Molecular cloning and biochemical characterization of a heat-stable type I pullulanase from Thermotoga neapolitana

Kang, Jeonggyu; Park, Kyung Min; Choi, Ki Young; Park, Cheon-Seok; Kim, Go-Eun; Kim, Doman; Cha, Jaeho

doi:10.1016/j.enzmictec.2010.11.006

Cited by 77 publications

(51 citation statements)

References 30 publications

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“…Several pullulanase-encoding genes have recently been cloned and characterized (12)(13)(14), and the crystal structures of a few pullulanases have been determined (19)(20)(21)(22). Pullulanase is usually composed of about 900 amino acids and has a molecular mass of about 100 kDa.…”

Section: ؊1mentioning

confidence: 99%

“…Although numerous pullulanases have been identified and heterologously expressed (12)(13)(14), reports have shown that the secretion ratio of recombinant pullulanase is low. Low extracellular productivity is currently the major factor limiting the large-scale production and widespread application of pullulanase.…”

mentioning

confidence: 99%

“…Although the recovery of enzymes secreted into the periplasmic space can be achieved by various approaches (16,17), its application on an industrial scale has been limited by the unavailability of efficient large-scale methods for the selective release of periplasmic proteins from the cell (18). Therefore, a method that allows the secretion of recombinant pullulanase directly into the culture medium is highly desirable.Several pullulanase-encoding genes have recently been cloned and characterized (12)(13)(14), and the crystal structures of a few pullulanases have been determined (19)(20)(21)(22). Pullulanase is usually composed of about 900 amino acids and has a molecular mass of about 100 kDa.…”

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Enhancing the Secretion Efficiency and Thermostability of a Bacillusderamificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

Duan

2015

Appl Environ Microbiol

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Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of ␣-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form of Bacillus deramificans pullulanase was recently constructed to enhance the thermostability and catalytic efficiency of the enzyme (X. Duan, J. Chen, and J. Wu, Appl Environ Microbiol 79:4072-4077, 2013, http://dx.doi.org/10.1128/AEM.00457-13). In the present study, three N-terminally truncated variants of this DM that lack the CBM41 domain (DM-T1), the CBM41 and X25 domains (DM-T2), or the CBM41, X25, and X45 domains (DM-T3) were constructed. Upon expression, DM-T3 existed as inclusion bodies, while 72.8 and 74.8% of the total pullulanase activities of DM-T1 and DM-T2, respectively, were secreted into the medium. These activities are 2.8-and 2.9-fold that of the DM enzyme, respectively. The specific activities of DM-T1 and DM-T2 were 380.0 ؋ 10 8 and 449.3 ؋ 10 8 U · mol ؊1, respectively, which are 0.94-and 1.11-fold that of the DM enzyme. DM-T1 and DM-T2 retained 50% of their activity after incubation at 60°C for 203 and 160 h, respectively, which are 1.7-and 1.3-fold that of the DM enzyme. Kinetic studies showed that the K m values of DM-T1 and DM-T2 were 1.5-and 2.7-fold higher and the K cat /K m values were 11 and 50% lower, respectively, than those of the DM enzyme. Furthermore, DM-T1 and DM-T2 produced D-glucose contents of 95.0 and 94.1%, respectively, in a starch saccharification reaction, which are essentially identical to that produced by the DM enzyme (95%). The enhanced secretion and improved thermostability of the truncation mutant enzymes make them more suitable than the DM enzyme for industrial processes. P ullulanase (EC 3.2.1.41), which catalyzes the hydrolysis of ␣-1,6-glucosidic linkages in pullulan, amylopectin, and the dextrins of amylopectin, is a well-known starch-debranching enzyme (1). It belongs to the ␣-amylase family, which is identified as glycoside hydrolase family 13 in the CAZy database. When combined with ␣-amylase, ␤-amylase, glucoamylase, or cyclodextrin glucosyltransferase, pullulanase can be used in the production of glucose, fructose, maltose, cyclodextrins, and amylose (2, 3). The addition of pullulanase allows the reaction time to be reduced, the substrate concentration to be increased, and the conversion rate to be improved (4, 5). Recently, pullulanase has been used as a biocatalyst for the production of fuel ethanol, low-calorie beers, resistant starch, maltotriose, and other important products (6, 7). With this plethora of potential industrial applications, pullulanase production has attracted a great deal of attention in recent years (8,9).A large number of microorganisms, including mesophiles, thermophiles, and hyperthermophiles, have been shown to produce pullulanase (10, 11). Although numerous pullulanases have been identified and heterologously expressed (12)(13)(14), reports have shown that the secretion ratio of recombinant pullulanase is low. Low extracellular prod...

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Section: ؊1mentioning

confidence: 99%

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confidence: 99%

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Enhancing the Secretion Efficiency and Thermostability of a Bacillusderamificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

Duan

2015

Appl Environ Microbiol

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“…1A). The structure model showed that region A (Asp503-Pro509), which is involved in van der Waals interaction with subsite Ϫ1 and Ϫ2 glucose residues in the substrate (9), is located in a loop (Phe490-Asp513, in green in Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

“…To date, many microorganisms, including mesophiles, thermophiles, and hyperthermophiles, have been shown to produce pullulanase, and a large number of pullulanases have been identified and characterized (3)(4)(5)(6). In addition, several pullulanase-encoding genes have been cloned and characterized (7)(8)(9), and their crystal structures have been determined (10)(11)(12).…”

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Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

Duan

Chen

2013

Appl Environ Microbiol

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b Pullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Its instability and low catalytic efficiency are the major factors preventing its widespread application. To address these issues, Asp437 and Asp503 of the pullulanase from Bacillus deramificans were selected in this study as targets for site-directed mutagenesis based on a structure-guided consensus approach. Four mutants (carrying the mutations D503F, D437H, D503Y, and D437H/D503Y) were generated and characterized in detail. The results showed that the D503F, D437H, and D503Y mutants had an optimum temperature of 55°C and a pH optimum of 4.5, similar to that of the wild-type enzyme. However, the half-lives of the mutants at 60°C were twice as long as that of the wild-type enzyme. In addition, the D437H/D503Y double mutant displayed a larger shift in thermostability, with an optimal temperature of 60°C and a half-life at 60°C of more than 4.3-fold that of the wild-type enzyme. Kinetic studies showed that the K m values for the D503F, D437H, D503Y, and D437H/D503Y mutants decreased by 7.1%, 11.4%, 41.4%, and 45.7% and the K cat /K m values increased by 10%, 20%, 140%, and 100%, respectively, compared to those of the wild-type enzyme. Mechanisms that could account for these enhancements were explored. Moreover, in conjunction with the enzyme glucoamylase, the D503Y and D437H/D503Y mutants exhibited an improved reaction rate and glucose yield during starch hydrolysis compared to those of the wild-type enzyme, confirming the enhanced properties of the mutants. The mutants generated in this study have potential applications in the starch industry.

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Cloning, purification, and functional characterization of recombinant pullulanase from Bacillus cereusATCC 14579 for improved detergent performance

Zafar,

Masood,

Rahman

et al. 2024

J Surfact & Detergents

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The present study outlines the approach that was employed for cloning, expression, and characterization of the recombinant pullulanase enzyme from Bacillus cereus ATCC 14579 into Escherichia coli BL21(DE3) using pET‐25b (+) expression vector. The recombinant pullulanase enzyme was purified using ammonium sulfate precipitation and immobilized metal ion affinity chromatography (IMAC). The molecular mass of the purified pullulanase enzyme was measured using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) as 95 kDa. The purified recombinant pullulanase enzyme demonstrated significant thermal stability, maintaining its structural integrity and functionality at temperatures as high as 90°C over a period of 4 h. The inclusion of divalent metal ions, specifically Ca2+ and Mg2+, had a positive effect on the activity of the pullulanase enzyme. Conversely, the presence of Co2+ and EDTA (Ethylene Diamine Tetra Acetic acid) resulted in suppression of the enzyme activity. The purified pullulanase enzyme demonstrated remarkable resistance when exposed to organic solvents. The enzyme activity was notably decreased in the presence of SDS (Sodium Dodecyl Sulfate) while β‐mercaptoethanol and tween‐60 did not substantially affect the enzyme activity and stability which suggest its potential applicability in the detergent sector. This discovery indicates a potential approach to improve the effectiveness of currently available detergents in the marketplace.

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Molecular cloning and biochemical characterization of a heat-stable type I pullulanase from Thermotoga neapolitana

Cited by 77 publications

References 30 publications

Enhancing the Secretion Efficiency and Thermostability of a Bacillusderamificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

Enhancing the Secretion Efficiency and Thermostability of a Bacillusderamificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

Cloning, purification, and functional characterization of recombinant pullulanase from Bacillus cereusATCC 14579 for improved detergent performance

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