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Recombinant production and characterization of full-length and truncated β-1,3-glucanase PglA from Paenibacillus sp. S09.

Cheng R, Chen J, Yu X, Wang Y, Wang S, Zhang J - BMC Biotechnol. (2013)

Bottom Line: Deletion of C-terminal domain resulted in obviously enhancing enzymatic thermostability.Carbohydrate-binding assay directly confirmed the binding capabilities of the N-and C-terminal domains.Activity comparison of full-length PglA and truncated forms revealed the negative effect of C-terminal region on thermal stability of the enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Molecular Metabolism, Nanjing University of Science & Technology, 200 Xiaolingwei, Nanjing 210094, China. jfzhang@mail.njust.edu.cn.

ABSTRACT

Background: β-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.

Results: A 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.

Conclusions: This 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.

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The nucleotide and amino acid sequences of PglA from Paenibacillus sp. S09. A vertical arrow indicates a cleavage site to produce the mature enzyme. The three N-terminal repeats are indicated by arrow line. The catalytic domain and Ig-like domain are shadowed by blue and red, respectively. The putative catalytic amino acids are circled. The stop codon is indicated by asterisk.
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Figure 2: The nucleotide and amino acid sequences of PglA from Paenibacillus sp. S09. A vertical arrow indicates a cleavage site to produce the mature enzyme. The three N-terminal repeats are indicated by arrow line. The catalytic domain and Ig-like domain are shadowed by blue and red, respectively. The putative catalytic amino acids are circled. The stop codon is indicated by asterisk.

Mentions: A β-1,3-glucanase-encoding gene designated PglA was identified from a 5053-bp fragment which was obtained by using degenerate PCR, I-PCR and SEFA-PCR. The deduced amino acid sequence of PglA (GenBank No.: AFO67889.1) comprises 876 amino acids with a predicted N-terminal signal peptide (residues 1-38) (Figure 2). The mature protein consists of 838 amino acid residues with a calculated pI of 4.5 and molecular mass of 90.4 kDa, respectively. The deduced amino acid sequence of PglA exhibited the highest identity (86%) with the hypothetical protein from Paenibacillus daejeonensis (NCBI Reference Sequence: WP_020617344.1), 77% identity with the glycoside hydrolase family 16 protein from Paenibacillus lactis 154 (GenBank No.: EHB65983.1), and 76% identity with the reported β-1,3-glucanase BglH from Bacillus circulans IAM1165 (GenBank No.: AAC60453.1). PglA showed only 45% identity with the well-characterized endo-β-1,3-glucanase LamA from Paenibacillus sp. CCRC 17245 (GenBank No.: ABJ15796.1). Combing the results of conserved domain search in the Conserved Domain Database (CDD) of NCBI, secondary structure prediction and tertiary structure homology modeling by SWISS-MODEL (See Additional file 3: Figure S2), PglA contains a GH16 laminarinase-like domain and a bacterial Ig-like domain (Figures 2 and 3A). The domain organization of PglA is not similar to LamA, which include a leader peptide, a threefold repeat of S-layer homologous module, a GH16 catalytic module, four repeats of CBM_4_9 and an analogue of coagulation factor Fa5/8C from N to C terminus [13]. PglA could be considered as a novel β-1,3-glucanase of Paenibacillus species. Phylogenetic tree showed that the catalytic region of PglA forms a distinct clade with BglH from B. circulans and putative glycoside hydrolases from Paenibacillus species (Figure 3B). Phylogenetic analysis based on the Ig-like domain sequence alignment indicated the relationships between PglA and other Ig-like domain containing proteins (Figure 3C).


Recombinant production and characterization of full-length and truncated β-1,3-glucanase PglA from Paenibacillus sp. S09.

Cheng R, Chen J, Yu X, Wang Y, Wang S, Zhang J - BMC Biotechnol. (2013)

The nucleotide and amino acid sequences of PglA from Paenibacillus sp. S09. A vertical arrow indicates a cleavage site to produce the mature enzyme. The three N-terminal repeats are indicated by arrow line. The catalytic domain and Ig-like domain are shadowed by blue and red, respectively. The putative catalytic amino acids are circled. The stop codon is indicated by asterisk.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219603&req=5

Figure 2: The nucleotide and amino acid sequences of PglA from Paenibacillus sp. S09. A vertical arrow indicates a cleavage site to produce the mature enzyme. The three N-terminal repeats are indicated by arrow line. The catalytic domain and Ig-like domain are shadowed by blue and red, respectively. The putative catalytic amino acids are circled. The stop codon is indicated by asterisk.
Mentions: A β-1,3-glucanase-encoding gene designated PglA was identified from a 5053-bp fragment which was obtained by using degenerate PCR, I-PCR and SEFA-PCR. The deduced amino acid sequence of PglA (GenBank No.: AFO67889.1) comprises 876 amino acids with a predicted N-terminal signal peptide (residues 1-38) (Figure 2). The mature protein consists of 838 amino acid residues with a calculated pI of 4.5 and molecular mass of 90.4 kDa, respectively. The deduced amino acid sequence of PglA exhibited the highest identity (86%) with the hypothetical protein from Paenibacillus daejeonensis (NCBI Reference Sequence: WP_020617344.1), 77% identity with the glycoside hydrolase family 16 protein from Paenibacillus lactis 154 (GenBank No.: EHB65983.1), and 76% identity with the reported β-1,3-glucanase BglH from Bacillus circulans IAM1165 (GenBank No.: AAC60453.1). PglA showed only 45% identity with the well-characterized endo-β-1,3-glucanase LamA from Paenibacillus sp. CCRC 17245 (GenBank No.: ABJ15796.1). Combing the results of conserved domain search in the Conserved Domain Database (CDD) of NCBI, secondary structure prediction and tertiary structure homology modeling by SWISS-MODEL (See Additional file 3: Figure S2), PglA contains a GH16 laminarinase-like domain and a bacterial Ig-like domain (Figures 2 and 3A). The domain organization of PglA is not similar to LamA, which include a leader peptide, a threefold repeat of S-layer homologous module, a GH16 catalytic module, four repeats of CBM_4_9 and an analogue of coagulation factor Fa5/8C from N to C terminus [13]. PglA could be considered as a novel β-1,3-glucanase of Paenibacillus species. Phylogenetic tree showed that the catalytic region of PglA forms a distinct clade with BglH from B. circulans and putative glycoside hydrolases from Paenibacillus species (Figure 3B). Phylogenetic analysis based on the Ig-like domain sequence alignment indicated the relationships between PglA and other Ig-like domain containing proteins (Figure 3C).

Bottom Line: Deletion of C-terminal domain resulted in obviously enhancing enzymatic thermostability.Carbohydrate-binding assay directly confirmed the binding capabilities of the N-and C-terminal domains.Activity comparison of full-length PglA and truncated forms revealed the negative effect of C-terminal region on thermal stability of the enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Molecular Metabolism, Nanjing University of Science & Technology, 200 Xiaolingwei, Nanjing 210094, China. jfzhang@mail.njust.edu.cn.

ABSTRACT

Background: β-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.

Results: A 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.

Conclusions: This 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.

Show MeSH
Related in: MedlinePlus