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

Abstract

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 α-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 α-amylase and pullulan production. In sucrose-grown cultures, pullulan yields increased over time while the molecular weight of pullulan generally decreased. However, no α-amylase activity was detected in these cultures. Low levels of α-amylase were present in starch-grown culture, but pullulan analysis was complicated by residual starch. To facilitate further studies on the role of α-amylase in the reduction of pullulan molecular weight, the α-amylase gene from A. pullulans NRRL Y-12974 was cloned and characterized. The coding region of the complete α-amylase gene contains 2,247 bp, including 7 introns and 8 exons. The putative mRNA was 1,878 bp long, encoding an α-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 α-amylase attack the minor maltotetraose subunits of pullulan and cause the reduction of molecular weight.