 100 70 50 60 60

Tris-HCl, 0.05M, pH 7.0 100 100 70 70 30 / K-HPO4, 1.0 IM, pH 7.0 (zncl2. Fed, 100 50 25 15 10 fK-HPO., 0.1 IM, pH 7.0 lcoCl2, MgSO, 100 40 40 0 0 | Ί <2ΗΡ04.0, IM, pH 7.0 100 160 130 8Q g0 \ MgCl2 / CaCl2 rK2HPO4, 0, IM, pH 7.0 JPeptone 0.5% \ n,. · noa 10 ° 560 650 600 550

Glucose 0.2% ^ MgCl2, CaCl2 H2O 100 60 40 40 20 a) the content of the inorganic salts MgCl2 / ZnCl2f CoCl2 »FeCl2 / CaCl2 and MnSO4 was 1 mM, b) the activity of the freshly prepared gel was defined as 100%.

A. simplex gel (0.5 g) was incubated in 9.0 ml of medium as shown and 0.5 ml of 20 mM cortisol dissolved in methanol was added. The suspension was shaken on a rotary shaker at 25 ° C and every 48 hours the medium was replaced with freshly prepared cortisol-containing medium. After the indicated intervals, the gel was removed by filtration, washed and analyzed for Δ 2 -dehydrogenase activity.

7 58941

Table III

Peptone / glucose activating effect on rolyacrylamide immobilized A.

a) 1 simplex for 3-ketosteroid-A dehydrogenase activity Medium Initial conversion rate0 '(%) 0 2 6 10 after 16 days

Peptone 1% 100 290 320 380 550 0.5% 100 460 500 650 530 0.1% 100 200 225 165 120 0.01% 100 125 30 0 0 (Peptone 0.1% θοθ 250 225 240 350 (Glucose 0, 2%

Peptone 0.01% iqO 110 55 0 0 ^ Glucose 0.2%

Glucose 0.2% 100 210 170 110 90 a) The experimental conditions are shown in Table II.

b) the activity of the freshly prepared gel is determined to be 100%.

Table XV

Repeated batch conversion of cortisol to prednisolone

Driving Conversion capacity Time to 100% conversion (No.) (mg prednisolone / h / g ,,, gel (wet weight)) 1 3.1 17.4 2 5.0 10.8 3 13.5 4.0 4 18.1 3.0 5 26.3 2.1 6 27.1 2.0 7 27.1 2.0 8 29.6 1.8 9 30.8 1.8 10 31.7 1.7 A. simplex gel (2.0 g) was suspended in 285 ml of 0.5% peptone, 1¾ 7.0 + 15 ml of 20 mM cortisol dissolved in methanol. The progress of the conversion was monitored spectrophotometrically, and when 100% conversion of prednisolone was achieved, the gel was washed and incubated again with fresh cortisol-containing medium. The whole experiment lasted 4 days.

8 58941

Example 3 In this experiment, the steroid-converting microorganism A. simplex was immobilized on calcium alginate. Immobilization was performed as follows: A. simplex (0.5 g wet weight), grown as in Example 1, was suspended in 9.5 ml of sodium alginate solution. This mixture was added dropwise to a 0.1 M CaCl 2 solution to form spherical bacterial particles (2 mm diameter). The particles were allowed to stand in 0.1 M. CaCl 2 for 20 hours. The gels were then activated with nutrients and the steroid-converting activity was determined at certain intervals. The results are shown in Table V.

Table V

Effect of nutrients on 3-ketosteroid β-dehydrogenase activity of A. simplex immobilized on a calcium alginate gel.

Medium Initial cell conversion rate (%) 0 1 2 4 6 After 11 days 0.5% Peptone 100 280 330 460 480 670 0.5% Peptone 100 260 310 500 560 930 0.2% Glucose H 2 O 100 90 70 35 20 a) The activity of the freshly prepared gel is 100%.

Example 4 In this example, A. simplex was immobilized on a 2% calcium alginate gel (diameter 1 mm). 1 g of alginate gel containing 10% A. simplex was incubated in 20 ml of medium on a shaking funnel. The medium contained 181 mg of cortisol, 5% methanol and either water or 0.5% peptone + 0.2% glucose and additionally contained 0.05 M CaCl 2. Samples were taken at regular intervals and analyzed by high performance liquid chromatography (HPLC). After 24 hours, the gels were collected and incubated with fresh medium. The results are shown in Table VI.

9 58941

Table VI

Activating effect of nutrients on the pseudo-crystal fermentation of cortisol to prednisolone using A. simplex immobilized on a calcium alginate gel.

Medium Conversion rate (=% prednisolone formed in 5 hours) in incubation No. 1 2 3 4 5 0.5% Peptone 11 23 38 45 53 0.2% Glucose H 2 O 22 17 15 12 11

Example 5 In this experiment, phenylacetic acid was cleaved from penicillin G with Escherichia coli immobilized on a calcium alginate gel.

E. coli was grown for 36 hours in medium containing 3% corn dissolution water, 0.15% ammonium sulfate and 0.4% phenylacetic acid; pH = 6.5. Bacteria were recovered by centrifugation. The bacteria were immobilized on 2% calcium alginate (bacterial content 15%). The obtained bacterial gel was used in the Akti well-being experiment using different media. After 24 hours of activation, penicillin acylase activity was measured (by titration), after which the activation was continued and determined qualitatively by high performance liquid chromatography (HPLC). The results are shown in Table VII.

Table VIi

Activation- Initial activity medium after 0123 days H 2 O 100 75 140 0 0.1% phenyl-100-50-acetic acid 0.5% Peptone 100 140 30 0 0.5% Peptone + 100 840 1100 1900 0.1% phenylacetic acid a) All activation media contained 0.05 M CaCl 2.

b) Activity was determined by titration at pH 7.5. The titration vessel contained 0.25 g of E. coli gel suspended in 3 ml of 0.05 M CaCl 2 + 5 mH penicillin G.

The activity of the fresh gel is 100%.

5 8 9 A 1 10

Example 6 In this example, the conversion of cortexolone to cortisol by immobilized Curvularia lunata was studied.

C. lunata spores were immobilized on a 3% calcium alginate gel (10,000 spores / ml gel). The gel was incubated for 24 hours in medium containing 1% corn leach water, 0.5% glucose, 0.05 M CaCl 2, pH 4.0. In this case, the spores germinated and grew to form an immobilized network structure.

Conversion experiments were performed on C. lunata as follows: 1 g of alginate gel was suspended in 25 ml of medium containing, in addition to 0.01 mM cortexolone, 2% methanol and 0.05 M CaCl 2, 0.5% peptone + 0.2% glucose , pH 4, or 1% corn dissolution water + 0.5% glucose. Samples were taken at regular intervals and analyzed by high performance liquid chromatography (HPLC).

After 48 hours, the gel was filtered off and a new incubation was performed with fresh medium. The results are shown in Table VIII.

Table VIII

8.) Medium Steroid-116 hydroxylase activity (=% cortisol formed in 24 hours) in various nutrient solutions in incubation No 12 3 1% Maize leaching water + 65 75 81 0,5% Glucose 0,5% Peptone + 59 23 - 0, 2% Glucose H 2 O 67 23 24 a) All media contained 0.1 mM cortexolone, 2% methanol, and 0.05 M CaCl 2.

58941 For use in the preparation of immobilized radioactive substances. The uptake of the compound is specifically designed for the activation of iminobilization of microorganisms using ΔΙ-dehydrogenation or 11β-hydroxylation with steroids and transformation with benzylpenicillin.

Under the present invention, microorganisms with iminobilization are used as a catalyst (Biotechnol. Bioeng. 17, 1797-1804 (1975); J. Appl. Chem. Biotechnol. 25, 115-141 (1975); Biotechnol. Bioeng. 12, 19 -27 (1970) The use of the word function as an iminobilized enzyme (FEBS Lett. 62. (supplement) E 80 - E 90 (1976)), which can be used in a continuous manner and is controlled under the control of the enzymes and is not In addition, the immobilized microorganisms are treated with an additional form, the enzymes and the separation of the enzymes in the boron, the enzymes being present in a stable manner, and in the case of a "solid". In addition, the iminobilized microorganisms can be treated with a catalyst, and the reaction can be eliminated.

In the context of the transformation process, drift of the transformation process is exacerbated by the activity of relatively small amounts of microorganisms. This problem has been reported in the presence of microorganisms.

In this case, the uptake process is specifically and specifically designed to be immobilized on a cell catalyst, which may be activated in situ by immobilizing enzymatic activities.

Lamp substrate for transformation and active activation with uptake can be used for 1) steroids, special corticosteroids, exempted cortisol

2) antibiotic, penicillin G

Activation of the domestic system, including corticosteroid transformation with cortisol N-dehydrogen-prednisolone. The reaction can be catalyzed by polyacrylamide in the presence of Corynebacterium simplex (Arthrobacter simplex).

12 5 894 1

Bland transformations are performed for activation with uptake of 1) Δ ^ -dehydrogenation. Införandet av en dubbelbindning i steroidmolekylens 1,2-ställning. Example: Δ

2) llg-hydroxylering. The 11B-position of the hydroxyl group and the steroid molecule. Exempel: 113-hydroxylation of cortexolone and derivative, exempted cortexolone to cortisol.

3) Penicillin G Transformation. Transformation of benzylpenicillin (penicillin G) to 6-aminopenicillans.

Lämpliga organismer is used for the preparation of samples containing up to 1) Arthrobacter simplex (även kallad Corynebacterium simplex, exempelvis ATCC 6946). This organism can be used for N-dehydrogenation.

2) Curvularia Lunata, exempelvis ATCC 12017. The organism is known to have Ιΐβ-hydroxylering.

3) Escherichia coli, exempted from ATCC 9637. This organism is derived from the 6-aminopenicillans species of penicillin G.

The microorganisms are immobilized on a warm, polyacrylamide, agar (2.5-15% v / v) or calcium alginate (1-5%).

Ändamälet med föreliggande uppfinning är attäkkomma et för-farand förhindrande av försvagningen av aktiviten av immo-biliserade levande microorganizmo oce höja hivja activities vid upp-repad ellin kontinuerlig användning. In particular, the uptake of the compound is carried out in addition to the activation of the immobilized system of microorganisms in the form of A1-dehydrogenation or of the addition of a steroid and the transformation of a benzylpenicillin to a compound of the same nature. In addition, the medium, immobilized microorganisms and substrates for transformation, for peptone, glycos or bleaching for peptone and glycos, for example 0.1-1.0% (v / v). Peptone in the form was 0.1-1.0% (v / v). The result is obtained with 0.1% (v / v) peptone and a column of 0.2% (v / v) glycos. The process temperature is 20-30 ° C and the process temperature is below the aerobic temperature.

The application of this Regulation does not affect the nature of the exemptions.

Exerapel 1 I performed an experiment studying a corticosteroid Transformations-system cortisol N-dehydrogenase prednisolone. Reaction catalysts with polyacrylamides in the form of Arthrobacter simplex (Kallas även Corynebacterium simplex).

A. simplexceller odlades i et 0,25%: igste extract extract. The Δ 2 -dehydrogenase activity induced the genome to form cortisol for about 12 times in the case of centrifugation at 10,000 g. The cell (5 g of water) is suspended in 20 ml of 0.1 M Tris-HCl buffer, pH 7.5 and treated with 25 ml of water to give 7.13 g of acrylamide and 0.38 g of Ν, β-methylene. bis-acrylamide. Blandningen is also used for laminating polymerization rollers (which are separated by a glass glaze of 20 x 20 x 0.2 cm, separated by a distance of 2 mm from a latex rotor) and a catalyst tower, potassium persulphate (50 mg) and tetramethylated diamine mg) was added to water (1 ml). This genome of the genome is suspended and polymerized for 2 minutes. The poly-acrylic plate is fragmented into a solid and yellow bead (0.2 mm base) with a three-buffer and three-fold gradient at -20 ° C, which can be stable at low temperatures.

The 3-ketosteroid-N-dehydrogenase activity of the immobilized A. simplex is analyzed spectrophotometrically and of its products, prednisolone is identified by flash chromatography. 40% of the dehydrogenase activity is present during immobilization (all of which are bacteria that are immobilized and in which the bacteria are present during incubation). Initial experiments were performed on 14,58941 emellers, which are relatively active in the field, and the present cortisol is transformed by the preparation of the dosage form and compensates for the presence of an unaffected genome of 4-electron-derived artifacts. . Däremot studerades den stabililiserande efekten av olika näringsmedel och salter. The results are set out in Table I and II. The medium is wattened or buffered with activity.

The medium of the peptone and glycosylated dermet is itself active after the action of the target until the active substance is present. 0.5%: igtpeptonmedium and 0.1%: igtpepton + 0.2%: igtglykosmedium utvaldes för fortsatt Studium och experi- utet utter fördes med upprepade satsvisa tranformationer. The result is that the starting material is effective and in Table III the result is 0.5% of peptonmedium. The transformation capacity is determined to be 100% or less, and the transformation capacity is determined to be 100% after 18 hours. The transformation capacity can be about 0.5 g of steroid / dose / g of gel (weight).

Exempel 2

Preliminära försöka har visat, att den s.k. pseudo-crystalline fermentation techniques and the presence of A. simplex. Cortisol is added in a mixture (3.6 g / l), which is further mixed with the medium and transformed into a solid medium and the mixture is mixed with a mixture of medium and medium. Prednisol-lon products are utilized when the genome is separated by genomic filtration, after which this granular granule can be obtained. In this case, the technician can reduce the media volume to the redundancy and the other account. Incubate 1 g of acrylamide with 10% A. simplex in 20 ml of medium. This includes 181 mg of cortisol, 5% methanol and 0.5% peptone + 0.2% glycos. The procedure is followed by analysis and analysis by high performance chromatography (HPLC). After 24 hours, the genome was filtered and incubated with medium. The results are shown in Table I.

Table I

58941

The active effect of the pseudo-crystalline fermentation of cortisol to prednisolone on the preparation of poly-acrylamide immobilized A. simplex.

iMedium Transformation rate (=% of prednisolone in 5 cases) with incubation No. 1 2 3 4 5 0.5% Peptone 38 50 67 77 77 0.2% Glycos H2C 32 21 20 15 13

Table II

Activated effects of nursing, buffering and buffering with S-ketosteroid-N-dehydrogenase activity in A. simplex are immobilized in polyacrylamides.

Medium3 Initial transformationshastighet; (%) efter 0 2 6 10 16 dagar

Peptone 0.5% 100 460 500 650 530

Glycos 0.2% 100 210 170 110 90 K 2 HPO 4, 0.1 M, pH 7.0 100 70 50 60 60

Tris-HCl, 0.05M, pH 7.0 100 100 70 70 30 (k2HPO4, 0.1M, pH 7.0 jznCl2, FeCl2 100 50 25 15 10 (k2HPO4, 0.1M, pH 7.0 (CoCl2, MgSO 4 100 40 40 0 0 iK ~ HPO ,, 0.1M, pH 7.0 „100 160 130 80 90

MgCl2, CaCl2 (K2HPO4, 0.1M, pH 7.0 jPeptone 0.5% <Λ „100 560 650 600 550 IGly.kos 0.2% ($ HqCl2t CaCl2 H2O 100 60 40 40 20 a) halogenated organic salt MgCl2 , ZnCl 2, CoCl 2, FeCl 2, CaCl 2 and MnSO 4 are 1 mM, b) the activity is prepared by gel preparation to 100%.

16 58941 A. Simplex gel (0.5 g) was incubated in 9.0 ml of medium, mixed with 0.5 ml of 20 mM cortisol in methanol. The suspension is stirred and rotated at 25 ° C and then diluted with medium cortisol in medium medium at 48 ° C. After this, an intervening interval was performed on the gel genome by filtration, analysis and analysis of N-dehydrogenase activities.

Table III

Activated effect of peptone / glycosyl. 3-Ketosteroid-N-dehydrogen activity in A. simplex3-immobilized polyacrylamides

Medium Initial transformationshastighet ^ (%) efter 0 2 6 10 16 dagar

Peptone 1% 100 290 320 380 550 0.5% 100 460 500 650 530 0.1% 100 200 225 165 120 0.01% 100 125 30 0 0 ^ Peptone 0.1%. P '100 250 225 240 350 (Glycos 0.2% {Peptone 0.01% F 100 110 55 0 0

Glycos 0.2%

Glycos 0,2% 100 210 170 110 90 (a) in accordance with Table II.

b) the activity of the gel preparation is 100%.

Table IV

Upprepad satsvis Transformation of cortisol to prednisolone Försök Transformationskapacitet Tid för 100%: igvversion (nr) (mg prednisolone / h / g gel ...

(vätvikt)) 1 3.1 17.4 2 5.0 10.8 3 13.5 4.0 ~ 4 iin n 5 26.3 2.1 _6_27.1_2.0 7 27.1 2.0 8 29 .6 1.8 9 30.8 1.8 10 31.7 1.7 17 5 89 41 By suspending A. simplex (2.0 g) in 285 ml of 0.5% peptone, pH 7.0 + 15 ml of 20 mM cortisol in methanol. Conversion of the Gang is performed spectrophotometrically and up to 100% of the conversion of prednisolone to a gel and incubation with a medium of cortisol. Hela försöket tog 4 dagar i anspräk.

Exemoel 3 _______-, ..

In this steroid test, the microorganism A. simplex immobilized in calcium alginate. Immobilization was performed according to the following conditions: A. simplex (0.5 g of water), added in Experiment 1, suspended in 9.5 ml of 2.1% sodium alginate solution. Density of 0.1 M CaCl 2, color spherical bacterial particles (diameter 2 mm). Particles were treated with 0.1 M CaCl 2 for 20 h. After further activation of the gel and steroid-free activity, the mixture was stirred. The results are shown in Table V.

Table V

Effect of näringsämnen pä 3-ketosteroid-A dehydrogenase activity on A. simplex immobilized with calcium alginate

Medium Initial transformation rate (%) efter 0 1 2 4 6 11 of 0,5% Peptone 100 280 330 460 480 670 0,5% Peptone 100 260 310 500 560 930 0,2% Glycos H2O 100 90 70 35 20 a) Activities hos nypreparerad gel is 100%.

Exempel 4 I performed an experiment by immobilizing A. simplex with 2% calcium alginate (diameter 1 mm). Incubate 1 g of alginate with 10% A. simplex incubate with 20 ml of medium. To this was added 181 mg of cortisol, 5% methanol and 0.5% peptone + 0.2% glycos and 0.05 M CaCl 2. This was followed by analysis and analysis by high performance chromatography (HPLC). After 24 hours of filtration through incubation and incubation with medium medium. The results are shown in Table VI.

18 58941

Table VI

The active effect of näringsämnen vid pseudocrystalline enzymes on cortisol to prednisolone with A. simplex is immobilized and caclium alginate.

Medium Transformationshastighet (=% bildad prednisolone in 5 timmar) under incubation no 1 2 3 4 5 0,5% Peptone 11 23 38 45 53 0,2% Glycos H2O 22 17 15 12 11

Exempel 5 I detta experiment spjälkades fenylättiksyra frän penicillin G med Eschericia coli immobilized in calcium alginate.

E. coli yields 36 timmars and 3% corn steep liquor, 0.15% ammonium sulfate and 0.4% phenylthix; pH = 6.5. Bacterierna skördades genome centrifugering. Bacterierna immobilized in 2% calcium alginate (15% bacterial). The bacteria were treated with an experimental experiment, which was mediated. After 24 hours of activation without penicillinase activity (titrimetry), the activation is carried out and verified by qualitative high performance chromatography (HPLC). The results are shown in Table VII.

Table VII

Activations- Initial activity after medium 0 1 23 for H20 100 75 140 0 0,1% phenyl- 100 - 50 - acetic acid 0.5% Peptone 100 140 30 0 0,5% Pepton + 100 840 1100 1900 0,1% phenylacetyl a) Samtliga aktiveringsraedier innehöll 0,05 M CaCl2 · b) Aktiviteten mättes titrimetriskt vid pH 7,5. The titration supernatant is suspended in 0.25 g of E. coli gel and 3 ml of 0.05 M CaCl2 + 5 mM penicillin G.

The activity of the gel gel is 100%.

19 58941

Exempel 6 I have been exempted from studying the cortexolone to cortisol with immobilized Curvularia Lunata.

Sporer av C. Immunize with 3% calcium alginate (10,000 Sporer / ml gel). The gel was incubated under 24 times in medium medium with 1% corn steep liquor, 0.5% glycos, 0.05 M CaCl2, pH 4.0. Därvid grodde sporerna och Växte ut till ett immobilerat nätverk.

The reaction medium is immobilized C. The redundant solution is prepared as follows: 1 g of alginate suspension in 25 ml of medium containing 0.1 μM of cortexolone, 2% of methanol, 0.05 M of CaCl 2 in 0.5% of peptone + 0.2% of glycos, pH 4, or 1% corn steep liquor + 0.5% glycos.

This was followed by analysis and analysis by high performance chromatography (HPLC). After 48 hours of filtration, the gel was incubated with a medium medium. The results are shown in Table VIII.

Table VIII

3}

Medium 'Steroid-11B-hydroxylasactivity (=% of cortisol after 24 hours) was mediated under incubation No. 12 3 1% corn steep liquor + 65 75 81 0.5% Glycos 0.5% Peptone + 59 23 - 0.2 % Glycos H2O 67 23 24 a) Samtliga medier innehöll 0.1 mM cortexolone, 2% methanol and 0.05 M CaCl2.