Identification and purification of O-acetyl-L-serine sulphhydrylase in Penicillium chrysogenum

We have demonstrated that Penicillium chrysogenum possesses the L-cysteine biosynthetic enzyme O-acetyI-L-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-L-serine sulphhydrylase and O-acetyl-L-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-L-serine as substrate for the formation of L-cysteine. The purified enzyme did not catalyse the formation of L-homocysteine from O-acetyl-L-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyI-L-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold in relation to the cell-free extract. Two bands, showing exactly the same intensity, were present on a sodium dodecyl sulphate/ polyacrylamide gel, and the molecular masses of these were estimated to be 59 kDa and 68 kDa respectively. The K-m value for O-acetyl-L-serine and V-max of O-acetyl-L-serine sulphhydrylase were estimated to be 1.3 mM and 14.9 mu mol/mg protein(-1) h(-1) respectively. The activity of the purified enzyme had a temperature optimum of approximately 45 degrees C, which is much higher than the actual temperature for penicillin synthesis. Furthermore, O-acetyl-L-serine sulphhydrylase activity was to have a maximum in the range of pH 7.0-7.4.