CH634348A5 - Process for the activation of immobilised microorganisms - Google Patents

Description

The invention relates to a method for activating immobilized microorganisms. In particular, the invention relates to a method for activating immobilized living microorganisms for the conversion of steroids, antibiotics and other compounds.

Immobilized microorganisms have found increasing interest as catalysts in recent years (Biotech-nol. Bioeng. 17, 1797-1804 (1975); J. Appi. Chem. Biotechnol. 25, 115-141 (1975); Biotechnol. Bioeng. 12 , 19-27 [1970]). They show the same procedural advantages as immobilized enzymes show (FEBS Lett. 62. (Supplement) E 80 - E 90 ([1976]); they are reusable, they are very well suited for continuous operation under controlled conditions, and further they are relatively resistant to microbial attack because they are protected by the polymer. Immobilized microorganisms offer the additional advantage that they avoid tedious and costly enzyme isolation, that the enzyme is more stable as a result of localization in its "natural environment" and that usually it is not Cofactor is required. Therefore, immobilized microorganisms are promising catalysts, provided that competitive reactions can be eliminated. However, in the case of continuous or repeated batch operations of transformation processes, the activity drops rapidly when immobilized microorganisms are used. This is related to The problem of immobilized living microorganisms has not yet been satisfactorily solved.

In the present invention, in particular, a property is considered that is inherent in immobilized whole-cell living catalysts, namely the possibility of in-situ activation of the immobilized enzyme activity.

The inventive method for activating immobilized living microorganisms for the conversion of steroids, antibiotics and other compounds is characterized in the preceding claim 1.

Suitable substrates for conversions in which the activation according to the method according to the invention can be used

1. steroids, especially corticosteroids, e.g. Cortisol,

2. antibiotics, such as penicillin G,

з. other compounds, e.g. a) alkaloids, e.g. Solasodine, tomatidine, b) organic acids, e.g. N-acetyl-L-amino acids, c) carbohydrates, e.g. Glucose, sorbose, and d) purine bases, nucleosides and nucleotides, e.g. 6-chloropurine and 6-chloropurine riboside.

Activation can be applied to various systems, such as corticosteroid conversion

Cortisol A'-dehydrogenase prednisolone

The reaction can be catalyzed by Corynebacterium simplex (Arthrobacter simplex) enclosed in polyacrylamide.

Among the conversions suitable for activation by the process according to the invention, the following may be mentioned as examples:

1. A'-dehydration. Introduction of a double bond in the 1,2 position of the steroid molecule. Example: À'-dehydration of cortisol and derivatives of cortisol, e.g. Cortisol to pred-nisols, 9a-fluoro-16ß-methylcortisol to 9a-fluoro-16ß-methyl-prednisolone (betametasone), 9a-fluoro-l 6a-methylcortisol to 9oc-fluoro-16a-methylprednisolone (dexametasone), 6a-methyl -cortisol to 6a-methylprednisolone, 6a-fluoro-16a-methylcortisol. to 6a-fluoro-16a-methylprednisolone (parametasone), 9a-fluoro-16a-hydroxycortisol to 9a-fluoro-16a-hydroxyprednisolone (triamcinolone), 9a-fluorocortisol to 9 <x-fluoroprednisolone, 6a, 9a-difluoro-16a, 17a -Isopropylidendioxycortisol to 6a, 9a-difluoro-16a, 17 a-isopropylidendioxyprednisolon.

2.1 la-hydroxylation. Introduction of a hydroxy group in the 1 la position of the steroid molecule. Example: Conversion of cortexolone and cortexolone derivatives into 1 la-hydroxycortexo-lon (epicortisol) and derivatives thereof.

3.1 β-hydroxylation. Introduction of a hydroxyl group into the 1β position of the steroid molecule. Example: 1β-hydroxylation of cortexolone and cortexolone derivatives, e.g. Cortexolone to cortisol, 9a-fluoro-16ß-methylcortexolone to 9a-fluoro-16ß-methylcortisol, 9a-fluoro-16ß-methylcortexolone to 9a-fluoro-16a-methylcortisol, 9a-fluorocortexolone to 9a-fluorocortisol, 6a-fluoro-16a- methylcortexolon to 6a-fluoro-16a-methylcorti-sol, 6a-methylcortexolon to 6a-methylcortisol, 6a, 9a-difluoro-cortexolon to 6a, 9a-difluorcortisol, 6a, 9a-difluor-16a, 17a-iso-propxolidonoxa 9a-difluoro-16a, 17a-isopropylidendioxycortisol.

4.16a hydroxylation. Introduction of a hydroxyl group in the 16a position of the steroid molecule. Example: 16a-hydroxylation of cortisol and cortisol derivatives, e.g. Cortisol to 16a-hydroxycortisol, 6a-fluorocortisol to 6a-fluoro-16a-hydro-xycortisol, 9a-fluorocortisol to 9a-fluoro-16a-hydroxycortisol, 6a, 9a-difluorocortisol to 6a, 9a-difluoro-16a-hydroxycortisol.

5. Penicillin-G conversion. Conversion of benzylpenicillin (penicillin G) to 6-aminopenicillanic acid.

6. Side chain splitting. Example: Conversion of Sito-sterol to A4-Androsten-3,17-dione, cholesterol to A1AA.ndrosta-dien-3,17-dione.

7.12a hydroxy cleavage. Example: Conversion of cholic acid to chenodeoxycholic acid.

Suitable organisms that can be used in connection with the method according to the invention are

и.a.

1. Arthrobacter simplex (also called Corynebacterium simplex, e.g. ATCC 6946). This organism can be used for A'-dehydration.

2. Rhizopus nigricans (also called Rhizopus stolinifer, e.g. ATCC 6227b). This organism can be used for 1 la hydroxylation.

3. Curvularia lunata, e.g. ATCC 12017. This organism can be used for 1β-hydroxylation.

4. Escherichia coli, e.g. ATCC 9637. This organism

2nd

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

3rd

634348

can be used for the production of 6-aminopenicillanic acid from penicillin G.

5. Aspergillus niger This organism can be used for 16a hydroxylation.

6. Streptomyces venezuelae. This organism can be used for the isomerization of glucose.

7. Brevibacterium ammoniogenes. This organism can be used to convert purine bases, nucleosides and nucleotides.

The microorganisms are immobilized in a suitable carrier, for example in polyacrylamide, agar (2.5-15% by weight), collagen (cell: collagen 1: 1) or calcium alginate (1-5%).

The present invention thus provides a method for preventing the reduction in activity of immobilized living microorganisms and even for increasing the activity in the case of repeated or continuous operation.

Preferred embodiments of the invention are explained in more detail below with the aid of examples.

example 1

In this experiment, the important corticosteroid conversion with A'-dehydrogenase cortisol - prednisolone was examined. The reaction was catalyzed by Arthrobacter simplex (also called Corynebacterium simplex), which is included in polyacrylamides.

A. simplex cells were grown in 0.25% yeast extract medium; A'-dehydrogenase activity was induced by adding cortisol to the culture; after 12 hours, the product was obtained by continuous centrifugation at 10,000 x g. The cells (5 g wet weight) were suspended in 20 ml ice-cold 0.1 M Tris-HCl buffer, pH 7.5 and with 25 ml ice-cold aqueous monomer solution containing 7.13 g acrylamide and 0.38 g N .N'-Methylbenbisacrylamid contained, mixed. The mixture was poured into a sandwich-like polymerization vessel (consisting of two glass plates 20x20x0.2, with a piece of rubber tubing spaced 2 mm apart) and the catalysts potassium persulfate (50 mg) and tetramethylethylenediamine (100 mg) were poured into water (1 ml ) added. Nitrogen was bubbled through the suspension and polymerization started within 2 minutes. The polyacrylamide gel sheet was cut in a mixer and the gel granules (average size 0.2 mm) were washed extensively with Tris buffer and then stored at -20 ° C; at this temperature the preparation was stable for several months.

The 3-ketosteroid A'-dehydrogenase activity of the immobilized A. simplex was determined in a conventional manner by a spectrophotometric method, and the sole product, prednisolone, was identified by thin layer chromatography. About 40% of the dehydrogenase activity was retained during the immobilization process (all added bacteria were immobilized and no release of bacteria was observed during incubations). Initial attempts, however, showed that the activity decreased quite rapidly with repeated batch conversions of large amounts of cortisol, and this could only be compensated for to a limited extent by adding the artificial electron acceptor menadione. Instead, the stabilizing influence of various nutrients and salts was examined. The results are shown in Tables I and II. The activity decreased in media consisting of water or buffer, while in media containing peptone and glucose, the activity was not only maintained but increased dramatically to a multiple of the original activity. The medium with 0.5% peptone and that with 0.1% peptone + 0.2% glucose was selected for further studies and a repeated batch conversion experiment was carried out. Both media were approximately equally effective and Table III shows the results obtained with the medium containing 0.5% peptone. As can be seen, the conversion capacity is increased significantly with each batch, whereas with the first batch 100% conversion was obtained after 18 hours, the last batch was completely converted in less than 2 hours. The conversion capacity at the end of the experiment was approximately 0.5 g steroid / day / gram io gel (wet weight).

Example 2

Preliminary tests recently carried out showed that the so-called pseudo-crystal fermentation technique can also be applied to included A. simplex. Cortisol was therefore added in an amount of 3.6 g per liter, which far exceeded the solubility of cortisol in the medium. Cortisol was converted at approximately the same rate as in trials with dissolved cortisol. The product, 20 prednisolone that failed, could simply be filtered off after allowing the rather dense gel granules to settle. This technique allows a reduction of the media volume by several orders of magnitude and therefore also a reduction in the nutrients.

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Table I

30 Activating effect of nutrients, buffers and salts on the 3-ketosteroid A'-dehydrogenase activity of immobilized Arthrobacter simplex

Medium3

35

Initial conversion rate (%) to

0 2 6 10 16 days

Peptone 0.5%

Glucose 0.2% 40 K2HP04.0.1 m, pH 7.0 Tris-HCl, 0.05 m, pH 7.0 K2HPO4.0.1 m, pH 7.0 ZnCh, FeCh K2HPO4.0.1 m , pH 7.0 45 COCl2, M9SO4

K2HPO4.0.1 m, pH 7.0 MgCl2, CaCl2 K2HP04.0.1 m, pH 7.0 peptone 0.5%

Glucose 0.2% MgCh.CaCk H2O

100 460 500 650 530 90

100 210 170 110 100 70 50 60

100 100 100 50 100 40

70 25 40

100 160 130

70 15 0 80

60 30

10th

0

90

50

100 560 650 600 550 100 60 40 40 20

a The concentration of the inorganic salts MgCk, ZnCb, 55 C0CI2, FeCl2, CaCk and MnS04 was 1 mmol.

b The activity of the freshly prepared gel was set to 100%.

A. simplex gel (0.5 g) was incubated in 9.0 ml of the indicated medium and 0.5 ml of 20 mmol cortisol (methanol) was added. The suspension was shaken on a rotary shaker at 25 ° C, and the medium was replaced with fresh cortisol-containing medium at 48 hour-65 den intervals. At the indicated intervals, the gel was filtered off, washed and examined for A'-dehydrogenase activity.

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Table II

Activation effect of peptone / glucose on 3-ketosteroid A'-dehydrogenase activity of immobilized Arthrobacter simplex3

Medium3 Initial conversion rate

(%)to

0

2nd

6

10th

16 days

Peptone 1%

100

290

320

380

550

Peptone 0.5%

100

460

500

650

530

Peptone 0.1%

100

200

225

165

120

Peptone 0.01%

100

125

30th

0

0

Peptone 0.1%

Glucose 0.2%

100

250

225

240

350

Peptone 0.01%

Glucose 0.2%

100

110

55

0

0

Glucose 0.2%

100

210

170

110

90

3 experimental conditions are given in Table I. b Activity of the freshly prepared gel = 100% set.

Table III

Repeated batch conversion of cortisol to prednisolone

Batch conversion capacity time for 100% conversion

5 (No.) (mg prednisolone / hour (hour)

Ig Gel wet weight)

1

3.1

17.4

2nd

5.0

10.8

3rd

13.5

4.0

4th

18.1

3.0

5

26.3

2.1

6

27.1

2.0

7

27.1

2.0

8th

29.6

1.8

9

30.8

1.8

10th

31.7

1.7

2o A. simplex gel (2.0 g) was suspended in 285 ml of a mixture of 0.5% peptone, pH 7.0, and 15 ml of 20 mmol of cortisol (methanol). The progress of the conversion was monitored spectrophotometrically, and with 100% conversion prednisolone, the gel was washed and incubated with fresh corti-25 sol-containing medium. The whole trial lasted 4 days.

Claims (7)

634348 PATENT CLAIMS 1. A method for activating immobilized living microorganisms for the conversion of steroids, antibiotics and other compounds, characterized in that peptone, glucose or a mixture of peptone and glucose is added to the reaction mixture. 2. The method according to claim 1, characterized in that cortisol or a derivative thereof is converted into prednisolone or a derivative thereof. 3. The method according to claim 1, characterized in that a steroid is hydroxylated in the 11- or 16-position. 4. The method according to claim 1, characterized in that the microorganism is Arthrobacter simplex. 5. The method according to claim 1, characterized in that a steroid is dehydrated in the 1-position. 6. The method according to claim 1, characterized in that the microorganism is enclosed in a polyacrylamide gel. 7. The method according to any one of claims 1 to 6, characterized in that peptone or glucose or a mixture of peptone and glucose in a concentration of 0.1 to 1.0% (w / v) is added to the reaction mixture.