The enzymatic hydrolysis of starch, consisting of linear (amylose) and branched (amylopectin) glucose polymers, is catalyzed by α-, β- and glucoamylases (γ-amylases), cyclodextrinases, α-glucosidases, and debranching enzymes. Saccharomyces cerevisiae cannot utilize starch. Our laboratory has previously co-expressed the Bacillus amyloliquefaciens α-amylase (AMY) and the Saccharomyces diastaticus glucoamylase (STA2) genes in S. cerevisiae. A gene encoding a debranching enzyme (pullulanase) from Klebsiella pneumoniae ATCC15050 was cloned and its nucleotide sequence determined. This gene will be co-expressed with the α- and γ-amylase to produce an amylolytic S. cerevisiae strain. Extensive data base comparisons of the K. pneumoniae pullulanase amino-acid sequence with the the amino-acid sequences of other debranching enzymes and α-, β- and γ-amylases (from bacteria, yeasts, higher fungi and higher eukaryotes), indicated that these debranching enzymes have amino-acid regions similar to those found in α-amylases. The conserved regions in α-amylases comprise key residues that are implicated in substrate binding, catalysis, and calcium binding and are as follows. Region 1: DVVINH; region 2: GFRLDAAKH and region 4: FVDNHD. When comparing conserved regions, no similarity could be detected between debranching enzymes and β- and γ-amylases. © 1993 Springer-Verlag.