Dataset Information

Characterization of Three Fungal Isomaltases Belonging to Glycoside Hydrolase Family 13 That Do not Show Transglycosylation Activity.

ABSTRACT: α-1,6-Glucosidase (isomaltase) belongs to glycoside hydrolase (GH) families 13 and 31. Genes encoding 3 isomaltases belonging to GH family 13 were cloned from filamentous fungi, Aspergillus oryzae (agl1), A. niger (agdC),and Fusarium oxysporum (foagl1), and expressed in Escherichia coli. The enzymes hydrolyzed isomaltose and α-glucosides preferentially at a neutral pH, but did not recognize maltose, trehalose, and dextran. The activity of AgdC and Agl1 was inhibited in the presence of 1 % glucose, while Foagl1 was more tolerant to glucose than the other two enzymes were. The three fungal isomaltases did not show transglycosylation when isomaltose was used as the substrate and a similar result was observed for AgdC and Agl1 when p-nitrophenyl-α-glucoside was used as the substrate.

SUBMITTER: Eisawa H

PROVIDER: S-EPMC8056888 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Characterization of Three Fungal Isomaltases Belonging to Glycoside Hydrolase Family 13 That Do not Show Transglycosylation Activity.

Eisawa Hiroki H Ogawa Shun S Yamazaki Nobuhiro N Maekawa Kohki K Yamaguchi Takahiro T Sato Shota S Shiota Kazuma K Yoshida Takashi T

Journal of applied glycoscience 20170220 1

α-1,6-Glucosidase (isomaltase) belongs to glycoside hydrolase (GH) families 13 and 31. Genes encoding 3 isomaltases belonging to GH family 13 were cloned from filamentous fungi, Aspergillus oryzae (agl1), A. niger (agdC),and Fusarium oxysporum (foagl1), and expressed in Escherichia coli. The enzymes hydrolyzed isomaltose and α-glucosides preferentially at a neutral pH, but did not recognize maltose, trehalose, and dextran. The activity of AgdC and ...[more]

PMID: 34354490

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Project description:Backgroundβ-mannanase can hydrolyze β-1,4 glycosidic bond of mannan by the manner of endoglycosidase to generate mannan-oligosaccharides. Currently, β-mannanase has been widely applied in food, medicine, textile, paper and petroleum exploitation industries. β-mannanase is widespread in various organisms, however, microorganisms are the main source of β-mannanases. Microbial β-mannanases display wider pH range, temperature range and better thermostability, acid and alkali resistance, and substrate specificity than those from animals and plants. Therefore microbial β-mannanases are highly valued by researchers. Recombinant bacteria constructed by gene engineering and modified by protein engineering have been widely applied to produce β-mannanase, which shows more advantages than traditional microbial fermentation in various aspects.ResultsA β-mannanase gene (Man1E), which encoded 731 amino acid residues, was cloned from Enterobacter aerogenes. Man1E was classified as Glycoside Hydrolase family 1. The bSiteFinder prediction showed that there were eight essential residues in the catalytic center of Man1E as Trp166, Trp168, Asn229, Glu230, Tyr281, Glu309, Trp341 and Lys374. The catalytic module and carbohydrate binding module (CBM) of Man1E were homologously modeled. Superposition analysis and molecular docking revealed the residues located in the catalytic module of Man1E and the CBM of Man1E. The recombinant enzyme was successfully expressed, purified, and detected about 82.5 kDa by SDS-PAGE. The optimal reaction condition was 55 °C and pH 6.5. The enzyme exhibited high stability below 60 °C, and in the range of pH 3.5-8.5. The β-mannanase activity was activated by low concentration of Co2+, Mn2+, Zn2+, Ba2+ and Ca2+. Man1E showed the highest affinity for Locust bean gum (LBG). The Km and Vmax values for LBG were 3.09 ± 0.16 mg/mL and 909.10 ± 3.85 μmol/(mL min), respectively.ConclusionsA new type of β-mannanase with high activity from E. aerogenes is heterologously expressed and characterized. The enzyme belongs to an unreported β-mannanase family (CH1 family). It displays good pH and temperature features and excellent catalysis capacity for LBG and KGM. This study lays the foundation for future application and molecular modification to improve its catalytic efficiency and substrate specificity.

Dataset Information

Characterization of Three Fungal Isomaltases Belonging to Glycoside Hydrolase Family 13 That Do not Show Transglycosylation Activity.

Publications

Characterization of Three Fungal Isomaltases Belonging to Glycoside Hydrolase Family 13 That Do not Show Transglycosylation Activity.

Similar Datasets

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