Glycosylation Status and Conformational Stability of Recombinant Stereum purpureum Endopolygalacturonase IVs Produced in an Aspergillus oryzae Expression System

摘要

Stereum purpureum endopolygalacturonase (EndoPG) IV was expressed in Aspergillus oryzae. Recombinant forms of EndoPG IV (rEndoPGs IV0, IV1, and IV2) were purified from the culture filtrate by ammonium sulfate precipitation and two-step column chromatography with DE52 and hydroxyapatite. Each of these three recombinants was shown to be homogeneous by SDS-PAGE. Since it was predicted that the differences between them would be based on the number of sugar chains, the changes in molecular mass after enzymatic cleavage of the N-linked oligosaccharide with EndoH were determined by MALDI-TOF MS. From the data, the numbers of N-linked oligosaccharides were estimated to be 0, 1, and 2 for rEndoPG IV0, IV1, and IV2, respectively. These sugar chains appeared to be a mixture of GlcNAc2Hexose5-10 and to include galactose as well as mannose. Mass analyses of IV0, IV1 and IV2 after deglycosylation with EndoH demonstrated a hexose carbohydrate, suggesting the presence of an O-linked oligosaccharide that is lacking in native EndoPG IVs. The thermal denaturation curves of rEndoPG IV0 and IV1 based on CD analysis indicated Tm values of 60 and 59 °C, with a one-step thermal denaturation curve, similar to that of native EndoPG IV.