Design of a process for improvement of phosphatidylglycerol-phosphatidylinositol transfer protein recovery from Aspergillus oryzae

A novel highly active phosphatidylglycerol-phosphatidylinositol transfer pr otein (PG/PI-TP) was isolated from a cell-free extract of Aspergillus oryza e LMTC 2.14 grown on lecithin fractions as sole carbon source. A protocol f or the extraction of the cytosolic PG/PI-TP that supports an extrapolation at industrial scale has been developed allowing the recovery of the active protein with high performance. Mycelium was harvested using a membrane pres s filter. A study of the cake permeability and compressibility which reflec ts the resistance of the cake to flow of liquid through pores was achieved in order to select the optimal working pressure (4 x 10(5) Pa). Mycelium di sruption was achieved using a high-pressure homogenizer, with semi-continuo us recycle. Two passes were necessary for an optimal recovery of PG/PI-TP. Removal of cell debris by filtration needed the use of 60 g 1(-1) Harbolite as filter aid. Tn the present growth conditions, mycelial intracellular li pid content was particularly high (approximate to 40% of the mycelial dry w eight) leading to major problems during chromatographic steps. In order to remove lipids, the crude extract was treated with hexane. Under these condi tions, the crude extract was significantly clarified without loss of PG/PI- TP activity. As molecular filtration is not a suitable first chromatographi c step in industry. ion-exchange chromatography was performed as a pre-puri fication step. Under these conditions, a recovery of 52% active PG/PI-TP wa s obtained, i.e. an improvement of 3.47-fold compared with previous work at labscale level. (C) 2000 Elsevier Science Ltd. All rights reserved.