Biochemical analysis of starch degradation by Ruminobacter amylophilus 70

Abstract

Ruminobacter amylophilus is an obligate anaerobe that uses only α-linked glucose molecules (i.e., maltose, maltodextrins, and starch) as a source of energy, making it an excellent model for the study of bacterial starch degradation. Constitutive amylase, amylopectinase, and pullulanase activities were found in intracellular and extracellular fractions of R. amylophilus. However, extracellular activities apparently resulted from cell lysis. Both soluble and membrane-bound polysaccharidase activities were detected. Most of the soluble polysaccharidase activity partitioned with the periplasmic cell fraction. No α-glucosidase or maltase activity was detected in either the cellular or extracellular fraction. In addition, intact cells of R. amylophilus bound U-14C-starch. This binding could be saturated and was constitutive and sensitive to proteinase K, indicating protein or protein complex mediation. Competition experiments showed that these starch-binding sites had equally high affinities for starch and maltodextrins larger than maltotriose. The sites had a reduced affinity for maltose and virtually no affinities for glucose and nonstarch polysaccharides. These findings suggest that R. amylophilus binds starch molecules to the cell surface as an initial step in transporting the molecule through the outer membrane and into the periplasmic space. Extracellular polysaccharidases do not appear to be involved in starch degradation.