Gene cloning, expression, and biochemical characterization of a recombinant trehalose synthase from Picrophilus torridus in Escherichia coli

Yi Shan Chen, Guan Chiun Lee, Jei Fu Shaw

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

A trehalose synthase (TSase) gene from a hyperacidophilic, thermophilic archaea, Picrophilus torridus, was synthesized using overlap extension PCR and transformed into Escherichia coli for expression. The purified recombinant P. torridus TSase (PTTS) showed an optimum pH and temperature of 6.0 and 45°C, respectively, and the enzyme maintained high activity at pH 5.0 and 60°C. Kinetic analysis showed that the enzyme has a 2.5-fold higher catalytic efficiency (kcat/KM) for maltose than for trehalose, indicating maltose as the preferred substrate. The maximum conversion rate of maltose into trehalose by the enzyme was independent of the substrate concentration, tended to increase at lower temperatures, and reached ≈71% at 20°C. Enzyme activity was inhibited by Hg2+, Al3+, and SDS. Five amino acid residues that are important for α-amylase family enzyme catalysis were shown to be conserved in PTTS (Asp203, Glu 245, Asp311, His106, and His310) and required for its activity, suggesting this enzyme might employ a similar hydrolysis mechanism.

Original languageEnglish
Pages (from-to)7098-7104
Number of pages7
JournalJournal of Agricultural and Food Chemistry
Volume54
Issue number19
DOIs
Publication statusPublished - 2006 Sep 20

Fingerprint

Thermoplasmales
Cloning
trehalose
Escherichia coli
Organism Cloning
molecular cloning
maltose
Genes
Maltose
Gene Expression
Enzymes
enzymes
catalytic activity
Trehalose
enzyme activity
Archaea
amylases
aluminum
Enzyme activity
Substrates

Keywords

  • High performance liquid chromatography
  • Maltose
  • Picrophilus torridus
  • Trehalose
  • Trehalose synthase

ASJC Scopus subject areas

  • Chemistry(all)
  • Agricultural and Biological Sciences(all)

Cite this

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title = "Gene cloning, expression, and biochemical characterization of a recombinant trehalose synthase from Picrophilus torridus in Escherichia coli",
abstract = "A trehalose synthase (TSase) gene from a hyperacidophilic, thermophilic archaea, Picrophilus torridus, was synthesized using overlap extension PCR and transformed into Escherichia coli for expression. The purified recombinant P. torridus TSase (PTTS) showed an optimum pH and temperature of 6.0 and 45°C, respectively, and the enzyme maintained high activity at pH 5.0 and 60°C. Kinetic analysis showed that the enzyme has a 2.5-fold higher catalytic efficiency (kcat/KM) for maltose than for trehalose, indicating maltose as the preferred substrate. The maximum conversion rate of maltose into trehalose by the enzyme was independent of the substrate concentration, tended to increase at lower temperatures, and reached ≈71{\%} at 20°C. Enzyme activity was inhibited by Hg2+, Al3+, and SDS. Five amino acid residues that are important for α-amylase family enzyme catalysis were shown to be conserved in PTTS (Asp203, Glu 245, Asp311, His106, and His310) and required for its activity, suggesting this enzyme might employ a similar hydrolysis mechanism.",
keywords = "High performance liquid chromatography, Maltose, Picrophilus torridus, Trehalose, Trehalose synthase",
author = "Chen, {Yi Shan} and Lee, {Guan Chiun} and Shaw, {Jei Fu}",
year = "2006",
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T1 - Gene cloning, expression, and biochemical characterization of a recombinant trehalose synthase from Picrophilus torridus in Escherichia coli

AU - Chen, Yi Shan

AU - Lee, Guan Chiun

AU - Shaw, Jei Fu

PY - 2006/9/20

Y1 - 2006/9/20

N2 - A trehalose synthase (TSase) gene from a hyperacidophilic, thermophilic archaea, Picrophilus torridus, was synthesized using overlap extension PCR and transformed into Escherichia coli for expression. The purified recombinant P. torridus TSase (PTTS) showed an optimum pH and temperature of 6.0 and 45°C, respectively, and the enzyme maintained high activity at pH 5.0 and 60°C. Kinetic analysis showed that the enzyme has a 2.5-fold higher catalytic efficiency (kcat/KM) for maltose than for trehalose, indicating maltose as the preferred substrate. The maximum conversion rate of maltose into trehalose by the enzyme was independent of the substrate concentration, tended to increase at lower temperatures, and reached ≈71% at 20°C. Enzyme activity was inhibited by Hg2+, Al3+, and SDS. Five amino acid residues that are important for α-amylase family enzyme catalysis were shown to be conserved in PTTS (Asp203, Glu 245, Asp311, His106, and His310) and required for its activity, suggesting this enzyme might employ a similar hydrolysis mechanism.

AB - A trehalose synthase (TSase) gene from a hyperacidophilic, thermophilic archaea, Picrophilus torridus, was synthesized using overlap extension PCR and transformed into Escherichia coli for expression. The purified recombinant P. torridus TSase (PTTS) showed an optimum pH and temperature of 6.0 and 45°C, respectively, and the enzyme maintained high activity at pH 5.0 and 60°C. Kinetic analysis showed that the enzyme has a 2.5-fold higher catalytic efficiency (kcat/KM) for maltose than for trehalose, indicating maltose as the preferred substrate. The maximum conversion rate of maltose into trehalose by the enzyme was independent of the substrate concentration, tended to increase at lower temperatures, and reached ≈71% at 20°C. Enzyme activity was inhibited by Hg2+, Al3+, and SDS. Five amino acid residues that are important for α-amylase family enzyme catalysis were shown to be conserved in PTTS (Asp203, Glu 245, Asp311, His106, and His310) and required for its activity, suggesting this enzyme might employ a similar hydrolysis mechanism.

KW - High performance liquid chromatography

KW - Maltose

KW - Picrophilus torridus

KW - Trehalose

KW - Trehalose synthase

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