OVEREXPRESSION AND CHARACTERIZATION OF ASPERGILLUS-AWAMORI WILD-TYPE AND MUTANT GLUCOAMYLASE SECRETED BY THE METHYLOTROPHIC YEAST PICHIA-PASTORIS - COMPARISON WITH WILD-TYPE RECOMBINANT GLUCOAMYLASE PRODUCED USING SACCHAROMYCES-CEREVISIAE AND ASPERGILLUS-NIGER AS HOSTS

Glucoamylase from Aspergillus niger (identical to Aspergillus awamori glucoamylase) is an industrially important, multidomain N- and O-glyco sylated starch-hydrolase. To produce protein-engineered glucoamylase, heterologous expression is established in the methylotrophic yeast Pic hia pastoris. Using the vector pHIL-D2, the cDNA encoding A. awamori g lucoamylase is inserted in the yeast genome downstream of the 5'AOX1 p romoter 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 glucoamyla se produced in P. pastoris, Saccharomyces cerevisiae, or A. niger disp layed similar catalytic properties, thiol content, and isoelectric poi nt, Glucoamylase from P. pastoris, however, has higher thermostability than the enzymes from S. cerevisiae, A. niger, or a commercial prepar ation of A. niger glucoamylase, The average M(r) determined by matrix- assisted laser desorption ionization mass spectrometry of these enzyme s is thus 82,327, 83,869, 82,839, and 80,370, respectively, and neutra l sugar analysis shows the differences to be due to variation in the e xtent of glycosylation, Compared to wild-type, single-residue mutation generally reduced the amount of secreted glucoamylase in S. cerevisia e 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 glu coamylase, while the corresponding single mutants have been produced a t 1 and 20% of the wild-type level in S. cerevisiae and A. niger, resp ectively. (C) 1997 Academic Press.