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 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.
Biological Sciences Program, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand.
Pennapa
P
Manitchotpisit
Christopher D
CD
Skory
Timothy D
TD
Leathers
Pongtharin
P
Lotrakul
Douglas E
DE
Eveleigh
Sehanat
S
Prasongsuk
Hunsa
H
Punnapayak
D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005936:Glucans / Q000096:biosynthesis* / Q000737:chemistry; D008970:Molecular Weight; D010802:Phylogeny; D013213:Starch / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
D005936:Glucans; C009109:pullulan; D013213:Starch; D000516:alpha-Amylases