Several authors as PISCHBACH et al. (1947), KING and HAMBLY (1950) already demonstrated the possible formation of o-hydroxyphenylacetic acid from phenylacetic acid in the course of a submerged cultivation of Penicillium chrysogenum. For the hydroxylation of phenylacetic acid, on the one hand the formation of free hydroxyl radicals and on the other hand, their introduction in the aromatic nucleus is required (MASSART and VERCAUTEREN 1959).
During their studies on the mechanism of formation of o-hydroxyphenylacetic acid ERDJ~LYI et al. (1964) observed that in the time of hydroxylation of phenylacetic acid a high catalase activity is present in the culture of Penicillium chrysogenum. The catalase activity of mycelia and filtered broth was measured gpectrophotometrically by the rate of decomposition of H,O, as described by BEERS and SIZER (1952).
In order to approach the mechanism of reaction firstly the conditions of hydroxylation of phenylacetic acid in nonezymatic system were investigated where the formation of free hydroxyl groups was already proved (BAXENDALE 1952). The utilisation of H,O, and Fez+ ion for this typo of reaction was reasonable since the aforementioned compound and ion are present in the culture broth of industrial scale penicillin biosynthesis -where the formation of o-hydroxyphenylacetic acid was first observed (FISCHBACH et al. 1947, KING andHAMBLY 1950).
In addition to detect the subsequent steps of reaction the enzymatic nature of phenylacetic acid hydroxylation was studied.
NISHIDA (1951), ISONO (1953), PAN (1955), P\TARASIMHACHARIet al. (1960) have
Methods
Mi c r o o r g a n i s m, c u l t u r e m e d i u m , c o n d i t i o n s of c u 1 t i v a t ion. Penicilliunz chrysogenuni DAD strain (isolated by Dr. K. P ~L Y A ) was used. The microorganism was grown in a synthetic medium of the composition: glucose 1.5 per cent; lactose 1.5 per cent; histidine 0.03 per cent; arginine 0.03 per cent; glutamine 0.4 per cent; KCI 0.05 per cent; KH,PO, 0.15 per cent; NaNO, 0.35 per cent; cysteine 0.01 per cent. The components were dissolved in distilled water. pH was adjusted t o 6.0 before sterilization. Medium was autoclaved at 1.2 atm for 30 minutes. Flasks containing 100 ml of synthetic medium and spores were put on a rotary shaker (290 r.p.m.) in the incubator, a t 25 "C. The microorganism was grown for various length of time. Mycelia were harvested, filtered and washed three times with distilled water. The mycelium mat was disrupted in 1/15 M phosphate buffer pH 7.0 (made 1 ) Part of this communication was presented before 6th Meeting of Hungarian Chemical Society, Tihany, 1964.