• Asian-Australasian Journal of Animal Sciences
• /
• 제17권11호
• /
• pp.1591-1598
• /
• 2004

To isolate the $\beta$-galactosidase producing thermophilic bacteria, samples of mud and water were collected from hot springs of avolcanic area near Golden Springs in New Zealand. Among eleven isolated strains, the strain of KNOUC112 produced the highest amounts of $\beta$-galactosidase at 40 h incubation time (0.013 unit). This strain was aerobic, asporogenic bacilli, immobile, gram negative, catalase positive, oxidase positive, and pigment producing. Optimum growth was at 70-72$^{\circ}C$, pH 7.0-7.2, and it could grow in the presence of 3% NaCl. The main fatty acids of cell components were iso-15:0 (30.26%), and iso-17:0 (31.31%). Based on morphological and biochemical properties and fatty acid composition, the strain could be identified as genus Thermus, and finally as Thermus thermophilus by phylogenetic analysis based on 16S rRNA sequence. So the strain is designated as Thermus thermophilus KNOUC112. A gene from Thermus thermophilus KNOUC112 encoding $\beta$-galactosidase was amplified by PCR using redundancy primers prepared based on the structure of $\beta$-galactosidase gene of Thermus sp. A4 and Thermus sp. strain T2, cloned and expressed in E. coli JM109 DE3. The gene of Thermus thermophilus KNOUC112 $\beta$-galactosidase(KNOUC112$\beta$-gal) consisted of a 1,938 bp open reading frame, encoding a protein of 73 kDa that was composed of 645 amino acids. KNOUC112$\beta$-gal was expressed as dimer and trimer in E. coli JM109 (DE3) via pET-5b.

• 한국식품과학회지
• /
• 제29권2호
• /
• pp.376-378
• /
• 1997

갈락토올리고당의 함량을 증가시키기 위하여 Thermus caldophilus와 Bacillus sp. 유래의 두 종류 ${\beta}-galactosidase$를 유당에 순차적으로 반응시킨 결과 고형분 중 갈락토올리고당의 함량이 60% 이상까지 증가하였다. 먼저 내열성 효소로 고온에서 반응이 진행되므로 유당의 농도를 높일 수 있는 장점이 있다.

• Journal of Microbiology and Biotechnology
• /
• 제16권2호
• /
• 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.

• KSBB Journal
• /
• 제27권4호
• /
• pp.257-262
• /
• 2012

The gene encoding Thermus thermophilus HJ6 laccase (Tt-laccase) was cloned, sequenced, and comprised of 1,389 nucleotides encoding a protein (462 amino acids) with a predicted molecular mass of 51,049 Da. The deduced amino acid sequence of Tt-laccase showed 99.7% and 44.3% identities to the Thermus thermophilus HB27 laccase and Synechococcus sp. RS9917 laccase, respectively. Tt-laccase gene was expressed as a fusion protein with six histidine residues in E. coli Rosetta-gami (DE3) cells, and the recombinant protein was purified to homogeneity. UV-Vis spectrum analysis revealed that the enzyme has copper atoms, a type I Cu(II) and a type III binuclear Cu(II). The optimum pH for the oxidation of guaiacol was 5.0 and the optimum temperature was $90^{\circ}C$ The half-life of heat inactivation was about 120 min at $90^{\circ}C$ The enzyme reaction was inhibited by sodium azide, L-cystein, EDTA, dithiothreitol, tropolone, and kojic acid. The enzyme oxidized various known laccase substrates, its lowest $K_m$ value being for 4-hydroxyindole, highest $k_{cat}$ value for syringaldazine, and highest $k_{cat}/K_m$ for guaiacol.

• BMB Reports
• /
• 제34권4호
• /
• 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
• /
• 제45권12호
• /
• pp.748-753
• /
• 2012

A sequence of 10 amino acids at the C-terminus region of methylglyoxal synthase from Escherichia coli (EMGS) provides an arginine, which plays a crucial role in forming a salt bridge with a proximal aspartate residue in the neighboring subunit, consequently transferring the allosteric signal between subunits. In order to verify the role of arginine, the gene encoding MGS from a thermophile species, Thermus sp. GH5 (TMGS) lacking this arginine was cloned with an additional 30 bp sequence at the 3'-end and then expressed in form of a fusion TMGS with a 10 residual segment at the C-terminus ($TMGS^+$). The resulting recombinant enzyme showed a significant increase in cooperativity towards phosphate, reflected by a change in the Hill coefficient (nH) from 1.5 to 1.99. Experiments including site directed mutagenesis for Asp-10 in TMGS and $TMGS^+$, two dimentional structural survey, fluorescence and irreversible thermoinactivation were carried out to confirm this pathway.

• 한국미생물생명공학회:학술대회논문집
• /
• 한국미생물생명공학회 2001년도 Proceedings of 2001 International Symposium
• /
• pp.165-168
• /
• 2001

Regulation of pyrimidine nucleotide synthesis has been studied extensively in enteric bacteria and Bacillus species. Varieties of control modes have been proposed for regulation of pyrimidine nucleotide biosynthetic (pyr) genes. In Bacillus caldolyticus and B. subtilis, it has been proved that pyrimidine de novo biosynthetic operon is controlled by a regulatory protein PyrR-mediated attenuation. Another Gram-positive bacteria including Enterococcus faecalis, Lactobacillus plantarum, and wctococcus lactis have been found to constitute a pyr gene cluster containing the pyrR gene. In addition, it has been proposed that the structure of the 5' leader region of the Gram-negative extreme thermophile Thermus strain Z05 pyr operon provides a novel mechanism of PyrR-dependent coupled transcription-translation attenuation. Bacterial genome sequencing projects have identified the PyrR homologues in Haemophilus influenzae, Synechocystis sp., Mycobacterium tuberculosis, Streptococcus pneumoniae, S. pyogenes, and Clostridium acetobutylicum, which are currently investigating for their physiological functions.

• 한국미생물·생명공학회지
• /
• 제11권1호
• /
• pp.5-13
• /
• 1983

고온균의 생리적 특성자 내열기구에 관한 기초 자료를 얻고자 온천토양에서 $\beta$-galactosidase를 생산하는 고온균을 분리하여 동정하고 몇가지 주요한 생리적 특성을 검토하였으며 그의 균체 지방산 조성을 gas chromatography로 분석하여 다음과 같은 결과를 얻었다. 1. $\beta$-galactosidase를 생산하는 공시균주는 Thermus sp. 으로 동정되었다. 2. 분리선정한 균주의 최적 생육온도는 $65^{\circ}C$이었고, 37$^{\circ}C$에서 생육하지 않는 절대고온균 이었으며 최적pH는 6.5내외이었고 pH에 민감하였다. 3. NaCl에 대한 내성은 NaCl 1% 이상에서는 생육하지 못했다. 4. 항생물질에 대한 내성은 penicillin G는 10$\mu\textrm{g}$/$m\ell$, chloramphenicol은 0.5$\mu\textrm{g}$/$m\ell$ 이었다. 5. Vitamin 요구성은 Ca-pantothenate와 pyridoxine-HCI를 절대적 생육인자로 요구하였고niacin을 자극적 생육인자로 요구하였다. 6. 공시균주의 균체 지방산 조성은 palmitic acid 60.20%, lauric acid 11.80%, myristic acid 7.56%. behenic acid 4.25%, Capric acid 1.77%, stearic acid 2.13%, arachidic acid 1.53% 이었다.