b-Fructosidases are a widespread group of enzymes that catalyze the hydrolysis of terminal fructosyl units from various substrates. These enzymes also exhibit transglycosylation activity when they function with high concentrations of @entity969 , which is used to synthesize fructooligosaccharides (FOS) in the food industry. A b-fructosidase (BfrA) with high transglycosylation activity was purified from @entity12799 FS4 as a monomeric glycoprotein. Compared with the most extensively studied @entity12799 spp. fructosidases that synthesize inulin-type b-(2-1)-linked FOS, BfrA has unique transfructosylating property of synthesizing levan- and neolevan-type b-(2-6)-linked FOS. The coding sequence (bfrAFS4, 1.86 kb) of BfrA was amplified and expressed in @entity526 and @entity10056 . Both native and recombinant proteins showed transfructosylation and hydrolyzation activities with broad substrate specificity. These proteins could hydrolyze the following linkages: Glc a-1, 2-b Fru; Glc a-1, 3-a Fru; and Glc a-1, 5-b Fru. Compared with the unglycosylated @entity526 -expressed BfrA (E.BfrA), the N-glycosylated native (N.BfrA) and the @entity10056 -expressed BfrA (P.BfrA) were highly stable at a wide pH range (pH 4 to 11), and significantly more thermostable at temperatures up to 50  C with a maximum activity at 55  C. Using @entity969 as substrate, the Km and kcat values for total activity were 37.19  5.28 mM and 1.0016  0.039*104 s-1 for N.BfrA. Moreover, 10 of 13 putative N-glycosylation sites were glycosylated on N.BfrA, and N-glycosylation was essential for enzyme thermal stability and optima activity. Thus, BfrA has demonstrated as a well-characterized A. oryzae fructosidase with unique transfructosylating capability of synthesizing levan- and neolevan-type FOS.

Purification, Cloning, Characterization, and N-Glycosylation Analysis of a Novel b-Fructosidase from XXXX FS4 Synthesizing Levan- and Neolevan-Type Fructooligosaccharides.

@entity526 :: ('562', 'Species') :: ['Escherichia coli', 'E. coli'] @entity969 :: ('MESH:D013395', 'Chemical') :: ['sucrose'] @entity10056 :: ('4922', 'Species') :: ['Pichia pastoris', 'P. pastoris'] @entity12799 :: ('5062', 'Species') :: ['Aspergillus oryzae', 'Aspergillus']
@entity12799 :: (5062,Species) :: ['Aspergillus oryzae']
