IE45676B1 - Immobilized microorganisms - Google Patents

Abstract

To activate immobilised living microorganisms for the conversion of steroids, antibiotics and other compounds, peptone, glucose or a mixture of peptone and glucose, advantageously in a concentration of 0.1 to 1.0 % (wt./vol.), is added to the reaction mixture. The microorganism, for example Arthrobacter simplex, can be trapped in a polyacrylamide gel. In the application of the process, for example, cortisol or a derivative thereof is converted into prednisolone or a derivative thereof or a steroid is hydroxylated in the 11 or 16 position or dehydrogenated in the 1 position.

Description

This invention relates to a method of activating immobilized microorganisms. Particularly this invention relates to a method for activating immobilized living microorganisms which may be applied to transformations of steroids, antibiotics and other compounds.

Immobilized microorganisms have attracted an increasing interest as catalysts in the last few years (see e.g. Biotechnol. Bioeng. 17, 1979-1804 (1975); J. Appl. Chem. Biotechnol. 25, 115-141 (1975); Biotechnol. Bioeng. 12, 19-27 (1970)). They exhibit the same operational advantages as those inherent in immobilized enzymes (PEBS Lett. 62. (supplement) E 80 E 90 (1976)); they are reusable, they are well suited for continuous operation under controlled conditions and further, when entrapped in a polymer, they are comparatively resistant to microbial attack, since they are protected by the polymer. Immobilized microorganisms offer the additional advantage that tedious and costly enzyme isolation is obviated, that the enzyme is more usually stable due to its localization in its natural environment, and that there is usually no co-factor requirement. Thus, provided competing reactions can be eliminated, immobilized microorganisms are very promising catalysts. However, in continuous or repeated batch operation of the transformation process, the activity declines rather rapidly when immobilized microorganisms 43676 are used. This problem has hitherto not been satisfactorily solved in connection with immobilized living microorganisms.

The present invention provides a method for activating immobilized living microorganisms which comprises bringing peptone, glucose or a mixture of peptone and glucose into contact with immobilized microorganisms which are entrapped in a polymer.

By means of the method of the present invention, it is possible to activate the immobilized enzymic activity in situ, something which is only possible in immobilized living whole cell catalysts.

The present invention also provides a method of carrying out a microbiological transformation in which a substrate is treated with immobilized microorganisms entrapped in a polymer, in the presence of peptone, glucose or a mixture of peptone and glucose.

Suitable substrates for transformations wherein the activation according to the invention can be applied are 1) steroids, particularly corticosteroids, for instance cortisol, 2) antibiotics, such as penicillin G, 3) other compounds such as a. alkaloids, e.g. solasodine, tomatidine b. organic acids, e.g. N-acetyl-L~amino acids c. carbohydrates, e.g. glucose, sorbose 43676 <3. purine bases, nucleosides and nucleotides; e.g. 6-chloropurine, 6-chloropurine riboside.

The activation can be applied to several systems, such as the corticosteroid transformation cortisol Δ^·-dehydrogenase, prednisolone. The reaction can be catalyzed ty polyacrylamide entrapped Corynebacterium simplex (Arthrobacter simplex).

Among transformations suitable for the activation according to the invention, particular examples are:1) Δ -dehydrogenation. Incorporation of a double bond in the 1,2-position of the steroid molecule. Example: Δ^-dehydrogenation of Cortisol and derivatives of Cortisol e.g. Cortisol to Prednisolone, 9a-Fluoro-16p-methylcortisol to 9a-Fluoro-16p-methylprednisolone (Betamethasone), 9a-Fluoro-16a-methylcortisol to 9a-Fluoro-16a-methylprednisolone (Dexamethasone), 6a-M£thylcortisol to 6«-Methylprednisolone 6a-Fluoro-16a-methylcortisol to 6a-Fluoro-16a-methylprednisolone (Paramethasone), 9a-Fluoro-16a-hydroxycortisol to 9a-Fluoro-16ahydrexyprednisolone (Triamcinolone), 9a-Pluorocortisol to 9a-Fluoroprednisolone, 6a,9«-Difluoro-16a,17a-isopropylidendioxycortisol to 6a,9a-Difluoro-16tt,17a-isopropylidendioxyprednisolone. 6 76 2, lla-hydroxylation. Incorporation of a hydroxy group in the lla-position of the steroid molecule. Example: transformation of cortexolone and derivatives of cortexolone to lla-hydroxy cortexolone {epicortisol) and derivatives of epicortisol. 3) Ιΐβ-hydroxylation. Incorporation of a hydroxy group in the Ιΐβ-position of the steroid molecule. Example: Ιΐβ-Hydroxylation of Cortexolone and derivatives of Cortexolone e.g. Cortexolone to Cortisol, 9a-Fluoro-16p-methylcortexolone to 9a-Fluoro-16pmethylcortisol, 9a-Fluoro-16a-methylcortexolone to 9a-Pluoro-16amethylcortisol, 9a-Fluorocortexolone to 9a-Fluorocortisol, 6a-Fluoro-16a-methylcortexolone to 6a-Fluoro-16amethylcortisol, 6a-Methylcortexolone to 6a-Methylcortisol, 6&,9a-Difluorocortexolone to 6a,9a~Difluoroeortisol. 6a,9a-Difluoro-16a,17a-isopropylidendioxycortexo~ lone to 6a,9a-Difluoro-16a,17a-isopropylidendioxycortisol. 4) 16a-hydroxylation. Incorporation of a hydroxy group in the 16a-position of the steroid molecule. Example: 16a-Hydroxylation of Cortisol and derivatives of Cortisol, e.g. Cortisol to 16a-Hydroxycortisol, 6a-Fluorocortisol to 6a-Fluoro-lSa-hydroxycortisol, 9a-3?luorocortisol to 9tt-Fluoro-16a-hydroxycortisol, 4S676 6α,9a-Difluorocortisol to 6a,9a-Difluoro-16a-hydroxycortisol. .) penicillin G transformation. Transformation of benzylpenicillin (penicillin G) to 6-aminopenicillanic acid, 6) Side-chain elimination. Example: transformation of sitosterol to A^-androstene-3,17-dione, cholesterol to A^,^l'-androstadiene-3,17-dione, 7) 12a-hydroxy elimination. Example: transformation of cholic acid to chenodesoxycholic acid.

Suitable organisms which can be used in connection with this invention are 1) Arthrobacter simplex (also called Corynebacterium simplex,· for instance ATCC 6946). This organism can be used for Δ^-dehydrogenation. 2) Rhizoaus nigricans (also called Khizopus stolinifer. for instance ATCC 6227 b). This organism can be used for all ά-hydroxylation. 3) Curvularia lunata. for instance ATCC 12017. This organism can be used for Ιΐβ-hydroxylation. 4) Escherichia coli. for instance ATCC 9637. This organism can be used for 6-aminopenicillanic acid production from penicillin G.

) Aspergillus niger. This organism can be used for 16a-hydroxylation. 6) Streptomyces venezuelae. This organism can be used for isomerization of glucose. 7, Brevibacterium ammonioqenes. This organism can be used for transformation of purine bases, nucleosides and nucleotides.

The microorganisms used in the present invention 5 are immobilized in a suitable polymer carrier, such as polyacrylamide gel, agar (2.5-15% w/v), collagen (cell : collagen 1:1) or calcium alginate (1-5%).

The activation method of the present invention makes it possible to prevent the decline of activity of •10 immobilized living microorganisms and even to enhance the activity by repeated or continuous operation.

Preferably the peptone, glucose or mixture thereof for the activation should be used in a concentration range of 0.1 to 1.0 g/100 ml. As an example, about 0.1 g/100 ml peptone and about 0.2 g/100 ml glucose in combination gives good results, as does about 0.5 g/100 ml peptone. The temperature of the process should preferably be 20 to 30°C and it should preferably be conducted under aerobic conditions.

The following Examples serve to illustrate the present invention.

Example 1 In this Example, the system studied was the important corticosteroid transformation cortisol Δ^-dehydrogenase^ prednisolone. The reaction was catalyzed by polyacrylamide-entrapped Arthrobacter simplex, (also called Corynebacterium simplex). 43676 A. simplex cells were grown in a medium of 0.25% yeast extract, the A^-dehydrogenase activity being induced by addition of cortisol to the culture 12 hours I prior to harvesting by continuous centrifugation at 5 10,000 x g. The cells (5 g wet weight) were suspended in 20 ml of ice-cold O.l M Tris-HCl buffer, pH 7.5 and mixed with 25 ml ice-cold aqueous monomer solution containing 7.13 g acrylamide and 0.38 g N.N^-methylenebis-acrylamide. The mixture was poured into a sandwich10 like polymerisation vessel (made of two glass plates x 20 x 0.2 cm, spaced 2 mm apart with a piece of latex tubing) and the catalysts potassium persulphate (50 mg) and tetramethylenediamine (100 mg) were added in water (1 ml). Nitrogen gas was bubbled through the suspension and the polymerisation started within 2 minutes. The polyacrylamide gel sheet was fragmented in a blender and the gel granules (average size 0.2 mm) were washed extensively with Tris buffer and then stored at -20°c, at which temperature the preparation was stable for several months.

The 3-ketosteroid- A^-dehydrogenase activity of the immobilized A. simplex was conveniently assayed by a spectrophotometric procedure and the sole product, prednisolone, was identified by thin-layer chromotography.

Approximately 40% of the dehydrogenase activity was retained during the immobilization procedure (all bacteria added were immobilized and no release of bacteria was observed during incubations). Initial experiments revealed, however, that the activity declined rather rapidly on repeated batchwise conversions of high loads of cortisol and this could only be compensated for to a limited degree by addition of the artificial electron acceptor menadione. Instead the stabilizing influence of various nutrients and salts was investigated; the results are given in the Tables I and II. In media consisting of water or buffer the activity decreased, whereas in peptone-and glucose-containing media the activity was not only preserved but also dramatically increased to several times that of the original activity. The 0.5% peptone medium and the 0.1% peptone + 0.2% glucose medium were selected for further study and an experiment with repeated batchwise transformation was conducted. Both media were approximately equally efficient and in Table III the results obtained with the 0.5% peptone medium are depicted. As can be seen, the transformation capacity increased remarkably with each run, thus while in the first batch 100% transformation was obtained after 18 hours, the last batch was completed in less than 2 hours. The transformation capacity at the end of the experiment was approximately 0.5 g steroid/day/g gel (wet weight).

Example 2 Recent preliminary experiments show that the so-called pseudo-crystallofermentation technique is applicable also to entrapped A. simplex. Cortisol was thus added in an amount (3.6 g/1) by far exceeding its solubility in the medium and was completely converted at approximately the same rate as in experiments with dissolved cortisol. The product’ prednisolone, which precipitated out,could be isolated simply by filtration after the rather dense gel granules had been allowed to settle. This technique allows reduction of media volumes by orders o'f magnitude and thus also of nutrients.

Table I Activating effect of nutrients, buffers and salts on 3ketosteroid-^-dehydrogenase activity of immobilized A, simplex Medium Initial transformation rate (%) after: 0 2 6 10 16 days Peptone 0.5% (i.e. g/100 ml) 100 460 500 650 530 10 Glucose 0.2% (i.e. g/100 ml) 100 210 170 110 90 KjHPO^, O.lM, pH 7.0 100 70 50 60 60 Tris-HCl, O.O5M, pH 7. (ΚΗΡΟ, , O.lM, pH 7.0 j 2 4 (ZnCl2< FeCl2 (X2HP04, O.lM, pH 7.0 (CoCl2, MgSO^ (Κ,ΗΡΟ, , O.lM, Ph 7.0 (MgCl2, CaCl2 (K2HP°4, O.lM, pH 7.0 (Peptone 0.5% ( ( (i.e. g/100 ml) ( (Glucose 0.2% ( ( (i.e. g/100 ml) (MgCl2, CaCl2 ,0 100 100 70 - 70 30 15 100 50 25 15 10 100 40 40 0 0 20 100 160 130 80 90 25 100 560 650 600 550 H20 100 60 40 40 20 a) the concentration of the inorganic salts MgCl^ZnCl^, CoCl2, PeCl2, CaCl2 and MnSO^ was ImM. b) activity of freshly prepared gel is set at 100%.

A, simplex gel (0‘.5 g) was incubated in 9.0 ml of medium as indicated and 0.5 ml 20 mM cortisol (methanol) was added. The suspension was shaken on a rotary shaker at 25°C and at 48 h intervals the medium was replaced by fresh cortisol-containing medium. At the intervals indicated the gel was filtered off, washed and assayed for ^-dehydrogenase activity.

Table XI Activating effect of peptone/glucose on 3-ketosteroid10 Δ^-dehydrogenase activity of immobilized A.simplex3^ Medium Initial transformation rate (%) after 0 2 6 10 16 days Peptone 1% (i.e. g/100 ml) 100 290 320 380 550 15 0.5% 100 460 500 650 530 0.1% 100 200 225 165 120 0.01% 100 125 30 0 0 (Peptone 0.1% ( (Glucose 0.2% 100 250 225 240 350 20 (Peptone 0.01% 11 ( (Glucose 0.2% loo 110 55 0 0 Glucose 0.2% 100 210 170 110 90 a) experimental conditions are as given in Table I. b) activity of freshly prepared gel is set at 100%.

Table III Repeated batch-wise transformation of cortisol to prednisolone.

Run (no.) Transformation capacity (mg prednisolone/hour/g gel (wet weight)) Time for 100% conversion (h) 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, pH 7.0 + 15 ml of 20 mM cortisol (methanol). The progress of the transformation was followed spectophotometrically and when 100% conversion to prednisolone was reached the gel was washed and again incubated with fresh cortisol-containing medium. The whole experiment lasted 4 days.

Claims (10)

1. A method for activating immobilized living microorganisms which comprises bringing peptone, glucose or a mixture of peptone and glucose into contact with immobilized microorganisms which are entrapped in a

2. A method according to claim 1, wherein the medium containing the immobilized microorganisms contains 0.1 to 1.0 g/100 ml peptone or glucose or mixture thereof. 10

3. A method according to claim 1 or 2, wherein the polymer is a polyacrylamide gel.

4. A method according to any one of claims 1 to 3, wherein the immobilized microorganism is Arthrobacter simplex. _1'5 5. A method according to claim 1 wherein the immobilized organism is Arthrobacter simplex entrapped in a polyacrylamide gel.

5. Mixture of peptone and glucose. 18. A method according to claim 17 wherein the medium containing the immobilized microorganisms contains about 0.5 g/100 ml peptone or a mixture of about 0.1 g/100 ml peptone and about 0.2 g/100 ml glucose. 5 polymer.

6. A method according to claim 5 wherein the medium containing the immobilized microorganism 20 contains about 0.5 g/100 ml peptone or a mixture of about 0.1 g/100 ml peptone and about 0.2 g/100 ml glucose.

7. A method for activating immobilized microorganisms according to claim 1, substantially as hereinbefore described with reference to either one of the Examples.

8. Activated microorganisms obtained by a method as claimed in any one of claims 1 to 4 and 7.

9. Activated microorganisms obtained by a method as claimed in claim 5 or 6. 10. A method of carrying out a microbiological transformation in which a substrate is treated with immobilized microorganisms entrapped in a polymer, in the presence of peptone, glucose or a mixture of peptone and glucose. 11. A method according to claim 10, wherein the medium containing the immobilized microorganisms contains 0.1 to 1.0g/100 ml peptone or glucose or mixture thereof. 12. A method according to claim 10 or 11, wherein the polymer is a polyacrylamide gel. 13. A method according to any one of claims 10 to 12, wherein the immobilized microorganism is Arthrobacter simplex. 14. A method according to any one of claims 10 to 13 wherein the substrate is cortisol or a derivative thereof and the substrate is transformed to prednisolone or a derivative thereof. 15. A method according to any one of claims 10 to 13 wherein the substrate is a steroid, which is transformed into a 11- or 16-hydroxy-steroid. 16. A method according to any one of claims 10 to 13, wherein the substrate is subjected to A^dehydrogenation. 48676 17. A method according to claim lo wherein cortisol is transformed to prednisolone by treatment with Arthrobacter simplex immobilized in a polyacrylamide gel, in the presence of peptone, glucose or a

10. 19. A method for carrying out a microbiological transformation according to claim 10 substantially as hereinbefore described with reference to either one of the Examples.