Glucoamylase from Aspergillus niger (identical to Aspergillus awamori glucoamylase) is an industrially important, multidomain N- and O-glycosylated starch-hydrolase. To produce protein-engineered glucoamylase, heterologous expression is established in the methylotrophic yeast Pichia pastoris. Using the vector pHIL-D2, the cDNA encoding A. awamori glucoamylase is inserted in the yeast genome downstream of the 5' AOX1 promoter to replace the AOX1 gene. Induction by 0.75% methanol for 48 h led to synthesis of secreted glucoamylase to give around 0.4 g/liter, as directed by the A. awamori signal sequence. Recombinant glucoamylase produced in P. pastoris, Saccharomyces cerevisiae, or A. niger displayed similar catalytic properties, thiol content, and isoelectric point. Glucoamylase from P. pastoris, however, has higher thermostability than the enzymes from S. cerevisiae, A. niger, or a commercial preparation of A. niger glucoamylase. The average Mr determined by matrix-assisted laser desorption ionization mass spectrometry of these enzymes is thus 82,327, 83,869, 82,839, and 80,370, respectively, and neutral sugar analysis shows the differences to be due to variation in the extent of glycosylation. Compared to wild-type, single-residue mutation generally reduced the amount of secreted glucoamylase in S. cerevisiae and A. niger. In P. pastoris, however, the Cys320 --> Ala/Glu400 --> Cys double mutant is produced at 0.3 g/liter, or 75% of wild-type glucoamylase, while the corresponding single mutants have been produced at l and 20% of the wild-type level in S. cerevisiae and A. niger, respectively.