Dataset Information

Biochemical properties of a native ?-1,4-mannanase from Aspergillus aculeatus QH1 and partial characterization of its N-glycosylation.

ABSTRACT: N-glycosylation plays critical roles in protein secretion, sorting, stability, activity modulation, and interactions to other molecules in the eukaryotic organisms. Fungal ?-1,4-mannanases have been widely used in the agri-food industry and contribute to the pathogenesis on plants. However, the information on N-glycosylation of a specific fungal carbohydrate-active enzyme (CAZyme) is currently limited. Herein, a cDNA was cloned from Aspergillus aculeatus QH1, displaying a full length of 1302 bp with an open reading frame of 1134 bp encoding for a GH5 subfamily 7 ?-1, 4-mannanase, namely AacMan5_7A. The enzyme was purified and exhibited an optimal activity at pH 4.6 and 60 °C, hydrolyzing glucomannan and galactomannan, but not yeast mannan. AacMan5_7A is an N-glycosylated protein decorated with a high-mannose type glycan. Further through UPLC-ESI-MS/MS analysis, one of the four predicted N-glycosylation sites at N255 position was experimentally verified. The present study expands the information of N-glycosylation in fungal CAZymes, providing scientific bases for enhancing the production of fungal enzymes and their applications in food, feed, and plant biomass conversions.

SUBMITTER: Ma L

PROVIDER: S-EPMC7887645 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Publications

Biochemical properties of a native β-1,4-mannanase from <i>Aspergillus aculeatus</i> QH1 and partial characterization of its N-glycosylation.

Ma Liqing L Jiang Heping H Li Weihua W Qin Hua H Lv Zhi Z Huang Jiujiu J Hou Xuewen X Wang Weijun W

Biochemistry and biophysics reports 20210212

N-glycosylation plays critical roles in protein secretion, sorting, stability, activity modulation, and interactions to other molecules in the eukaryotic organisms. Fungal β-1,4-mannanases have been widely used in the agri-food industry and contribute to the pathogenesis on plants. However, the information on N-glycosylation of a specific fungal carbohydrate-active enzyme (CAZyme) is currently limited. Herein, a cDNA was cloned from <i>Aspergillus aculeatus</i> QH1, displaying a full length of 1 ...[more]

PMID: 33644418

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Project description:BackgroundThe study of coffee polysaccharides-degrading enzymes from the coffee berry borer Hypothenemus hampei, has become an important alternative in the identification for enzymatic inhibitors that can be used as an alternative control of this dangerous insect. We report the cloning, expression and biochemical characterization of a mannanase gene that was identified in the midgut of the coffee berry borer and is responsible for the degradation of the most abundant polysaccharide in the coffee bean.MethodsThe amino acid sequence of HhMan was analyzed by multiple sequence alignment comparisons with BLAST (Basic Local Alignment Search Tool) and CLUSTALW. A Pichia pastoris expression system was used to express the recombinant form of the enzyme. The mannanase activity was quantified by the 3,5-dinitrosalicylic (DNS) and the hydrolitic properties were detected by TLC.ResultsAn endo-1,4-β-mannanase from the digestive tract of the insect Hypothenemus hampei was cloned and expressed as a recombinant protein in the Pichia pastoris system. This enzyme is 56% identical to the sequence of an endo-β-mannanase from Bacillus circulans that belongs to the glycosyl hydrolase 5 (GH5) family. The purified recombinant protein (rHhMan) exhibited a single band (35.5 kDa) by SDS-PAGE, and its activity was confirmed by zymography. rHhMan displays optimal activity levels at pH 5.5 and 30°C and can hydrolyze galactomannans of varying mannose:galactose ratios, suggesting that the enzymatic activity is independent of the presence of side chains such as galactose residues. The enzyme cannot hydrolyze manno-oligosaccharides such as mannobiose and mannotriose; however, it can degrade mannotetraose, likely through a transglycosylation reaction. The K(m) and k(cat) values of this enzyme on guar gum were 2.074 mg ml(-1) and 50.87 s(-1), respectively, which is similar to other mannanases.ConclusionThis work is the first study of an endo-1,4-β-mannanase from an insect using this expression system. Due to this enzyme's importance in the digestive processes of the coffee berry borer, this study may enable the design of inhibitors against endo-1,4-β-mannanase to decrease the economic losses stemming from this insect.

Dataset Information

Biochemical properties of a native ?-1,4-mannanase from Aspergillus aculeatus QH1 and partial characterization of its N-glycosylation.

Publications

Biochemical properties of a native β-1,4-mannanase from <i>Aspergillus aculeatus</i> QH1 and partial characterization of its N-glycosylation.

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