• Journal of Microbiology and Biotechnology
• /
• v.16 no.12
• /
• pp.2000-2003
• /
• 2006

A gene (GenBank AF096282) coding for a $\alpha$-glucosidase (TcaAG, EC 3.2.1.20) from Thermus caldophilus GK24 was expressed in Saccharomyces cerevisiae, a generally recognized as safe (GRAS) host. The thermostable $\alpha$-glucosidase was produced inside of the GRAS host at 0.04 unit/mg-dry cell by the constitutively expressing ADH1 promoter and at 1.2 unit/mg-dry cell by the inductively expressing GALl0 promoter, respectively. No $\alpha$-glucosidase activities were found in the medium when the MF-alpha signal sequence from S. cerevisiae or $\alpha$-amylase signal sequence from Aspergillus oryzae were fused before the $\alpha$-glucosidase gene for the secretion.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.2
• /
• pp.272-279
• /
• 2006

The gene encoding Thermus sp. T351 alkaline phosphatase (T351 APase) was cloned and sequenced. The gene consisted of 1,503 bp coding for a protein with 500 amino acid residues including a signal peptide. The deduced amino acid sequence of T351 APase showed relatively low similarity to other Thermus APases. The T351 APase gene was expressed under the control of the T7lac promoter on the expression vector pET-22b(+) in Escherichia coli BL21 (DE3). The expressed enzyme was purified by heat treatment, and $UNO^{TM}$ Q and $HiTrap^{TM}$ Heparin HP column chromatographies. The purified enzyme exhibited high activity at extremely alkaline pHs, reaching a maximum at pH 12.0. The optimum temperature of the enzyme was $80^{\circ}C$, and the half-life at $85^{\circ}C$ was approximately 103 min. The enzyme activity was found to be dependent on metal ions: the addition of $Mg^{2+}$ and $CO^{2+}$ increased the activity, whereas EDTA inhibited it. With p-nitrophenyl phosphate as the substrate, T351 APase had a Michaelis constant ($K_{m}$) of $3.9{\times}10^{-5}M$. The enzyme catalyzed the hydrolysis of a wide variety of phosphorylated compounds.

• Microbiology and Biotechnology Letters
• /
• v.25 no.3
• /
• pp.298-304
• /
• 1997

Thermus caldophilus GK24 was selected as sources of thermostable $\beta$-galactosidase from a survey of genus Thermus. T. caldophilus GK24 (Tca) $\beta$-galactosidase was found to be inducible. The enzyme was optimally active at 75$\circ$C. Enzyme induction was achieved by addition of lactose, galactose and cellobiose to basal media. The addition of glucose to culture media had a repressive effect on further enzyme synthesis. T caldophilus GK24 was tested for production of $\beta$-galactosidase by addition of various concentration of lactose, galactose and cellobiose to standard media. Cellobiose was found to be effective for the $\beta$-galactosidase induction. The optimal induction medium for production of $\beta$-galactosidase was composed of 0.2% cellobiose, 0.3% bactotryptone, 0.3% yeast extract, basal salts and Tris/HCI(pH 7.8). The activity of the enzyme in the optimal induction medium increased nearly 16.5-fold compared to the standard medium. Tca $\beta$-galactosidase was detected when cell extracts was subjected to electrophoresis in a nondenaturing polyacryamide gel and stained for activity with 6-bromo-2-naphtyl-$\beta$-D-galactopyranoside(BNG).

• Journal of Microbiology and Biotechnology
• /
• v.13 no.1
• /
• pp.139-145
• /
• 2003

The gene encoding Thermus caldophilus GK24 polyphosphate kinase (Tca PPK) was cloned and sequenced. The gene contains an open reading frame encoding 608 amino acids with a calculated molecular mass of 69,850 Da. The deduced amino acid sequence of Tca PPK showed a 40% homology to Escherichia coli PPK, and $39\%$ to Klebsiella aerogenes PPK. The Tca ppk gene was expressed under the control of the T7lac promoter on pET-22b(+) in E. coli and its enzyme was purified about 70-fold with $36\%$ yield, following heating and HiTrap chelating HP column chromatography. The native enzyme was found to have an approximate molecular mass of 580,000 Da and consisted of eight subunits. The optimum pH and temperature of the enzyme were 5.5 and $70^{\circ}C$, respectively. A divalent cation was required for the enzyme activity, with $Mg^2+$ being the most effective.

• Journal of Microbiology and Biotechnology
• /
• v.13 no.4
• /
• pp.628-631
• /
• 2003

The recombinant alanine dehydrogenase (ADH) from E. coli containing Thermus caldophilus ADH was purified to homogeneity from a cell-free extract. The enzyme was purified 38-fold with a yield of 68% from the starting cell-free extract. The purified enzyme gave a single band in polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 45 kDa. The pH optimum was 8.0 for reductive amination of pyruvate and 12.0 for oxidative deamination of L-alanine. The enzyme was stable up to $70^{\circ}C$. The activity of the enzyme was inhibited by 1 mM $Zn^{2+}$, 20% hexane, and 20% $CHCl_3$. However, 10 mM $Mg^{2+}$ and 40% propanol had no effect on the enzyme activity. The Michaelis constants ($K_m$) for the substrates were $50\;\mu\textrm{M}$ for NADH, 0.2 mM for pyruvate, 39.4 mM for $NH_4+$, 2.6 mM for L-alanine, and 1.8 mM for $NAD^+$.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.8
• /
• pp.1401-1407
• /
• 2008

The roles of conserved amino acid residues (Va1329-Ala330-Asn331-Glu332), constituting an extra sugar-binding space (ESBS) of Thermus maltogenic amylase (ThMA), were investigated by combinatorial saturation mutagenesis. Various ThMA mutants were firstly screened on the basis of starch hydrolyzing activity and their enzymatic properties were characterized in detail. Most of the ThMA variants showed remarkable decreases in their hydrolyzing activity, but their specificity against various substrates could be altered by mutagenesis. Unexpectedly, mutant H-16 (Gly-Leu-Val-Tyr) showed almost identical hydrolyzing and transglycosylation activities to wild type, whereas K-33 (Ser-Gly-Asp-Glu) showed an extremely low transglycosylation activity. Interestingly, K-33 produced glucose, maltose, and acarviosine from acarbose, whereas ThMA hydrolyzed acarbose to only glucose and acarviosine-glucose. These results propose that the substrate specificity, hydrolysis pattern, and transglycosylation activity of ThMA can be modulated by combinatorial mutations near the ESBS.

• BMB Reports
• /
• v.34 no.4
• /
• pp.347-354
• /
• 2001

$\alpha$-Glucosidase of an extreme thermophile, Thermus caldophilus GK24 (TcaAG), was purified 80-fold from cells to a homogeneous state and characterized. The enzyme exhibited optimum activity at pH 6.5 and $90^{\circ}C$, and was stable from pH 6.0 to 85 and up to $90^{\circ}C$. The enzyme had a half-life of 85 minutes at $90^{\circ}C$. An analysis of the substrate specificity showed that the enzyme hydrolyzed the non-reducing terminal unit of $\alpha$-1,6-glucosidic linkages of isomaltosaccharides and panose, $\alpha$-1,3-glycosidic bond of nigerose and turanose, and $\alpha$-1,2-glycosidic bond of sucrose. The gene encoding the TcaAG was cloned, sequenced, and sequenced in E. coli. The nucleotide sequence of the gene encoded a 530 amino acid polypeptide and had a G+C content of 68.4% with a strong bias for G or C in the third position of the codons (93.6%). A sequence analysis revealed that TcaAG belonged to the $\alpha$-amylase family. We suggest that this monomeric, thermostable, and broad-acting $\alpha$-glucosidase is a departure from previously exhibited specificities. It is, therefore, a novel $\alpha$-glucosidase.

• BMB Reports
• /
• v.31 no.6
• /
• pp.600-603
• /
• 1998

A highly thermostable xylose isomerase from Thermus thermophilus has been expressed in Escherichia coli and crystallized. The purified enzyme shows its optimum temperature at $90^{\circ}C$. It has been crystallized at room temperature using polyethylene glycol 4000 as the precipitant. The crystal belongs to the orthorhombic space group $P2_12_12_1$, with unit cell parameters of a = 73.34 ${\AA}$, b = 144.05 ${\AA}$, c = 155.07 ${\AA}$. The presence of one molecule of tetrameric xylose isomerase in the asymmetric unit gives a crystal volume per protein mass ($V_m$) of 2.32 ${\AA}^3/Da$ and the solvent content of 47.0% by volume. The diffraction pattern extends to 1.9 ${\AA}$ Bragg spacing with synchrotron radiation and a set of native data has been collected to 2.3 ${\AA}$.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.3
• /
• pp.513-517
• /
• 2010

A thermostable trehalose synthase (TtTSase) from Thermus thermophilus HJ6 was immobilized on chitosan activated with glutaraldehyde. The yield of immobilization was evaluated as 39.68%. The optimum pH of the immobilized enzyme was similar to that of the free enzyme. However, the optimal temperature ranges were shifted by about $4^{\circ}C$ owing to better thermal stability after immobilization. The half-life of heat inactivation for free and immobilized enzymes was 5.7 and 6.3 days at $70^{\circ}C$, respectively, thus showing a lager thermostability of the immobilized enzyme. When tested in batch reaction, the immobilized enzyme retained its relative activity of 53% after 30 reuses of reaction within 12 days, and still retained 82% of its initial activity even after 150 days at $4^{\circ}C$. A packed-bed bioreactor with immobilized enzyme showed a maximum yield of 56% trehalose from 100 mM maltose in a continuous recycling system (bed volume: 10 ml) under conditions of pH 7.0 and $70^{\circ}C$.

• Journal of Microbiology
• /
• v.41 no.2
• /
• pp.152-156
• /
• 2003

Through enrichment on two nutrient agars, 57 Thermus isolates were recovered from 15 hot spring samples taken from the Rehai geothermal area, Tengchong, China. Unique growth characteristics were observed when the strains were transferred from YIM14 medium to Thermus medium. Phylogenetic analysis showed that the 16S rDNA sequences of the isolates and clones from the Rehai geothermal area farmed a monophyletic group on the phylogenetic tree. A secondary structure comparison showed that their 16S rRNAs have unique secondary structure characteristics.