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Gene Cloning and Expression of Trehalose Synthase from Thermus thermophilus HJ6 (Thermus thermophilus HJ6 유래 내열성 Trehalose Synthase의 유전자 클로닝 및 발현)

  • Kim, Hyun-Jung;Kim, Han-Woo;Jeon, Sung-Jong
    • Microbiology and Biotechnology Letters
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    • v.36 no.3
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    • pp.182-188
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    • 2008
  • A hyperthermophilic bacteria (strain HJ6) was isolated from a hot springs located in the Arima-cho, Hyogo, Japan. The cells were long-rod type ($2-4{\mu}m$), about $0.4{\mu}m$ in diameter. The pH and temperature for optimal growth were 6.5 and $80^{\circ}C$, respectively. Phylogenetic analysis based on the 16S rDNA sequence and biochemical studies indicated that HJ6 belonged to the genus Thermus thermophilus (Tt). The gene encoding the Trehalose synthase (TS) was cloned and sequenced. The open reading frame (ORF) of the TtTS gene was composed of 2,898 nucleotides and encoded a protein (975 amino acids) with a predicted molecular weight of 110.56 kDa. The deduced amino acid sequence of TtTS showed 99% and 83% identities to the Thermus caldophilus TS and Meiothermus ruber TS, respectively. TtTS gene was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for Trehalose synthase activity were found to be $80^{\circ}C$ and 7.5, respectively. The half-life of heat inactivation was about 40 min at $90^{\circ}C$. The maximum trehalose conversion rate of maltose into trehalose by the enzyme increased as the substrate concentration increased, and reached 55.7% at the maltose concentration of 500 mM, implying that the enzyme conversion was dependent of the substrate concentration.

Spectrofluorometric Characteristics of the N-Terminal Domain of Riboflavin Synthase (아미노-말단 리보플라빈 생성효소 단백질의 형광 특성)

  • Kim, Ryu-Ryun;Yi, Jeong-Hwan;Nam, Ki-Seok;Ko, Kyung-Won;Lee, Chan-Yong
    • Korean Journal of Microbiology
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    • v.47 no.1
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    • pp.14-21
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    • 2011
  • Riboflavin synthase catalyzes the formation of one molecule of each riboflavin and 5-amino-6-ribitylamino-2,4-pyrimidinedione by the transfer of a 4-carbon moiety between two molecules of the substrates, 6,7-dimetyl-8-ribityllumazine. The most remarkable feature is the sequence similarity between the N-terminal half (1-97) and the C-terminal half domain (99-213). To investigate the structure and fluorescent characteristics of the N-terminal half of riboflavin synthase (N-RS) in Escherichia coli, more than 10 mutant genes coding for the mutated N-terminal domain of riboflavin synthase were generated by polymerase chain reaction. The genes coding for the proteins were inserted into pQE vector designed for easy purification of protein by 6X-His tagging system, expressed, and the proteins were purified. Almost all mutated N-terminal domain of riboflavin synthases bind to 6,7-dimethyl-8-ribityllumazine and riboflavin as fluorescent ligands. However, N-RS C47D and N-RS ET66,67DQ mutant proteins show colorless, indicating that fluorescent ligands were dissociated during purification. In addition, most mutated proteins show low fluorescent intensity comparing to N-RS wild type, whereas N-RS C48S posses stronger fluorescent intensity than that of wild type protein. Based on this result, N-RS C48S can be used as the tool for high throughput screening system for searching for the compound with inhibitory effect for the riboflavin synthase.

Molecular Cloning of cDNA Encoding a Putative Eugenol Synthase in Tomato (Solanum lycopersicum 'Micro-Tom') and Prediction of 3D Structure and Physiochemical Properties (토마토 'Micro-Tom' 과실의 eugenol synthase 유전자 클로닝, 단백질의 3차 구조 및 생리화학적 특성 예측)

  • Kang, Seung-Won;Seo, Sang-Gyu;Lee, Tai-Ho;Lee, Gung-Pyo
    • Journal of agriculture & life science
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    • v.46 no.4
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    • pp.9-20
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    • 2012
  • Eugenol is a volatile compound synthesized by eugenol synthase in various plants and belongs to phenylpropene compounds. However, characteristics of eugenol synthase in tomato has not been known. Therefore, we cloned a full length cDNA of a putative eugenol synthase from tomato 'Micro-Tom' using rapid amplification of cDNA ends (RACE) technique and named a clone SlEGS. Open reading frame of SlEGS was 921bp long and its deduced amino acid sequence was 307bp. The BLAST analysis indicated that SlEGS shared high similarity with PhEGS1 (67.1%) and CbEGS2 (69.4%). Amino acid composition of SlEGS was determined by CLC genomics workbench tool and 3D structure of SlEGS was constructed by homology modeling using Swiss-PDB viewer and validated using PROCHECK and ProSA-web tool. In addition, the physiochemical properties of SlEGS was evaluated using ExPASy's ProtParam tool. Molecular weight was 33.93kDa and isoelectric point was 5.85 showing acidic nature. Other properties such as extinction coefficient, instability index, aliphatic index, and grand average hydropathy was also analyzed.

Cloning and Analysis of a Type II Polyketide Synthase Gene Cluster from Streptomyces toxytricini NRRL 15,443

  • Yoo An-Na;Demirev Atanas V.;Lee, Ji-Seon;Kim, Sang-Dal;Nam Doo-Hyun
    • Journal of Microbiology
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    • v.44 no.6
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    • pp.649-654
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    • 2006
  • A standard type II polyketide synthase (PKS) gene cluster was isolated while attempting to clone the biosynthetic gene for lipstatin from Streptomyces toxytricini NRRL 15,443. This result was observed using a Southern blot of a PstI-digested S. toxytricini chromosomal DNA library with a 444 bp amplified probe of a ketosynthase (KS) gene fragment. Four open reading frames [thioesterase (TE), $\beta$-ketoacyl systhase (KAS), chain length factor (CLF), and acyl carrier protein (ACP)], were identified through the nucleotide sequence determination and analysis of a 4.5 kb cloned DNA fragment. In order to confirm the involvement of a cloned gene in lipstatin biosynthesis, a gene disruption experiment for the KS gene was performed. However, the resulting gene disruptant did not show any significant difference in lipstatin production when compared to wild-type S. toxytricini. This result suggests that lipstatin may not be synthesized by a type II PKS.

Effects of Ginseng radix on Alcohol-induced Decrease in New Cell Formation and Nitric Oxide Synthase Expression in Dentate Gyrus of Rats (인삼이 흰쥐의 치상회에서 알코올에 의한 새로운 신경세포 생성 및 nitric oxide synthase 발현에 미치는 영향)

  • Min-Chul, Shin;Ee-Hwa, Kim;Youn-Hee, Kim
    • The Journal of Korean Medicine
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    • v.23 no.3
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    • pp.26-32
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    • 2002
  • 목적 : 본 연구에서는 알코올 독성에 대하여 흰쥐의 치상회에서 새로운 신경세포의 생성 및 nitric oxide synthase (NOS) 발현에 인삼이 미치는 영향을 5-bromo-2-deoxyuridine (BrdU) 면역 조직 화학법 및 nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) 조직화학법을 통해서 관찰하고자 한다. 방법 : 실험동물을 정상군, 인삼처치군, 알코올처치군 및 알코올-인삼 처치군으로 분류하여 각각의 실험군에 3일간 BrdU (50mg/kg)를 복강주사하였다. 인삼처치군은 30mg/kg 용량의 인삼 전탕액을 중완혈에 약침주사하였고, 알코올 처치군은 2 g/kg 용량의 알코올을 투여하였으며. 알코올-인삼 처치군은 2 g/kg 용량의 알코올 및 30mg/kg 용량의 인삼 전탕액을 투여한 후 각각의 BrdU 양성 세포수와 NADPH-d 양성세포수를 관찰하였다. 결과 : 알코올 투여군은 BrdU 양성세포 및 NADPH-d 양성세포 발현이 감소하였으나, 인삼 및 알코올 인삼처치군에서는 알코올 투여군에 비해서 모두 증가하였다. 결론 : 인삼은 알코올에 의해서 유발된 새로운 신경세포 생성의 감소에 대하여 보호효과가 있으며, 알코올에 의해서 부가적으로 영향 받는 산화질소는 세포생성 조절에 중요한 역할을 하는 것으로 사려된다.

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