HIGH-YIELD PRODUCTION OF SACCHAROMYCOPSIS-FIBULIGERA GLUCOAMYLASE IN ESCHERICHIA-COLI, REFOLDING, AND COMPARISON OF THE NONGLYCOSYLATED ANDGLYCOSYLATED ENZYME FORMS

The truncated GLA1 gene encoding the mature form of glucoamylase from the yeast Saccharomycopsis fibuligera has been over-expressed in Esche richia coli using the IPTG inducible pET system. Over-expression has l ed to the accumulation of insoluble glucoamylase in inclusion bodies f rom which an electrophoretically homogeneous active enzyme has been pr epared yielding 30 mg per litre medium. This protein represents an N-t erminus Met-free, non-glycosylated product which displays the identica l specific activity of 45 units/mg and reduced thermal stability when compared to glycosylated enzymes isolated from Saccharomyces cerevisia e carrying the GLA1 gene. These data suggest that S. cerevisiae glycos ylation of S. fibuligera glucoamylase does not play a critical role in enzymatic activity but that it does contribute to its thermal stabili ty. (C) 1996 Academic Press, Inc.