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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

SDS-PAGE analysis of (a) the cell-free extracts and (b) the purified recombinant enzymes. For cell-free extracts, lane 1–9 meant the total soluble protein of the construct PUL, PULΔN5, PULΔN22, PULΔN45, PULΔN64, PULΔN78, PULΔN106, PULΔC9 and PULΔC36, respectively. For purified enzymes, lane 1–9 meant the purified PUL, PULΔN5, PULΔN22, PULΔN45, PULΔN78, PULΔN64, PULΔN106, PULΔC9 and PULΔC36, respectively. Lane M meant the protein molecular weight marker. The amount of loaded sample was 10 μL in each lane.
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f3: SDS-PAGE analysis of (a) the cell-free extracts and (b) the purified recombinant enzymes. For cell-free extracts, lane 1–9 meant the total soluble protein of the construct PUL, PULΔN5, PULΔN22, PULΔN45, PULΔN64, PULΔN78, PULΔN106, PULΔC9 and PULΔC36, respectively. For purified enzymes, lane 1–9 meant the purified PUL, PULΔN5, PULΔN22, PULΔN45, PULΔN78, PULΔN64, PULΔN106, PULΔC9 and PULΔC36, respectively. Lane M meant the protein molecular weight marker. The amount of loaded sample was 10 μL in each lane.

Mentions: To understand the possible reason leading to the difference of total pullulanase activities among the truncated and full-length enzymes, SDS-PAGE analysis of the cell-free extracts after expression was conducted for the mutants and the wild-type enzyme. The results indicated that all the truncated proteins could be expressed as soluble form and compared with the wild-type PUL, high-level expression of target protein was obviously achieved for the mutants of PULΔN5 and PULΔC9 (Fig. 3a). Additionally, the recombinant proteins were purified to homogeneity (Fig. 3b), and then the specific activities of the purified enzymes were measured. Of the truncated mutants, activities in the cells of PULΔN5 and PULΔN106 were 573 U mL−1 and 519 U·mL−1 with the amounts of purified enzymes were 1.77 mg mL−1 and 1.36 mgmL−1, respectively. Thus, PULΔN5 and PULΔN106 performed their specific activities as 324 U mg−1 and 382 U mg−1, which were 1.18- and 1.38-fold increase of the PUL, respectively (Fig. 2). Associating with the results from SDS-PAGE analysis of the expressed proteins of the constructs, the increased total activities of the mutants would be mainly caused by the improvement of the specific activity of the corresponding mutants. Therefore, truncation of the disordered region of the enzyme would be helpful to make the protein more active in catalyzing hydrolysis reaction, involving the acting behavior of binding substrate and molecular interactions. Taken together, it was worth to note that truncation of the first five amino acid residues at N-terminus significantly improved both the expression level and the enzymatic activity of the pullulanase.


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)

SDS-PAGE analysis of (a) the cell-free extracts and (b) the purified recombinant enzymes. For cell-free extracts, lane 1–9 meant the total soluble protein of the construct PUL, PULΔN5, PULΔN22, PULΔN45, PULΔN64, PULΔN78, PULΔN106, PULΔC9 and PULΔC36, respectively. For purified enzymes, lane 1–9 meant the purified PUL, PULΔN5, PULΔN22, PULΔN45, PULΔN78, PULΔN64, PULΔN106, PULΔC9 and PULΔC36, respectively. Lane M meant the protein molecular weight marker. The amount of loaded sample was 10 μL in each lane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: SDS-PAGE analysis of (a) the cell-free extracts and (b) the purified recombinant enzymes. For cell-free extracts, lane 1–9 meant the total soluble protein of the construct PUL, PULΔN5, PULΔN22, PULΔN45, PULΔN64, PULΔN78, PULΔN106, PULΔC9 and PULΔC36, respectively. For purified enzymes, lane 1–9 meant the purified PUL, PULΔN5, PULΔN22, PULΔN45, PULΔN78, PULΔN64, PULΔN106, PULΔC9 and PULΔC36, respectively. Lane M meant the protein molecular weight marker. The amount of loaded sample was 10 μL in each lane.
Mentions: To understand the possible reason leading to the difference of total pullulanase activities among the truncated and full-length enzymes, SDS-PAGE analysis of the cell-free extracts after expression was conducted for the mutants and the wild-type enzyme. The results indicated that all the truncated proteins could be expressed as soluble form and compared with the wild-type PUL, high-level expression of target protein was obviously achieved for the mutants of PULΔN5 and PULΔC9 (Fig. 3a). Additionally, the recombinant proteins were purified to homogeneity (Fig. 3b), and then the specific activities of the purified enzymes were measured. Of the truncated mutants, activities in the cells of PULΔN5 and PULΔN106 were 573 U mL−1 and 519 U·mL−1 with the amounts of purified enzymes were 1.77 mg mL−1 and 1.36 mgmL−1, respectively. Thus, PULΔN5 and PULΔN106 performed their specific activities as 324 U mg−1 and 382 U mg−1, which were 1.18- and 1.38-fold increase of the PUL, respectively (Fig. 2). Associating with the results from SDS-PAGE analysis of the expressed proteins of the constructs, the increased total activities of the mutants would be mainly caused by the improvement of the specific activity of the corresponding mutants. Therefore, truncation of the disordered region of the enzyme would be helpful to make the protein more active in catalyzing hydrolysis reaction, involving the acting behavior of binding substrate and molecular interactions. Taken together, it was worth to note that truncation of the first five amino acid residues at N-terminus significantly improved both the expression level and the enzymatic activity of the pullulanase.

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