SUMMARYAmong 20 locally isolated fungi, Aspergillus niger isolate no. 58 proved to be suitable for the production of active extracellular milk-clotting enzyme. The addition of acetate buffer to the reaction mixture enhanced the clotting of milk by the enzyme whereas citrate +phosphate buffer hindered the process. The general properties of the crude enzyme were studied. Precipitation with ammonium sulphate, ethanol, acetone and tannin showed that ammonium sulphate was unsuitable for precipitation while the other precipitantgproduced sufficiently active fractions. I N T R O D U C T I O NStudies on the production of milk-clotting enzymes from microbial sources have gained interest during the last decade; the production of milk-clotting enzymes by different types of micro-organisms has been reported (Arima & Iwasaki, I 964; Veselov, Tipograf & Petina, I 965 ; Knight, I 966). The increasing demands on rennin for cheese making in Egypt and the prohibition of the slaughter of calves justified a study aiming to search for rennin substitutes from sources other than animals and in particular from micro-organisms. Cultivation. Transfers were made from the stock slants to Dox agar plates which were then incubated at 30' for 7 days. Two discs (0.5 cm. diam.) were cut from the 7-day culture and inoculated into 50 ml. sterile medium in 250 ml. flat bottomed flasks. Unless otherwise specified, the flasks were incubated for 3 to 4 days at 3 0 ' . At the end of the incubation period the culture medium was filtered off and its volume adjusted to 50 ml.
The use of corticosteroids as anti-inflammatory agents requires the introduction of an oxygen function a t position 11 of the steroid nucleus. Since the introduction of this oxygen is too difficult chemically, efforts were made to introduce it microbiologically. PETERSON (1952) was the first to report the introduction of a hydroxyl group a t C-11 in progesterone by Rhizopus arrhizus with the formation of 1 la-hydroxyprogesterone and a quantitative yield was obtained when Rhixopus nigricans was used ( PETERSON et al. 1952). Other examples of such a transformation were given by DULANEY et al. (1955), M C A L E E R~~ al. (1958) andCAPEK et al. (1962). The influence of certain factors which affect the rate of this conversion was given by KAROW et al. (1956) and by WEAVER et aZ. (1960). Continuous addition of substrate has also been reported (RUSSER et al. 1961) as an effective technique.In the present work, progesterone hydroxylation was studied using two strains of fungi. The first was Rhizopus nigricans which was found to give 1 la-hydroxyprogesterone in 73.3% optimal yield in addition to another transformation product identified as 6@, 1 la-dihydroxyprogesterone. The effects of progesterone concentration (applied in the forms of a solution and as a suspension), transformation period and age of culture were studied. The second fungus was Penicilliuna oxalicum which produced 1 Ig-hydroxyprogesterone as the sole product of transformation. JIaterial and methodsThe organisms used were Rhizopus nigricans (isolated by the present authors) and Penicillium oxalicum (obtained from the Centre of Cultures, National Research Centre). The culture rneclinm nscd for Rhizopus nigricans was composed of bactotryptone 20 8. ml\icose 50 g and corn steep solids 3 g in one liter of tap water and the p H was adjusted to 475-4.6 before sterilization by concentrated hydrochloric acid. I n the case of Penicillium oxnlicum the culture medium was composed of glucose 3 O g , KH,PO, 0.2g,NaNO, 4 3 and RIgSO, 2g in one liter of distilled water and the pH was adjusted to 5.4 bcfore stcrilization by loo,:) NaOH.Transformation of progesterone : In the Rhizopus izigricans experiments, 500 rnl ERLEN-MEYER-flasks (each containing 50 ml sterile nutritive medium) were inoculated and placed on a rotatory shaer (220 rpm) for 24hr, then progesterone (dissolved in 9676 ethanol) was added. The transformation was left to proceed for the specified time in each experiment.I n the Penicillium oxalicunz experiments, the organism was first cultivated for 48 hr. From this culture a series of ERLENMEYER-flasks (each containing 50 ml sterile nutritive medium) were inoculated and placed on t,he shaker (220 rpm) for 48 hr. Progesterone 13 Zeitschrift f. Allg. Yikrobiologie, Bd. 9, H. 3
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