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 ± 26 U·mg-1) over soluble starch (254 ± 3 U·mg-1), amylopectin (46.1 ± 2.6 U·mg-1) and pullulan (72.5 ± 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-β-cyclodextrin (G2-β-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.