• 한국미생물·생명공학회지
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• 제25권4호
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• pp.367-373
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• 1997

One of the three components of the phenanthrene dioxygenase which is required for conversion of phenanthrene to cis-phenanthrene dihydrodiol, Rieske-type ferredoxin encoded by phnR has been cloned and sequenced from Pseudomonas sp. strain DJ77. The gene phnR is positioned at the downstream of phnQ encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase. The PhnR ferredoxin contains 108 amino acids with a Mr of 11,355. The deduced amino acid sequence of the PhnR ferredoxin is 35-79% identical to those of homologous ferredoxins encoded by various genes.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.625-628
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• 2001

토양으로부터 특이적인 광학 이성질체에 활성을 갖는 두 효소원을 선별한 후, 동정하여 Pseudomonas sp. S34와 B. stearothermophilus JY144로 명명하였고, genecloning과 sequencing을 통해 유전자의 특성 및 관련효소들과의 유연관계를 규명하였다. 규명된 정보의 분석과 비교를 통해 ketoprofen ethyl ester에 활성을 갖는 효소들의 구조적 특성을 추론할 수 있었고, 이를 바탕으로 발현시스템을 구축하였다. 대량생산된 효소를 활용한 반응의 결과 높은 수율과 순도를 갖는 각각의 광학이성질체를 경제적으로 생산할 수 있었다.

• 원예과학기술지
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• 제31권4호
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• pp.496-502
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• 2013

Grapes (Vitis vinifera) are one of the most important fruits worldwide and are eaten raw or after conversion to jelly, jam, juice and wine. Grape skins are a major source of resveratrol (3,5,4'-trihydroxystilbene), which has the ability to reduce blood sugar as well as anticancer, anti-inflammatory, and other beneficial cardiovascular effects. In this study, we investigated the increased accumulation of resveratrol in grape skin and leaves following ultrasonication treatment, which has been shown to induce resveratrol accumulation in several plants. Various ultrasonication treatment times and incubation periods were employed to identify the optimum conditions for the maximum accumulation of resveratrol. Treatment and further incubation led to increased resveratrol in both grape skins and leaves, with the highest increases of 7.7-fold and 1.9-fold occurring in response to 5 min ultrasonication treatment followed by 6 hour incubation and 15 min ultrasonication treatment followed by 3 hour incubation, respectively. The underlying mechanism for the increased amounts of resveratrol were studied by employing a semi-quantitative RT-PCR to monitor the expression levels of the resveratrol synthase (RS) gene in response to ultrasonication treatment. The RS gene increased the expression in response to ultrasonication treatment, suggesting that up-regulation of the RS gene by ultrasonication treatment triggers increased amounts of resveratrol. Taken together, these data indicate that this simple ultrasonication treatment of grapes can be an efficient post-harvest technology for increasing resveratrol in grape skins in addition to cleaning the fruits.

• Journal of Microbiology and Biotechnology
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• 제18권7호
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• pp.1216-1220
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• 2008

Sequence analysis of the metabolically rich genome of Streptomyces peucetius ATCC 27952 revealed a 2,199 bp sesquiterpene alcohol (germacradienol) synthase-encoding gene from the germacradienol synthase/terpene cyclase gene cluster. The gene was named spterp13, and its putative function is as a germacradienol synthase/terpene cyclase. The amino acid sequence of Spterp13 shows 66% identity with SAV2163 (GeoA) from S. avermitilis MA4680 and 65% identity with SCO6073 from S. coelicolor A3(2), which produces germacradienol/geosmin. The full-length recombinant protein was heterologously expressed as a his-tagged fusion protein in Escherichia coli, purified, and shown to catalyze the $Mg^{2+}$-dependent conversion of farnesyl diphosphate to the germacradienol, which was verified by gas chromatography/mass spectrometry.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2022년도 추계학술대회
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• pp.24-24
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• 2022

This present study was to identify a novel candidate gene that contribute to the elevated α-linolenic acid (ALA, ω-3) concentration in PE2166 from mutagenesis of Pungsannamul. Major loci qALA5_1 and qALA5_2 were detected on chromosome 5 of soybean through quantitative trait loci mapping analyses of recombinant inbred lines. With next generation sequencing of parental lines and Pungsannamul, and recombinant analyses, a potential gene, Glyma. 05g221500 (HD) controlling elevated ALA concentration was identified. HD is a homeodomain-like transcriptional regulator that may regulate the expression level of microsomal ω-3 fatty acid desaturase (FAD3) genes responsible for the conversion of linoleic acid into ALA in the fatty acid biosynthetic pathway. In addition, we hypothesized that combination of mutant alleles, HD and either of microsomal delta-12 fatty acid desaturase 2-1 (FAD2-1\ could reduce the ω-6/ω-3 ratio. In populations where HD, and FAD2-1A and FAD2-1B genes were segregated, combination of a hd allele from PE2166 and either of the variant FAD2-1 alleles were sufficient to reduce the ω-6/ω-3 ratio in seeds.

• Asian-Australasian Journal of Animal Sciences
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• 제28권2호
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• pp.151-157
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• 2015

Fatness qualities in pigs measured by the amount of fat deposition and composition of fatty acids (FAs) in pork have considerable effect on current breeding goals. The stearoyl-CoA desaturase (SCD) gene plays a crucial role in the conversion of saturated FAs into monounsaturated FAs (MUFAs), and hence, is among the candidate genes responsible for pig fatness traits. Here, we identified a single nucleotide polymorphism (SNP, $c.^*2041T$ >C) in the 3' untranslated region by direct sequencing focused on coding and regulatory regions of porcine SCD. According to the association analysis using a hundred of Berkshire pigs, the SNP was significantly associated with FA composition (MUFAs and polyunsaturated FAs [PUFAs]), polyunsaturated to saturated (P:S) FA ratio, n-6:n-3 FA ratio, and extent of fat deposition such as intramuscular fat and marbling (p<0.05). In addition, the SNP showed a significant effect on the SCD mRNA expression levels (p = 0.041). Based on our results, we suggest that the SCD $c.^*2041T$ >C SNP plays a role in the gene regulation and affects the fatness qualities in Berkshire pigs.

• Archives of Pharmacal Research
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• 제27권5호
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• pp.570-575
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• 2004

2-Hydroxymuconic semialdehyde (2-HMS) dehydrogenase catalyzes the conversion of 2-HMS to 4-oxalocrotonate, which is a step in the meta cleavage pathway of aromatic hydrocarbons in bacteria. A tomC gene that encodes 2-HMS dehydrogenase of Burkholderia cepacia G4, a soil bacterium that can grow on toluene, cresol, phenol, or benzene, was overexpressed into E. coli HB 101, and its gene product was characterized in this study. 2-HMS dehydrogenase from B. cepacia G4 has a high catalytic efficiency in terms of V$_{max}$K$_{max}$ towards 2-hydroxy-5-methyl-muconic semialdehyde followed by 2-HMS but has a very low efficiency for 5-chloro-2-hydroxymuconic semialdehyde. However, the enzyme did not utilize 2-hydroxy-6-oxo-hepta 2,4-dienoic acid and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid as substrates. The molecular weight of 2-HMS dehydrogenase from B. cepacia G4 was predicted to be 52 kDa containing 485 amino acid residues from the nucleotide sequence of the tomC gene, and it exhibited the highest identity of 78% with the amino acid sequence of 2-HMS dehydrogenase that is encoded in the aphC gene of Comamonas testosteroni TA441. 2-HMS dehydrogenase from B. cepacia G4 showed a significant phylogenetic relationship not only with other 2-HMS dehydrogenases, but also with different dehydrogenases from evolutionarily distant organisms.sms.

• 한국미생물·생명공학회지
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• 제36권3호
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• pp.182-188
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• 2008

내열성 Trehalose synthase를 생산하는 초고온성 균주 HJ6은 일본 Arima 온천수에서 분리하였다. 세포의 길이는 $2{\sim}4\;um$, 직경 0.4 um의 간균으로 생육최적 pH와 온도는 각각 6.5와 $80^{\circ}C$이였다. 분리된 균주의 16s rRNA 염기서열을 분석하고 계통학적으로 분류한 결과, HJ6 균주는 Thermus thermophilus에 속하는 것으로 동정되었다. PCR법을 이용하여 trehalose synthase(TS) 유전자를 클로닝하고 염기서열을 분석한 결과, ORF는 2,898개의 뉴클레오타이드로 구성되고 915개의 아미노산을 암호화하였다. 아마노산 서열을 바탕으로 상동성을 분석한 결과, Thermus caldophilus GK24 유래 TS와 99%, Meiothermus ruber 유래 TS와 83%의 identity를 나타내었다. 이 유전자를 온도감수성 프로모터를 포함하는 pJLA503 벡터를 이용하며 대장군에서 발현하고 정제하여 약 110 kDa 단백질을 얻을 수 있었다. 정제된 효소는 트레할로스 전환활성에 대한 최적 pH가 7.5이고, 최적온도는 $80^{\circ}C$이며, 활성의 반감기는 $90^{\circ}C$에서 40분으로 확인되어 높은 내열성을 가지는 것으로 확인되었다. 본 효소의 트레할로스 최대 전환율은 기질농도 500mM에서 55.7%를 나타내었고, 기질 농도가 증가함에 따라 더불어 증가하였기 때문에 본 효소의 트레할로스 전환율을 기질농도에 의존적인 것으로 생각되었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.457-460
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• 2002

In an effort to isolate novel strains expressing a thermostable esterase that hydrolyzed the rac-ketoprofen ethyl ester to ketoprofen in the stereospecific manner, we screened various soils and composts from broad ecological niches in which the activity was expected to be found. Three hundreds of microbial strains were tested to determine their ester-hydrolyzing activity by using an agar plate containing insoluble tributyrin as an indicative substrate, and then further screened by activity on the (R,S)-ketoprofen ethyl ester. Twenty-six strains were screened primarily at high growth and incubation temperature and further compared the ability to ethyl ester-hydrolyzing activity in terms of conversion yield and chiral specificity. Consequently, a strain JYl44 was isolated as a novel strain that produced a (R)-stereospecific esterase with high stability and systematically identified as a Bacillus stearothermophilus JY144. The enzyme indeed stables at a broad range of temperature, upto 65 $^{\circ}C$, and pH ranging from 6.0 to 10.0. The optimal temperature and pH for enzymatic conversion were 50 $^{\circ}C$ and 9.0, respectively. Based on the observations that resulted a poor cell growth, and enzyme expression in wild type strain, we further attempted the gene cloning into a general host Escherichia coli and determined its primary structure, concomitantly resulting a high level expression of the enzyme. The cloned gene had an open reading frame (250 amino acids) with a calculated molecular mass of 27.4 kDa, and its primary structure showed a relative high homology (45-52 %) to the esterases from Streptomyces and Bacillus strains. The recombinant whole cell enzyme could efficiently convert the rac-ketoprofen ethyl ester to (R)-ketoprofen, with optical purity of 99 % and yield of 49 %.

• 한국미생물·생명공학회지
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• 제26권1호
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• pp.15-22
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• 1998

일반적으로 Streptomyces류는 성장이 늦고 유지가 어려운 반면, E. coli는 배양기간이 짧고 유전자 조작도 간편한 장점이 있기 때문에, E. coli를 이용하여 유용단백질을 생산하는 연구가 일반적인 흐름이다. 그러나 E. coli에서 외래유전자를 도입하여 대량으로 생산을 시키는 경우에 비용해성의 inclusion body를 형성하는 경우가 많으므로 용해성의 활성형 단백질을 생산하기 위하여는 여러 가지 조건을 고려하여야 한다. 본 논문에서는 aklavlnone 11-hydroxylase gene(dnrF)을 E. coli BL2l에서 발현시킬 때의 배양조건을 배양온도와 IPTG농도의 두가지 요소를 조합하여 변형시키는 방법으로, 활성형 단백질의 생산을 최대화하고 inclusion body의 형성을 최소화하는 배양조건을 조사하였다. 그 결과, 37$^{\circ}C$에서 배양했을 때에는 0.02mM의 IPTG를 첨가하였을 때 inclusion body를 가장 적게 만들고, 그에 따라 생산되는 효소활성도 가장 높았다. 반면, 28$^{\circ}C$로 배양온도를 낮추었을 때에는 0.06mM의 IPTG를 첨가하였을 때 aklavinone 11-hydroxylase효소가 최대로 생산됨을 SDS-PAGE 및 효소 활성측정으로 확인하였다. IPTG농도를 0.1 mM로 높인 경우에는 28$^{\circ}C$, 37$^{\circ}C$에서 모두 aklavinone 11-hydroxylase효소가 과발현되어 Inclusion body를 가장 많이 생성하였음을 알 수 있었다. 방선균에서 동일 유전자를 대량발현시키는 경우 발현된 단백질의 효소 활성은 있으나 SDS-PAGE상에서의 단백질의 관찰이 불가능하였고, 동시에 단백질의 정제시 효소활성이 소실되어 정제가 불가능하였다. 이러한 효소 활성의 소실의 원인은 세포내의 어떤 저분자물질일 것으로 추정되며 본 연구에서 제작한 antibody를 이용하면, 효소의 정제가 용이하게 수행될 것이다. 특히 대장균계에서의 활성형 효소의 최적발현조건에서 세포를 배양하거나, inclusion body의 refolding을 실시한 후, 항체를 이용한 Western blot assay를 지표로 효소를 정제하면, 미지의 cofactor의 정체도 밝혀지고 aklavinone 11-hydroxylase류의 효소 특성 연구에 큰 도움이 될 것이다. 또한 이 효소를 이용한 다양한 종류의 bioconversion을 실시하여 그동안 background 때문에 생성된 product의 검출이 불가능했었던 소량의 생성산물의 분석도 가능할 것으로 판단되어 금후의 bioconversion연구에 기대하는 바가 크다.