GB1560850A

GB1560850A - Immobilized microorganisms

Info

Publication number: GB1560850A
Application number: GB28177/77A
Authority: GB
Original assignee: Fermenta AB
Current assignee: Fermenta AB
Priority date: 1976-07-06
Filing date: 1977-07-05
Publication date: 1980-02-13
Also published as: IE45676B1; FI58941B; CA1101348A; FR2357507A1; BE856358A; AT372403B; FI772012A; NL7707449A; FR2357507B1; DE2729490A1; ATA465577A; SE7607698L; CH634348A5; ES460368A1; IT1079745B; DK292677A; JPS5326383A; SE427116B; IE45676L; FI58941C

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

(54) IMMOBILIZED MICROORGANISMS (71) We, AKTIEBOLAGET FERMENTA, a Swedish Body Corporate of, Fack, 15200 Strangnas, Sweden, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- 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, 1797-1804(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 (FEBS 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 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 b. carbohydrates, e.g. glucose, sorbose d. purine bases, nucleosides and nucleotides, e.g. 6-chloropurine, 6 chloropurine riboside.

The activation can be applied to several systems, such as the corticosteroidtransformation cortisol A1 ehydro enase prednisolone. The reaction can be catalyzed by polyacrylamide entrapped Corynebacterium simplex (Arthrobacter simplex).

Among transformations suitable for the activation according to the invention, particular examples are: 1) A1-dehydrogenation. Incorporation of a double bond in the 1,2-position of the steroid molecule. Example: A1-dehydrogenation of Cortisol and derivatives of Cortisol e.g. Cortisol to Prednisolone, 9a-Fluor-16P-m ethylcortisol to 9α-Fluor-16ss-methylprednisolone (B etamethasone), 9 - Fluor - 16 - methylcortisol to 9a - Fluor - 16α methylprednisolone (Dexa- methasone), 6a - Methylcortisol to 6a - Methylprednisolone, 6a - Fluor - 16cr - methylcortisol to 6a - Fluor - 16α - methylprednisolone (Paramethasone), 9 - Fluor - 16α - hydroxicortisol to 9e - Fluor - 16α - hydroxiprednisolone (Triamcinolone), 9cr-Fluorcortisol to 9cr-Fluorprednisolone, 6α,9α-Difluor-16α,17α-isopropylidenedioxicortisol to 6a,9cr-Difluor- 16α,17α-isopropylidendioxiprednisolone.

2) 1 la-hydroxylation. Incorporation of a hydroxy group in the 1 la-position of the steroid molecule. Example: transformation of cortexolone and derivatives of cortexolone to 1 la-hydroxy cortexolone (epicortisol) and derivatives of epicortisol.

3) I l,B-hydroxylation. In corporation of a hydroxy group in the l l,B-position of the steroid molecule. Example: -Hydroxylation of Cortexolone and derivatives of Cortexolone e.g. Cortexolone to Cortisol, 9α-Fluor-16ss-methylcortexolone to 9α-Fluor-16ss-methylcortisol, 9a-Fluor-16cr-methylcortexolone to 9a-Fluor-16er-methylcortisol, 9cr- Fluorocortexol one to 9cr-Fluorcortisol, 6a-Fluor- 16α-methylcortexolone to 6a-Fluor- 16-methylcortisol, 6α-Methylcortexolone to 6cr-Methylc ortisol, 6a,9a-Difluorocortexolone to 6a,9a-Difluorcortisol, 6cr,9cr-Difluor- 16α,17α-isopropylidendioxicortexolone to 6a,9a-Difluor-16a,17cr-isopropyldendioxicortisol.

4) 16cr-hydroxylation. Incorporation of a hydroxy group in the 16-position of the steroid molecule. Example: 16cr-Hydroxylation of Cortisol and derivatives of Cortisol, e.g. Cortisol to 16cr- Hydroxicortisol, 6er-Fluorocortisol to 6α-Fluor-16α-hydroxicortisol, 9cr-Fluorcortisol to 9α-Fluor-16α-hydroxicortisol, 6cr,9cr-Difluorcortisol to 6α,9α-Difluor-16α-hydroxicortisol.

5) penicillin G transformation. Transformation of benzylpenicillin (penicillin G) to 6-aminopenicillanic acid, 6) Side-chain elimination. Example: transformation of sitosterol to A4-androstene 3,17-dione, cholesterol to A #1,4-androstadiene-3,17-dione, 7) 12α-hydroxy elimination. Example: transformation of cholic acid to chenodesoxycholic acid.

Suitable organisms which can be used in connection with this invention are 1) Arthrabacter simplex (also called Corynebacterium simplex, for instance ATCC 6946). This organism can be used for #-dehydrogenation.

2) Rhizopus nigricans (also called Rhizopus stolinifer, for instance ATCC 6227 b).This organism can be used for 11 cr-hydroxylation.

3) Curvularia lunata, for instance ATCC 12017. This organism can be used for 11 p-hydroxylation.

4) Escherichia coli, for instance ATCC 9637. This organism can be used for 6aminopenicillanic acid production from penicillin G.

5) Aspergillus niger. This organism can be used for 16cr-hydroxylation.

6) Streptomyces venezulae. This organism can be used for isomerization of glucose.

7) Brevibacterium ammoniogenes. This organism can be used for transformation of purine bases, nucleosides and nucleotides.

The microorganisms used in the present invention are immobilized in a suitable polymer carrier, such as polyacrylamide gel, agar (2.515% 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 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 g100 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 300C 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 å-dehydrogenase prednisolone. The reaction was catalyzed by polyarylamide-entrapped Arthrobacter simplex, (also called Corynebacterium simplex).

A. simplex cells were grown in a medium of 0.25% yeast extract, the Al dehydrogenase activity being induced by addition of cortisol to the culture 12 hours prior to harvesting by continuous centrifugation at 10,000xg. The cells (5 g wet weight) were suspended in 20 ml of ice-cold 0.1 M Tris-HC1 buffer, pH 7.5 and mixed with 25 ml ice-cold aqueous monomer solution containing 7.13 g acrylamide and 0.38 g N,N1-methylene-bis-acrylamide. The mixture was poured into a sandwich-like polymerisation vessel (made of two glass plates 20x20x0.2 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 200 C, at which temperature the preparation was stable for several months.

The 3-ketosteroid-A1-dehydrogenase activity of the immobilized A. simplex was conveniently assayed by a spectrophotometric procedure and the sole product, prednisolone, was identified by thin-layer chromatography. 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 batch-wise 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/get (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 gfl) 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 of magnitude and thus also of nutrients.

TABLE I Activating effect of nutrients, buffers and salts on 3-ketosteroid-å'-dehydrogenase activity of immobilized A. Simplex Mediums Initial transformation rates (%) after 0 2 6 10 16 days Peptone 0.5% (i.e. g/100 ml) 100 460 500 650 530 Glucose 02% (i.e. g/100 ml) 100 210 170 110 90 K2HPO4, 0.1M, 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 # ZnCl2, FeCl2 100 50 25 15 10 K2HPO4, 0.1M, pH 7.0 CoCl2, M9SO4 100 40 40 0 0 K2HPO4, 0.1M, pH 7.0 MgCl2, CaCI2 100 160 130 80 90 K2HPO4, 0.1 M, pH 7.0 Peptone 0.5% (i.e. g/100 ml) Glucose 0.2% (i.e. g/100 ml) MgCl2, CaCl2 100 560 650 600 550 H2O 100 60 40 40 20 a) the concentration of the inorganic salts MgCl2, ZnCl2, CoCI2, FeCl2, CaCl2 and MnSO4 was 1 mM.

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 lnM cortisol (methanol) was added. The suspension was shaken on a rotary shaker at 250C 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 A1-dehydrogenase activity.

TABLE II Activating effect of peptone/glucose on 3-ketosteroid-å'-dehydrogenase activity of immobilized A. simple Medium Initial transformation rateb) (%) after 0 2 6 10 16 days Peptone 1% % (i.e. g/100 ml) 100 290 320 380 550 0.5% " 100 460 500 650 530 ,, 100 200 225 165 120 0.01% ,, 100 125 30 0 0

peptone 0.1% ,, 100 250 225 240 350 Glucose 0.2% Peptone0.01% " 100 110 55 0 0 Glucose 0.2% 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 Transformation capacity Run (mg prednisolone"hour/g Time for 100% conversion (no) gel (wet weight) (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 spectrophotometrically 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.

WHAT WE CLAIM IS: 1. A method for activating immobilized living microorganisms which comprises bring peptone, glucose or a mixture of peptone and glucose into contact with immobilized microorganisms which are entrapped in a polymer.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. TABLE II Activating effect of peptone/glucose on 3-ketosteroid-å'-dehydrogenase activity of immobilized A. simple Medium Initial transformation rateb) (%) after 0 2 6 10 16 days Peptone 1% % (i.e. g/100 ml) 100 290 320 380 550 0.5% " 100 460 500 650 530 ,, 100 200 225 165 120 0.01% ,, 100 125 30 0 0 peptone 0.1% ,, 100 250 225 240 350 Glucose 0.2% Peptone0.01% " 100 110 55 0 0 Glucose 0.2% 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 Transformation capacity Run (mg prednisolone"hour/g Time for 100% conversion (no) gel (wet weight) (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 spectrophotometrically 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.

2. A method according to claim 1, wherein the medium containing the

immobilized microorganisms contains 0.1 tod 1.0 g/100 ml peptone or glucose or mixture thereof.

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 I to 3, wherein the immobilized microorganism is Arthrobacter simplex.

5. A method according to claim 1 wherein the immobilized organism is Arthrobacter simplex entrapped in a polyacrylamide gel.

6. A method according to claim 5 wherein the medium containing the immobilized microorganism 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 clainis 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 1.0 g/100 ml peptone or glucose 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 A1-dehydrogenation.

17. A method according to claim 10 wherein cortisol is transformed to prednisolone by treatment with Arthrobacter simplex immobilized in a polyacrylamide gel, in the presence of peptone, glucose or a 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.

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.