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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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Sequence analysis of PglA. A, Schematic representation of PglA with the functional domains. The numbers indicated amino acid positions. The GH16 catalytic domain was indicated in black, signal peptide was indicated in gray, the bacterial Ig-like domain was shown by a stippled box. B and C, Phylogenetic analysis of the GH16 and Ig-like domains from different proteins, respectively. The amino acid sequence of the GH16 or Ig-like domain was used in BLASTP search and matched sequences were extracted and aligned by ClustalX2. The starting and ending amino acid positions are shown. The unrooted dendrogram was constructed by neighbor-joining method. The percentages of bootstrap values (based on 1000 bootstraps) are shown at the internal nodes. GenBank accession numbers: P. lactis 154, EHB65983.1; B. circulans BglH, AAC60453.1; P. vortex, EFU40835.1; C. butyricum DKU-01, EMU55784.1; S. frigidimarina NCIMB 400, ABI71172.1; Sphingomonas sp. SKA58, EAT10234.1, M. maris MCS10, ABI64540.1; Enterobacter sp. Ag1, EJF31699.1; Pantoea sp. At-9b, ADU69429.1; F. johnsoniae UW101, ABQ04240.2; E.coli, EMX44285.1; Clostridium sp. CAG:1024, CCX42470.1.
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Figure 3: Sequence analysis of PglA. A, Schematic representation of PglA with the functional domains. The numbers indicated amino acid positions. The GH16 catalytic domain was indicated in black, signal peptide was indicated in gray, the bacterial Ig-like domain was shown by a stippled box. B and C, Phylogenetic analysis of the GH16 and Ig-like domains from different proteins, respectively. The amino acid sequence of the GH16 or Ig-like domain was used in BLASTP search and matched sequences were extracted and aligned by ClustalX2. The starting and ending amino acid positions are shown. The unrooted dendrogram was constructed by neighbor-joining method. The percentages of bootstrap values (based on 1000 bootstraps) are shown at the internal nodes. GenBank accession numbers: P. lactis 154, EHB65983.1; B. circulans BglH, AAC60453.1; P. vortex, EFU40835.1; C. butyricum DKU-01, EMU55784.1; S. frigidimarina NCIMB 400, ABI71172.1; Sphingomonas sp. SKA58, EAT10234.1, M. maris MCS10, ABI64540.1; Enterobacter sp. Ag1, EJF31699.1; Pantoea sp. At-9b, ADU69429.1; F. johnsoniae UW101, ABQ04240.2; E.coli, EMX44285.1; Clostridium sp. CAG:1024, CCX42470.1.

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)

Sequence analysis of PglA. A, Schematic representation of PglA with the functional domains. The numbers indicated amino acid positions. The GH16 catalytic domain was indicated in black, signal peptide was indicated in gray, the bacterial Ig-like domain was shown by a stippled box. B and C, Phylogenetic analysis of the GH16 and Ig-like domains from different proteins, respectively. The amino acid sequence of the GH16 or Ig-like domain was used in BLASTP search and matched sequences were extracted and aligned by ClustalX2. The starting and ending amino acid positions are shown. The unrooted dendrogram was constructed by neighbor-joining method. The percentages of bootstrap values (based on 1000 bootstraps) are shown at the internal nodes. GenBank accession numbers: P. lactis 154, EHB65983.1; B. circulans BglH, AAC60453.1; P. vortex, EFU40835.1; C. butyricum DKU-01, EMU55784.1; S. frigidimarina NCIMB 400, ABI71172.1; Sphingomonas sp. SKA58, EAT10234.1, M. maris MCS10, ABI64540.1; Enterobacter sp. Ag1, EJF31699.1; Pantoea sp. At-9b, ADU69429.1; F. johnsoniae UW101, ABQ04240.2; E.coli, EMX44285.1; Clostridium sp. CAG:1024, CCX42470.1.
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Figure 3: Sequence analysis of PglA. A, Schematic representation of PglA with the functional domains. The numbers indicated amino acid positions. The GH16 catalytic domain was indicated in black, signal peptide was indicated in gray, the bacterial Ig-like domain was shown by a stippled box. B and C, Phylogenetic analysis of the GH16 and Ig-like domains from different proteins, respectively. The amino acid sequence of the GH16 or Ig-like domain was used in BLASTP search and matched sequences were extracted and aligned by ClustalX2. The starting and ending amino acid positions are shown. The unrooted dendrogram was constructed by neighbor-joining method. The percentages of bootstrap values (based on 1000 bootstraps) are shown at the internal nodes. GenBank accession numbers: P. lactis 154, EHB65983.1; B. circulans BglH, AAC60453.1; P. vortex, EFU40835.1; C. butyricum DKU-01, EMU55784.1; S. frigidimarina NCIMB 400, ABI71172.1; Sphingomonas sp. SKA58, EAT10234.1, M. maris MCS10, ABI64540.1; Enterobacter sp. Ag1, EJF31699.1; Pantoea sp. At-9b, ADU69429.1; F. johnsoniae UW101, ABQ04240.2; E.coli, EMX44285.1; Clostridium sp. CAG:1024, CCX42470.1.
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