High-yield extraction of periplasmic asparaginase produced by recombinant Pichia pastoris harbouring the Saccharomyces cerevisiaeASP3 gene

Abstract

The enzyme asparaginase is used for the treatment of haematopoietic diseases, such as acute lymphoblastic leukaemia and non-Hodgkin lymphomas. The extraction of the periplasmic asparaginase produced in high levels by a recombinant Pichia pastoris strain harbouring the Saccharomyces cerevisiae ASP3 gene was studied. We submitted the yeast cells to freeze–thaw cycles, ethanol treatment and alkaline extraction in the presence and absence of cysteine. The use of six freeze–thaw cycles, followed by extraction with 20 mM potassium phosphate buffer pH 7.0 for 20 h, resulted in 85% enzyme recovery whereas the alkaline extraction using 500 mM potassium phosphate at pH 11.5 in the presence of 10 mM cysteine allowed 100% enzyme recovery. The protein and asparaginase concentrations in the crude extract for the alkaline cysteine treatment (1220 mg L −1 protein; 19,134 U L −1 asparaginase) were higher than those observed for the freeze–thaw procedure (840 mg L −1; 13,274 U L −1). The activities of the two aforementioned asparaginase crude preparations were stable upon storage at −18 °C for several months. SDS-PAGE analysis of the two extracts displayed two major protein bands from each extraction protocol, that were both identified as asparaginase II from S. cerevisiae by mass spectrometric analyses.