Abstract:
A variety of habitats were sampled for the presence of Aureobasidium black yeasts with the objective to find pullulan-producing strains in Thailand. Aureobasidium spp. Were successfully isolated from distinct diverse habitats: from leaves to painted surfaces. Parameters for the identification of the isolates were a balance of morphology, nutritional parameters, exopolysaccharides (EPSs) production, and rDNA Internal Transcribed Spacer (ITS) sequencing. The isolates were polymorphic with blastospores, chlamydospores, and hyphae, typically black yet with color variant strains. Their ITS sequences were very similar to each other and showed strong correlation with the GenBank A. pullulans, alignment using the BLAST program. Nutritional assimilation patterns of all isolates corresponded well to A. pullulans var. pullulans. All isolates produced pullulan EPSs as deduced from anthrone carbohydrate analysis, pullulanase sensitivity, infrared analysis, and ¹³C-NMR spectroscopy. The five higher-yielding strains, BK4, BK6, LB3, NRM2 and SK3, were subjected to EPS optimization. Parameters for optimization included various carbon and nitrogen sources. The maximal EPS yield (25 g.1⁻¹) was obtained from strain NRM2 under the optimal conditions (sucrose and peptone) after 7 days. The higher-molecular weight EPSs were from strain BK6 (2,450,000 Da) and NRM2 (1,770,000 Da) after 3 days culture, as analyzed using high performance size exclusion chromatography. The molecular weight (and viscosities) of EPSs from all strains decreased in late culture presumably as a result of A. pullulans producing extracellular hydrolytic enzymes such as alpha-amylase and pullulanase. In assays for alpha-amylase and pullulanase, all A. pullulans strains were positive in both solid and liquid starch-based media. Alpha-amylase and pullulanase were also detected in the A. pullulans culture grown on sucrose as the sole carbon source. Culture on sucrose still resulted in synthesis of alpha-amylase and pullulanase. Two approaches have been considered to obtain the higher-molecular weight pullulan: preparation of an amylase-negative mutant, or the alternative use of an amylase inhibitor acarbose. The degradation of late culture EPSs from the alpha-amylase negative mutant was similar to that of the wild type. The EPS from strain NRM2 cultured in the presence of acarbose, in late culture showed higher molecular weight. This result implied that part of the reduction in the molecular weight of EPS was due to alpha-amylase.
