α-Amylase activity during pullulan production and α-amylase gene analyses of Aureobasidium pullulans

Aureobasidium pullulans is the source of commercially produced pullulan, a high molecular weight polysaccharide that is used in the manufacture of edible films. It has been proposed that alpha-amylase decreases the molecular weight of pullulan in late cultures. Based on a recent phylogenetic analysis, five representative strains were chosen to study the relationship between alpha-amylase and pullulan production. In sucrose-grown cultures, pullulan yields increased over time while the molecular weight of pullulan generally decreased. However, no alpha-amylase activity was detected in these cultures. Low levels of alpha-amylase were present in starch-grown culture, but pullulan analysis was complicated by residual starch. To facilitate further studies on the role of alpha-amylase in the reduction of pullulan molecular weight, the alpha-amylase gene from A. pullulans NRRL Y-12974 was cloned and characterized. The coding region of the complete alpha-amylase gene contains 2,247 bp, including 7 introns and 8 exons. The putative mRNA was 1,878 bp long, encoding an alpha-amylase of 625 amino acid residues. Southern blot analysis indicated that there was only one copy of this gene in the genome. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that the gene was transcribed in both sucrose- and starch-grown cultures. It is possible that very low levels of alpha-amylase attack the minor maltotetraose subunits of pullulan and cause the reduction of molecular weight.