Limits...
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

Total enzyme activities and specific activities of the PUL and its truncated mutants with auto-induction.Total activity was defined as the sum of extracellular and intracellular activity. All the values of enzymatic activities were averaged from three replicates with standard deviations, and significant differences (p < 0.05) were measured.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835747&req=5

f2: Total enzyme activities and specific activities of the PUL and its truncated mutants with auto-induction.Total activity was defined as the sum of extracellular and intracellular activity. All the values of enzymatic activities were averaged from three replicates with standard deviations, and significant differences (p < 0.05) were measured.

Mentions: With the primers designed according to the results of disorder prediction and the principle of codon usage (Table S1), the genes encoding these N- or C-terminally truncated pullulanases were subcloned into the recombinant expression vector pET-28a-PelB and expressed in E. coli BL21 (DE3) by auto-induction. As known, heterologous protein expressed in secretory recombinant E. coli system is generally transported to the periplasmic space by the available signal peptide17. Therefore, most activities were detected in the periplasmic space but not in the supernatant. In this work, supplementation of glycine was not applied for extracellular accumulation of the pullulanase. Thus, the total pullulanase activities from both the culture supernatant and the periplasmic space were measured to evaluate the effect of truncations on the expression of the PUL. Of these mutants, PULΔN5 and PULΔN106 exhibited the total enzyme activities of 768 U mL−1 and 633 U mL−1, which were 42% and 17% higher than that of the wild-type PUL, respectively. However, other truncated mutants performed somewhat lower total pullulanase activities, compared with the wild-type PUL (Fig. 2).


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)

Total enzyme activities and specific activities of the PUL and its truncated mutants with auto-induction.Total activity was defined as the sum of extracellular and intracellular activity. All the values of enzymatic activities were averaged from three replicates with standard deviations, and significant differences (p < 0.05) were measured.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Total enzyme activities and specific activities of the PUL and its truncated mutants with auto-induction.Total activity was defined as the sum of extracellular and intracellular activity. All the values of enzymatic activities were averaged from three replicates with standard deviations, and significant differences (p < 0.05) were measured.
Mentions: With the primers designed according to the results of disorder prediction and the principle of codon usage (Table S1), the genes encoding these N- or C-terminally truncated pullulanases were subcloned into the recombinant expression vector pET-28a-PelB and expressed in E. coli BL21 (DE3) by auto-induction. As known, heterologous protein expressed in secretory recombinant E. coli system is generally transported to the periplasmic space by the available signal peptide17. Therefore, most activities were detected in the periplasmic space but not in the supernatant. In this work, supplementation of glycine was not applied for extracellular accumulation of the pullulanase. Thus, the total pullulanase activities from both the culture supernatant and the periplasmic space were measured to evaluate the effect of truncations on the expression of the PUL. Of these mutants, PULΔN5 and PULΔN106 exhibited the total enzyme activities of 768 U mL−1 and 633 U mL−1, which were 42% and 17% higher than that of the wild-type PUL, respectively. However, other truncated mutants performed somewhat lower total pullulanase activities, compared with the wild-type PUL (Fig. 2).

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