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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

Circular dichroism analysis of the secondary structures of the wild-type PUL and the truncated mutants.The far-UV spectra of the wild-type PUL and the truncated mutants were recorded at 20 °C.
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f4: Circular dichroism analysis of the secondary structures of the wild-type PUL and the truncated mutants.The far-UV spectra of the wild-type PUL and the truncated mutants were recorded at 20 °C.

Mentions: In this study, specific activity, substrate affinity and catalytic efficiency of the PUL were improved by disorder prediction-based truncation at the N- or C-terminus of the enzyme. To investigate the effects of these truncations on the structural elements, especially the composition of secondary structures, circular dichroism spectrum was applied to analyze the change of secondary structures of the truncated mutants, compared with the wild-type enzyme. As shown in Fig. 4, most of the truncated mutants performed slight difference from the PUL in the circular dichroism spectrum and the composition of secondary structures. Associating with the kinetic parameters of the mutants, change of secondary structures, especially the content of unordered loop, generated from truncation of disordered region would cause significant changes of kcat and Vmax values, while the mutants with the percentage of unordered loop close to that of the wild-type enzyme, such as PULΔN5 and PULΔN106, would be more favorable for the catalytic activity of the enzyme (Table S2).


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)

Circular dichroism analysis of the secondary structures of the wild-type PUL and the truncated mutants.The far-UV spectra of the wild-type PUL and the truncated mutants were recorded at 20 °C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Circular dichroism analysis of the secondary structures of the wild-type PUL and the truncated mutants.The far-UV spectra of the wild-type PUL and the truncated mutants were recorded at 20 °C.
Mentions: In this study, specific activity, substrate affinity and catalytic efficiency of the PUL were improved by disorder prediction-based truncation at the N- or C-terminus of the enzyme. To investigate the effects of these truncations on the structural elements, especially the composition of secondary structures, circular dichroism spectrum was applied to analyze the change of secondary structures of the truncated mutants, compared with the wild-type enzyme. As shown in Fig. 4, most of the truncated mutants performed slight difference from the PUL in the circular dichroism spectrum and the composition of secondary structures. Associating with the kinetic parameters of the mutants, change of secondary structures, especially the content of unordered loop, generated from truncation of disordered region would cause significant changes of kcat and Vmax values, while the mutants with the percentage of unordered loop close to that of the wild-type enzyme, such as PULΔN5 and PULΔN106, would be more favorable for the catalytic activity of the enzyme (Table S2).

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