CZ303607B6 - Codeine-hydrolyzing strain of Rhizobium radiobacter R89-1 CCM 7947 microorganism to 14-OH-codeine and 14-OH-codeinone - Google Patents

Abstract

The present invention relates to a strain of Rhizobium radiobacter R89-1 CCM 7947 microorganism, which is capable of hydrolyzing codeine in position 14 with 100 percent conversion of the starting codeine of the general formula 1 to 14-hydroxycodeine of the general formula 2 and 14-hydroxycodeinone of the general formula 3. The present invention further relates to the use of the Rhizobium radiobacter R89-1 CCM 7947 microorganism for hydroxylation of codeine of the general formula 1 and related alkaloids in position 14 as well as to a process for preparing biomass of the strain of the Rhizobium radiobacter R89-1 CCM 7947 microorganism, where the microorganism is cultured in a simple or inflow cultivation at a temperature ranging from 20 to 35 degC on the LB complex medium or on LBTE medium at the initial pH value in the range of 4 to 9.

Description

Technical field

The present invention relates to the hydroxylation of codeine of formula 1 to 14-hydroxycodeine of formula 2 and 14-hydroxycodeinone of formula 3 with a strain of Rhizobium radiobacter R89-1 CCM 7947.

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The reaction provides 100% conversion of codeine to 14-hydroxycodein of formula 2 and 14-hydroxycodeinone of formula 3 at an initial codeine concentration of 1.5 g / l, 90% at 3.0 g / l, and at an initial concentration of 6 g / l the conversion is 47% .

BACKGROUND OF THE INVENTION

Codeine, morphine and thebaine are natural alkaloids produced by poppy (Papaver somruje rum). Introduction of the hydroxyl group at the C-14 position of the morphine alkaloid structure is used for the preparation of oxycodone, naloxone, oxymorphone and naltrexone, which are used in medicine as analgesics, antitussives and drugs for drug and alcohol dependence cessation. The most commonly used starting material for the chemical production of these opiates is thebaine, a minor component of the natural opium extract. This is converted by oxidation (peroxyacid) to 14-hydroxycodeinone (Lopez, D, Quioo, E., Riguera, R. 1994 Tetrahedron Lett., 31,5727-5730). Since codeine and morphine are much more accessible than thebaine, they are therefore promising precursors for the preparation of 14-hydroxy opiates. The chemical methods described show low yields, the accumulation of hardly separable by-products and the necessity of using unwanted heavy metals (Zhang, Q., Rich, JO, Cotterill, C., Pantaleone DP, Michels, P. C., 2005 J. Am. Chem. Soc. 127,7286-7287).

The beginnings of interest in obtaining more effective analgesics from natural opiates through biotransformation date back to the 1960s. The first work describing the ability of the basidiomycetes Trametes sanguinea to transform thebaine by allylic oxygenation and demethylation to 14-hydroxycodeinone and subsequent reduction to 14-hydroxycodein was described by Iizuka et al. (Iizuka, K., Okuda, S., Aida, K., Asai, T., Tsuda, K., Yamada, M., Seki, 1, 1960 I. Chem. Pharm. Bull. 8, 1056-7, and Iizuka, K., Yamada, M., Suzuki, J., Seki, I., Aida, K., Okuda, S., Assay, T., Tsuda, K. 1962, Chem. Pharm. Bull. 10, 6770 ).

Tsuda, K. (1964, AA, M. Symposium on Microbiology No. 6, Institute of Appl. Microbiol., University of Tokyo, Tokyo, Japan) described in Trametes sanguinea (and another 120 basidiomycetes strains from 1200 different bacterial strains tested). and fungal strains) converting thebaine to 14-hydroxycodeinone and 14-hydroxycodein. Similar results were published in another independent study with Trametes cinnabarina (Gröger, D and Schmauder, ma P. 1969, Experentia 25, 95-96).

- 1 GB 303607 B6

Microbial transformation of morphine alkaloids has also been described in bacterial strains. Liras, P. and Umbreit, W.W. (1975, Appl. Microbiok 30, 262-266) described the conversion of morphine to 14-hydroxymorphine by resting Arthobacter sp.

Low concentrations of 14-hydroxymorphine as a biotransformation product have also been found in Pseudomonas testosteronii. In this strain, α- and β-hydroxysteroid dehydrogenases have been described which produced morphinone and codeinone from inine or morphine code in the presence of NAD ⁺ (Liras, P., Kasparian, SS Umbreit, WW 1975, Appl. Microbiol. 30, 650-656 ).

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Also in Bacillus sp. the transformation of morphine and codeine into 14-hydroxymorphinone and 14-hydroxycodeinone has been described (Madyastha, M., Reddy, G.V. 1994, J. Chem. Soc. Perkin Trans. 1 (8), 911-912).

Harder, P.A. and Kunz, D.A. (1989 United States Patent No. 4,798,792) patented bacterial hydroxylation of codeine and its water-soluble salts to 14-hydroxycodeine strains of the genus Streptomyces. Streptomyces griseus NRRL B8090 showed the highest conversion. Using a rich medium containing soy flour during a 30 day culture and at an initial codeine concentration of 1 mM (0.29 mg / ml). After 27 days, 0.2 mM (63 mg / l) of 14-OH codeine and 0.03 mM (8.55 mg / l) of norodeine were obtained in this strain.

Zhang, Q., Rich, J.O., Cotterill, I.C., Pantaleone, D.P., Michels, P.C. 2005, J. Am. Chem. Soc. 127, 7286-7287 described in Mycobacterium neoaurum (MTP650) the ability of hydroxy codeine to 14-hydroxycodeine and the mechanism of reactions.

Biconversion of codeine to semi-synthetic opioid derivatives (6-acetyl codeine, oxycodone, norcodeine, morphine) has been described by Nostoc muscorum Niknam et al. (Nikam, S., Faramazi, M.A., Abdi, K., Yazdi, T. T., Amini, M., Rastegar, H. 2010, World J. Microbiol. Biotechnol. 26, 119-123). The 14-OH codeine derivative was not detected. Optimal codeine concentration for whole biotransformation

3 cells were 0.5 to 1.0 mg / ml; derivatives were identified after conversion for 5 to 10 days.

Wick, A., Wagner, M., Ternem, T. A. 2011, Environ. Sci. Technol. 46, 3374-3385 described codeine transformation pathways by activated sludge from a sewage treatment plant under aerobic conditions. One of the pathways described is the hydroxylation of codeine at the C-14 position. The initial codeine concentration was 5 mg / l.

There is no description in the literature of the hydroxylation of codeine 1 to 14-hydroxycodein 2 and 14-hydroxycodeinone 3 by strains of a microorganism of the genus Rhizobium.

SUMMARY OF THE INVENTION

The present invention provides a new strain of Rhizobium radiobacter R89-1 CCM 7947 which is capable of hydroxylating codeine of formula 1 at position 14

to 14-hydroxycodein of formula 2 and 14-hydroxycodeinone of formula 3

- 2 GB 303607 B6

The hydroxylation produces predominantly 14-hydroxycodeine.

DETAILED DESCRIPTION OF THE INVENTION

The Rhizobium radiobacter strain R89-1 CCM 7947 was obtained by extensive screening of more than 2000 collection and naturally isolated microorganisms (yeasts, molds and bacteria). Two bacterial strains showing the given properties were screened. However, only strain R89-1 converted codeine (1.5 g / l) with 100% efficiency. This microorganism was classified taxonometrically and designated Rhizobium radiobacter R89-1. Its bioehemic-morphological classification was confirmed by sequence analysis of 16S rDNA. The bacterial strain described does not fall within the taxonometrically described strains with the ability to hydroxylate codeine of formula 1 to 14-hydroxy codeine of formula 2 and 14-hydroxy code of ionon of formula 3.

The subject of the present invention is a novel strain of the microorganism Rhizobium radiobacter R89-1 CCM 7947 capable of hydroxylating codeine 1.

to 14-hydroxycodein 2 and 14-hydroxycodeinone 3

The hydroxylation produces predominantly the 14-hydroxycodeine of formula 2.

The advantage of said process is 100% conversion of codeine at an initial codeine concentration of 1.5 g / L, 90% at 3.0 g / L and 47% at 6 g / L. The advantage of conversion by a given microorganism is the absence of other biotransformation intermediates.

-3GB 303607 B6

The novel microorganism of the present invention is deposited with the Czech Collection of Microorganisms at Masaryk University in Brno, Tvrdého 14, under the designation CCM 7947.

The strain characteristics are shown in Table 1.

Table 1 Characteristics of Rhizobium radiobacter strain R89-1 CCM 7947

A. Morphology (microscopy) Cell shape sticks, individually in irregular clusters Scanty asporogenni Gram-staining negative Mobility positive B. Morphology of colonies round, smooth, glossy, slightly convex, continuous edge, 2-3 mm in diameter C. Growth conditions Temperature grows well at 20 - 35 ° C, grows well at 37 ° C, does not grow at 42 ° C pH 6.0-8.5 Relationship to oxygen aerobic Growth in the presence of 6.5% NaCl negative D. Physiological characteristics Hydrolysis of starch negative Casein hydrolysis negative Hydrolysis of esculin positive Hydrolysis of tyrosine negative Hydrolysis of o-nitrophenyl-β-D-galactoside positive Hydrolysis of lecithin negative Hydrolysis of gelatin negative Hydrolysis of Tween 80 negative DNA hydrolysis negative Decarboxylation of omitin negative Reduction of nitrates negative Reduction of nitrites negative Glucose acid oxidatively positive Glucose acid fermentatively negative Xylose acid positive Mannitol acid negative Maltose acid negative Fructose acid positive Growth on Simons Citrate negative D. Physiological characteristics Urease positive Catalase positive Oxidase positive Arginine dihydrolase negative Fluorescein production negative

The composition of the culture medium was optimized and the influence of various carbon and energy sources, complex media components and various physical conditions of culture on the growth of culture and its ability to codeot biotransformation was tested.

Unless otherwise noted, strain R89-1 was cultured in shake flasks on Luria-Bertani (LB) complex medium and trace medium supplemented (LBTE medium), at a temperature of up to 35 ° C and an initial pH of 4-9. is shown in Example 2. Optimal conditions for growth of the cells under which the strain was cultured in a stirred bioreactor were selected.

In a single or fed-batch culture condition, it exhibits the ability of codeine bioconversion. Single cultures were performed without pH regulation and dissolved oxygen concentration. The fed-batch cultures were conducted under control of pH and dissolved oxygen concentration in the medium. The feed solution contained glucose or sucrose at a concentration of 20 to 50 wt% as a source of carbon and energy.

The degree of conversion was determined for the culture samples under the following conditions: the samples taken were mixed with tert-butyl methyl ether (TBME) and NH ₄ OH (pH adjustment) and substances 1, 2 and 3 were extracted by shaking for 20 min at 28 ° C. The conversion of compound 1 to derivatives 2 and 3 was determined by HPLC.

The biotransformation of morphine 4 and thebaine 5 under the same conditions as codeine except that the morphine and thebaine concentrations were 0.2 g / l. At 96 hours, both the morphine and thebaine conversion values were 100%.

The properties of strain CCM 7947, i.e. 100% conversion of codeine of formula 1 to 14-hydroxycodein of formula 2 and 14-hydroxycodeinone of formula 3, suggest a realistic assumption of utilizing the subject strain for hydroxylation of codeine of formula 1, morphine of formula 4 and thebaine of formula 5. 14-hydroxy derivatives in the pharmaceutical industry.

The following examples describe in detail a method for obtaining a Rhizobium radiobacter R89-1 strain, a method for culturing it, aimed at, but not limited to, biotransformation activity of codeine and optimizing below 25 mins of biotransformation.

BRIEF DESCRIPTION OF THE DRAWINGS

Giant. 1 Time course of biomass concentration (X 1) and biotransformation activity (conversion, ♦) in a single culture of Rhizobium radiobacter R89-1 CCM 7947 strain on LBTE medium according to Example 4.

Giant. 2 Time course of biomass concentration (X 1) and biotransformation activity (conversion, ♦) in a single culture of Rhizobium radiobacter strain R89-1 CCM 7947 on LBTE medium with glucose (0.5%) in a stirred bioreactor according to Example 5.

Giant. 3 Time course of biomass concentration (X,) and biotransformation activity (conversion, ♦) in a fed batch of Rhizobium radiobacter strain R89-1 CCM 7947 in a stirred bioreactor at a culture temperature of 30 ° C in LBTE medium with glucose (1.0 %) according to Example 6.

Examples

Example 1

Isolation of Rhizobium radiobacter R89-1

-5GB 303607 B6

The Rhizobium radiobacter R89-I strain was obtained from compost soil where poppy residue was composted. 10 g of soil was resuspended in 100 ml of saline (9 g / l NaCl) and shaken (200 rpm) for 1 hour. The autochthonous microflora was appropriately diluted in saline and plated on LB agar. Individual morphologically different colonies of isolates, grown in 3 days at 28 ° C, were inoculated onto fresh LB agar and, after rising at 28 ° C, were assessed for the ability of hydroxylation of codeine 1 to 14-OH-codeine 2 and 14OH-codeinone 3 ml of LB medium supplemented with codeine at a concentration of 0.1 g / t in 12-well culture plates (mcwp) were inoculated with the appropriate isolate and after 72 hours of culture (28 ° C and 200 rpm) to 1 ml. 0.1 mL of NH ₄ OH and 0.5 mL of TBME was added to the sample and the culture liquid was extracted for 20 min. After centrifugation (15 min, 10,000 rpm), the organic phase was used to quantify the codeine of formula 1,14 hydroxy code of formula 2 and 14-hydroxycodeinone of formula 3 by HPLC under the following conditions:

Column: C-18 Hibar Lichrom man-fix

Elution mixture: water + 0.1% TFA: MeOH (4: 1 v / v)

Flow rate: 1.3 ml / min

Application volume: 0.05 ml

Detection at wavelength: 220 to 214 nm

Column temperature: 40 ° C Analysis time: 5 min Retention times:

Substance 1 codeine 2.6 min,

Substance 2 14-hydroxycodeine 2.2 min

Substance 3 14-hydroxycodeinone 3.6 min

From the 2000 isolates tested (collection strains and isolates from natural materials), 2 bacterial strains were obtained showing the ability to hydroxylate the compound of formula 1 to the products of formulas 2 and 3. These strains were cultured in 50 ml LB medium in 500 ml culture flasks as follows: inoculated with LB agar-colonized strains and the flasks were incubated on a rotary shaker (200 rpm, 28-30 ° C) for 24 hours (first vegetative generation), 2 ml of the grown culture was used to inoculate three parallel 50-well flasks. ml of LBTE broth with the addition of codeine at a concentration of 0.1 g / l (second vegetative generation) which were cultivated under the same conditions. After 48 hours of culture (28 ° C and 200 rpm) the culture sample was extracted with TBME (see above) and the organic phase was used for quantitative determination of bioconversion by HPLC (see above). From both strains tested, a natural isolate was selected from a sample of poppy compost waste, which showed 100% conversion. The strain was designated R89-1 and deposited in the collection of microorganisms under number CCM7947.

Example 2

Optimization of Rhizobium radiobacter R89-1 CCM 7947 culture medium composition

Optimization of the media composition for growth and conversion was performed in 12 well mcwp as shown in Example 1.

Composition of culture media: mineral medium (M9):

Na ₂ HPO ₄

KH ₂ PO ₄

NaCl

NH4Cl

MgSO ₄ .7H ₂ O distilled water

14.6 g / l 3.0 g / l 0.5 g / l L0 g / l 0.2 g / l

1000 ml carbon and energy source:

glucose, sucrose, xylose, glycerol, sorbitol at concentrations of: 1; 5; 10; 15 and 20 g / l complex substrates:

casein hydrolyzate (CH) 20 and 50 g / l yeast extract (YE) 20 and 50 g / l trypton (TRY) 20 and 50 g / l 10 Luria-Bertani (LB) complex medium: trypton 10 g / l yeast extract 5 g / l

NaCl 10 g / l distilled water 1000 ml trace elements (TE)

MgSO ₄ .7H ₂ O 0.2 g / l

CaCl ₂ .2H ₂ O 50 mg / 1

FeSO ₄ .7H ₂ O 10 mg / l

Table 2 Effect of medium and complex source on Rhizobium radiobacter R.89-1 CCM 7947 increase (OD 600) and codeine conversion (0.2 g / l)

Media type Complex substrate (g / l) ODoo 24. h cultivation Conversions 48. h (%) M9 YE 20 5.4 0 M9 YE 50 5.5 0 M9 TR20 5.5 50 M9 TRY 50 4.1 20 May M9 CH 20 3.7 10 M9 CH 50 1,8 0 LB YE5aTRY10 3.2 100 ALIGN! LB aTE YE 5, TRY 10 and TE 4.6 100 ALIGN!

Based on the results shown in Table 2, LB + TE (LTBE) broth was selected for further optimization. The effect of carbon and energy sources is shown in Table 3.

Table 3 Effect of carbon source and energy on growth (OD ₆₀ o) of Rhizobium radiobacter R89-1 CCM 7947 and codeine conversion (0.2 g / l) in LBTE medium

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Source of carbon and energy (g / 1) OD (> oo 24. h cultivation Conversions 48. h (%) glucose 1 5.4 100 ALIGN! glucose 5 6.8 100 ALIGN! glucose 10 7.5 99 glucose 15 7.0 30 glucose 20 6.8 12 sucrose 1 5.7 100 ALIGN!

Source of carbon and energy (g / 1) Οθόοο 24. h cultivation Conversions 48. h (%) sucrose 5 6.2 100 ALIGN! sucrose 10 6.0 70 sucrose 15 6.5 15 Dec sucrose 20 6.3 10 glycerol 1 4.8 50 glycerol 5 4,5 50 glycerol 10 4.0 30 glycerol 15 4.1 20 May glycerol 20 4.2 15 Dec

Optimal sources of carbon and energy are glucose and sucrose in 0.5% concentration. Xylose and sorbitol are also a useful source of carbon and energy.

Example 3 io

Optimization of culture conditions for Rhizobium radiobacter R89-1 CCM 7947

The effect of pH on cell growth was monitored in 12 well mcwp at 29 ° C; 2.0 ml of LBTE medium with appropriate initial pH was inoculated with 40 μϊ of culture (24 h, 29 ° C, LTBE medium of appropriate pH). The effect of pH on codeine conversion was also monitored in 12 well mcwp at 29 ° C, except that a 10% cell suspension prepared as follows: LBTE grown culture (24 h, pH 7.0, 29 ° C) was centrifuged. and resuspended in fresh LBTE medium at the appropriate initial pH. After 48 h conversion, concentrations of 1.2 and 3 were determined as described in Example 1.

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Table 4 Effect of initial pH of LTBE medium on increase (OD ₆ <m) of Rhizobium radiobacter R89-1 CCM 7947 culture and codeine conversion (0.2 g / L)

PH ODéoo 24. h cultivation Conversions 48. h (%) 3.0 0.3 0 4.0 0.5 20 May 5.0 4,5 70 6.0 4.4 100 ALIGN! 7.0 4.6 100 ALIGN! 8.0 3.7 100 ALIGN! 9.0 2.5 100 ALIGN! 10.0 0.2 0

The effect of temperature on cell growth was monitored in 12 well mcwp at an initial pH of 7.0: 2.0 ml of LBTE medium was inoculated with 40 μΐ cultures grown in LTBE medium (24 h, 29 ° C). The effect of temperature on codeine conversion was also observed in 12 well mcwp at an initial pH of 7.0, except that a 10% cell suspension prepared as in pH monitoring (this example) was used. After 48 h conversion at different temperatures, concentrations of 1.2 and 3 were determined as described in Example 1.

Table 4 Effect of temperature on Rhizobium radiobacter R89-1 CCM 7947 growth (ODo) and codeine conversion (0.2 g / L)

Temperature ODeoo Conversions 48. h (° C) 24. h cultivation (%) 20 May 3.9 100 ALIGN! 25 4.3 100 ALIGN! 30 4.3 100 ALIGN! 35 3.6 100 ALIGN!

Example 4

Single culture of Rhizobium radiobacter R89-1 CCM 7947 strain on LBTE medium in a 25 stirred bioreactor

Single cultivation on LBTE medium was performed in a Biostat MD stirred bioreactor: 6 L working volume, 6 L air / min aeration, 300 Rpm stirring start at 30 ° C and an initial pH of 7.2. The medium was inoculated with 5% second generation culture (see Example 1, except that there were 100 ml of LBTE medium in 500 ml culture flasks). pH of medium and concentration

The dissolved oxygen was not regulated during cultivation. During culture in the bioreactor, culture samples were taken at regular time intervals and the biomass concentration (dry cell weight, X) and the ability of the cells to hydroxylate codeine of formula 1 to 14OH-codeine of formula 2 and 14-OH-codeinone of formula 3 were determined. ml of culture liquid was stored at -30 [deg.] C. after completion of the culture all samples were resuspended in 2 ml of LBTE medium with codeine at a concentration of 0.2 g / l and after 48 h conversion the concentration of the compound of formula 1, 2 and 3 was determined. in Example 1). The maximum dry mass concentration of biomass (1.7 g / l) was reached at 16 hours of cultivation, maximum conversion of compound of formula 1 (82%) only at the end of cultivation (25 hours). The time course of biomass concentration (X 1) and biotransformation activity is also shown in Fig. 2. 1.

Example 5

Single culture of Rhizobium radiobacter R89-1 CCM 7947 strain on LTBE medium with glucose in a stirred bioreactor.

A single culture was performed under the same conditions as in Example 4 except that glucose at a concentration of 0.5% was added to the medium at the beginning of the culture. The maximum dry mass concentration of biomass (5.7 g / l) was achieved at 4 pm, the maximum conversion of compound 1 (100%) at 18 hours of culture. The time course of biomass concentration (X 1) and biotransformation activity (conversion, ♦) in the stirred bioreactor is shown in Fig. 2. 2.

Example 6

Fed-batch (40% glucose solution) culture of Rhizobium radiobacter R89-1 CCM 7947 on LTBE medium with glucose in a stirred bioreactor

The fed-batch cultivation was carried out under the same conditions as in Example 5 except that the initial glucose concentration was 1.0% and from 1pm to 10pm a 40% glucose solution was added to the reactor to maintain dissolved oxygen concentration in the reactor. medium (pO ₂ ) ranging from 25 to 35% of the maximum oxygen saturation value of the medium. The pH was controlled with ammonia to pH = 7.5. Giant. 3 shows the time course of biomass concentration and biotransformation activity. Under this cultivation regimen, both the maximum biomass dry weight concentration (11.9 g / L) and the maximum conversion of the compound of Formula 1 (100%) at 20 hours of culture were achieved.

Example 7

Effect of initial concentration of codeine 1 on its biotransformation by 10% cell suspension

The cells from the end of the culture described in Example 6 were centrifuged, washed with saline, and resuspended in LBTE medium to a final cell concentration of 10% (wet cell weight). Biotransformations were performed in 500 ml culture flasks with 50 ml cell suspension on a rotary shaker at 200 rpm and 29 ° C. The initial substrate 1 concentration was 0.2 to 0.2

6.0 g / l.

- 10GB 303607 B6

Initial concentration 1 (g / 1) conversion (%) 24. h 48. h 72. h 96. h 120. h 0.2 76 100 ALIGN! - 0.5 75 100 ALIGN! - 1.0 46 100 ALIGN! - 1.5 29 85 100 ALIGN! 3.0 23 53 53 52 51 6.0 10 24 24 23 21

Example 8

Influence of initial concentration of code 1 in on its biotransformation by 20% cell suspension

The bioconversion was carried out under the same conditions as in Example 7 except that the final cell concentration was 20% (wet cell weight) and the starting compound of Formula 1 was 1.5; 3 to or 6 g / l.

initial concentration 1 (g / 1) conversion (%) 24. h 48. h 72. h 96. h 1.5 95 100 ALIGN! - - 3.0 46 80 87 90 6.0 21 36 46 47

is Example 9

Biotransformation of morphine and thebaine by 20% cell suspension

The biotransformation of morphine 4 and thebaine 5 under the same conditions as 20 in Example 8 except that the concentration of both morphine and thebaine was 0.2 g / l. At 96 hours, both the morphine and thebaine conversion values were 100%.

Claims (7)

PATENT CLAIMS 5 1. Rhizobium radiobacter strain R89-1 CCM 7947 capable of hydroxylating codeine at position 14 with 100% conversion of the starting codeine of formula 10 to 14-hydroxycodeine of formula 2 and 14-hydroxycodeinone of formula 3 Use of the microorganism Rhizobium radiobacter R89-1 CCM 7947 according to claim 1 for hydrogenation at position 14 of the codeine of formula 1 and related alkaloids which are morphine of formula 4 and thebaine of formula 5 20 May A method of preparing a biomass of a strain of Rhizobium radiobacter R89-1 CCM 7947 according to claim 1, wherein the microorganism is cultured in a single or fed-batch culture at 20 to 35 ° C on LB complex medium or LBTE medium at an initial pH 4 to 9. The method of preparing biomass according to claim 3, characterized in that the single cultivation is carried out without controlling the pH and the dissolved oxygen concentration. A method for preparing biomass according to claim 3, characterized in that the fed-batch cultivation is carried out under the control of the pH and the dissolved oxygen concentration in the feed medium The solution comprises a source of carbon and energy. - 12GB 303607 B6 A method for preparing biomass according to claim 5, characterized in that the feed solution contains glucose or sucrose as the carbon and energy source. A method for preparing biomass according to claim 5, characterized in that the fed-in solution contains glucose or sucrose in a concentration of 20 to 50% by weight. 2 drawings - 13GB 303607 B6