Thermus thermophilus HJ6 유래 내열성 Trehalose Synthase의 유전자 클로닝 및 발현
Gene Cloning and Expression of Trehalose Synthase from Thermus thermophilus HJ6.
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초록
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 μm), about 0.4 μm in diameter. The pH and temperature for optimal growth were 6.5 and 80oC, 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 80oC and 7.5, respectively. The half-life of heat inactivation was about 40 min at 90oC. 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.
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 μm), about 0.4 μm in diameter. The pH and temperature for optimal growth were 6.5 and 80oC, 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 80oC and 7.5, respectively. The half-life of heat inactivation was about 40 min at 90oC. 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.
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