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 -acetyl- 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 -acetyl- 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 μmol/mg protein −1 h −1 respectively. The activity of the purified enzyme had a temperature optimum of approximately 45 °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.