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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

SDS-PAGE analysis of isoamylase expression and purification in E. coli BL21 (DE3) and Rosetta (DE3).(a)Lanes: M, markers; B, BL21 host; R, Rosetta host; S, the supernatant of the cell lysate of E. coli Rosetta; T, the cell lysate of E. coli Rosetta; P: pellets of the cell lysate of E. coli Rosetta; HT, the supernatant of the heat-treated cell lysate of E. coli Rosetta; and His, the purified isoamylase by using Ni-charged resins. Effect of pH on the isoamylase activity (b). Buffer concentration was 40 mM and 0.5 mM MgCl2: acetate buffer (pH 4–6) and phosphate buffer (pH 5–8). Data represent the mean ± S.D. from triplicate experiments. Effect of temperature on the isoamylase activities (c). Reaction conditions were 40 mM acetate buffer (pH 5.5) containing 0.5 mM MgCl2. Data represent the mean ± S.D. from triplicate experiments.
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f3: SDS-PAGE analysis of isoamylase expression and purification in E. coli BL21 (DE3) and Rosetta (DE3).(a)Lanes: M, markers; B, BL21 host; R, Rosetta host; S, the supernatant of the cell lysate of E. coli Rosetta; T, the cell lysate of E. coli Rosetta; P: pellets of the cell lysate of E. coli Rosetta; HT, the supernatant of the heat-treated cell lysate of E. coli Rosetta; and His, the purified isoamylase by using Ni-charged resins. Effect of pH on the isoamylase activity (b). Buffer concentration was 40 mM and 0.5 mM MgCl2: acetate buffer (pH 4–6) and phosphate buffer (pH 5–8). Data represent the mean ± S.D. from triplicate experiments. Effect of temperature on the isoamylase activities (c). Reaction conditions were 40 mM acetate buffer (pH 5.5) containing 0.5 mM MgCl2. Data represent the mean ± S.D. from triplicate experiments.

Mentions: The ST0928 was sub-cloned into the T7-promoter plasmid pET20b by restriction enzyme-free, ligase-free Simple Cloning technique25. Two E. coli strains BL21(DE3) and Rosetta (DE3) were tested to express the recombinant IA with a His tag on its C terminus. Apparently, E. coli Rosetta was a better host than BL21 to express the soluble targeted enzyme (Fig. 3A, the left gel) because this gene contained a lot of rare codons in E. coli, including one three-rare codon cluster and several two-rare codon clusters. Although the host Rosetta can co-express the tRNAs for rare codons, a clear band with a molecular weight of ~81 kDa was observed in the pellet fraction by SDS-PAGE (Fig. 3a, Lane P), suggesting a significant amount of inclusion body formed. The His-tag enzyme was purified by affinity adsorption on nickel-charged resins. Alternatively, the cell lysate containing this enzyme was treated at 80 oC for 30 min, to denature E. coli cellular proteins. After centrifugation, the targeted protein was the predominant band in the supernatant, being approximately 85% purity (Fig. 3a, Lane HT). The protein recovery efficiency for nickel resin adsorption and heat precipitation were 81% and 98%, respectively. Approximately 10 mg of the purified His-tagged enzyme was purified from 200 mL of the cell culture grown in the LB media. This His-tagged enzyme had a specific activity of 6.4 IU/mg on amylopectin at 80 oC based on the reducing ends generated. The specific activity of heat precipitated enzyme was approximately 89% of that purified from nickel resin adsorption, in consistent of SDS-PAGE data. Heat precipitation is the easiest approach for purifying relatively pure thermostable enzymes suitable for in vitro biocatalysis2627.


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)

SDS-PAGE analysis of isoamylase expression and purification in E. coli BL21 (DE3) and Rosetta (DE3).(a)Lanes: M, markers; B, BL21 host; R, Rosetta host; S, the supernatant of the cell lysate of E. coli Rosetta; T, the cell lysate of E. coli Rosetta; P: pellets of the cell lysate of E. coli Rosetta; HT, the supernatant of the heat-treated cell lysate of E. coli Rosetta; and His, the purified isoamylase by using Ni-charged resins. Effect of pH on the isoamylase activity (b). Buffer concentration was 40 mM and 0.5 mM MgCl2: acetate buffer (pH 4–6) and phosphate buffer (pH 5–8). Data represent the mean ± S.D. from triplicate experiments. Effect of temperature on the isoamylase activities (c). Reaction conditions were 40 mM acetate buffer (pH 5.5) containing 0.5 mM MgCl2. Data represent the mean ± S.D. from triplicate experiments.
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Related In: Results  -  Collection

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f3: SDS-PAGE analysis of isoamylase expression and purification in E. coli BL21 (DE3) and Rosetta (DE3).(a)Lanes: M, markers; B, BL21 host; R, Rosetta host; S, the supernatant of the cell lysate of E. coli Rosetta; T, the cell lysate of E. coli Rosetta; P: pellets of the cell lysate of E. coli Rosetta; HT, the supernatant of the heat-treated cell lysate of E. coli Rosetta; and His, the purified isoamylase by using Ni-charged resins. Effect of pH on the isoamylase activity (b). Buffer concentration was 40 mM and 0.5 mM MgCl2: acetate buffer (pH 4–6) and phosphate buffer (pH 5–8). Data represent the mean ± S.D. from triplicate experiments. Effect of temperature on the isoamylase activities (c). Reaction conditions were 40 mM acetate buffer (pH 5.5) containing 0.5 mM MgCl2. Data represent the mean ± S.D. from triplicate experiments.
Mentions: The ST0928 was sub-cloned into the T7-promoter plasmid pET20b by restriction enzyme-free, ligase-free Simple Cloning technique25. Two E. coli strains BL21(DE3) and Rosetta (DE3) were tested to express the recombinant IA with a His tag on its C terminus. Apparently, E. coli Rosetta was a better host than BL21 to express the soluble targeted enzyme (Fig. 3A, the left gel) because this gene contained a lot of rare codons in E. coli, including one three-rare codon cluster and several two-rare codon clusters. Although the host Rosetta can co-express the tRNAs for rare codons, a clear band with a molecular weight of ~81 kDa was observed in the pellet fraction by SDS-PAGE (Fig. 3a, Lane P), suggesting a significant amount of inclusion body formed. The His-tag enzyme was purified by affinity adsorption on nickel-charged resins. Alternatively, the cell lysate containing this enzyme was treated at 80 oC for 30 min, to denature E. coli cellular proteins. After centrifugation, the targeted protein was the predominant band in the supernatant, being approximately 85% purity (Fig. 3a, Lane HT). The protein recovery efficiency for nickel resin adsorption and heat precipitation were 81% and 98%, respectively. Approximately 10 mg of the purified His-tagged enzyme was purified from 200 mL of the cell culture grown in the LB media. This His-tagged enzyme had a specific activity of 6.4 IU/mg on amylopectin at 80 oC based on the reducing ends generated. The specific activity of heat precipitated enzyme was approximately 89% of that purified from nickel resin adsorption, in consistent of SDS-PAGE data. Heat precipitation is the easiest approach for purifying relatively pure thermostable enzymes suitable for in vitro biocatalysis2627.

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