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Purification, cloning, characterization, and N-glycosylation analysis of a novel ?-fructosidase from Aspergillus oryzae FS4 synthesizing levan- and neolevan-type fructooligosaccharides.

ABSTRACT: ?-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 ?-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 ?-(2-1)-linked FOS, BfrA has unique transfructosylating property of synthesizing levan- and neolevan-type ?-(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 ?-1, 2-? Fru; Glc ?-1, 3-? Fru; and Glc ?-1, 5-? 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.

SUBMITTER: Xu L

PROVIDER: S-EPMC4264766 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Purification, cloning, characterization, and N-glycosylation analysis of a novel β-fructosidase from Aspergillus oryzae FS4 synthesizing levan- and neolevan-type fructooligosaccharides.

Xu Li L Wang Dongxue D Lu Lili L Jin Lan L Liu Jiawei J Song Deyong D Guo Zhongwu Z Xiao Min M

PloS one 20141212 12

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 Asp ...[more]

PMID: 25501957

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Project description:BackgroundFungal allergy is considered as serious health problem worldwide and is increasing at an alarming rate in the industrialized areas. Rhizopus oyzae is a ubiquitously present airborne pathogenic mold and an important source of inhalant allergens for the atopic population of India. Here, we report the biochemical and immunological features of its 44 kDa sero-reactive aspartic protease allergen, which is given the official designation 'Rhi o 1'.MethodThe natural Rhi o 1 was purified by sequential column chromatography and its amino acid sequence was determined by mass spectrometry and N-terminal sequencing. Based on its amino acid sequence, the cDNA sequence was identified, cloned and expressed to produce recombinant Rhi o 1. The allergenic activity of rRhi o 1 was assessed by means of its IgE reactivity and histamine release ability. The biochemical property of Rhi o 1 was studied by enzyme assay. IgE-inhibition experiments were performed to identify its cross-reactivity with the German cockroach aspartic protease allergen Bla g 2. For precise characterization of the cross-reactive epitope, we used anti-Bla g 2 monoclonal antibodies for their antigenic specificity towards Rhi o 1. A homology based model of Rhi o 1 was built and mapping of the cross-reactive conformational epitope was done using certain in silico structural studies.ResultsThe purified natural nRhi o 1 was identified as an endopeptidase. The full length allergen cDNA was expressed and purified as recombinant rRhi o 1. Purified rRhi o 1 displayed complete allergenicity similar to the native nRhi o 1. It was recognized by the serum IgE of the selected mold allergy patients and efficiently induced histamine release from the sensitized PBMC cells. This allergen was identified as an active aspartic protease functional in low pH. The Rhi o 1 showed cross reactivity with the cockroach allergen Bla g 2, as it can inhibit IgE binding to rBla g 2 up to certain level. The rBla g 2 was also found to cross-stimulate histamine release from the effector cells sensitized with anti-Rhi o 1 serum IgE. This cross-reactivity was found to be mediated by a common mAb4C3 recognizable conformational epitope. Bioinformatic studies revealed high degree of structural resemblances between the 4C3 binding sites of both the allergens.Conclusion/significanceThe present study reports for the first time anew fungal aspartic protease allergen designated as Rhi o 1, which triggers IgE-mediated sensitization leading to various allergic diseases. Here we have characterized the recombinant Rhi o 1 and its immunological features including cross-reactive epitope information that will facilitate the component-resolved diagnosis of mold allergy.

Dataset Information

Purification, cloning, characterization, and N-glycosylation analysis of a novel ?-fructosidase from Aspergillus oryzae FS4 synthesizing levan- and neolevan-type fructooligosaccharides.

Publications

Purification, cloning, characterization, and N-glycosylation analysis of a novel β-fructosidase from Aspergillus oryzae FS4 synthesizing levan- and neolevan-type fructooligosaccharides.

Similar Datasets

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