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Paenibacillus sp. strain E18 bifunctional xylanase-glucanase with a single catalytic domain.

ABSTRACT: Xylanases are utilized in a variety of industries for the breakdown of plant materials. Most native and engineered bifunctional/multifunctional xylanases have separate catalytic domains within the same polypeptide chain. Here we report a new bifunctional xylanase (XynBE18) produced by Paenibacillus sp. E18 with xylanase and beta-1,3-1,4-glucanase activities derived from the same active center by substrate competition assays and site-directed mutagenesis of xylanase catalytic Glu residues (E129A and E236A). The gene consists of 981 bp, encodes 327 amino acids, and comprises only one catalytic domain that is highly homologous to the glycoside hydrolase family 10 xylanase catalytic domain. Recombinant XynBE18 purified from Escherichia coli BL21(DE3) showed specificity toward oat spelt xylan and birchwood xylan and beta-1,3-1,4-glucan (barley beta-glucan and lichenin). Homology modeling and molecular dynamic simulation were used to explore structure differences between XynBE18 and the monofunctional xylanase XynE2, which has enzymatic properties similar to those of XynBE18 but does not hydrolyze beta-1,3-1,4-glucan. The cleft containing the active site of XynBE18 is larger than that of XynE2, suggesting that XynBE18 is able to bind larger substrates such as barley beta-glucan and lichenin. Further molecular docking studies revealed that XynBE18 can accommodate xylan and beta-1,3-1,4-glucan, but XynE2 is only accessible to xylan. These results indicate a previously unidentified structure-function relationship for substrate specificities among family 10 xylanases.

SUBMITTER: Shi P

PROVIDER: S-EPMC2876455 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Paenibacillus sp. strain E18 bifunctional xylanase-glucanase with a single catalytic domain.

Shi Pengjun P Tian Jian J Yuan Tiezheng T Liu Xin X Huang Huoqing H Bai Yingguo Y Yang Peilong P Chen Xiaoyan X Wu Ningfeng N Yao Bin B

Applied and environmental microbiology 20100409 11

Xylanases are utilized in a variety of industries for the breakdown of plant materials. Most native and engineered bifunctional/multifunctional xylanases have separate catalytic domains within the same polypeptide chain. Here we report a new bifunctional xylanase (XynBE18) produced by Paenibacillus sp. E18 with xylanase and beta-1,3-1,4-glucanase activities derived from the same active center by substrate competition assays and site-directed mutagenesis of xylanase catalytic Glu residues (E129A ...[more]

PMID: 20382811

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Project description:BACKGROUND: ?-1,3-Glucanases catalyze the hydrolysis of glucan polymers containing ?-1,3-linkages. These enzymes are of great biotechnological, agricultural and industrial interest. The applications of ?-1,3-glucanases is well established in fungal disease biocontrol, yeast extract production and wine extract clarification. Thus, the identification and characterization of novel ?-1,3-glucanases with high catalytic efficiency and stability is of particular interest. RESULTS: A ?-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 ?-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 ?-1,3-glucan. Insoluble ?-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. CONCLUSIONS: This study provides new insight into the impacts of non-catalytic modules on enzymatic properties of ?-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 ?-1,3-glucan, and could be classified into CBM families.

Dataset Information

Paenibacillus sp. strain E18 bifunctional xylanase-glucanase with a single catalytic domain.

Publications

Paenibacillus sp. strain E18 bifunctional xylanase-glucanase with a single catalytic domain.

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