Gene Cloning and Expression of Trehalose Synthase from Thermus thermophilus HJ6

Thermus thermophilus HJ6 유래 내열성 Trehalose Synthase의 유전자 클로닝 및 발현

  • Kim, Hyun-Jung (Department of Biomaterial Control (Brain korea 21 program), Dong-Eui University) ;
  • Kim, Han-Woo (Research Institute of Cell Engineering, National Institute of Advanced Industrial Science and Technology (AISI)) ;
  • Jeon, Sung-Jong (Department of Biomaterial Control (Brain korea 21 program), Dong-Eui University)
  • 김현정 (동의 대학교 바이오물질제어학과) ;
  • 김한우 (일본 산업기술종합연구소) ;
  • 전숭종 (동의 대학교 바이오물질제어학과)
  • Published : 2008.09.28

Abstract

A hyperthermophilic bacteria (strain HJ6) was isolated from a hot springs located in the Arima-cho, Hyogo, Japan. The cells were long-rod type ($2-4{\mu}m$), about $0.4{\mu}m$ in diameter. The pH and temperature for optimal growth were 6.5 and $80^{\circ}C$, respectively. Phylogenetic analysis based on the 16S rDNA sequence and biochemical studies indicated that HJ6 belonged to the genus Thermus thermophilus (Tt). The gene encoding the Trehalose synthase (TS) was cloned and sequenced. The open reading frame (ORF) of the TtTS gene was composed of 2,898 nucleotides and encoded a protein (975 amino acids) with a predicted molecular weight of 110.56 kDa. The deduced amino acid sequence of TtTS showed 99% and 83% identities to the Thermus caldophilus TS and Meiothermus ruber TS, respectively. TtTS gene was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for Trehalose synthase activity were found to be $80^{\circ}C$ and 7.5, respectively. The half-life of heat inactivation was about 40 min at $90^{\circ}C$. The maximum trehalose conversion rate of maltose into trehalose by the enzyme increased as the substrate concentration increased, and reached 55.7% at the maltose concentration of 500 mM, implying that the enzyme conversion was dependent of the substrate concentration.

Keywords

Trehalose;trehalose synthase;Thermus thermophilus;thermostability

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