유전자 조작 , 균주분리 재조합 균주 Escherichia coli 가 생산하는 Bacillus stearothermophilus NO.236 β-Xylosidase B의 정제 및 특성

장욱진,조쌍구,최용진 ( Uck Jin Chang,Ssang Goo Cho,Yong Jin Choi ) 한국미생물생명공학회 1998 한국미생물·생명공학회지 Vol.26 No.4

Molecular Colning and Ewpression of the $\alpha$-L-Arabinofuranosidase Gene of Bacillus stearothermophilus in Escherichia coli

엄수정,김희선,조쌍구,최용진,Eom, Soo-Jung,Kim, Hee-Sun,Cho, Ssang-Goo,Choi, Yong-Jin 한국미생물 · 생명공학회 1994 한국미생물·생명공학회지 Vol.22 No.6

The Bacillus stearothermophilus arfI gene encoding a-arabinofuranosidase was isolated from the genomic library, cloned into pBR322, and subsequently transferred into the Escherichia coli HB101. The recombinant E. coli was selected from approximately 10,000 transformants screened by making use of its ability to produce a yellow pigment around the colony on the selective medium supplemented with p-nitrophenyl-$\alpha$-L-arabinofuranoside (pNPAf), a chromogenic substrate. The functional clone was found to harbor a recombinant plasmid, pKMG11 with an insertion of about 5 kb derived from the B. stearothermophilus chromosomal DNA. Identity of the arfI gene on the insert DNA was confirmed by a zymogram with 4-methylumbelliferyl-$\alpha$-L-arabinofuranoside as the enzyme substrate. The $\alpha$-arabinofuranosidase from the recombinant E. coli strain showed very high substrate specificity; the enzyme displayed high activity only with pNPAf among many other p- or $o$-nitrophenyl derivatives of several sugars, and acted only on arabinoxylan among various natural arabinose containing polysaccharides tested.

Molecular Cloning and Expression of the Acetyl Xylan Esterase Gene(estII) of Bacillus Stearothermophilus in Escherichia coli

김희선,엄수정,조쌍구,최용진,Kim, Hee-Sun,Eom, Soo-Jung,Cho, Ssang-Goo,Choi, Yong-Jin 한국미생물 · 생명공학회 1994 한국미생물·생명공학회지 Vol.22 No.6

Bacillus stearothermomophilus, a strong xylan degrader, was confirmed to express multiple esterase activities in addition to the major xylanolytic enzymes. One of the genes encoding the esterases was isolated from the genomic library of B. stearothermophilus constructed with EcoRl restriction endonuclease and pBR322 plasmid. Three recombinant plasmids showing the tributyrin degrading activity were selected from approximately 7, 000 E. coli HB101 transformants, and were found to have the same insert of a 3.2 kb DNA fragment. Restriction mapping and hybridization studies revealed that the gene(estII) on the hybrid plasmid (pKMG7) had originated from the B. stearothermophilus chromosome, and was distinct from the estl, another esterase gene of B. stearothermophilus isolated in the previous work. The E. coli cells harboring pKMG7 produced an acetylxylan esterase that exibited similar substrate specificity to the esterase encoded by the estI gene.

Modulation of Apoptosis in HaCaT keratinocytes via Differential Regulation of ERK Signaling Pathway by Flavonoids

Eung-Ryoung Lee(이응룡),Yong-Jin Kang(강용진),Jung-Hyun Kim(김정현),Ssang-Goo Cho(조쌍구) 한국생물공학회 2005 한국생물공학회 학술대회 Vol.2005 No.10

Bacillus stearothermophilus로부터 Endo-xylanase 유전자의 클로닝 및 Escherichia coli에서의 발현

조쌍구,박성수,박영인,최용진 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.3

내알카리성 및 내열성 xylanase를 생산하는 토양 분리균인 Bacillus stearothermophilus의 chromosomal DNA와 pBR322 plasmid DNA의 HindⅢ 절단 DNA 단편을 ligation시켜 E. coli HB101을 형질전환, 약 5천개의 형질전환체를 얻었으며 이들 중에서 세개의 xylanase 양성 형질전환체를 분리하였다. 상기 세 xylanase 양성 형질전환체로부터 분리한 제조합 plasmid(pMG11, pMG12 및 PMG13)는 다같이 xylanase활성과 관계되는 B. stearothermophilus 유래의 동일 4kb 외래 DNA을 가지고 있었으나, pMG13은 외래 DNA의 삽입 방향만이 다름을 확인하였다. B. stearothermophilus 균주는 최소한 세개 이상의 xylanase 활성단백질을 생산하는 것으로 관찰되었으며, xylanase 양성 형질전환체는 그 중 분자량이 가장 큰 효소단백질을 생산하는 것으로 판단되었다. 한편, 형질전환체 xylanase는 xylotetraose 이상의 xylo-oligosaccharide와 xylan 기질에 작용하여 최종 산물로서 xylobiose와 xylotriose을 생산하는 endo-acting 효소로서, trans-xylosidase 활성도 가지고 있는 것으로 추측되었다. Genomic DNA of Bacillus stearothermophilus, which expressed alkalophilic and thermophilic xylanases, was partially digested with HindⅢ, cloned into pBR322, and subsequently transferred into the Escherichia coli HB101 cells. Three among 5,000 transformants screened formed clear zones around their colonies. From the functional clones, three recombinant plasmids(pMG11, pMG12 and pMG13) had been isolated, and they were identified to carry the same 4 kb HindⅢ fragment originated from B. stearothermophilus which was responsible for the xylanase activity. pMG13, however, had the foreign DNA of opposite orientation compared to the other two recombinant plasmids. This recombinant plasmid gave much lower xylanase activity. B. stearothermophilus was observed to produce at least three xylanase activities as evidenced by the PAGE-xylan zymogram. The xylanase from E. coli HB101/pMG12 was judged to correspond to the largest among the three B. stearothermophilus xylanases observed in the zymogrom. The enzyme hydrolyzed xylooligosaccharides larger than xylotriose and degraded xylan to produce xylobiose and xylotriose as major products. The xylanase was considered to have trans-xylosidase activity, too.

Xylan 분해균주인 Bacillus stearothermophilus의 오탄당 이용

이효선,조쌍구,최용진 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.4

강력한 xylan 분해 토양 분리균인 Bacillus stearothermophilus의 xylan 분해 주 산물인 xylose와 arabinose등의 pentose 이용과 pentose이용에 미치는 glucose와 기타 maltose 및 cellobiose 등의 효과를 분석하였다. 본 균주는 유일 탄소원으로 glucose를 가장 효율적으로 이용하였으며 다음으로 xylose와 ribose와 같은 pentose 그리고 maltose 등의 이당류도 잘 이용하였으나 glycerol은 전혀 이용하지 못하였다. 또한 glucose와 pentose 또는 xylooligomer와의 혼합 탄소원을 첨가했을 때도 B. stearothermophilus는 glucose를 우선적으로 이용하는 순서적 이용(sequential utilization) 현상과 더불어 전형적인 diauxic growth 양상을 나타내었다. 이에 비해 maltose와 pentose 혼합기질의 경우는 동시이용(co-utilization) 현상을 보였고 cellobios와 pentose 혼합물은 순서적 이용, 그리고 xylose와 arabinose의 pentose 혼합 탄소원인 경우는 두 탄소원을 동시에 이용하였다. 이와 같은 B. stearothermophilus의 탄소원 이용 특성은 glucose 대사관련 주요 효소인 hexokinase의 구성적 생산(constitutive production)과 이에 반한 ^D-xylose isomerase, ^D-xylulokinase 및 ^L-arabinose isomerase 등의 pentose 대사 주 관련 효소의 유도 생산(induced production)과 관계되는 효소 생산 조절 기작 내지는 inducer exclusion 현상을 비롯한 catabolite regulatory mechanism에 기인하는 것으로 설명할 수 있었다. Bacillus stearothermophilus, a potent xylanolytic bacterium isolated from soil, was tested for the strain's strategies of pentose utilization and the evidence of substreate preferences. The strain metabolized glucose, xylose, ribose, maltose, cellobiose, sucrose, arabinose and xylitol. The efficacy of the sugars as a carbon and energy source in this strain was of the order named above. The organism, however, could not grow on glycerol as a sole growth substrate. During cultivation on a mixture of glucose and xylose or arabinose, the major hydrolytic products of xylan, B. stearothermophilus displayed classical diauxic growth in which glucose was utilized during the first phase. On the other hand, the pentose utilization was prevented immediately upon addition of glucose. Cellobiose was preferred over xylose or arabinose. In contrast, maltose and pentose were co-utilized, and also no preference on between xylose and arabinose. Enzymatic studies indicated that B. stearothermophilus possessed constitutive hexokinase, a key enzyme of the glucose metabolic system. While, the production of ^D-xylose isomerase, ^D-xylulokinase and ^D-arabinose isomerase essential for pentose phosphate pathway were induced by xylose, xylan, and xylitol but repressed by glucose. Taken together, the results suggested that the sequential utilization of B. stearothermophilus would be mediated by catabolite regulatory mechanisms such as catabolite inhibition or inducer exclusion.

플라보노이드의 세포 신호전달 조절

이응룡,강근호,강용진,김우열,최혜연,김봉우,정효순,조쌍구 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10

Many studies revealed the neuroprotective, cardioprotective, and chemopreventive actions of dietary flavonoids. The plausible mechanistic interpretation of the various effects of flavonoids was concentrated on the anti-oxidant or free radical-scavenging properties of these phytochernicals, both in model systems and under in vivo conditions. While there has been a major focus on the anti-oxidant properties. there is an emerging view that flavonoids and their in vivo metabolites. do not act as conventional hydrogen-donating anti-oxidants. but they may exert regulatory functions in cells through actions at protein kinase or lipid kinase signaling pathways. Flavonoids and more recently their metabolites. have been reported to act at phosphoinositide 3-kinase(PI 3-kinase). Akt/protein kinase B(Akt/PKB), protein kinase C (PKC), mitogen activated protein kinase(MAP kinase), and various tyrosine kinases signaling cascades. Inhibitory or stimulatory actions at these pathways are likely to affect cellular function profoundly by altering the phosphorylation state of target molecules and by modulating gene expression. A clear understanding of the mechanisms of action of flavonoids, either as anti-oxidants or modulators of cellular signaling pathways, and the influence of their metabolism on these properties are key to the evaluation of these potent biomolecules as anti-cancer agents, cardio-protectants, and inhibitors of neurodegeneration.

재조합 균주 Escherichia coli가 생산하는 Bacillus stearothermophilus α-L-Arabinofuranosidase의 정제 및 특성

엄수정,조쌍구,최용진 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.4

Bacillus stearothermophilus의 α-arabinofuranosidase 생산 유전자 (arfI)가 클로닝된 재조합 플라스미드(pKMG11)를 가지고 있는 E. coli HB101/pKMG11 균주에 의한 α-arabinofuranosidase 생산 최적 배양 조건을 조사해 본 결과, 탄소원으로 0.5% arabinose를 첨가한 LB 배지에서 약 20시간 배양했을 때 가장 높은 생산량을 보였다. 또한 상기 배양 조건에서 다량의 효소를 생산하고 생산 α-arabinofuranosidase를 ammonium sulfate 분획, DEAE-Sepharose CL-6B ion exchange column chromatography 및 Sepharose 6B-100 gel 여과 등의 과정을 거쳐 단일 단백질로 정제하였다. 정제 효소는 pH 6.5와 55℃에서 가장 높은 효소 활성을 보였고, 자연기질로는 arabinoxylan, 합성기질은 pNPAf에만 작용하는 매우 높은 기질특이성을 보이면서 pNPAf에 대한 K_m 값은 2.99 mM, V_max 값은 0.43 μ㏖e/min(319.74 μ㏖e/min/㎎)로 측정되었다. 또한 본 효소의 pI 값은 4.5 정도였으며 분자량은 SDS-polyacrylamide gel 영동법으로는 약 108 kDa, gel 여과법에 의해서는 약 289 kDa 정도로 측정됨으로써, arfI 생산 α-arabinofuranosidase는 trimer 인 것으로 확인되었으며 N-말단 아미노산 서열은 X-Ser-Thr-Ala-Pro-Arg(?)-Ala-Thr-Met-Val-Ile-Asp-X-Ala-Phe으로 결정되었다. α-Arabinofuranosidase was produced by E. coli HB101 haboring the recombinant plasmid pKMG11 which contained the arfI gene of Bacillus stearothermophilus. The maximum production of the enzyme was observe when E. coli HB101 cells were grown at 37℃ for 20 hours in the medium containing 0.5% arabinose, 1.0% tryptone, 0.5% yeast extract, and 1% NaCl. The α-arabinofuranosidase produced was purified to homogeneity using a combination of 20∼50% ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion exchange column chromatography and Sepharose 61B-100 gel filtration. The purified enzyme was most active at 55℃ and pH 6.5. The K_m and V_max values of the enzyme on ρ-nitrophenyl-α-arabinofuranoside was determined to be 2.99 mM and 0.43 μ㏖e/min (319.74 μ㏖e/min/㎎), respectively. The pI value was 4.5. The molecular weight of the native protein was estimated to be 289 kDa. The SDS-polyacrylamide gel electrophoresis analysis suggested that the functional protein was a trimer of the 108 kDa identical subunits. The N-terminal amino acid sequence of the α-arabinofuranosidase was identified as X-Ser-Thr-Ala-Pro-Arg(?)-Ala-Thr-Met-Val-Ile-Asp-X-Ala-Phe.

Bacillus stearothermophilus Acetyl Xylan Esterase 유전자의 크로닝과 Escherichia coli에서의 발현

김인숙,조쌍구,최용진 한국산업미생물학회 1993 한국미생물·생명공학회지 Vol.21 No.6

토양에서 분리한 강력한 xylan 분해 균주인 Bacillus stearothermophilus의 HindIII 절단 genomic DNA 단편을 pBR322에 크로닝하여 약 4000개의 E. coli HB101 형질전환체를 얻었으며 이 중 한 개의 형질전환체가 tributyrin 분해능을 나타내었다. 본 연구에서는 상기 형질전환체를 선별, tributyrin 분해 활성 관련 유전자를 조사 분석하였던 바 본 형질전환체가 tributyrin 분해능과 관련된 약 5.1 kb의 외래 DNA가 삽입된 재조합 plasmid를 가지고 있음을 확인하고 이 plasmid를 pKMG5로 명명하였다. 다음 5.1 kb 삽입 DNA 상의 restriction site를 결정하고 subcloning을 실시하여 높은 tributyrin 분해 활성을 나타내는 새로운 재조합 균주로부터 3.5 kb의 외래 DNA를 가지는 pKMG6를 분리하였다. 또한 pKMG6에 삽입된 tributyrin 분해 활성 관련 상기 3.5 kb 외래 DNA는 Southern blotting 실험 결과, B. stearothermophilus chromosome 유래의 DNA 단편임을 확인할 수 있었으며 동시에 삽입 DNA 단편상의 tributyrin 분해 활성 관련 유전자의 발현 산물이 일종의 acetyl xylan esterase로 분류할 수 있는 xylan 분해계 효소임을 알았다. Bacillus stearothermophilus was shown to express multiple xylanolytic enzymes including acetyl xylan esterase. Genomic DNA of the strain partially digested with HindIII was ligated into theh HindIII site of pBR322, and expressed in E. coli HB010 cells in order to clone the gene for acetyl xylan esterase. One transformant among 4000 screened formed a clear zone around its colony on the LB agar supplemented with 1.0% tributyrin. The functional clone harbored the recombinant plasmid pKMG5 with an insert of 5.1 kb. Subcloning yielded the recombinant molecule pKMG6 with 3.5 kb HindIII fragment derived from the B. stearothermophilus chromosomal DNA as determined by restriction enzyme analysis and Southern hybridization. The tributyrin degrading activity produced by the cloned gene also revealed acetyl residue cleavage activity on oat spelt acetyl xylan. The results of substrate specificity confirmed that the esterase from E. coli HB101/pKMG6 was an acetyl xylan esterase.

Bacillus stearothermophilus로부터 α-L- Arabinofuranosidase 유전자의 클로닝 및 Escherichia coli에서의 발현

엄수정,김희선,조쌍구,최용진 한국산업미생물학회 1994 한국미생물·생명공학회지 Vol.22 No.6

강력한 xylan 분해 균주인 Bacillus stearothermophilus의 EcoRI 절단 genomic library, pBR322 플라스미드 및 E. coli HB101 숙주세포 등을 이용하여 B. stearothermophilus의 α-arabinofuranosidase 생산유전자(arfI)를 분리, 클로닝하였다. arfI 유전자 클론의 분리는 이 유전자 생산효소의 높은 p-nitrophenyl-α-L-arabinofuranoside 분해 활성에 의한 황색 색소형성 현상을 활용하였으며 약 10,000개의 형질전환체를 검색하여 한개의 형질전환체가 arfI 유전자를 포함하고 있는 5 kb 외래 DNA가 삽입된 재조합 플라스미드(pKMGll)을 가지고 있는 것으로 분석되었다. Southern blotting과 zymogram 기술을 이용하여, 상기 arfI 클론이 B. stearothermophilus chromosome으로부터 유래된 매우 높은 기질특이성을 가진 α-arabinofuranosidase 생산 유전자임을 확인함과 동시에 B. stearothermophilus는 arfI 효소 외에도 이보다 분자량도 크고 효소 활성도 높은 최소한 두개 이상의 다른 α-arabinofuranosidase 관련 효소를 생산하고 있음을 발견하였다. The Bacillus stearothermophilus arfI gene encoding α-arabinofuranosidase was isolated from the genomic library, cloned into pBR322, and subsequently transferred into the Escherichia coli HB101. The recombinant E. coli was selected from approximately 10,000 transformants screened by making use of its ability to produce a yellow pigment around the colony on the selective medium supplemented with p-nitrophenyl-α-L-arabinofuranoside (pNPAf), a chromogenic substrate. The functional clone was found to harbor a recombinant plasmid, pKMG11 with an insertion of about 5 kb derived from the B. stearothermophilus chromosomal DNA. Identity of the arfI gene on the insert DNA was confirmed by a zymogram with 4-methylumbelliferyl-α-L-arabinofuranoside as the enzyme substrate. The α-arabinofuranosidase from the recombinant E. coli strain showed very high substrate specificity; the enzyme displayed high activity only with pNPAf among many other p- or o-nitrophenyl derivatives of several sugars, and acted only on arabinoxylan among various natural arabinose containing polysaccharides tested.