@article {1413, title = {Bioinformatic and biochemical analysis of a novel maltose-forming α-amylase of the GH57 family in the hyperthermophilic archaeon Thermococcus sp. CL1.}, journal = {Enzyme Microb Technol}, volume = {60}, year = {2014}, month = {2014 Jun 10}, pages = {9-15}, abstract = {

Maltose-forming \α-amylase is a glycoside hydrolase family 57 (GH57) member that is unique because it displays dual hydrolysis activity toward \α-1,4- and \α-1,6-glycosidic linkages and only recognizes maltose. This enzyme was previously identified only in Pyrococcus sp. ST04 (PSMA); however, we recently found two homologs subgroups in Thermococcus species. One subgroup (subgroup A) showed relatively high amino acid sequence similarity to PSMA (\>71\%), while the other subgroup (subgroup B) showed lower homology with PSMA (\<59\%). To characterize the subgroup B maltose-forming \α-amylase from Thermococcus species (TCMA), we cloned the CL1_0868 gene from Thermococcus sp. CL1 and then successfully expressed the gene in Escherichia coli. Although TCMA has a different oligomeric state relative to PSMA, TCMA showed similar substrate specificity. However, TCMA was shown to hydrolyze maltooligosaccharides more easily than PSMA. Also, TCMA displayed different optimum conditions depending on the glycosidic linkage of the substrate. TCMA had the highest activity at 85\°C and at pH 5.0 for \α-1,4-glycosidic linkage hydrolysis whereas it showed its maximal activity to cleave \α-1,6-glycosidic linkages at 98\°C and pH 6.0.

}, issn = {1879-0909}, doi = {10.1016/j.enzmictec.2014.03.009}, author = {Jeon, Eun-Jung and Jung, Jong-Hyun and Seo, Dong-Ho and Jung, Dong-Hyun and Holden, James F and Park, Cheon-Seok} } @article {1200, title = {Complete genome sequence of hyperthermophilic archaeon Thermococcus sp. ES1.}, journal = {J Biotechnol}, volume = {174C}, year = {2014}, month = {2014 Jan 25}, pages = {14-15}, abstract = {

Thermococcus sp. strain ES1 is an anaerobic, hyperthermophilic archaeon from a hydrothermal vent that catabolizes sugars and peptides and produces H2S from S{\textdegree}, H2, acetate and CO2 as its primary metabolites. We present the complete genome sequence of this strain (1,957,742bp) with a focus on its substrate utilization and metabolite production capabilities. The sequence will contribute to the development of heterotrophic archaea for bioenergy production and biogeochemical modeling in hydrothermal environments.

}, issn = {1873-4863}, doi = {10.1016/j.jbiotec.2014.01.022}, author = {Jung, Jong-Hyun and Kim, You-Tae and Jeon, Eun-Jung and Seo, Dong-Ho and Hensley, Sarah A and Holden, James F and Lee, Ju-Hoon and Park, Cheon-Seok} } @article {1201, title = {Identification and Characterization of an Archaeal Kojibiose Catabolic Pathway in the Hyperthermophilic Pyrococcus sp. Strain ST04.}, journal = {J Bacteriol}, volume = {196}, year = {2014}, month = {2014 Mar}, pages = {1122-31}, abstract = {

A unique gene cluster responsible for kojibiose utilization was identified in the genome of Pyrococcus sp. strain ST04. The proteins it encodes hydrolyze kojibiose, a disaccharide product of glucose caramelization, and form glucose-6-phosphate (G6P) in two steps. Heterologous expression of the kojibiose-related enzymes in Escherichia coli revealed that two genes, Py04_1502 and Py04_1503, encode kojibiose phosphorylase (designated PsKP, for Pyrococcus sp. strain ST04 kojibiose phosphorylase) and β-phosphoglucomutase (PsPGM), respectively. Enzymatic assays show that PsKP hydrolyzes kojibiose to glucose and β-glucose-1-phosphate (β-G1P). The Km values for kojibiose and phosphate were determined to be 2.53 {\textpm} 0.21 mM and 1.34 {\textpm} 0.04 mM, respectively. PsPGM then converts β-G1P into G6P in the presence of 6 mM MgCl2. Conversion activity from β-G1P to G6P was 46.81 {\textpm} 3.66 U/mg, and reverse conversion activity from G6P to β-G1P was 3.51 {\textpm} 0.13 U/mg. The proteins are highly thermostable, with optimal temperatures of 90{\textdegree}C for PsKP and 95{\textdegree}C for PsPGM. These results indicate that Pyrococcus sp. strain ST04 converts kojibiose into G6P, a substrate of the glycolytic pathway. This is the first report of a disaccharide utilization pathway via phosphorolysis in hyperthermophilic archaea.

}, issn = {1098-5530}, doi = {10.1128/JB.01222-13}, author = {Jung, Jong-Hyun and Seo, Dong-Ho and Holden, James F and Park, Cheon-Seok} } @article {1203, title = {Maltose-forming α-amylase from the hyperthermophilic archaeon Pyrococcus sp. ST04.}, journal = {Appl Microbiol Biotechnol}, volume = {98}, year = {2014}, month = {2014 Mar}, pages = {2121-31}, abstract = {

The deduced amino acid sequence from a gene of the hyperthermophilic archaeon Pyrococcus sp. ST04 (Py04_0872) contained a conserved glycoside hydrolase family 57 (GH57) motif, but showed <13~\% sequence identity with other known Pyrococcus GH57 enzymes, such as 4-α-glucanotransferase (EC 2.4.1.25), amylopullulanase (EC 3.2.1.41), and branching enzyme (EC 2.4.1.18). This gene was cloned and expressed in Escherichia coli, and the recombinant product (P yrococcus sp. ST04 maltose-forming α-amylase, PSMA) was a novel 70-kDa maltose-forming α-amylase. PSMA only recognized maltose (G2) units with α-1,4 and α-1,6 linkages in polysaccharides (e.g., starch, amylopectin, and glycogen) and hydrolyzed pullulan very poorly. G2 was the primary end product of hydrolysis. Branched cyclodextrin (CD) was only hydrolyzed along its branched maltooligosaccharides. 6-O-glucosyl-β-cyclodextrin (G1-β-CD) and β-cyclodextrin (β-CD) were resistant to PSMA suggesting that PSMA is an exo-type glucan hydrolase with α-1,4- and α-1,6-glucan hydrolytic activities. The half-saturation value (K m) for the α-1,4 linkage of maltotriose (G3) was 8.4~mM while that of the α-1,6 linkage of 6-O-maltosyl-β-cyclodextrin (G2-β-CD) was 0.3~mM. The k cat values were 381.0~min(-1) for G3 and 1,545.0~min(-1) for G2-β-CD. The enzyme was inhibited competitively by the reaction product G2, and the K i constant was 0.7~mM. PSMA bridges the gap between amylases that hydrolyze larger maltodextrins and α-glucosidase that feeds G2 into glycolysis by hydrolyzing smaller glucans into G2 units.

}, issn = {1432-0614}, doi = {10.1007/s00253-013-5068-6}, author = {Jung, Jong-Hyun and Seo, Dong-Ho and Holden, James F and Park, Cheon-Seok} } @article {1204, title = {Molecular cloning and enzymatic characterization of cyclomaltodextrinase from hyperthermophilic archaeon Thermococcus sp. CL1.}, journal = {J Microbiol Biotechnol}, volume = {23}, year = {2013}, month = {2013 Aug}, pages = {1060-9}, abstract = {

Genome organization near cyclomaltodextrinases (CDases) was analyzed and compared for four different hyperthermophilic archaea: Thermococcus, Pyrococcus, Staphylothermus, and Thermofilum. A gene (CL1_0884) encoding a putative CDase from Thermococcus sp. CL1 (tccd) was cloned and expressed in Escherichia coli. TcCD was confirmed to be highly thermostable, with optimal activity at 85{\textcelsius}. The melting temperature of TcCD was determined to be 93oC by both differential scanning calorimetry and differential scanning fluorimetry. A size-exclusion chromatography experiment showed that TcCD exists as a monomer. TcCD preferentially hydrolyzed α-cyclodextrin (α-CD), and at the initial stage catalyzed a ring-opening reaction by cleaving one α-1,4-glycosidic linkage of the CD ring to produce the corresponding single maltooligosaccharide. Furthermore, TcCD could hydrolyze branched CDs (G1-α-CD, G1-β- CD, and G2-β-CD) to yield significant amounts (45\%, 40\%, and 46\%) of isomaltooligosaccharides (panose and 6(2)-α-maltosylmaltose) in addition to glucose and maltose. This enzyme is one of the most thermostable maltogenic amylases reported, and might be of potential value in the production of isomaltooligosaccharides in the food industry.

}, issn = {1738-8872}, author = {Lee, Jae-Eun and Kim, In-Hwan and Jung, Jong-Hyun and Seo, Dong-Ho and Kang, Sung-Gyun and Holden, James F and Cha, Jaeho and Park, Cheon-Seok} } @article {1207, title = {Complete genome sequence of the hyperthermophilic archaeon Pyrococcus sp. strain ST04, isolated from a deep-sea hydrothermal sulfide chimney on the Juan de Fuca Ridge.}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 Aug}, pages = {4434-5}, abstract = {

Pyrococcus sp. strain ST04 is a hyperthermophilic, anaerobic, and heterotrophic archaeon isolated from a deep-sea hydrothermal sulfide chimney on the Endeavour Segment of the Juan de Fuca Ridge in the northeastern Pacific Ocean. To further understand the distinct characteristics of this archaeon at the genome level (polysaccharide utilization at high temperature and ATP generation by a Na(+) gradient), the genome of strain ST04 was completely sequenced and analyzed. Here, we present the complete genome sequence analysis results of Pyrococcus sp. ST04 and report the major findings from the genome annotation, with a focus on its saccharolytic and metabolite production potential.

}, keywords = {Adenosine Triphosphate, Anaerobiosis, DNA, Archaeal, Genome, Archaeal, Heterotrophic Processes, Hydrothermal Vents, Molecular Sequence Data, Pacific Ocean, Polysaccharides, Pyrococcus, Seawater, Sequence Analysis, DNA, Sodium Chloride, Sulfides}, issn = {1098-5530}, doi = {10.1128/JB.00824-12}, author = {Jung, Jong-Hyun and Lee, Ju-Hoon and Holden, James F and Seo, Dong-Ho and Shin, Hakdong and Kim, Hae-Yeong and Kim, Wooki and Ryu, Sangryeol and Park, Cheon-Seok} } @article {1205, title = {Complete genome sequence of the hyperthermophilic archaeon Thermococcus sp. strain CL1, isolated from a Paralvinella sp. polychaete worm collected from a hydrothermal vent.}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 Sep}, pages = {4769-70}, abstract = {

Thermococcus sp. strain CL1 is a hyperthermophilic, anaerobic, and heterotrophic archaeon isolated from a Paralvinella sp. polychaete worm living on an active deep-sea hydrothermal sulfide chimney on the Cleft Segment of the Juan de Fuca Ridge. To further understand the distinct characteristics of this archaeon at the genome level, its genome was completely sequenced and analyzed. Here, we announce the complete genome sequence (1,950,313 bp) of Thermococcus sp. strain CL1, with a focus on H(2)- and energy-producing capabilities and its amino acid biosynthesis and acquisition in an extreme habitat.

}, keywords = {Animals, Base Sequence, Chromosome Mapping, DNA, Archaeal, DNA, Ribosomal, Genome, Bacterial, Hydrothermal Vents, Molecular Sequence Data, Phylogeny, Polychaeta, Sequence Analysis, DNA, Thermococcus}, issn = {1098-5530}, doi = {10.1128/JB.01016-12}, author = {Jung, Jong-Hyun and Holden, James F and Seo, Dong-Ho and Park, Kwan-Hwa and Shin, Hakdong and Ryu, Sangryeol and Lee, Ju-Hoon and Park, Cheon-Seok} } @article {1208, title = {Production of hydrogen from α-1,4- and β-1,4-linked saccharides by marine hyperthermophilic Archaea.}, journal = {Appl Environ Microbiol}, volume = {77}, year = {2011}, month = {2011 May}, pages = {3169-73}, abstract = {

Nineteen hyperthermophilic heterotrophs from deep-sea hydrothermal vents, plus the control organism Pyrococcus furiosus, were examined for their ability to grow and produce H$_{2}$ on maltose, cellobiose, and peptides and for the presence of the genes encoding proteins that hydrolyze starch and cellulose. All of the strains grew on these disaccharides and peptides and converted maltose and peptides to H$_{2}$ even when elemental sulfur was present as a terminal electron acceptor. Half of the strains had at least one gene for an extracellular starch hydrolase, but only P. furiosus had a gene for an extracellular β-1,4-endoglucanase. P. furiosus was serially adapted for growth on CF11 cellulose and H$_{2}$ production, which is the first reported instance of hyperthermophilic growth on cellulose, with a doubling time of 64 min. Cell-specific H$_{2}$ production rates were 29 fmol, 37 fmol, and 54 fmol of H$_{2}$ produced cell$^{-}${\textonesuperior} doubling$^{-}${\textonesuperior} on α-1,4-linked sugars, β-1,4-linked sugars, and peptides, respectively. The highest total community H$_{2}$ production rate came from growth on starch (2.6 mM H$_{2}$ produced h$^{-}${\textonesuperior}). Hyperthermophilic heterotrophs may serve as an important alternate source of H$_{2}$ for hydrogenotrophic microorganisms in low-H$_{2}$ hydrothermal environments, and some are candidates for H$_{2}$ bioenergy production in bioreactors.

}, keywords = {Archaea, Carbohydrate Metabolism, Hot Springs, Hydro-Lyases, Hydrogen, Peptides, Seawater}, issn = {1098-5336}, doi = {10.1128/AEM.01366-10}, author = {Oslowski, Daniel M and Jung, Jong-Hyun and Seo, Dong-Ho and Park, Cheon-Seok and Holden, James F} }