DEVELOPING BIOCONVERSION PROCESSES FOR HIGH-VALUE CARBOHYDRATE PRODUCTS
Location: Renewable Product Technology Research Unit
Title: a-Amylase activity during pullulan production and a-Amylase gene analyses of Aureobasidium pullulans
Submitted to: Journal of Industrial Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 29, 2010
Publication Date: August 15, 2011
Citation: Manitchotpisit, P., Skory, C.D., Leathers, T.D., Lotrakul, P., Eveleigh, D.E., Prasongsuk, S., Punnapayak, H. 2011. a-Amylase activity during pullulan production and a-Amylase gene analyses of Aureobasidium pullulans. Journal of Industrial Microbiology and Biotechnology. 38:1211-1218. DOI: 10.1007/s10295-010-0899-y.
Interpretive Summary: This research examined the effect of the enzyme amylase on the molecular weight of pullulan, a commercially produced polysaccharide, made by the fungus Aureobasidium, that is used in the manufacture of edible films (e.g., food coatings, flavor strips), cosmetics, and drug delivery composites. High molecular weight pullulan is usually preferred for most applications. It has been proposed that the enzyme amylase produced by Aureobasidium during growth leads to degradation of pullulan. Fundamental information is needed to determine the cause of this degradation so strategies can be implemented to control molecular weight of the product. Results of this showed that only low levels of amylase are expressed during pullulan degradation and likely are not solely responsible for this effect. Results are important to researchers developing improved methods to produce microbial polysaccharides.
The fungus 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 negatively affects the molecular weight of pullulan in late cultures. Based on a recent phylogenetic analysis, five representative strains were chosen to study the relationship between a-amylase and pullulan production. In sucrose-grown cultures, pullulan yields increased over time while the molecular weight of pullulan generally decreased. However, no a-amylase activity was detected in these cultures. Low levels of a-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 a-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. 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. Alternatively, other as yet uncharacterized enzymes or other factors may play a role.