Construction of a recombinant thermostable β-amylase-trehalose synthase bifunctional enzyme for facilitating the conversion of starch to trehalose

Jia Hung Wang, Meng Yin Tsai, Guan Chiun Lee, Jei Fu Shaw

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29 Citations (Scopus)

Abstract

A fusion gene that encoded a polypeptide of 1495 amino acids was constructed from the β-amylase (BA) gene of Clostridium thermosulfurogenes and trehalose synthase (TS) gene of Thermus thermophilus. The fused gene was overexpressed in Escherichia coli, and a recombinant bifunctional fusion protein with BA at the N-terminal (BATS) or C-terminal (TSBA) of TS having both β-amylase and trehalose synthase activities with an apparent molecular mass of 164 kDa was obtained. BATS or TSBA catalyzes the sequential reaction in which maltose is formed from starch and then is converted into trehalose. The K m values of the BATS and TSBA fusion enzymes for the reaction from starch to trehalose were smaller than those of an equimolar mixture of BA and TS (BA/TS). On the other hand, the k cat value of BATS approximated that of the BA/TS mixture, but that of TSBA exceeded it. TSBA showed much higher sequential catalytic efficiency than the separately expressed BA/TS mixture. The catalytic efficiency of TSBA or BATS was 3.4 or 2.4 times higher, respectively, than that of a mixture of individual enzymes, showing the kinetic advantage of the fusion enzyme. The thermal stability readings of the recombinant fusion enzymes BATS and TSBA were better than that of the mixture of individual recombinant enzymes. These results apparently demonstrate that fusion enzymes catalyzing sequential reactions have kinetic advantages over a mixture of both enzymes.

Original languageEnglish
Pages (from-to)1256-1263
Number of pages8
JournalJournal of Agricultural and Food Chemistry
Volume55
Issue number4
DOIs
Publication statusPublished - 2007 Feb 21

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Keywords

  • Bifunctional enzyme
  • Starch
  • Thermus thermophilus trehalose synthase
  • Trehalose

ASJC Scopus subject areas

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

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