Characterization of a novel extracellular α-amylase from Ruminococcus bromii ATCC 27255 with neopullulanase-like activity.

Ruminococcus is one of the keystone bacteria of the human colonic microbiota and is highly specific for utilization of resistant starch via formation of amylosomes. Here, we present the characteristics of an extracellular amylase, Rbamy5, in Ruminococcus bromii. In an in silico study, it showed low homology with any other known amylases, but it was evolutionarily classified as a GH13_36 subfamily intermediary amylase. Recombinant Rbamy5 exhibited maximum activity toward amylose (384 ±â€¯26 U·mg-1) over soluble starch (254 ±â€¯3 U·mg-1), amylopectin (46.1 ±â€¯2.6 U·mg-1) and pullulan (72.5 ±â€¯0.41 U·mg-1) at 45 °C and pH 5.0. It was also able to degrade small substrates such as maltotriose (G3), maltotetraose (G4), and maltopentaose (G5) into maltose (G2). Despite lacking a specific N-terminal domain, Rbamy5 opened the cyclodextrin ring, which resembles those of neopullulanase. Moreover, it accumulated short α-(1 → 6)-branched maltooligosaccharides from soluble starch and maltosyl-ß-cyclodextrin (G2-ß-CD), while panose was solely derived from pullulan. These results suggest that Rbamy5 may have a role to provide branched maltooligosaccharides to stimulate the growth of beneficial microorganisms in the human intestine.

Diversity analysis of Saccharomyces cerevisiae isolated from natural sources by multilocus sequence typing (MLST).

We used multilocus sequence typing (MLST) to analyze the diversity of natural isolates of Saccharomyces cerevisiae, the most important microorganism in alcoholic fermentation. Six loci, ADP1, RPN2, GLN4, ACC1, MET4, and NUP116, in S. cerevisiae genome were selected as MLST markers. To investigate genetic diversity within S. cerevisiae, 42 S. cerevisiae isolated from natural sources in Korea as well as six S. cerevisiae obtained from Genbank and four industrial S. cerevisiae were examined using MLST. Twenty-six polymorphic sites were found in the six loci. Among them, ACC1 had the most genetic variation with eight polymorphic sites. MLST differentiated the 52 strains into three clades. Alcohol fermentation results revealed that S. cerevisiae in Clade III produced less alcohol than those in Clades I and II. These results suggested that MLST is a powerful tool to differentiate S. cerevisiae and can potentially be used to select S. cerevisiae suitable for industrial use.