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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

Effects of (a) pH value and (b) temperature on the enzyme activities of the PUL and its truncated mutants. Activity was measured in 0.1 M sodium acetate buffer from pH 3.0 to pH 5.5 or at the temperatures ranging from 45–70 °C. The highest activity was taken as 100%. The error bars showed the standard deviations of three replicates.
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f6: Effects of (a) pH value and (b) temperature on the enzyme activities of the PUL and its truncated mutants. Activity was measured in 0.1 M sodium acetate buffer from pH 3.0 to pH 5.5 or at the temperatures ranging from 45–70 °C. The highest activity was taken as 100%. The error bars showed the standard deviations of three replicates.

Mentions: Molecular modification by mutation or truncation would generally introduce the change of structure and function of the enzyme. For the truncated mutants involving deletion at N- or C-terminus, the effects of pH value and temperature on the enzyme activity were investigated for these mutants, respectively. As shown in Fig. 6, for the activity and catalytic efficiency-increased mutants, such as PULΔN5 and PULΔN106, the values of optimal pH and temperature were 4.5 and 60 °C, respectively, indicating that these truncations did not change the pH and temperature profile of the pullulanase. According to the requirements of saccharification process involving pullulanase, the enzyme properties of optimal pH and temperature fit well the working conditions. Therefore, with respect to the application potential, the positive candidates with enhanced enzyme activity and catalytic efficiency were successfully achieved by truncation of the disordered regions of the PUL.


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)

Effects of (a) pH value and (b) temperature on the enzyme activities of the PUL and its truncated mutants. Activity was measured in 0.1 M sodium acetate buffer from pH 3.0 to pH 5.5 or at the temperatures ranging from 45–70 °C. The highest activity was taken as 100%. 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

f6: Effects of (a) pH value and (b) temperature on the enzyme activities of the PUL and its truncated mutants. Activity was measured in 0.1 M sodium acetate buffer from pH 3.0 to pH 5.5 or at the temperatures ranging from 45–70 °C. The highest activity was taken as 100%. The error bars showed the standard deviations of three replicates.
Mentions: Molecular modification by mutation or truncation would generally introduce the change of structure and function of the enzyme. For the truncated mutants involving deletion at N- or C-terminus, the effects of pH value and temperature on the enzyme activity were investigated for these mutants, respectively. As shown in Fig. 6, for the activity and catalytic efficiency-increased mutants, such as PULΔN5 and PULΔN106, the values of optimal pH and temperature were 4.5 and 60 °C, respectively, indicating that these truncations did not change the pH and temperature profile of the pullulanase. According to the requirements of saccharification process involving pullulanase, the enzyme properties of optimal pH and temperature fit well the working conditions. Therefore, with respect to the application potential, the positive candidates with enhanced enzyme activity and catalytic efficiency were successfully achieved by truncation of the disordered regions of the PUL.

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