Biochemical characterization of a beta-fructofuranosidase from Rhodotorula dairenensis with transfructosylating activity.
An extracellular beta-fructofuranosidase from the yeast Rhodotorula dairenensis was characterized biochemically. The enzyme molecular mass was estimated to be 680 kDa by analytical gel filtration and 172 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of which the N-linked carbohydrate accounts for 16% of the total mass. It displays optimum activity at pH 5 and 55-60 degrees C. The enzyme shows broad substrate specificity, hydrolyzing sucrose, 1-kestose, nystose, leucrose, raffinose and inulin. Although the main reaction catalyzed by this enzyme is sucrose hydrolysis, it also exhibits transfructosylating activity that, unlike other microbial beta-fructofuranosidases, produces a varied type of prebiotic fructooligosaccharides containing beta-(2-->1)- and beta-(2-->6)-linked fructose oligomers. The maximum concentration of fructooligosaccharides was reached at 75% sucrose conversion and it was 87.9 g L(-1). The 17.0% (w/w) referred to the total amount of sugars in the reaction mixture. At this point, the amounts of 6-kestose, neokestose, 1-kestose and tetrasaccharides were 68.9, 10.6, 2.6 and 12.7 g L(-1), respectively.
Gutiérrez-Alonso
Patricia
P
Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma Madrid, Cantoblanco, Madrid, Spain.
Fernández-Arrojo
Lucía
L
Plou
Francisco J
FJ
Fernández-Lobato
María
M
D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005632:Fructose / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D055503:Protein Multimerization; D021122:Protein Subunits; D012248:Rhodotorula / Q000201:enzymology*; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D013696:Temperature; D043324:beta-Fructofuranosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D005656:Fungal Proteins; D009844:Oligosaccharides; D021122:Protein Subunits; C116580:fructooligosaccharide; D005632:Fructose; D013395:Sucrose; D043324:beta-Fructofuranosidase