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

研究成果: 雜誌貢獻期刊論文同行評審

60 引文 斯高帕斯(Scopus)

摘要

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.

原文英語
頁(從 - 到)7098-7104
頁數7
期刊Journal of Agricultural and Food Chemistry
54
發行號19
DOIs
出版狀態已發佈 - 2006 九月 20

ASJC Scopus subject areas

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

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