REGIONAL SEQUENCE HOMOLOGIES IN STARCH-DEGRADING ENZYMES

The enzymatic hydrolysis of starch, consisting of linear (amylose) and branched (amylopectin) glucose polymers, is catalyzed by alpha-, beta - and glucoamylases (gamma-amylases), cyclodextrinases, alpha-glucosid ases, and debranching enzymes. Saccharomyces cerevisiae cannot utilize starch. Our laboratory has previously co-expressed the Bacillus amylo liquefaciens alpha-amylase (AMY) and the Saccharomyces diastaticus glu coamylase (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 alpha- and gamma-amylase to produce an amylolytic S. cerevis iae strain. Extensive data base comparisons of the K. pneumoniae pullu lanase amino-acid sequence with the the amino-acid sequences of other debranching enzymes and alpha-, beta- and gamma-amylases (from bacteri a, yeasts, higher fungi and higher eukaryotes), indicated that these d ebranching enzymes have amino-acid regions similar to those found in a lpha-amylases. The conserved regions in alpha-amylases comprise key re sidues that are implicated in substrate binding, catalysis, and calciu m binding and are as follows. Region 1: DVVINH; region 2: GFRLDAAKH an d region 4: FVDNHD. When comparing conserved regions, no similarity co uld be detected between debranching enzymes and beta- and gamma-amylas es.