Abstract
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.
Highlights
Cloning of the β-1,3-glucanase gene and sequence analysis A β-1,3-glucanase-encoding gene designated Full-length Paenibacillus sp. S09 β-1 (PglA) was identified from a 5053-bp fragment which was obtained by using degenerate PCR, Inverse PCR (I-PCR) and SEFA-PCR
Combing the results of conserved domain search in the Conserved Domain Database (CDD) of National Center for Biotechnology Information (NCBI), secondary structure prediction and tertiary structure homology modeling by SWISS-MODEL (See Additional file 3: Figure S2), PglA contains a glycoside hydrolase family 16 (GH16) laminarinase-like domain and a bacterial immunoglobulin like (Ig-like) domain (Figures 2 and 3A)
In the work reported here, a novel β-1,3-glucanase gene PglA was cloned from Paenibacillus sp.S09
Summary
Media, vectors and chemicals The strain Paenibacillus sp. S09 (CCTCC accession no: M2012196; China Center for Type Culture Collection, Wuhan, China) was originally isolated from the rhizosphere of oat plants. Luria-Berntani (LB) broth supplemented with 0.5% pachyman was used for the production of β-1,3-glucanase by strain S09. E. coli competent cell DH5α (Takara, Otsu, Japan) and the plasmid pCR2.1 vector (Invitrogen, Carlsbad, CA, USA) were used for gene cloning. E. coli BL21 (DE3) and the pET-29a(+) vector (Novagen, San Diego, CA, USA) were used for gene expression. Recombinant E. coli was cultured in LB broth supplemented with kanamycin (50 μg/ml) at 37°C. Recombinant E. coli BL21 (DE3) harboring the expression vector was cultivated in 2 × YT medium which containing 1.0% (w/v) yeast extract, 1.6% (w/v) peptone and 0.5% (w/v) NaCl. The His6-tagged protein was purified by Ni-sepharose 6 fast flow column (GE healthcare, Sweden). The DNA purification kit, restriction endonucleases, T4 DNA ligase, pfu and LA Taq DNA polymerase with GC buffer and dNTPs were purchased from Takara. Isopropyl-β-Dthiogalactopyranoside (IPTG) and 5-bromo-4-chloro3-indolyl β-D-glucuronide (X-Gal) were purchased from Takara
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