• 한국미생물·생명공학회지
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• 제13권1호
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• pp.19-25
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• 1985

Saccharomyces cerevisiae HIS 5 유전자는 histidinol phosphate aminotransferase (EC: 2, 6, 1,9)를 code하는 아미노산 합성유전자이다. 이 유전자는 plasmid pSH 530에 cloning되어 E. coli와 Saccharomyces cerevisiae 숙주에서 promoter로서 전사하였다. HIS 5 유전자의 총염기 수는 736개이였고 5' 상류영역에는 긴 reading frame, directed repeat, 전사개시점, 그리고 Pribnow box염기배열이 있었다. 특히 HIS 5 유전자의 ATG 주변 염기배열은 -A-A-A-T-T-A-C-A-C-T-A-T-G-G-T-T-T-T-T-G-A-T-였으며 C block은 없었다.

• 생명과학회지
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• 제22권3호
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• pp.417-427
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• 2012

대장균 야생주와 프로판올 내성 변이주에서 프로판올 스트레스에 의해 발현이 크게 변화하는 유전자를 DNA microarray 기술을 이용하여 비교 분석하였다. 프로판올 첨가한 야생주와 무첨가한 야생주 사이의 RNA 발현 연관값은 0.949이며, 50개의 유전자 발현이 2배 이상 변화하였다. 프로판올을 첨가한 내성변이주와 무첨가한 변이주 사이의 연관값은 0.952이며, 71개의 유전자 발현이 크게 변화하였다. 그러나, 야생주와 변이주 사이의 연관값은 프로판올 무첨가한 조건과 첨가한 조건에서 각각 0.992 및 0.974로 매우 높았으며, 2배 이상의 발현차이를 나타내는 유전자는 각각 1개 및 2개로, 두 균주는 매우 유사한 발현양상을 보였다. 야생주 또는 변이주에서 프로판올 스트레스에 반응하는 대표적인 유전자들의 특징은 많은 열충격 반응에 관여하는 유전자들의 발현이 크게 증가하였으며, 리보소옴 합성에 필요한 많은 유전자들의 발현이 감소하였다. 또한, 전사조절인자들인 ArcA, CRP, FNR, H-NS, GatR, PurR에 의해 조절받는 유전자들의 발현이 크게 변화하였으며, 시그마인자들 중에서는 RpoH에 의해서 발현되는 유전자들의 발현이 크게 증가하였다. rpoH가 정상적으로 발현되지 못하는 변이주와 야생주를 이용한 프로판올 내성정도를 측정한 결과, RpoH는 대장균에서 프로판올 스트레스에 적응하는데 중요한 기능을 하는 것으로 확인되었다.

• Journal of Microbiology and Biotechnology
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• 제15권2호
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• pp.330-336
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• 2005

Lactococcus lactis A164 is a nisin Z-producing strain isolated from kimchi. Its antimicrobial spectrum has been found to be active against most Gram-positive bacteria tested, yet inactive against Gram-negative bacteria [3]. Accordingly, to overcome this drawback, the current study attempted to express human $\beta$-defensin-l (hBD-l), which kills both Gram-positive and Gram-negative bacteria in L. lactis AI64. When the hBD-l cDNA was introduced using a nisin Z-controlled expression cassette, the L. lactis A164 transformants grew very poorly, due to the bactericidal effect of the expressed hBD-l against the transformants. Therefore, a gene fusion system was designed to reduce the toxicity of the expressed heterologous protein against the host cells. As such, the hBD-l gene was fused to the DsbC- Tag of pET -40b(+), then introduced to L. lactis A 164. The transformants expressed an intracellular 35.6-kDa DsbC-hBD-l fusion protein that exhibited slight activity against the host cells, yet not enough to strongly inhibit the cell growth. To obtain the recombinant hBD-l, the DsbC-hBD-l fusion protein was purified by nickel-affinity column chromatography, and the DsbC-Tag removed by cleaving with enterokinase. The cleaved mature hBD-l exhibited strong bactericidal activity against E. coli JM109, indicating that the recombinant L. lactis A 164 produced a biologically active hBD-I. In addition, the recombinant L. lactis A 164 was also found to produce the same level of nisin Z as the wild-type.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.257-264
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• 2009

A $\beta$-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli $DH5{\alpha}$ as a host. The purified rAgaB34 was a $\beta$-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at $30^{\circ}C$ and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to $50^{\circ}C$. Its specific activity and $k_{cat}/K_m$ value for agarose were 242 U/mg and $1.7{\times}10^6/sM$, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT, $\beta$-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.

• Journal of Microbiology and Biotechnology
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• 제18권8호
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• pp.1386-1392
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• 2008

A gene (sll0158) putatively encoding a glycogen branching enzyme (GBE, E.C. 2.4.1.18) was cloned from Synechocystis sp. PCC6803, and the recombinant protein expressed and characterized. The PCR-amplified putative GBE gene was ligated into a pET-21a plasmid vector harboring a T7 promoter, and the recombinant DNA transformed into a host cell, E. coli BL21(DE3). The IPTG-induced enzymes were then extracted and purified using Ni-NTA affinity chromatography. The putative GBE gene was found to be composed of 2,310 nucleotides and encoded 770 amino acids, corresponding to approx. 90.7 kDa, as confirmed by SDS-PAGE and MALDI-TOF-MS analyses. The optimal conditions for GBE activity were investigated by measuring the absorbance change in iodine affinity, and shown to be pH 8.0 and $30^{\circ}C$ in a 50 mM glycine-NaOH buffer. The action pattern of the GBE on amylose, an $\alpha$-(1,4)-linked linear glucan, was analyzed using high-performance anion-exchange chromatography (HPAEC) after isoamylolysis. As a result, the GBE displayed $\alpha$-glucosyl transferring activity by cleaving the $\alpha$-(1,4)-linkages and transferring the cleaved maltoglycosyl moiety to form new $\alpha$-(1,6)-branch linkages. A time-course study of the GBE reaction was carried out with biosynthetic amylose (BSAM; $M_p{\cong}$8,000), and the changes in the branch-chain length distribution were evaluated. When increasing the reaction time up to 48 h, the weight- and number-average DP ($DP_w$ and $DP_n$) decreased from 19.6 to 8.7 and from 17.6 to 7.8, respectively. The molecular size ($M_p$, peak $M_w{\cong}2.45-2.75{\times}10^5$) of the GBE-reacted product from BSAM reached the size of amylose (AM) in botanical starch, yet the product was highly soluble and stable in water, unlike AM molecules. Thus, GBE-generated products can provide new food and non-food applications, owing to their unique physical properties.