Glucoamylase fromAspergillus niger(identical toAspergillus awamoriglucoamylase) is an industrially important, multidomain N- and O-glycosylated starch-hydrolase. To produce protein-engineered glucoamylase, heterologous expression is established in the methylotrophic yeastPichia pastoris.Using the vector pHIL-D2, the cDNA encodingA. awamoriglucoamylase is inserted in the yeast genome downstream of the5′AOX1promoter to replace theAOX1gene. Induction by 0.75% methanol for 48 h led to synthesis of secreted glucoamylase to give around 0.4 g/liter, as directed by theA. awamorisignal sequence. Recombinant glucoamylase produced inP. pastoris, Saccharomyces cerevisiae,orA. nigerdisplayed similar catalytic properties, thiol content, and isoelectric point. Glucoamylase fromP. pastoris,however, has higher thermostability than the enzymes fromS. cerevisiae, A. niger,or a commercial preparation ofA. nigerglucoamylase. The averageM r 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 inS. cerevisiaeandA. niger.InP. 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 1 and 20% of the wild-type level inS. cerevisiaeandA. niger,respectively.