NDLI: Purification, Cloning, Characterization, and N-Glycosylation Analysis of a Novel β-Fructosidase from Aspergillus oryzae FS4 Synthesizing Levan- and Neolevan-Type Fructooligosaccharides

Content Provider	PubMed Central
Author	Xu, Li Wang, Dongxue Lu, Lili Jin, Lan Liu, Jiawei Song, Deyong Guo, Zhongwu Xiao, Min
Editor	Tyagi, Anil Kumar
Copyright Year	2014
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.
Related Links	http://dx.doi.org/10.1371/journal.pone.0114793
Starting Page	114793
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	12
Volume Number	9
Language	English
Publisher	Public Library of Science
Publisher Date	2014-12-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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