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Disorder prediction-based construct optimization improves activity and catalytic efficiency of Bacillus naganoensis pullulanase.

Wang X, Nie Y, Mu X, Xu Y, Xiao R - Sci Rep (2016)

Bottom Line: Kinetic studies showed that substrate affinities of the mutants were improved in various degrees and the catalytic efficiency of PULΔN5, PULΔN45, PULΔN78, PULΔN106 and PULΔC9 were enhanced.However, the truncated mutations did not change the advantageous properties of the enzyme involving optimum temperature and pH for further application.Therefore, Disorder prediction-based truncation would be helpful to efficiently improve the enzyme activity and catalytic efficiency.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.

ABSTRACT
Pullulanase is a well-known starch-debranching enzyme. However, the production level of pullulanase is yet low in both wide-type strains and heterologous expression systems. We predicted the disorder propensities of Bacillus naganoensis pullulanase (PUL) using the bioinformatics tool, Disorder Prediction Meta-Server. On the basis of disorder prediction, eight constructs, including PULΔN5, PULΔN22, PULΔN45, PULΔN64, PULΔN78 and PULΔN106 by deleting the first 5, 22, 45, 64, 78 and 106 residues from the N-terminus, and PULΔC9 and PULΔC36 by deleting the last 9 and 36 residues from the C-terminus, were cloned into the recombinant expression vector pET-28a-PelB and auto-induced in Escherichia coli BL21 (DE3) cells. All constructs were evaluated in production level, specific activities and kinetic parameters. Both PULΔN5 and PULΔN106 gave higher production levels of protein than the wide type and displayed increased specific activities. Kinetic studies showed that substrate affinities of the mutants were improved in various degrees and the catalytic efficiency of PULΔN5, PULΔN45, PULΔN78, PULΔN106 and PULΔC9 were enhanced. However, the truncated mutations did not change the advantageous properties of the enzyme involving optimum temperature and pH for further application. Therefore, Disorder prediction-based truncation would be helpful to efficiently improve the enzyme activity and catalytic efficiency.

No MeSH data available.


Related in: MedlinePlus

Comparison between the N-terminal domain of PUL and TmCBM41 from structure and sequence analysis.(a) Structure overlap of the N-terminal domain of PUL (grey) and TmCBM41 (yellow) with carbohydrate-binding sites in stick and the ligand in line. (b) Amino acid sequence alignment of the N-terminal domain of PUL and TmCBM41 with the secondary structures, respectively. The carbohydrate-binding sites were indicated above and below the sequences with red and blue triangles for TmCBM41 and PUL, respectively.
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f5: Comparison between the N-terminal domain of PUL and TmCBM41 from structure and sequence analysis.(a) Structure overlap of the N-terminal domain of PUL (grey) and TmCBM41 (yellow) with carbohydrate-binding sites in stick and the ligand in line. (b) Amino acid sequence alignment of the N-terminal domain of PUL and TmCBM41 with the secondary structures, respectively. The carbohydrate-binding sites were indicated above and below the sequences with red and blue triangles for TmCBM41 and PUL, respectively.

Mentions: As known, 3D structure of pullulanase generally consists of several domain structures, e.g. CBM41-X45a-X25-X45b-CBM48-GH13 in BaPul13A, N1-N2-N3-A-C in the K. pneumoniae pullulanase and N1-N2-A-C in the pullulanase from Anoxybacillus sp. (PulA)181920. The N-terminal domain CBM41 or N1 has been identified as the carbohydrate-binding module, interacting with oligosaccharide molecules. Functioning like a lid, the N-terminal domain had a conformational change for substrate accommodation and played an important role in assisting substrate binding for catalytic activity20. On the basis of the knowledge on function of the N-terminal domain of known pullulanases, the N-terminal sequence of PUL (residues 1–110) was analyzed by homology searching in NCBI using the database of PDB. The searching result revealed that the N-terminal sequence of PUL was homologous to the conserved domain of CBM41 pullulanase superfamily (Fig. S2). Additionally, a family 41 carbohydrate-binding module from Thermotoga maritima pullulanase (TmCBM41) (PDB ID: 2J71) was found to perform the highest identity (45%) to the N-terminal sequence of PUL. Then the structure of the N-terminal domain of PUL was predicted by homology modeling using TmCBM41 structure as the template. As shown in Fig. 5, the backbone structures of the N-terminal domain of PUL and TmCBM41 matched in the structure alignment and the key residues for ligand binding, e.g. Trp, Lys and Asp recognizing the α-D-glucosyl-maltotriose unit in pullulan, were conserved in the N-terminal domain of PUL21. Furthermore, the architectures of the active sites of TmCBM41 were structurally well conserved with the N-terminal domain (N1 domain) of the K. pneumoniae pullulanase, for which the structure-function relationship has been clarified by solving the complex structures of the enzyme1921. Therefore, the N-terminal domain of PUL comprising obvious disordered regions would be involved in substrate recognition, and substrate binding would cause significant conformational change of the domain to accommodate substrate.


Disorder prediction-based construct optimization improves activity and catalytic efficiency of Bacillus naganoensis pullulanase.

Wang X, Nie Y, Mu X, Xu Y, Xiao R - Sci Rep (2016)

Comparison between the N-terminal domain of PUL and TmCBM41 from structure and sequence analysis.(a) Structure overlap of the N-terminal domain of PUL (grey) and TmCBM41 (yellow) with carbohydrate-binding sites in stick and the ligand in line. (b) Amino acid sequence alignment of the N-terminal domain of PUL and TmCBM41 with the secondary structures, respectively. The carbohydrate-binding sites were indicated above and below the sequences with red and blue triangles for TmCBM41 and PUL, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835747&req=5

f5: Comparison between the N-terminal domain of PUL and TmCBM41 from structure and sequence analysis.(a) Structure overlap of the N-terminal domain of PUL (grey) and TmCBM41 (yellow) with carbohydrate-binding sites in stick and the ligand in line. (b) Amino acid sequence alignment of the N-terminal domain of PUL and TmCBM41 with the secondary structures, respectively. The carbohydrate-binding sites were indicated above and below the sequences with red and blue triangles for TmCBM41 and PUL, respectively.
Mentions: As known, 3D structure of pullulanase generally consists of several domain structures, e.g. CBM41-X45a-X25-X45b-CBM48-GH13 in BaPul13A, N1-N2-N3-A-C in the K. pneumoniae pullulanase and N1-N2-A-C in the pullulanase from Anoxybacillus sp. (PulA)181920. The N-terminal domain CBM41 or N1 has been identified as the carbohydrate-binding module, interacting with oligosaccharide molecules. Functioning like a lid, the N-terminal domain had a conformational change for substrate accommodation and played an important role in assisting substrate binding for catalytic activity20. On the basis of the knowledge on function of the N-terminal domain of known pullulanases, the N-terminal sequence of PUL (residues 1–110) was analyzed by homology searching in NCBI using the database of PDB. The searching result revealed that the N-terminal sequence of PUL was homologous to the conserved domain of CBM41 pullulanase superfamily (Fig. S2). Additionally, a family 41 carbohydrate-binding module from Thermotoga maritima pullulanase (TmCBM41) (PDB ID: 2J71) was found to perform the highest identity (45%) to the N-terminal sequence of PUL. Then the structure of the N-terminal domain of PUL was predicted by homology modeling using TmCBM41 structure as the template. As shown in Fig. 5, the backbone structures of the N-terminal domain of PUL and TmCBM41 matched in the structure alignment and the key residues for ligand binding, e.g. Trp, Lys and Asp recognizing the α-D-glucosyl-maltotriose unit in pullulan, were conserved in the N-terminal domain of PUL21. Furthermore, the architectures of the active sites of TmCBM41 were structurally well conserved with the N-terminal domain (N1 domain) of the K. pneumoniae pullulanase, for which the structure-function relationship has been clarified by solving the complex structures of the enzyme1921. Therefore, the N-terminal domain of PUL comprising obvious disordered regions would be involved in substrate recognition, and substrate binding would cause significant conformational change of the domain to accommodate substrate.

Bottom Line: Kinetic studies showed that substrate affinities of the mutants were improved in various degrees and the catalytic efficiency of PULΔN5, PULΔN45, PULΔN78, PULΔN106 and PULΔC9 were enhanced.However, the truncated mutations did not change the advantageous properties of the enzyme involving optimum temperature and pH for further application.Therefore, Disorder prediction-based truncation would be helpful to efficiently improve the enzyme activity and catalytic efficiency.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.

ABSTRACT
Pullulanase is a well-known starch-debranching enzyme. However, the production level of pullulanase is yet low in both wide-type strains and heterologous expression systems. We predicted the disorder propensities of Bacillus naganoensis pullulanase (PUL) using the bioinformatics tool, Disorder Prediction Meta-Server. On the basis of disorder prediction, eight constructs, including PULΔN5, PULΔN22, PULΔN45, PULΔN64, PULΔN78 and PULΔN106 by deleting the first 5, 22, 45, 64, 78 and 106 residues from the N-terminus, and PULΔC9 and PULΔC36 by deleting the last 9 and 36 residues from the C-terminus, were cloned into the recombinant expression vector pET-28a-PelB and auto-induced in Escherichia coli BL21 (DE3) cells. All constructs were evaluated in production level, specific activities and kinetic parameters. Both PULΔN5 and PULΔN106 gave higher production levels of protein than the wide type and displayed increased specific activities. Kinetic studies showed that substrate affinities of the mutants were improved in various degrees and the catalytic efficiency of PULΔN5, PULΔN45, PULΔN78, PULΔN106 and PULΔC9 were enhanced. However, the truncated mutations did not change the advantageous properties of the enzyme involving optimum temperature and pH for further application. Therefore, Disorder prediction-based truncation would be helpful to efficiently improve the enzyme activity and catalytic efficiency.

No MeSH data available.


Related in: MedlinePlus