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Functional Analysis of a Novel β-(1,3)-Glucanase from Corallococcus sp. Strain EGB Containing a Fascin-Like Module.

ABSTRACT: A novel β-(1,3)-glucanase gene designated lamC, cloned from Corallococcus sp. strain EGB, contains a fascin-like module and a glycoside hydrolase family 16 (GH16) catalytic module. LamC displays broad hydrolytic activity toward various polysaccharides. Analysis of the hydrolytic products revealed that LamC is an exo-acting enzyme on β-(1,3)(1,3)- and β-(1,6)-linked glucan substrates and an endo-acting enzyme on β-(1,4)-linked glucan and xylan substrates. Site-directed mutagenesis of conserved catalytic Glu residues (E304A and E309A) demonstrated that these activities were derived from the same active site. Excision of the fascin-like module resulted in decreased activity toward β-(1,3)(1,3)-linked glucans. The carbohydrate-binding assay showed that the fascin-like module was a novel β-(1,3)-linked glucan-binding module. The functional characterization of the fascin-like module and catalytic module will help us better understand these enzymes and modules.IMPORTANCE In this report of a bacterial β-(1,3)(1,3)-glucanase containing a fascin-like module, we reveal the β-(1,3)(1,3)-glucan-binding function of the fascin-like module present in the N terminus of LamC. LamC displays exo-β-(1,3)/(1,6)-glucanase and endo-β-(1,4)-glucanase/xylanase activities with a single catalytic domain. Thus, LamC was identified as a novel member of the GH16 family.

SUBMITTER: Zhou J

PROVIDER: S-EPMC5541203 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Functional Analysis of a Novel β-(1,3)-Glucanase from Corallococcus sp. Strain EGB Containing a Fascin-Like Module.

Zhou Jie J Li Zhoukun Z Wu Jiale J Li Lifeng L Li Ding D Ye Xianfeng X Luo Xue X Huang Yan Y Cui Zhongli Z Cao Hui H

Applied and environmental microbiology 20170801 16

A novel β-(1,3)-glucanase gene designated <i>lamC</i>, cloned from <i>Corallococcus</i> sp. strain EGB, contains a fascin-like module and a glycoside hydrolase family 16 (GH16) catalytic module. LamC displays broad hydrolytic activity toward various polysaccharides. Analysis of the hydrolytic products revealed that LamC is an exo-acting enzyme on β-(1,3)(1,3)- and β-(1,6)-linked glucan substrates and an endo-acting enzyme on β-(1,4)-linked glucan and xylan substrates. Site-directed mutagenesis o ...[more]

PMID: 28625980

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Project description:Backgroundβ-1,3-Glucanases catalyze the hydrolysis of glucan polymers containing β-1,3-linkages. These enzymes are of great biotechnological, agricultural and industrial interest. The applications of β-1,3-glucanases is well established in fungal disease biocontrol, yeast extract production and wine extract clarification. Thus, the identification and characterization of novel β-1,3-glucanases with high catalytic efficiency and stability is of particular interest.ResultsA β-1,3-glucanase gene designated PglA was cloned from a newly isolated strain Paenibacillus sp. S09. The gene PglA contained a 2631-bp open reading frame encoding a polypeptide of 876 amino acids which shows 76% identity with the β-1,3-glucanase (BglH) from Bacillus circulans IAM1165. The encoded protein PglA is composed of a signal peptide, an N-terminal leader region, a glycoside hydrolase family 16 (GH16) catalytic domain and a C-terminal immunoglobulin like (Ig-like) domain. The Escherichia coli expression system of PglA and five truncated derivatives containing one or two modules was constructed to investigate the role of catalytic and non-catalytic modules. The pH for optimal activity of the enzymes was slightly affected (pH 5.5-6.5) by the presence of different modules. However, the temperature for optimal activity was strongly influenced by the C-terminal domain and ranged from 50 to 60°C. Deletion of C-terminal domain resulted in obviously enhancing enzymatic thermostability. Specific activity assay indicated that PglA specifically hydrolyzes β-1,3-glucan. Insoluble β-1,3-glucan binding and hydrolysis were boosted by the presence of N-and C-terminal domains. Kinetic analysis showed that the presence of N-and C-terminus enhances the substrate affinity and catalytic efficiency of the catalytic domain toward laminarin. Carbohydrate-binding assay directly confirmed the binding capabilities of the N-and C-terminal domains.ConclusionsThis study provides new insight into the impacts of non-catalytic modules on enzymatic properties of β-1,3-glucanase. Activity comparison of full-length PglA and truncated forms revealed the negative effect of C-terminal region on thermal stability of the enzyme. Both the N-and C-terminal domains exerted strong binding activity toward insoluble β-1,3-glucan, and could be classified into CBM families.

Dataset Information

Functional Analysis of a Novel β-(1,3)-Glucanase from Corallococcus sp. Strain EGB Containing a Fascin-Like Module.

Publications

Functional Analysis of a Novel β-(1,3)-Glucanase from Corallococcus sp. Strain EGB Containing a Fascin-Like Module.

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