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 d irect sulphhydrylation pathway. The finding of this enzyme, and thus the pr esence of the direct sulphhydrylation pathway in P. chrysogenum, creates th e potential for increasing the overall yield in penicillin production by en hancing the enzymatic activity of this microorganism. Only O-acetyl-L-serin e sulphhydrylase and O-acetyl-L-homoserine sulphhydrylase (EC 4.2.99.10) ha ve been demonstrated to use O-acetyl-L-serine as substrate for the formatio n of L-cysteine. The purified enzyme did not catalyse the formation of L-ho mocysteine from O-acetyl-L-homoserine and sulphide, excluding the possibili ty that the purified enzyme was O-acetyI-L-homoserine sulphhydrylase with m ultiple substrate specificity. The purification enhanced the enzymatic spec ific activity 93-fold in relation to the cell-free extract. Two bands, show ing 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-m ax 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 enz yme 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.