• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.367-372
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• 2019

Deactivation of aminoglycosides by their modifying enzymes, including a number of aminoglycoside O-phosphotransferases, is the most ubiquitous resistance mechanism in aminoglycoside-resistant pathogens. Nonetheless, in a couple of biosynthetic pathways for gentamicins, fortimicins, and istamycins, phosphorylation of aminoglycosides seems to be a unique and initial step for the creation of a natural defensive structural feature such as a 3',4'-dideoxy scaffold. Our aim was to elucidate the biochemical details on the beginning of these C3',4'-dideoxygenation biosynthetic steps for aminoglycosides. The biosynthesis of istamycins must surely involve these 3',4'-didehydroxylation steps, but much less has been reported in terms of characterization of istamycin biosynthetic genes, especially about the phosphotransferase-encoding gene. In the disruption and complementation experiments pointing to a putative gene, istP, in the genome of wild-type Streptomyces tenjimariensis, the function of the istP gene was proved here to be a phosphotransferase. Next, an in-frame deletion of a known phosphotransferase-encoding gene forP from the genome of wild-type Micromonospora olivasterospora resulted in the appearance of a hitherto unidentified fortimicin shunt product, namely 3-O-methyl-FOR-KK1, whereas complementation of forP restored the natural fortimicin metabolite profiles. The bilateral complementation of an istP gene (or forP) in the ${\Delta}forP$ mutant (or ${\Delta}istP$ mutant strain) successfully restored the biosynthesis of 3',4'-dideoxy fortimicins and istamycins, thus clearly indicating that they are interchangeable launchers of the biosynthesis of 3',4'-dideoxy types of 1,4-diaminocyclitol antibiotics.

• 미생물학회지
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• 제41권1호
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• pp.8-12
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• 2005

S. chungbukensis DJ77의 genome project수행 결과 다당류 생 합성 에 관련된 유전자들의 염기서열을 찾아내었다. 본 논문에서는 이러한 유전자들 중 sphigan형 다당류 생합성에 관여하는 glucosyl-isoprenyl phosphate-transferase를 암호화 하는 유전자의 완전한 서열을 결정하였고, spsB로 명명하였다. 이 유전자는 ATG를 개시코돈으로 사용하며, TGA를 종결코돈으로 사용하고 있다. 또한 총 1392 bp의 open reading frame을 포함하며, 463개의 아미노산으로 구성되어있다. SpsB를 구성하는 아미노산 서열은 동일한 속의 sphingan 형성 균주인 Sphingomonas spp S88의 SpsB와 $50\%$, Sphingomonas paucimobilis ATCC 31461의 GelB와 $48\%$의 유사성을 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제28권5호
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• pp.776-783
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• 2018

The agarase gene gaa16a was identified from a draft genome sequence of Gilvimarinus agarilyticus JEA5, an agar-utilizing marine bacterium. Recently, three agarase-producing bacteria, G. chinensis, G. polysaccharolyticus, and G. agarilyticus, in the genus Gilvimarinus were reported. However, there have been no reports of the molecular characteristics and biochemical properties of these agarases. In this study, we analyzed the molecular characteristics and biochemical properties of agarases in Gilvimarinus. Gaa16A comprised a 1,323-bp open reading frame encoding 441 amino acids. The predicted molecular mass and isoelectric point were 49 kDa and 4.9, respectively. The amino acid sequence of Gaa16A showed features typical of glycosyl hydrolase family 16 (GH16) ${\beta}$-agarases, including a GH16 domain, carbohydrate-binding region (RICIN domain), and signal peptide. Recombinant Gaa16A (excluding the signal peptide and carbohydrate-binding region, rGaa16A) was expressed as a fused protein with maltose-binding protein at its N-terminus in Escherichia coli. rGaa16A had maximum activity at $55^{\circ}C$ and pH 7.0 and 103 U/mg of specific activity in the presence of 2.5 mM $CaCl_2$. The enzyme hydrolyzed agarose to yield neoagarotetraose as the main product. This enzyme may be useful for industrial production of functional neoagaro-oligosaccharides.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.331.3-332
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• 2002

A novel penicillin G acylase (PGA)-producing bacterial strain was isolated from soil by using the Serratia marcescens overlay technique. The isolated strain was identified as Leclercia adecarboxylata based on the analyses of the biochemical characteristics (API 20E). the cellular fatty acid profile. and the 16S rDNA sequences. The gene encoding the PGA (pac gene) was cloned into the pHSG399 vector and the recombinant E. coliHB101 clones harboring the pac gene were isolated on agar plates containing phenylacetyl-L -leucine and penicillin G. (omitted)

• 한국식품영양학회지
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• 제9권2호
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• pp.123-136
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• 1996

IPSKCDNA gene(1.8 kbp) encoding rat brain IP3K enzyme contained Not I restric site in open reading frame. The Not I sequence, GCGGCCGC, was converted to GCAGCCGC by site-directed mutagenesis. The mutated IP3KcDNA was digested with EcoR I and ligated with EcoR I-restricted psp72·Not2 vector. The resulting psp72 · Not2-IP3KCDNA was digested with the Not I restriction enzyme and then subcloned into the Not I -digested PZIP · NeoSV(X) mammalian expression vector. The PZIP · NeoSV(X) -IPSKCDNA was transfected into CCL39 hamster lung fibroblast cells. The efficiency of the expressed IPSKCDNA gene was significantly higher than expected generally, not only a mean 5-fold increase in the amount of enzyme, but also 16-fold increase in enzyme activity from tractsfected CCL39 cells by the method of Western blot using anti-lP3K antibodies. Both distribution of IPSK in various rat tissues and biochemical properties were discussed.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.334-337
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• 2008

A tightly regulated gene expression system composed of a single-copy target gene under the control of a lac promoter derivative and lacI gene in a multicopy plasmid is proposed, and its ability to control the flux of a metabolic pathway is demonstrated. A model system to control the flux of acetyl-CoA to acetyl phosphate was constructed by integrating pta, a gene encoding phosphotransacetylase, under a tac promoter into the chromosome of E. coli with a pta-negative background and transforming a multicopy plasmid containing the $lacI^Q$ gene into the strain. The production rate of acetate was shown to be tightly controlled when varying the concentration of the inducer (IPTG) in he model system.

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.77-83
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• 2006

A gene encoding a $\gamma-butyrolactone$ autoregulator receptor was cloned from Saccharopolyspora erythraea, and the biochemical characteristics, including the autoregulator specificity, were determined with the purified recombinant protein. Using primers designed for the conserved amino acid sequence of Streptomyces $\gamma-butyrolactone$ autoregulator receptors, a 120 bp S. erythraea DNA fragment was obtained by PCR. Southern and colony hybridization with the 120 bp fragment as a probe allowed to select a genomic clone of S. erythraea, pESG, harboring a 3.2 kb SacI fragment. Nucleotide sequencing analysis revealed a 615 bp open reading frame (ORF), showing moderate homology (identity, $31-34\%$; similarity, $45-47\%$) with the $\gamma-butyrolactone$ autoregulator receptors from Streptomyces sp., and this ORF was named seaR (Saccharopolyspora erythraea autoregulator receptor). The seaR/pET-3d plasmid was constructed to overexpress the recombinant SeaR protein (rSeaR) in Escherichia coli, and the rSeaR protein was purified to homogeneity by DEAE-Sephacel column chromatography, followed by DEAE-ion-exchange HPLC. The molecular mass of the purified rSeaR protein was 52 kDa by HPLC gel-filtration chromatography and 27 kDa by SDS-polyacrylamide gel electrophoresis, indicating that the rSeaR protein is present as a dimer. A binding assay with tritium-labeled autoregulators revealed that rSeaR has clear binding activity with a VB-C-type autoregulator as the most effective ligand, demonstrating for the first time that the erythromycin producer S. erythraea possesses a gene for the $\gamma-butyrolactone$autoregulator receptor.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.644-648
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• 2005

Dextranase (${\alpha}$-1,6-D-glucan-6-glucanogydrolase:E.C. 3.2.1.11) catalyzes the hydrolysis of ${\alpha}$-(1.6) linkages of dextran. A lsd1 gene encoding an extracellular dextranase was isolated from the genomic DNA of L. starkeyi. The lsd11 gene is a synthetic dextranase (lsd1) after codon optimization for gene expression with Pichia pastoris system. A open reading frame of lsd11 gene was 1827 bp and it was inserted into the pPIC3.5K expression vector. The plasmid linearized by Sac I was integrated into the 5'AOX region of the chromosomal DNA of P. pastoris. The lsd11 gene fragment encoding a mature protein of 608 amino acids with a predicted molecular weight of 70 kDa, was expressed in the methylotrophic yeast P. pastoris by controling the alcohol oxidase-1 (AOX1) promoter. The recombinant lds11 was optimized by using the shake-flask expression and upscaled using fermentation technology. More than 9.8 mg/L of active dextranase was obtained after induction by methanol. The optimum pH of LSD11 was found to be 5.5 and the optimum temperature $28^{\circ}C$.

• 미생물학회지
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• 제46권2호
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• pp.207-212
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• 2010

전통 발효식품인 된장으로부터 mannanase의 생산균으로 분리된 WL-8 균주는 형태적 특성, 생화학적 성질 및 16S rRNA의 염기서열에 근거하여 Bacillus subtilis로 동정되었다. B. subtilis WL-8은 locust bean gum 보다는 밀기울이 첨가된 LB 배지에서 mannanase 생산성이 높았으며, 24시간 배양하였을 때 약 20 U/ml에 이르렀다. 분리균 WL-8의 mannanase 유전자를 클로닝하여 그 염기서열을 결정한 결과 mannanase 유전자는 362 아미노산으로 구성된 단백질을 코드하며 1,086 뉴클레오티드로 이루어졌다. 아미노산 잔기배열을 분석한 결과 WL-8의 mannanase는 GH family 26에 속하며 B. subtilis의 mannanases와 매우 상동성이 높았다. B. subtilis WL-8의 mannanase 유전자를 함유한 재조합 대장균의 배양상등액과 균체파쇄상등액을 사용하여 반응특성을 조사한 결과 pH 5.5와 $60^{\circ}C$에서 최대 반응활성을 보였으며, $60^{\circ}C$보다 높은 온도에서 배양상등액보다는 균체파쇄상등액에 존재하는 mannanase가 더 높은 활성을 보였다.

• Journal of Applied Biological Chemistry
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• 제65권4호
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• pp.383-386
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• 2022

Thermotoga maritima cellulose B 유전자의 The open reading frame (ORF)은 825 bp이고, cellulase B는 분자량이 약 31,732 kDa인 274개의 아미노산으로 구성되어 있다 이 중 N-말단의 17개 아미노산은 signal peptide로 추정된다. Signal peptide를 제외한 ORF를 pRSET 대장균 발현벡터에 삽입하여 pRBTmCelB를 제조하였다. 6-His tag을 포함하는 TmCelB 융합단백질의 cellulose 분해활성과 생화학적 특성을 분석하기 위해 pRBTmCelB를 대장균 BL21에서 과다발현하였고 순수분리하였다. TmCelB 융합단백질은 cellulose 분해활성을 나타내었고 이 있는 것으로 분석되었다. TmCelB 융합단백질은 약 95 ℃ 부근에서 가장 높은 효소 활성을 나타내었고, 100 ℃에서 최대 활성을 80% 이상 유지하는 것을 확인하였다. 또한 TmCelB 융합단백질은 약 pH 4.5 부근에서 최대 활성을 나타내었고, pH 4.0-6.0 범위에서 최대 활성을 60% 이상 유지하였다. TmCelB 융합단백질은 100 ℃에서 180분 후에도 효소활성을 80% 이상 유지하였다.