• Journal of Life Science
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• v.14 no.3
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• pp.435-440
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• 2004

The gene encoding a extremely thermostable 4-$\alpha$-glucanotransferase from a hyperthermophilic archaeon, Thermococcus litoralis, was cloned, sequenced and expressed in Escherichia coli. The amino acid sequence of the enzyme was distantly related to other functionally-related ones, such as D-enzymes. The enzyme is of industrial interest because of a novel activity of producing cycloamylose and is also important for fundamental studies of protein, sugar-metabolizing enzymes. In this paper, the overexpression of 4-$\alpha$-glucanotransferase in E. coli was carried out expression vector system with lac and T7 promoters. The enzyme was successfully overexpressed, and purified by the heat treatment of a cell-free extract, successive Butyl-Toyopearl and Mono Q chromatographies. The purified recombinant enzyme showed the same specific activity and the same mobility in SDS-PAGE as natural enzyme.

• Korean Journal of Microbiology
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• v.48 no.1
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• pp.42-47
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• 2012

Hydrothermal vents (HTV) provide special environments for evolution of lives independent on solar energy. HTV samples were gained from Tofua arc trench in Tonga, South Pacific. We investigated archaeal diversity enriched using combinations of various electron donors (yeast extract and $H_2$) and electron acceptors [Iron (III), elemental sulfur ($S^0$) and nitrate. PCR amplification was performed to detect archaeal 16S rRNA genes after the cultures were incubated $65^{\circ}C$ and $80^{\circ}C$ for 2 weeks. The cultures showing archaeal growth were transferred using the dilution-to-extinction method. 16S rRNA gene PCR-Denaturing Gradient Gel Electrophoresis was used to identify the enriched archaea in the highest dilutions where archaeal growth was observed. Most of cultured archaea belonged to genus of Thermococcus (T. alcaliphilius, T. litoralis, T. celer, T. barossii, T. thoreducens, T. coalescens) with 98-99% 16S rRNA gene similarities. Interestingly, archaeal growth was observed in the cultures with Iron (III) and nitrate as an electron acceptor. It was supposed that archaea might use the elemental sulfur generated from oxidation of the reducing agent, sulfide. To cultivate diverse archaea excluding Thermococcus, it would be required to use other reducing agents instead of sulfide.