PATHWAY ENGINEERING OF FUNGI FOR IMPROVED BIOPROCESS APPLICATIONS
Location: Renewable Product Technology Research Unit
Title: Relationship Between a-amylase Activity and Pullulan Profiles, and a-amylase Gene Analyses of the Fungus Aureobasidium Pullulans
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: December 12, 2009
Publication Date: December 12, 2009
Citation: Manitchotpisit, P., Leathers, T.D., Skory, C.D., Lotrakul, P., Prasongsuk, S., Punnapayak, H. 2009. Relationship between a-amylase activity and pullulan profiles, and a-amylase gene analyses of the fungus Aureobasidium pullulans [abstract]. Biological Sciences Graduate Congress. Abstract #BT-OR 04. p. 47.
Tropical isolates of Aureobasidium pullulans isolated from various habitats in Thailand were classified based on multilocus phylogenetic analyses using concordance analysis of DNA sequences. This fungus is the major source of commercially produced pullulan, a high molecular weight polysaccharide that is used in the manufacture of edible films. It has been proposed that a-amylase negatively affects the molecular weight of pullulan in late cultures. To study the relationship between the activities of a-amylase and pullulan profiles, 5 representative strains were selected and grown in pullulan production medium. During cultivation, each culture broth was collected to assay the a-amylase and pullulanase activities and determine the pullulan profiles, including a-amylase sensitivity, pullulanase sensitivity, molecular weight, and viscosity. Pullulan yields gradually increased over time in individual strains, while the molecular weight of pullulan decreased. Adiitionally, no pullulanase activities were detected for all cultures. Even though the a-amylase activity of each strain was very low and pullulanse activity was not readily detected, it is possible that very low levels of a-amylase hydrolyze the minor maltotetraose structure of pullulan and cause the reduction of molecular weight; it is also possible that other as yet uncharacterized enzymes or other factors play a role. Thus, further studies are needed to determine whether a-amylase is important in the reduction of pullulan molecular weight. To facilitate such studies, the a-amylase gene from A. pullulans NRRL Y-12974 was cloned and characterized. The coding region of the complete a-amylase gene contains 2,247-bp, including 7 introns and 8 exons. The putative mRNA was 1,878-bp long, encoding an a-amylase of 625 amino acid residues. Southern blot analysis indicated that there was only one copy of this gene in the genome. Finally, a-amylase mRNA was detected using rtPCR with samples obtained between 4-8 days of cultivation on production medium.