GLYCAN MODIFICATION OF A THERMOSTABLE RECOMBINANT (1-3,1-4)-BETA-GLUCANASE SECRETED FROM SACCHAROMYCES-CEREVISIAE IS DETERMINED BY STRAIN AND CULTURE CONDITIONS

High level biosynthesis and secretion of the thermostable hybrid (1-3, 1-4)-beta-glucanase H(A16-M) has been achieved in Saccharomyces cerevi siae by means of the yeast vacuolar endoprotease B promoter (PRB1(p)) and the Bacillus macerans (1-3,1-4)-beta-glucanase signal peptide. The N-glycans present on the yeast-secreted H(A16-M), denoted H(A16-M)-Y, were released by endoglycosidase H, and identified by proton NMR spec troscopy to be a homologous series of Man(8-13)GlcNAc(2), although onl y traces of Man(9)GlcNAc(2) were found. Therefore, processing of N-gly cans on H(A16-M)-Y is similar to that on homologous proteins. Most of the N-glycans (88%) were neutral while the remainder were charged due to phosphorylation. Site-directed mutagenesis of Asn to Gin in two of the N-glycosylation sequons, and subsequent analysis of the N-glycans on the yeast-secreted proteins together with analysis of the N-glycans from the individual sites of H(A16-M)-Y suggest the presence of steri c hindrance to glycan modification by the glycans themselves. H(A16-M) -Y produced under control of either the yeast protease B or the yeast 3'-phosphoglycerate kinase promoter, each in two different Saccharomyc es strains revealed a dependence of N-glycan profile on both strain an d culture conditions. The extent of O-glycosylation was found to be ni ne mannose units per H(A16-M)-Y molecule. An attempt to identify the l inkage-sites for the O-glycans by amino acid sequencing failed, sugges ting non-stoichiometric or heterogeneous O-glycosylation. The possible modes in which N-glycans might contribute to resistance of H(A16-M)-Y to irreversible thermal denaturation are discussed with respect to st ructural information available for H(A16-M)-Y.