Cloning and Expression of the Isoflavone Synthase Gene (IFS-Tp) from Trifolium pratense

안중훈,Bong Gyu Kim,Song-Young Kim,Hee Su Song,이찬,Hor-Gil Hur,Su-Il Kim 한국분자세포생물학회 2003 Molecules and cells Vol.15 No.3

대두 Bowman - Birk trypsin isoinhibitor PI Ⅳ의 cDNA 유전자 분리

안중훈,김희진,최양도,김수일 ( Joohg Hoon Ahn,Hee Jin Kim,Yang Do Choi,Su Il Kim ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.2

Three different kinds of proteinase inhibitors are known in soybeans; Kunitz trypsin inhibitor, Bowman-Birk trypsin inhibitor (BBTI) and its isoinhibitors. A cDNA clone for the soybean trypsin inhibitor was isolated from a soybean λgt11 cDNA library by plaque hybridization with a oligonucleotide probe which corresponds to the N-terminal region of the BBTI. The cDNA clone λB13 was 446bp long and the nucleotide sequencing reveals that there were one open reading frame of 243 bp, the 5` leader sequences of 70 bp and the 3`-untranslating region of 133 bp in the cDNA clone λB13. Nucleotide sequences at the 5` and 3` noncoding regions were highly homologous, respectively, to those of the BBTI and isoinhibitor C-2. Compared the deduced amino acid sequences to the amino acid sequences of proteinase inhibitor, it corresponds to the Bowman-Birk trypsin isoinhibitor PI IV. Sequences around the reactive site of Arg-Ser were repeated twice, giving the double headed structure. Northern blot analysis demonstrated that the size of the mRNA was about 700 nucleotides. It was expressed only in soybean seed maintaining tissue specific gene expression pattern. Expression level was increased with the maturation of seed and reached maximum in full size seed. It suggests that the expression of the isoinhibitor PI IV could be regulated at the transcriptional level.

Bacillus stearothermophilus가 생산하는 Cyclodextrin Glucanotransferase: Affinity Chromatography를 이용한 정제 및 성질

안중훈,황진봉,김승호 한국미생물 · 생명공학회 1990 한국미생물·생명공학회지 Vol.18 No.6

Bacillus stearothermophilus가 생산하는 cyclodextrin glucanotransferase (CGTase)를 ammonium sulfate 법과 affinity chromatography법에 의해 정제하였다. 이 CGTase의 비활성은 11.5U/mg protein에서 33445.0U/mg protein으로 증가하였다. 이 효소의 분자량을 측정하기 위해 SDS-PAGE를 실시한 결과 분자량은 약 7,800이었다. 이 효소의 최적 pH와 온도는 각각 6.0과 $60^{\circ}C$이었다. 이 효소는 pH5.5와 10.0에서 안정하였다. 이 효소에 calcium ion을 첨가하였더니 열안정성이 증가하였다. 이 효소의 등전점은 약 4.8 이었다. The cyclodextrin glucanotransferase (CGTase) was purified from the culture broth of Bacillus stearothermophilus by ammonium sulfate precipitation and affinity chromatography. The specific activity of the CGTase increased by about 31-fold from 111.5 U/mg protein to 3445.0 Ulmg protein. The SDSPAGE indicated that the purified CGTase was homogeneous and the molecular weight of the purified CGTase was about 78,000. The optimum pH and temperature was 6.0 and $60^{\circ}C$, respectively. This enzyme was stable from pH 5.5-10.0. The enzyme retained its full activity at the incubation temperature up to $60^{\circ}C$ and calcium ion increased the thermal stability. The isoelectric point was about 4.8.

여러 첨가물의 용매가 Bacillus stearothermophilus가 생산하는 Cyclodextrin Glucanotransferase의 열안정성에 미치는 영향

안중훈,황진봉,김승호 한국미생물 · 생명공학회 1991 한국미생물·생명공학회지 Vol.19 No.4

Ethylene glycol, glycerol, sorbitol 그리고 sucrose가 Bacillus stearothermophilus가 생산하는 cyclodextrin glucanotransferase(CGYase)의 열안정성에 미치는 영향을 조사하였다. Glycerol, sorbitol 그리고 sucrose가 CGTase의 열안정성에 효과가 있었다. 이 효과는 첨가물의 농도와 밀접한 관계가 있었다. n-Butanol, 1,4-dioxane그리고 n-octane과 같은 유기 용매에서 CGTase의 열안정성을 조사하였다. 1,4-dioxane 과 n-octane은 CGTase의 열안정성을 증가시켰다. 특히, n-octane에서 CGTase를 $75^{\circ}C$에서 90분간 정치시킨 후에도 원래 효소활성의 81를 유지하였다. The influence of ethylene glycol, glycerol, sorbitol and sucrose on the thermostability of Bacilus stearothermophzlus cyclodextrin glucanotransferase (CGTase) was investigated. Glycerol, sorbitol and sucrose had effect on thermostability of the CGTase. The effects appeared to be strongly dependent on concentration of additives. The thermostability of CGTase also was assayed in organic solvents such as n-butanol, l, &dioxane, n-octane. The therrnostability of CGTase increased in l, 4-dioxane and n-octane. Particularly, in n-octane, the CGTase retained the 81% of the initial activity after incubation at $75^{\circ}C$ for 90 min.

Molecular Cloning and Nucleotide Sequencing of cDNA encoding Soybean Bowman-Birk Trypsin Isoinhibitor PI IV

안중훈,김희진,최양도,김수일,Ahn, Joong-Hoon,Kim, Hee-Jin,Choi, Yang-Do,Kim, Su-Il 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.2

대두의 cDNA 유전자 은행을 선별한 결과 Bowman-Birk trypsin isoinhibitor PI IV의 cDNA 유전자를 분리하였다. Probe 는 Bowman-Birk trypsin inhibitor (BBTI)의 아미노 말단 부위에 해당하는 oligonucleotide를 합성 사용하였다. 이 유전자의 크기는 446 bp 였다. 염기서열을 결정한 결과 243 bp 의 open reading frame 과 70 bp 의 5'-leader sequence 그리고 133 bp의 3'-untranslating sequence로 이루어져 있었다. 5'-과 3'-noncoding 부위 각각의 염기서열은 BBTI 와 isoinhibitor C-2의 경우와 매우 유사하였다. 이 유전자에 의해 code되는 단백질은 6 개 아미노산의 signal peptide 를 포함한 Bowman-Birk iscinhibitor PI IV로 나타났다. 아미노산 Arg-Ser을 포함한 활성부 위는 두번 반복하고 있어서 double headed structure를 이루고 있었다. Northern blot을 실시한 결과 이 유전자의 mRNA는 길이가 약 700 nucleotide 였으며 대두 종자에서만 발현되었다. 종자가 성숙하면서 유전자 발현은 증가하여 완숙된 대두에서 최고치를 나타내었다. 이 결과 PI IV는 전사 단계에서 발현 조절이 이루어지는 것을 알 수 있다. Three different kinds of proteinase inhibitors are known in soybeans; Kunitz trypsin inhibitor, Bowman-Birk trypsin inhibitor (BBTI) and its isoinhibitors. A cDNA clone for the soybean trypsin inhibitor was isolated from a soybean ${\lambda}$gt11 cDNA library by plaque hybridization with a oligonucleotide probe which corresponds to the N-terminal region of the BBTI. The cDNA clone ${\lambda}$ B13 was 446bp long and the nucleotide sequencing reveals that there were one open reading frame of 243 bp, the 5' leader sequences of 70 bp and the 3'-untranslating region of 133 bp in the cDNA clone ${\lambda}$B13. Nucleotide sequences at the 5' and 3' noncoding regions were highly homologous, respectively, to those of the BBTI and isoinhibitor C-2. Compared the deduced amino acid sequences to the amino acid sequences of proteinase inhibitor, it corresponds to the Bowman-Birk trypsin isoinhibitor PI IV. Sequences around the reactive site of Arg-Ser were repeated twice, giving the double headed structure. Northern blot analysis demonstrated that the size of the mRNA was about 700 nucleotides. It was expressed only in soybean seed maintaining tissue specific gene expression pattern. Expression level was increased with the maturation of seed and reached maximum in full size seed. It suggests that the expression of the isoinhibitor PI IV could be regulated at the transcriptional level.

단보 : Affinity chromatography 를 이용한 Cyclodextrin Glucanotransferase 의 정제

안중훈(Joong - Hoon Ahn),황진봉(Jin - Bong Hwang),김경은(Kyoung - Eun Kim),김승호(Seung - Ho Kim) 한국미생물생명공학회(구 한국산업미생물학회) 1991 한국미생물·생명공학회지 Vol.19 No.3

Cyclodextrin Glucanotransferase from Bacillus Stearothermophilus : Purification by Affinity Chromatography and Its Properties

안중훈(Joong - Hoon Ahn),황진봉(Jin - Bong Hwang),김승호(Seung - Ho Kim) 한국미생물생명공학회 1990 한국미생물·생명공학회지 Vol.18 No.6

Effect of Various Additives and Solvents on Thermostability of Cyclodextrin Glucanotransferase from Bacillus stearothermophilus

Ahn, Joong Hoon,Hwang, Jin Bong,Kim, Seung Ho 한국산업미생물학회 1991 한국미생물·생명공학회지 Vol.19 No.4

Ethylene glycol, glycerol, sorbitol 그리고 sucrose가 Bacillus stearothermophilus가 생산하는 cyclodextrin glucanotransferase(CGTase)의 열안정성에 미치는 영향을 조사하였다. Glycerol, sorbitol 그리고 sucrose가 CGTast의 열안정성에 효과가 있었다. 이 효과는 첨가물의 농도와 밀접한 관계가 있었다. n-Butanol, 1,4-dioxane 그리고 n-octane과 같은 유기 용매에서 CGTase의 열안정성을 조사하였다. 1,4-dioxane과 n-octane은 CGTase의 열안정성을 증가시켰다. 특히, n-octane에서 CGtase를 75℃에서 90분간 정치시킨 후에도 원래 효소활성의 81%를 유지하였다. The influence of ethylene glycol, glycerol, sorbitol and sucrose on the thermostability of Bacilus stearothermophilus cyclodextrin glucanotransferase (CGTase ) was investigated. Glycerol, sorbitol and sucrose had effect on thermostability of the CGTase. The effects appeared to be strongly dependent on concentration of additives. The thermostability of CGTase also was assayed in organic solvents such as n-butanol, 1,4-dioxane, n-octane. The thermostability of CGTase increased in 1,4-dioxane and n-octane. Particularly, in n-octane, the CGTase retained the 81% of the initial activity after incubation at 75℃ for 90 min.

비 기능성 부신피질암 1례

전윤수,안중훈,박영호,장대수 大韓泌尿器科學會 1988 Korean Journal of Urology Vol.29 No.1

포플러로부터 flavone synthase I 유전자의 클로닝 및 생화학적 특성

김봉규,안중훈 한국응용생명화학회 2009 Journal of Applied Biological Chemistry (J. Appl. Vol.52 No.1

포플러는 naringenin, kaempferol, myricetin, apigenin, luteolin, rhamnetin, quercetin과 같은 플라보노이드를 가지고 있다. 이러한 플라보노이드들은 여러 가지 효소에 의해 naringenin으로부터 합성된다. 그러나, 포플러에서는 플라보노이드 합성에 관여하는 효소들의 연구가 거의 이루어 지지 않았다. 포플러 에서 flavone synthase I 유전자를 RT-PCR방법을 이용하여 클로닝 하였다. PFNS I-1 유전자의 기질 특성을 알아보기 위하여 대장균에 과발현하여 glutathione S-transferase 친화 크로마토그래피를 이용하여 정제하였다. 정제한 PFNS I-1 재조합 단백질은 flavanone인 2S-naringenin기질을 이용하여 flavone인 apigenin을 생성함을 알 수 있었다. 또한 cofactor인 oxoglutarate, FeSO4, ascorbate, catalase가 PFNS I-1의 반응성에 중대한 영향을 미치는 것을 알 수 있었다. 이상의 결과를 종합해 볼 때 PFNS I-1 유전자는 flavone synthase I을 암호화하는 유전자임을 확인하였다. Poplar contains various flavonoids including naringenin, kaempferol, myricetin, apigenin, luteolin,rhamnetin, and quercetin. These flavonoids are synthesized from naringenin with various enzymes. However, none of genes from poplar involved in flavonoid biosynthesis have been biochemically characterized. We cloned PFNS I-1 from Populus deltoids by RT-PCR method. The open reading frame of PFNS I-1 consisted of 1,017-bp and it showed high similarity with other FNS genes. The purified recombinant PFNS I-1, expressed in Escherichia coli, catalyzed the reaction from flavanone (naringenin) to flavone (apigenin). The reaction of PFNS I-1 was enhanced by cofactors such as oxoglutarate, Fe2+, ascorbate and catalase. Thus, it is concluded that PFNS I-1 encodes a flavone synthase I.