title: Purification, cloning, characterization, and N-glycosylation analysis of a novel β-fructosidase from Aspergillus oryzae FS4 synthesizing levan- and neolevan-type fructooligosaccharides.
abstract: β-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 sucrose, which is used to synthesize fructooligosaccharides (FOS) in the food industry. A β-fructosidase (BfrA) with high transglycosylation activity was purified from Aspergillus oryzae FS4 as a monomeric glycoprotein. Compared with the most extensively studied Aspergillus spp. fructosidases that synthesize inulin-type β-(2-1)-linked FOS, BfrA has unique transfructosylating property of synthesizing levan- and neolevan-type β-(2-6)-linked FOS. The coding sequence (bfrAFS4, 1.86 kb) of BfrA was amplified and expressed in Escherichia coli and Pichia pastoris. Both native and recombinant proteins showed transfructosylation and hydrolyzation activities with broad substrate specificity. These proteins could hydrolyze the following linkages: Glc α-1, 2-β Fru; Glc α-1, 3-α Fru; and Glc α-1, 5-β Fru. Compared with the unglycosylated E. coli-expressed BfrA (E.BfrA), the N-glycosylated native (N.BfrA) and the P. pastoris-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 sucrose 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.
authors.affiliation: State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong University, Jinan, PR China.
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.affiliation: State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong University, Jinan, PR China.
authors.forename: Dongxue
authors.initials: D
authors.lastname: Wang
authors.affiliation: State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong University, Jinan, PR China.
authors.forename: Lili
authors.initials: L
authors.lastname: Lu
authors.affiliation: State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong University, Jinan, PR China.
authors.forename: Lan
authors.initials: L
authors.lastname: Jin
authors.affiliation: State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong University, Jinan, PR China.
authors.forename: Jiawei
authors.initials: J
authors.lastname: Liu
authors.affiliation: State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong University, Jinan, PR China.
authors.forename: Deyong
authors.initials: D
authors.lastname: Song
authors.affiliation: State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong University, Jinan, PR China.
authors.forename: Zhongwu
authors.initials: Z
authors.lastname: Guo
authors.affiliation: State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong University, Jinan, PR China.
authors.forename: Min
authors.initials: M
authors.lastname: Xiao
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D005630:Fructans / Q000737:chemistry*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013696:Temperature; D043324:beta-Fructofuranosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D005630:Fructans; D009844:Oligosaccharides; C116580:fructooligosaccharide; C072599:levan; D043324:beta-Fructofuranosidase