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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

Diagrammatic sketch of the full-length PUL and its truncated mutants.The sites for truncation were determined by analyzing the amino acid sequence using both the methods of disorder prediction algorithms and sequence analysis tools. The up- and downstream sequences of the pul in expression plasmid were also presented, involving the expression elements in pET-28a-PelB, N-terminal PelB signal sequence and C-terminal His-tag sequence.
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f1: Diagrammatic sketch of the full-length PUL and its truncated mutants.The sites for truncation were determined by analyzing the amino acid sequence using both the methods of disorder prediction algorithms and sequence analysis tools. The up- and downstream sequences of the pul in expression plasmid were also presented, involving the expression elements in pET-28a-PelB, N-terminal PelB signal sequence and C-terminal His-tag sequence.

Mentions: The N- and C-terminus of the PUL were predicted to be disordered by the majority of algorithms, whereas the conserved sequences were predicted to be ordered (Fig. S1). The results of secondary structure consensus generated from PROFsec and PSIPred indicated several β-sheet structures at both the N- and C-terminus of the enzyme, with the intervals of loop structures. It could be obviously observed that there were five distinct loops covering the residues 1–5, 13–22, 27–45, 64–78 and 99–110 at the N-terminus and two distinct loops covering the residues 918–927 and 891–904 at the C-terminus. Thus, it would be feasible to truncate the flexible loops at the corresponding sits to investigate the influence of these disordered regions on the activity and catalytic efficiency of the enzyme. In addition to the secondary structure analysis for identification of disordered loops, the graph of disorder consensus further indicated the possible disordered sites in the protein sequence. As shown in Fig. S1, with the plot showing disorder levels of involved sites in disorder consensus, the sites of much disorder in the N- and C-terminal sequences were consistent with the predicted loop structures, covering the residues 1–5, 16–22, 33–45, 74–78 and 100–106 at the N-terminus and 891–903 and 918–927 at the C-terminus. For the fragment 46–64, although its secondary structure was predicted as two β-sheets, the disorder levels of these sites were somewhat high and hence the fragment was also taken into account for truncation. Therefore, the cutting sites to truncate the disordered regions at the N- or C-terminus of the PUL were proposed as 5, 22, 45, 64, 78, 106, 891 and 918. Consequently, on the basis of the disorder prediction consensus, combined with secondary structure prediction and domain boundary identification, eight truncated mutants were constructed, 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 (Fig. 1).


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)

Diagrammatic sketch of the full-length PUL and its truncated mutants.The sites for truncation were determined by analyzing the amino acid sequence using both the methods of disorder prediction algorithms and sequence analysis tools. The up- and downstream sequences of the pul in expression plasmid were also presented, involving the expression elements in pET-28a-PelB, N-terminal PelB signal sequence and C-terminal His-tag sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Diagrammatic sketch of the full-length PUL and its truncated mutants.The sites for truncation were determined by analyzing the amino acid sequence using both the methods of disorder prediction algorithms and sequence analysis tools. The up- and downstream sequences of the pul in expression plasmid were also presented, involving the expression elements in pET-28a-PelB, N-terminal PelB signal sequence and C-terminal His-tag sequence.
Mentions: The N- and C-terminus of the PUL were predicted to be disordered by the majority of algorithms, whereas the conserved sequences were predicted to be ordered (Fig. S1). The results of secondary structure consensus generated from PROFsec and PSIPred indicated several β-sheet structures at both the N- and C-terminus of the enzyme, with the intervals of loop structures. It could be obviously observed that there were five distinct loops covering the residues 1–5, 13–22, 27–45, 64–78 and 99–110 at the N-terminus and two distinct loops covering the residues 918–927 and 891–904 at the C-terminus. Thus, it would be feasible to truncate the flexible loops at the corresponding sits to investigate the influence of these disordered regions on the activity and catalytic efficiency of the enzyme. In addition to the secondary structure analysis for identification of disordered loops, the graph of disorder consensus further indicated the possible disordered sites in the protein sequence. As shown in Fig. S1, with the plot showing disorder levels of involved sites in disorder consensus, the sites of much disorder in the N- and C-terminal sequences were consistent with the predicted loop structures, covering the residues 1–5, 16–22, 33–45, 74–78 and 100–106 at the N-terminus and 891–903 and 918–927 at the C-terminus. For the fragment 46–64, although its secondary structure was predicted as two β-sheets, the disorder levels of these sites were somewhat high and hence the fragment was also taken into account for truncation. Therefore, the cutting sites to truncate the disordered regions at the N- or C-terminus of the PUL were proposed as 5, 22, 45, 64, 78, 106, 891 and 918. Consequently, on the basis of the disorder prediction consensus, combined with secondary structure prediction and domain boundary identification, eight truncated mutants were constructed, 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 (Fig. 1).

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