• Biotechnology and Bioprocess Engineering:BBE
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• 제11권4호
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• pp.344-350
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• 2006

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at $59^{\circ}C$ and pH 2. Along with another thermoacidophilic archaeon, Sulfolobus solfataricus, it is known to metabolize glucose by the non-phosphorylated Entner-Doudoroff (nED) pathway. In the course of these studies, the specific activities of glyceraldehyde dehydrogenase and glycerate kinase, two enzymes that are involved in the downstream part of the nED pathway, were found to be much higher in T. acidophilum than in S. solfataricus. To characterize glycerate kinase, the enzyme was purified to homogeneity from T. acidophilum cell extracts. The N-terminal sequence of the purified enzyme was in exact agreement with that of Ta0453m in the genome database, with the removal of the initiator methionine. Furthermore, the enzyme was a monomer with a molecular weight of 49kDa and followed Michaelis-Menten kinetics with $K_m$ values of 0.56 and 0.32mM for DL-glycerate and ATP, respectively. The enzyme also exhibited excellent thermal stability at $70^{\circ}C$. Of the seven sugars and four phosphate donors tested, only DL-glycerate and ATP were utilized by glycerate kinase as substrates. In addition, a coupled enzyme assay indicated that 2-phosphoglycerate was produced as a product. When divalent metal ions, such as $Mn^{2+},\;CO^{2+},\;Ni^{2+},\;Zn^{2+},\;Ca^{2+},\;and\;Sr^{2+}$, were substituted for $Mg^{2+}$ the enzyme activities were less than 10% of that obtained in the presence of $Mg^{2+}$. The amino acid sequence of T. acidophilum glycerate kinase showed no similarity with E. coli glycerate kinases, which belong to the first glycerate kinase family. This is the first report on the biochemical characterization of an enzyme which belongs to a member of the second glycerate kinase family.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2001년도 추계학술대회
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• pp.39-45
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• 2001

Extremophiles are unique microorganisms that are adapted to survive in ecological niches such as high or low temperatures, extremes of pH, high salt concentrations and high pressure. These unusual microorganisms have unique biochemical features which can be exploited for use in the biotechnological industries. Due to the high biodiversity of extremophilic archaea and bacteria and their existence in various biotopes a variety of biocatalysts with different physicochemical properties have been discovered. The extreme molecular stability of their enzymes, membranes and the synthesis of unique organic compounds and polymers make extremophiles interesting candidates for basic and applied research. Some of the enzymes from extremophiles, especially hyperthermophilic marine microorganisms (growth above $85^{\circ}C$), have already been purified in our laboratory. These include the enzyme systems from Pyrococcus, Pyrodictium, Thermococcus and Thermotoga sp. that are involved in polysacharide modification and protein bioconversion. Only recently, the genome of the thermoalkaliphilic strain. Anaerobranca gottschalkii has been completely sequenced providing a unique resource of novel biocatalysts that are active at high temperature and pH. The gene encoding the branching enzyme from this organism was cloned and expressed in a mesophilic host and finally characterized. A novel glucoamylase was purified from an aerobic archaeon which shows optimal activity at $90^{\circ}C$ and pH 2.0. This thermoacidophilic archaeon Picrophilus oshimae grows optimally at pH 0.7 and $60^{\circ}C$. Furthermore, we were able to detect thermoactive proteases from two anaerobic isolates which are able to hydrolyze feather keratin completely at $80^{\circ}C$ forming amino acids and peptides. In addition, new marine psychrophilic isolates will be presented that are able to secrete enzymes such as lipases, proteases and amylases possessing high activity below the freezing point of water.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.327-333
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• 2005

Dihydroxy-acid dehydratase (DHAD, 2,3-dihydroxy-acid hydrolyase, EC 4.2.1.9) is one of the key enzymes involved in the biosynthetic pathway of the branched chain amino acid isoleucine and valine. Although the enzyme have been purified and characterized in various mesophiles including bacteria and eukarya, the biochemical properties of DHAD has bee not yet reported from hyperthermophilic archaea. In this study, we cloned, expressed, and purified a DHAD homologue from the thermoacidophilic archaeon Sulfolobus solfataricus P2, which grows optimally at $80\;^{\circ}C$ and pH 3, in E. coli. Characterization of the recombinant S. solfataricus DHAD (rSso_DHAD) revealed that it is the dimeric protein with a subunit molecular weight of 64,000 Da in native structure. rDHAD showed the highest activity toward 2,3-dihydroxyisovaleric acid among 17 aldonic acid substrates Interestingly, this enzyme also displayed 50 % activities toward some pentonic acids and hexonic acids when compared with the activity of this enzyme to the natural substrate. Moreover, rSso_DHAD indicated relatively higher activity toward D-gluconate than any other hexonic acids tested in substrates. $K_m$ and $V_{max}$ values of rSso_DHAD were calculated as $0.54\;{\pm}\;0.04\;mM$ toward 2,3dihydroxyisovalerate and $2.42\;{\pm}\;0.19\;mM$ toward D-gluconate, and as $21.6\;{\pm}\;0.4\;U/mg$ toward 2,3-dihydroxyisovalerate and $13.8\;{\pm}\;0.4\;U/mg$ toward D-gluconate, respectively. In the study for biochemical properties, the enzyme shows maximal activity between $70^{\circ}C$ and $80^{\circ}C$, and the pH range of pH 7.5 to 8.5. The half life time at $80^{\circ}C$ was 30 min. A divalent metal ion, $Mn^{2+}$, was only powerful activators, whereas other metal ions made the enzyme activity reduced. $Hg^{2+}$, organic mercury, and EDTA also strongly inhibited enzyme activities. Particularly, the rSso_DHAD activity was very stable under aerobic condition although the counterparts reported from mesophiles had been deactivated by oxygen.