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Doubling Power Output of Starch Biobattery Treated by the Most Thermostable Isoamylase from an Archaeon Sulfolobus tokodaii.

Cheng K, Zhang F, Sun F, Chen H, Percival Zhang YH - Sci Rep (2015)

Bottom Line: This enzyme was characterized and required Mg(2+) as an activator.This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 (o)C in the presence of 0.5 mM MgCl2, suitable for simultaneous starch gelatinization and isoamylase hydrolysis.The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, 450002, China.

ABSTRACT
Biobattery, a kind of enzymatic fuel cells, can convert organic compounds (e.g., glucose, starch) to electricity in a closed system without moving parts. Inspired by natural starch metabolism catalyzed by starch phosphorylase, isoamylase is essential to debranch alpha-1,6-glycosidic bonds of starch, yielding linear amylodextrin - the best fuel for sugar-powered biobattery. However, there is no thermostable isoamylase stable enough for simultaneous starch gelatinization and enzymatic hydrolysis, different from the case of thermostable alpha-amylase. A putative isoamylase gene was mined from megagenomic database. The open reading frame ST0928 from a hyperthermophilic archaeron Sulfolobus tokodaii was cloned and expressed in E. coli. The recombinant protein was easily purified by heat precipitation at 80 (o)C for 30 min. This enzyme was characterized and required Mg(2+) as an activator. This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 (o)C in the presence of 0.5 mM MgCl2, suitable for simultaneous starch gelatinization and isoamylase hydrolysis. The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated.

No MeSH data available.


Related in: MedlinePlus

Photos of iodine dyed amylopectin and isoamylase treated amylopectin.(a) and light absorption spectrum of the iodine-stained amylopectin compared with isoamylase treated amylopectin. Reaction conditions were 0.75% amylopectin in 40 mM acetate buffer (pH 5.5) containing 0.5 mM MgCl2 and 7.5 μg/ml isoamylase incubated at 80 °C for 30 min. The stained samples were diluted by a factor of 10 in water.
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f4: Photos of iodine dyed amylopectin and isoamylase treated amylopectin.(a) and light absorption spectrum of the iodine-stained amylopectin compared with isoamylase treated amylopectin. Reaction conditions were 0.75% amylopectin in 40 mM acetate buffer (pH 5.5) containing 0.5 mM MgCl2 and 7.5 μg/ml isoamylase incubated at 80 °C for 30 min. The stained samples were diluted by a factor of 10 in water.

Mentions: Amylopectin was hydrolyzed by this enzyme under its optimal condition (e.g., acetate buffer (pH 5.5) containing 5 mM MgCl2 and 80 oC) (Fig. 4). The branched amylopectin shows a typical brown-blue color after the iodine dying (Fig. 4a) because branched amylopectin cannot form coils and thus it does not form a complex with iodine. After this enzyme treatment, the solution turned a purple color (Fig. 4a), suggesting that linear amylodextrin forms a representative starch/iodine color – purple/deep blue. Figure 4b shows the changes in absorption spectra of the iodine-staining solution for the amylopectin before and after the treatment of this enzyme. The absorbance increased and the maximum wavelength of absorption shifted to a longer wavelength from 530 to 560 nm. These results suggest that the enzyme hydrolyzed the 1,6-alpha-glycosidic linkage of branched amylopectin. This enzyme exhibited a very low activity on amylose (~5%) relative to that on amylopectin, indicating that this enzyme preferred hydrolyzing alpha-1,6-glycosidic bonds. This very low activity on amylose could be due to the high-sensitivity reducing end assay based on the BCA assay instead of the commonly-used Somogyi assay and/or some minor branches in natural amylose. Also, this enzyme can generate new reducing ends on long-chain maltodextrin (DE 4.0–7.0) but no new ends generated on short-chain maltodextrin (DE 16.5–19.5), suggesting that it cannot hydrolyze alpha-1,4,6-D-glucose branch-points for short maltodextrin, different from pullulanase. The above results seemed appropriate to refer to this enzyme as an isoamylase but its weak alpha-1,4-hydrolytic activity was not eliminated completely. This enzyme had a specific activity of 6.4 IU/mg on amylopectin at 80 oC based on the reducing ends generated.


Doubling Power Output of Starch Biobattery Treated by the Most Thermostable Isoamylase from an Archaeon Sulfolobus tokodaii.

Cheng K, Zhang F, Sun F, Chen H, Percival Zhang YH - Sci Rep (2015)

Photos of iodine dyed amylopectin and isoamylase treated amylopectin.(a) and light absorption spectrum of the iodine-stained amylopectin compared with isoamylase treated amylopectin. Reaction conditions were 0.75% amylopectin in 40 mM acetate buffer (pH 5.5) containing 0.5 mM MgCl2 and 7.5 μg/ml isoamylase incubated at 80 °C for 30 min. The stained samples were diluted by a factor of 10 in water.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4542469&req=5

f4: Photos of iodine dyed amylopectin and isoamylase treated amylopectin.(a) and light absorption spectrum of the iodine-stained amylopectin compared with isoamylase treated amylopectin. Reaction conditions were 0.75% amylopectin in 40 mM acetate buffer (pH 5.5) containing 0.5 mM MgCl2 and 7.5 μg/ml isoamylase incubated at 80 °C for 30 min. The stained samples were diluted by a factor of 10 in water.
Mentions: Amylopectin was hydrolyzed by this enzyme under its optimal condition (e.g., acetate buffer (pH 5.5) containing 5 mM MgCl2 and 80 oC) (Fig. 4). The branched amylopectin shows a typical brown-blue color after the iodine dying (Fig. 4a) because branched amylopectin cannot form coils and thus it does not form a complex with iodine. After this enzyme treatment, the solution turned a purple color (Fig. 4a), suggesting that linear amylodextrin forms a representative starch/iodine color – purple/deep blue. Figure 4b shows the changes in absorption spectra of the iodine-staining solution for the amylopectin before and after the treatment of this enzyme. The absorbance increased and the maximum wavelength of absorption shifted to a longer wavelength from 530 to 560 nm. These results suggest that the enzyme hydrolyzed the 1,6-alpha-glycosidic linkage of branched amylopectin. This enzyme exhibited a very low activity on amylose (~5%) relative to that on amylopectin, indicating that this enzyme preferred hydrolyzing alpha-1,6-glycosidic bonds. This very low activity on amylose could be due to the high-sensitivity reducing end assay based on the BCA assay instead of the commonly-used Somogyi assay and/or some minor branches in natural amylose. Also, this enzyme can generate new reducing ends on long-chain maltodextrin (DE 4.0–7.0) but no new ends generated on short-chain maltodextrin (DE 16.5–19.5), suggesting that it cannot hydrolyze alpha-1,4,6-D-glucose branch-points for short maltodextrin, different from pullulanase. The above results seemed appropriate to refer to this enzyme as an isoamylase but its weak alpha-1,4-hydrolytic activity was not eliminated completely. This enzyme had a specific activity of 6.4 IU/mg on amylopectin at 80 oC based on the reducing ends generated.

Bottom Line: This enzyme was characterized and required Mg(2+) as an activator.This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 (o)C in the presence of 0.5 mM MgCl2, suitable for simultaneous starch gelatinization and isoamylase hydrolysis.The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, 450002, China.

ABSTRACT
Biobattery, a kind of enzymatic fuel cells, can convert organic compounds (e.g., glucose, starch) to electricity in a closed system without moving parts. Inspired by natural starch metabolism catalyzed by starch phosphorylase, isoamylase is essential to debranch alpha-1,6-glycosidic bonds of starch, yielding linear amylodextrin - the best fuel for sugar-powered biobattery. However, there is no thermostable isoamylase stable enough for simultaneous starch gelatinization and enzymatic hydrolysis, different from the case of thermostable alpha-amylase. A putative isoamylase gene was mined from megagenomic database. The open reading frame ST0928 from a hyperthermophilic archaeron Sulfolobus tokodaii was cloned and expressed in E. coli. The recombinant protein was easily purified by heat precipitation at 80 (o)C for 30 min. This enzyme was characterized and required Mg(2+) as an activator. This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 (o)C in the presence of 0.5 mM MgCl2, suitable for simultaneous starch gelatinization and isoamylase hydrolysis. The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated.

No MeSH data available.


Related in: MedlinePlus