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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

The half-lives of the PUL and its truncated mutants at 60 °C.The error bars showed the standard deviations of three replicates.
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f7: The half-lives of the PUL and its truncated mutants at 60 °C.The error bars showed the standard deviations of three replicates.

Mentions: In addition to the features of optimal pH and temperature, stability as another important property of pullulanase was further evaluated towards the mutants derived from disorder prediction-based construct optimization. The residual activities of the truncated mutants and the wild-type enzyme were measured after incubation at 60 °C and pH 4.5 for different periods of time. As shown in Fig. 7, PULΔN5, PULΔN78, PULΔN106 and PULΔC9 of the mutants performed higher stability than the PUL. Compared with the wild-type enzyme, the half-lives (T1/2) were increased to 143.7, 139.8, 150.0 and 155.2 h for the positive mutants of PULΔN5, PULΔN78, PULΔN106 and PULΔC9, respectively. Correspondingly, the half-life of the enzyme was prolonged to 1.16 folds improvement of the original level (133.8 h). This finding was consistent with the observation that deletion or replacement of unstable domains or peptides would enhance the stability of proteins23242526. Therefore, with the unchanged features suitable for saccharification process, the pullulanase mutants, such as PULΔN5, were created to be more active and stable by disorder prediction-based construct optimization and it would be more feasible to use the developed enzyme in potential applications.


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)

The half-lives of the PUL and its truncated mutants at 60 °C.The error bars showed the standard deviations of three replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: The half-lives of the PUL and its truncated mutants at 60 °C.The error bars showed the standard deviations of three replicates.
Mentions: In addition to the features of optimal pH and temperature, stability as another important property of pullulanase was further evaluated towards the mutants derived from disorder prediction-based construct optimization. The residual activities of the truncated mutants and the wild-type enzyme were measured after incubation at 60 °C and pH 4.5 for different periods of time. As shown in Fig. 7, PULΔN5, PULΔN78, PULΔN106 and PULΔC9 of the mutants performed higher stability than the PUL. Compared with the wild-type enzyme, the half-lives (T1/2) were increased to 143.7, 139.8, 150.0 and 155.2 h for the positive mutants of PULΔN5, PULΔN78, PULΔN106 and PULΔC9, respectively. Correspondingly, the half-life of the enzyme was prolonged to 1.16 folds improvement of the original level (133.8 h). This finding was consistent with the observation that deletion or replacement of unstable domains or peptides would enhance the stability of proteins23242526. Therefore, with the unchanged features suitable for saccharification process, the pullulanase mutants, such as PULΔN5, were created to be more active and stable by disorder prediction-based construct optimization and it would be more feasible to use the developed enzyme in potential applications.

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