The present invention relates to a microbial method of 1 lα-hydroxyiation of steroids

Microbial 1 l -hydroxylation of steroids is a well known process, in vivo as well as in vitro For instance 1 lα-hydroxylation of progesterone by cell-free preparations of Aspergillus ochraceus has been reported by Shibahara et al Biochim Biophys Acta, 202 ( 1970), 172-179 It has also been known that microbial 1 lα-hydroxylation reactions of steroids are unpredictable, and invariably lead to incomplete transformations Typicallv conversion degrees of 80-85% are obtained For industrial applications it is however of importance to obtain high predictable conversion rates, which preferably lead to higher than 95% yields of l lα- hydroxylated steroids Mathematical models in the optimization of such fermentation processes are discussed by Deshayes et al , Bull Soc Chim Fr , ( 1980), II 24-34 For example, in the l lα-hydroxylation of canrenone, Deshayes disclosed that under optimum conditions better than 95% yields could be attained when Aspergillus ochraceus was used in a medium containing I a 10 g/1 of glucose m a matπx of malt-extract and trypticase Under these conditions up to 1 5 g/1 of canrenone could be transformed, which was considered to be an improvement in the art, for instance as disclosed by Blunt et al , J Chem Soc , 6 ( 1971 ), 1 136 who were not able to obtain more than 90% yield of 1 lα-hvdroxylated canrenone using at the most 0 5 g 1 of substrate

Since it can be reasoned that contaminations will have a detrimental influence on the microbial process, it can be expected that further optimization could be obtained by increasing the purity of the starting materials Surprisingly however, it has now been found that a further improvement is obtained by using less than pure substrate, in particular by using a substrate having a purity of less than 97% The invention therefore relates to a microbial method of in vitro transformation of a steroid into its corresponding 1 l -hydroxy analogue using oxygen and a micro-organism selected from A speigiJlus oclvaceus Aspergillus niger Rhizopus stolonifer, Rhizopus nigricans, Rhizopus art hizits, and strains of ^' Pestelolia, characterized in that a steroid having a purity of less than 97% is used

Preferably the micro-organism is Aspergillus ochraceus The purity of the steroid is preferably more than 90% More preferably the purity of the steroid is between 90 and 95 % The present method using impure substrates affords microbial transformations at substrate concentrations, which are substantially greater than the maximum concentrations as disclosed by Blunt or Deshayes

The present microbial method can be used with steroids having an unsubstituted 1 1- position. Preferred examples are estr-4-ene-3, 17-dione and canrenone

The surprising effect of impurities on the conversion degree is illustrated in the following tables:

Table I

Conversion of estr-4-ene-3, 17-dione by A. ochraceus purity substrate concentration conversion degree

(%) (g/i) (%)

99 15 78

99 10 83

98 25 85

94* 15 91

15 91

94*

15 94

94* 25 97

93 10 98

92

various natural impurities added to pure substrate

Table II

Conversion of canrenone by A. ochraceus purity substrate concentration conversion degree

(%) (g i) (%)

100 5 74

100 10 78

100 20 73

100 35 72

96 10 98

94 15 96

96 22 96

95 22 95

The invention is further illustrated by the following examples.

EXAMPLES

Example 1

A shake flask containing a mineral growth medium with glucose was inoculated with spores of A. ochraceus and placed on a reciprocal shaker at 28 °C for 15 h A stirred fermentor containing 5 1 of medium was subsequently inoculated with 250 ml of germinated spore suspension. The used medium contained a glucose/yeast extract medium (glucose 40 g/1, yeast extract 10 g/1, pH 5 0) The culture conditions were as follows stirrer speed 750 rpm, airflow 0 2 1/1/m, temp 28 °C Foaming was measured with an antifoam electrode and controlled by automatic addition of a silicon based antifoam agent When the culture had a biomass concentration of at least 2 g/1 a small portion of estr-4-ene-3, 17-dione ( 1 g/1) was added to induce the synthesis of the hydroxylation enzymes Three hours later higher concentrations of steroid were added to reach the desired concentrations as given in Table I The steroid trans¬ formation was stopped when repeatedly no increase in conversion was observed

Example 2

In a shake flask the same culture as described in Example 1 was prepared When the culture had a biomass concentration of at least 4 g 1 the canrenone was added at once to reach the desired concentrations as given in Table II The steroid transformation was stopped when repeatedly no increase in conversion was observed