• 농업과학연구
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• 제41권3호
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• pp.245-249
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• 2014

Among dozens of DNA polymerases cloned from thermophilic bacteria, Taq DNA polymerase from Thermus aquaticus has been most frequently used in polymerase chain reaction (PCR) that is being applied to gene cloning, DNA sequencing, gene expression analysis, and detection of infectious and genetic diseases. Since native Taq DNA polymerase is expressed at low level in T. aquaticus, recombinant Escherichia coli system was used to produce Taq DNA polymerase in a large amount. Taq DNA polymerase was expressed as a soluble form under the control of tac promoter in E. coli, and purified by heat treatment and ion exchange chromatographies. The purified Taq DNA polymerase was nearly homogeneous and exhibited a similar DNA amplification activity with a commercial Taq DNA polymerase.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1201-1205
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• 2009

Corn starches with different amylose contents were enzymatically modified using Thermus aquaticus 4-$\alpha$-glucanotransferase ($TA{\alpha}GTase$). Upon the enzyme treatment, the chain-length distributions of isoamylolytically debranched products became broader [degree of polymerization (DP): 3-40] than those of untreated corn starches. In addition, a variety of cycloamyloses (CAs) with different sizes were formed by the glucanotransfer activity of $TA{\alpha}GTase$. CAs with DP 5-40 were detectable in all of the $TA{\alpha}GTase$-treated corn starches. From the results of high-performance anion-exchange chromatography and high-performance size-exclusion chromatography analyses, it was suggested that the amount of CAs produced by the enzyme treatment increased as the amylose content of the starches increased. Thus, we concluded that the extent of modification of starch molecules was enhanced in proportion to amylose content by the transfer activity of $TA{\alpha}GTase$. This finding could be useful for developing an efficient process of CA production using this enzyme.

• Applied Biological Chemistry
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• 제31권3호
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• pp.274-283
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• 1988

Aqualysin I is an alkaline serine protease which is secretet into the culture medium by Thermus aquaticus YT-1, an extreme thermophile. Aqualysin I was purified, and its partial amino acid sequence was determined. The gene encoding aqualysin I was cloned into E. coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequenc, agreed with the determid amino acid sequences, including the $NH_2-$ and COOH terminal sequence of the tryptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin type serine protease, and 43% identity with proteinase K, 37-30% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active-site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'. Aqualysin I contains two disulfide bonds, Cys67-Cys99 and Cys163-Cys194, and these disulfide bonds seem to contribute to the heat stability of the enzyme. The determined positions of the twe disulfide bonds of aqualysin I agreed with those predicted previously on the basis of computer graphics of the crystallographic data for subtilisin BPN'. Therefore, these findings sugests that the three-dimensional structure of aqualysin I is similar to that of subtilisin BPN' Aqualysin I is produced as a lage precursor, which contains $NH_2-$ and COOH- terminal portions besides the mature protease sequence.

• Food Science and Biotechnology
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• 제17권4호
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• pp.705-712
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• 2008

Sweet potato starch was modified using Thermus aquaticus $\alpha$-1,4-glucanotransferase ($Ta{\alpha}GT$), and its structural and rheological properties were investigated. $Ta{\alpha}GT$-modified starch had a lower amylose level and molecular weight than raw starch. The chain length distribution showed an increased number of short and long branched chains and the formation of cycloamyloses. Compared with raw starch, $Ta{\alpha}GT$-modified starch displayed a lower gelatinization enthalpy and a wider melting temperature range. The X-ray diffraction of $Ta{\alpha}GT$-modified starch was a weak V-type pattern with distinct sharp peaks at 13 and $20^{\circ}$. Scanning electron micrographs of modified starch exhibited big holes on the surface and the loss of granular structure. The frequency sweep measurement revealed that the gel of $Ta{\alpha}GT$-modified starch was more rigid than raw starch gel. However, the structure of modified starch gel was destroyed by heating at $75^{\circ}C$, and a firm gel was re-formed by subsequent storage at $5^{\circ}C$, indicating thermoreversible property.

• 한국미생물·생명공학회지
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• 제37권1호
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• pp.17-23
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• 2009

PCR법을 이용하여 Thermus thermophilus HJ6 유래 DNA polymerase(Tod) 유전자를 클로닝하고 염기서열을 분석한 결과, ORF는 2,505개의 뉴클레오타이드로 구성되고 834개의 아미노산을 암호화하였다. 아마노산 서열을 바탕으로 상동성을 분석한 결과, Thermus thermophilus HB8 유래 DNA polymerase와 98%, Thermus aquaticus 유래 DNA polymorase와 86%의 identity를 나타내었다. 이 유전자를 박테리오파지 $\lambda$ 유래 온도감수성 프로모터(PR, PL)를 포함하는 pJLA503 벡터를 이용하여 대장균에서 발현하였다. 발현된 효소는 열처리, $HiTrap^{TM}$ Q column과 $HiPrep^{TM}$ Sephacryl S-200 HR 26/60 columun으로 정제하여 94 kDa의 단백질을 얻을 수 있었다. 정제된 효소의 DNA 중합 활성에 대한 최적온도는 $75{\sim}80^{\circ}C$이고 최적 pH가 9.0이었다. $Mg^{2+}$ and $Mn^{2+}$에 대한 최적 농도는 각각 2.5mM과 1mM이었고 효소활성은 2가 양이온의 존재 하에서는 활성화 되지만 1가양이온에서는 저 해되었다. Tod DNA 중합효소를 이용한 PCR 실험결과, Tod DNA 중합효소는 DNA 증폭 및 PCR 관련 기술에 응용 가능할 것으로 생각된다.

• BMB Reports
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• 제30권4호
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• pp.262-268
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• 1997

The thermophilic and thermostable alkaline phosphatase was purified to near homogeneity from the osmotic lysis of Thermus caldophilus GK24, The purified enzyme had an apparent molecular mass of 108, 000 Da and consisted of two subunits of 54,000 Da. lsoelectric-focusing analysis of the purified enzyme showed a pi of 7.3. The enzyme contained two Cys residues, and its amino acids composition was quite different from that of Thermus aquaticus YT-1 alkaline phosphatase and Escherichia coli alkaline phosphatase, The optimum pH and temperature of the enzyme were 11.0-11.5 and $80^{\circ}C$ respectively. The enzyme was stable in the pH range of 9.0-12.0 at $25^{\circ}C$ for 36 h. and the half-life at $80^{\circ}C$ (pH 11.0) was 6 h. The enzyme was activated by $MgCl_2$ and inhibited by EDTA. With ${\rho}-nitrophenyl\;phosphate\;({\rho}NPP)$ as the substrate, the enzyme had a Michaelis constant $(K_m) $of $3.6{\times}10^{-5}M$, The enzyme preferentially hydrolyzed the phosphomonoester bond of AMP in ribonucleotides and glycerophosphate.

• Journal of Microbiology and Biotechnology
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• 제9권4호
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• pp.381-385
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• 1999

Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in a polymerase chain reaction. Taq DNA polymerase has a domain at the amino terminus (residues 1 to 290) that has 5'-3' exonuclease activity and a domain at the C-terminus that catalyzes the polymerase reaction. Taq DNA polymerase is classified into the Pol I family, which is represented by E. coli DNA polymerase I. The alignment of amino acid sequences for the 5'-3' exonuclease domains of the Pol I family DNA polymerases shows ten highly conserved carboxylic amino acids. Crystallographic studies suggested that six of the carboxylic amino acids are clustered within a 7 $\AA$ radius by chelating three metal ions in the active site. Those six carboxylic residues are mutagenized to alanines in order to better understand their function. All six carboxylic residues, Asp l8, Glu1l7, Asp1l9, Asp120, Asp142, and Aspl44, are crucial for catalysis of 5'-3' exonuclease.

• Journal of Microbiology and Biotechnology
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• 제8권5호
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• pp.471-477
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• 1998

Taq DNA polymerase from Thermus aquaticus is very useful in the polymerase chain reaction. Taq DNA polymerase is classified in the pol I family, represented by E. coli DNA polymerase I. The three-dimensional structural alignment of 3'-5'exonuclease domains from the pol I family DNA polymerases explains why Taq DNA polymerase does not carry out proofreading in polymerase chain reactions. Three sequence motifs, Exo I, II, and III, must exist to carry out 3'-5'exonuclease activity for proof- reading by a 3'-5'exonuclease reaction, but these are abolished in Taq DNA polymerase. The key catalytic module in 3'-5'exonuclease is two metal ions chelated by four active-site carboxylic amino acids. Taq DNA polymerase was mutagenized to construct the catalytic module in the active site. The circular dichroism technique supported the formation of the catalytic module, and the radioactive assay showed that the 3'-5'exonuclease activity doubled in the mutant Taq DNA polymerase.

• BMB Reports
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• 제29권1호
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• pp.38-44
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• 1996

Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in the polymerase chain reaction method, which is being used for amplifying DNA. Not only does Taq DNA polymerase have high commercial value commercial value for the polymerase chain reaction application, but it is also important in studying DNA replication, because it is apparently an homologue to E. coli DNA polymerase I, which has long been used for DNA replication study (Lawyer et ai., 1993). The crystal structure determination of Taq DNA polymerase was initiated. An X-ray diffraction pattern breaks down a crystal structure into discrete sine waves in a Fourier series. The original shape of a crystal object in terms of electron density may be represented as the sum of those sine waves with varying amplitudes and phases in three dimensions. The molecular replacement method was initially employed to provide phase information for the structure of Taq DNA polymerase. The rotation search using the program MERLOT resulted in a solution peak with 5.4 r.m.s. PC-refinement of the X-PLOR program verified the result and also optimized the orientation angles. Next, the translation search using the X-PLOR program resulted in a unique solution peak with 7.35 r.m.s. In addition, the translation search indicated $P3_121$ to be the true space group out of two possible ones. The phase information from the molecular replacement was useful in the MIR phasing experiment.