폴리비닐 알콜 분해균 Xanthomonas campestris J2Y의 Polyvinyl alcohol oxidase 정제 및 성질

권대준,조윤래,Kwoen, Dae-Jun,Jo, Youl-Lae 한국응용생명화학회 1996 Applied Biological Chemistry (Appl Biol Chem) Vol.44 No.3

생물학적 난분해성 물질인 폴리비닐 알콜(PVA : Polyvinyl alcohol)을 탄소원 및 에너지원으로 이용하는 Xanthomonas campestris J2Y로부터 PVA oxidase를 생산하여 정제하기 위하여 PVA가 탄소원으로 첨가된 배지에서 진탕배양한 배양액을 원심분리한 후 상등액을 10 mM phosphate buffer(pH 7.5)로 평형시킨 DEAE -cellulose를 통과시켜 얻은 분획을 사용하여 DEAE-cellulose와 Sephadex G-150을 이용한 Gel filtration을 통하여 PVA oxidase를 정제하였다. 정제된 PVA oxidase는 polyacrylamide gel 전기영동으로 단일밴드로 확인되었으며 SDS-polyacrylamide gel 전기영동과 Sephadex G-150 column chromatography를 통해 측정한 결과 55,000 daltons 이었다. PVA oxidase의 최적 pH는 7이고 최적온도는 $37^{\circ}C$였다. 열 안정성은 $50^{\circ}C$까지는 70% 이상의 안정성을 나타내었고, pH에 대한 안정성은 5-11에서 비교적 안정하였다. 금속이온에 대한 영향은 $Ag^{2+},\;Hg^{2+},\;Sn^{2+}$ 등에서는 아주 강한 저해를 받았고, $Co^{2+},\;Fe^{2+},\;Zn^{2+},\;Pb^{2+}$에서는 50% 정도의 저해를 받았다. 반면에 $Mn^{2+},\;Cu^{2+}$는 효소활성을 증가시켰다. PVA에 정제 PVA oxidase의 Km치는 $7.04{\times}10\;^2mmol/{\ell}$이었다. The Polyvinyl alcohol(PVA) oxidase involved in PVA degradation by microorganism has been purified to homogeneity from culture broth of Xanthomonas campestris J2Y grown in a minimal medium containing PVA as a sole carbon source. The enzyme was purified by DEAE-cellulose chromatograpy and Sephadex G-150 gel filtration. The purified PVA oxidase was electrophoretically homogeneous both in the absence and presence of SDS. The molecular weight of the enzyme was estimated to be about 55,000 daltons by SDS-polyacrylamide gel electrophoresis and Sephadex G-150 gel filtration. The native enzyme existed as a monomer. The optimal pH and temperature was shown to be pH 7 and $37^{\circ}C$ respectively. The activity of enzyme was stable below $55^{\circ}C$ and between pH range of $5{\sim}11$. The enzyme activity was significantly inhibited by metal compounds such as $Ag^{2+},\;Hg^{2+}$. While, metal ions such as $Mn^{2+},\;and\;Cu^{2+}$ stimulated the reaction. Km value of the enzyme for PVA was $7.04{\times}10^{-2}mmol/{\ell}$.

폴리비닐 알콜 분해균 Xanthomonas campestris J2Y 의 Polyvinyl alcohol oxidase 정제 및 성질

권대준(Dae Jun Kwoen),조윤래(Youl Lae Jo) 한국응용생명화학회 1996 Applied Biological Chemistry (Appl Biol Chem) Vol.39 No.5

The Polyvinyl alcohol(PVA) oxidase involved in PVA degradation by microorganism has been purified to homogeneity from culture broth of Xanthomonas campestris J2Y grown in a minimal medium containing PVA as a sole carbon source. The enzyme was purified by DEAE-cellulose chromatograpy and Sephadex G-150 gel filtration. The purified PVA oxidase was electrophoretically homogeneous both in the absence and presence of SDS. The molecular weight of the enzyme was estimated to be about 55,000 daltons by SDS-polyacrylamide gel electrophoresis and Sephadex G-150 gel filtration. The native enzyme existed as a monomer. The optimal pH and temperature was shown to be pH 7 and 37℃ respectively. The activity of enzyme was stable below 55℃ and between pH range of 5∼11. The enzyme activity was significantly inhibited by metal compounds such as Ag^(2+), Hg^(2+). While, metal ions such as Mn^(2+) and Cu^(2+) stimulated the reaction. Km value of the enzyme for PVA was 7.04×10^(-2)mmol/ℓ.

Bacillus sp. SSA3 균주의 Expression Vector 開發

조윤래,김종규,권대준 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.6

한국 재래식 된장·간장 발효균 Bacillus sp. SSA3 균주의 expression vector를 개발하기 위해 Bacillus sp. SSA3의 chromosomal DNA로부터 유전자의 promoter 부위를 cloning하였다. Recombinant plasmid를 제작하기 위해 Bacillus sp. SSA3의 chromosomal DNA을 HindⅢ로 절단한 단편을 pGR71 plasmid의 CAT gene과 pUC18 plasmid의 β-galactosidase gene의 전방에 삽입시킨 후, E. coli JM109에 형질 전환하였다. E. coli JM109의 chloramphenicol 내성 clones으로부터 6 recombinant plasmid를 선별하였다. 이들 선별된 plasmid는 Bacillus sp. SSA3와 E. coli JM109에서 promoter의 활성이 확인되었다. 그리고 실제 간장·된장 발효시 여러 농도의 염이 첨가되므로 이 선별된 재조합 plasmid를 Bacillus sp. SSA3의 expression vector로 사용시 각 재조합 plasmid 중에 삽입된 promoter의 염에 대한 영향의 정도를 확인하기 위해 10% NaCl이 첨가된 LB medium상에서 배양하였을 때, 이들 중 Bacillus sp. SSA3의 4 clones은 융합 CAT gene의 발현이 강하게 감소되었으나 2 clones은 약하게 저해되었다. The promoter regions from chromosomal DNA of Bacillus sp. SSA3 which is responsible for fermentation of Korean traditional soy sauce, were cloned for construction of expression vector of Bacillus sp. SSA3. Recombinant plasmids were constructed by insertion of HindⅢ-cleaved Bacillus sp. SSA3 chromosomal DNA fragments in front of the CAT gene of pGR71 plasmid and β-galactosidase gene of pUC18 plasmid. 6 recombinant plasmids were isolated from chloramphenicol resistant E. coli JM109 clones. All these plasmids were found to have promoter activity in Bacills sp. SSA3 and E. coli JM109. When these 6 clones of Bacills sp. SSA3 were cultivated in LB agar medium supplemented with 10% NaCl, fused CAT gene expression of 4 clones was significantly decreased in common. But the others were poorly inhibited.

Polyvinyl Alcohol 이용 공생균 Pseudomonas J2W 균주와 Xanthomonas J2Y 균주의 배양조건

조윤래 영남대학교 자원문제연구소 1992 資源問題硏究 Vol.11 No.-

In preceding studies, two strains Pseudomonos J2W and Xanthomonos J2Y which can utilize polyvinyl alcohol (PVA) as a sole carbon source were isolated from soil. PVA was utilized symbiotically by mixed culture of these two strains. The optimum culture conditions for the symbiotic PVA utilization of the strains were investigated. The optimum temperature and pH for growth of the mixed culture were 28-30℃ and 7.0, respectively. And then, when the agitation speed of the mixed culture of symbionts was increased, the degradation rate of PVA of theirs was increased. Ammonium sulfate, urea and peptone. were effective as nitrogen sources. and then, vitamin BH was appeared to have a significant influence on growth of the mixed culture of symbionts. When the symbionts were cultivated in the rich medium, the degradation rate of PVA of theirs was significantly decreased.

잡초성벼의 superoxide dismutase cDNA cloning과 재배벼로의 형질전환

박상규,박종석,이승인,서석철,김병극,조윤래,서학수 한국환경농학회 2002 한국환경농학회지 Vol.21 No.2

냉해나 한발등의 환경스트레스에 대해 저항성을 유발하는 유전자를 환경스트레스에 강한 잡초성벼로부터 선발하고 이들 유전자를 재배벼에 도입하여 도입유전자 산물의 과량 발현을 통해 냉해나 한발 등에 대한 저항성이 향상된 벼를 선발하고자 하였다. 잡초성벼인 Bhutan 14Ad로부터 냉해 및 한발 저항성 유전자로 알려진 superoxide dismutase (SOD) cDNA를 분리하고자 mRNA를 분리하고 이 분리된 mRNA를 이용해 reverse transcriptase PCR방법으로 SOD cDNA를 cloning 하였다. 그 결과 2종의 SOD cDNA가 cloning되어 SOD-A, SOD-B로 명명하였다. 이들 cDNA의 염기서열을 결정한 결과 이들은 아미노산 서열 상동성이 88.4%를 나타내었으며, SOD-A는 oryza sativa 계열의 Cu/Zn SOD유전자인 GenBank accession No. L36320와 99.3%로 동일하였으며, SOD-B는 accession No. D01000과 100% 동일하였다. 이들 SOD-A와 SOD-B cDNA를 재배벼인 낙동벼에 형질전환하여 형질전환체 벼를 선발하였으며, 이들 형질전환체 벼의 냉해저항성 및 한발저항성 검정을 통해 저항성이 향상된 형질전환체 벼를 선발하고 있다. Two different cDNA clones for superoxide dismutase (SOD) were isolated from an weedy rice variety (Oryza sativa, cv. Bhutanl4Ad) and were introduced into a cultivated rice variety (Oryza sativa, cv. Nakdong) in order to develop the environmental stress-resistant rice plants. Sequence analysis of the cloned cDNAs indicated that the deduced amino acid sequence of SOD-A is 88.4% identical to that of SOD-B. Furthermore, the nucleotide sequence of SOD-A is 99.3% identical to that of a Cu/Zn SOD gene of Oryza sativa (GenBank accession No. L36320). The nucleotide sequence of SOD-B was identical to that of the previously published SOD gene (Accession No. D01000). A cultivated rice variety, Nakdong-byeo, was transformed with chimenc SOD genes containing a actin promoter of rice and pin2 terminator using a particle bombardment technique. Transformed calli were selected on an selection medium containing phosphinothricin (PPT). Transgenic rice plants were regenerated from the PPT-resistant calli. PCR analysis with genomic DNAs from transgenic plants revealed that transgenes are introduced into rice genome.

폴리비닐 알콜 분해균주의 분리 및 특성

정선용,조윤래,조무환,김정목 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.1

폐수중의 PVA를 생물학적 처리방법에 의한 제거를 위해 자연계로부터 PVA를 분해할 수 있는 세균을 분리하였으며, 그 중 균 성장율이 높고 PVA를 강력하게 분해, 자화할 수 있는 G5Y 균주와 PW 균주를 선별하여 동정 및 생육 특성을 조사하였다. 분리된 두 균주 G5Y와 PW는 혼합배양에 의해서만 PVA를 분해,자화할 수 있으며, 두 균은 PVA 분해에 있어서 공생관계에 있음을 확인하였다. G5Y 균주와 PW 균주의 형태학적, 생화학적 등 제특성에 따라 각각 Pseudomonas cepacia와 Pseudomonas pseudomallei로 동정되었다. 이들 두 균주에 의한 PVA 분해에 있어서 PVA의 농도는 큰 영향을 미치지 않았으며, PVA 중합도에 따른 PVA 분해율도 큰 차이가 없었다. 또한 혼합균주가 접종량의 증가에 따라 균 성장 및 PVA 분해속도는 현저히 증가하였으며, 10% 접종시 48시간만에 80% 이상의 매우 높은 PVA 분해율을 나타내었다. Two strains of polyvinyl alcohol (PVA) utilizing bacteria were isolated from the waste water and soil. These strains, G5Y and PW, were able to utilize PVA symbiotically as a carbon source, but could not utilize PVA separately. In the mixed culture of these strains, 0.5 percent of PVA was almost completely degraded in 3 days. Effect of degree of PVA polymerization on the its utilization was examined, and there was no remarkable difference among three kind of PVA (PVA 500, 1500, and 2000). These bacteria were able to utilize PVA in the desizing waste water of factory as well as enrichment PVA medium. These strains, G5Y and PW, were identified as Pseudomonas cepacia and Pseudomonas pseudomallei, respectively, based on morphological and biological characteristics.

폴리비닐 알콜 분해균 Pseudomonas cepacia G5Y의 Polyvinyl alcohol oxidase 정제 및 특성

장대균,조윤래 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.2

폴리비닐 알코올(PVA : Polyvinyl alcohol) 분해균인 Pseudomonas cepacia G5Y 균주로부터 PVA oxidase를 생산하여 이 효소를 정제하기 위하여 PVA가 첨가된 배지에서 진탕배양한 배양액을 원심 분리한 후 ammonium sulfate를 가하여 얻은 침전물을 phosphate 완충용액에 녹여 조효소로 사용하여 DEAE-Cellulose와 Sephadex G-150을 이용한 gel filtration을 통하여 PVA oxidase를 정제하였다. 정제된 PVA oxidase는 polyacrylamide gel 전기영동을 통해서 단일밴드를 확인할 수 있었으며, SDS-polyacrylamide gel 전기영동을 통해 분자량을 측정한 결과 약 60,000 daltons이었다. 이 효소의 최적온도는 45℃였으며 최적 pH는 8.5였다. 열안정성은 55℃ 까지는 안정하였으나, 그 이상에서는 효소의 활성이 급격히 실활되었다. pH 안정성은 pH 6에서 10까지는 거의 100%의 잔존 활성을 나타내었다. 금속 이온에 대한 영향은 Ag^(2+), Hg^(2+) 등에 의해 강한 저해를 받았으나, Mg^(2+), Ba^(2-) 등에는 영향을 받지 않았다. 나머지 대부분의 금속이온에 의해서는 20∼50% 정도의 활성이 저해되었다. The Polyvinyl alcohol (PVA) oxidase is a key enzyme involved in degradation of PVA with PVA hydrolase. The PVA oxidase has been purified to homogeneity from the culture broth of PVA grown Pseudomonas cepacia G5Y strain by ammonium sulfate precipitation. DEAE-cellulose column chromatography, and Sephadex G-150 gel filtration. The molecular weight of the purified enzyme was estimated about 60,000 daltons by SDS-polyacrylamid gel electrophoresis. The enzyme is most active at 45℃ and at pH 8.5, and is stable below 55℃ and between pH 6 and 9. The enzyme activity was strongly inhibited by Ag^(2+) and Hg^(2+).

한국 간장·된장 발효균주 Bacillus sp. SSA3의 형질전환

朴鉉甲,趙閏來,金鍾奎,鄭善容 영남대학교 자원문제연구소 1993 資源問題硏究 Vol.12 No.-

A protoplast transformation system has been developed for Bacillus sp. SSA3 strain which is responsible for Korean soy sauce fermentation. Plasmid pGR71 DNA was introduced into Bacillus sp. SSA3 by this method. The protoplasts of B. sp SSA3 were prepared with treatment of 200Hgfm1 Iysozyme in SMMP buffer. Optimal temperature, pH and incubation time for its protoplast formation was 37℃,7.0 and 120min, respectively. The best transformation efficiency was obtained with cells cultured for 3 hours(OD574=0.5) , adding 10% (w/v) of PEG 4,000 and 300ng of plasmid DNA. Plasmids introduced by this method were stably maintained by 3. sp. SSA3.

Pseudomonas sp. PW와 Pseudomonas sp. G5Y에 의한 Polyvinyl Alcohol의 공생적 이용

정선용,권대준,조윤래 영남대학교 자원문제연구소 1997 資源問題硏究 Vol.16 No.-

Properties and roles of fgrudouθne5 sp. GSY and Pseudomonas sp. PW in polyvinyl alcohol (PVA) utilization were studied. It was shown that PVA was utilized symbiotically by these strains which could not grow in each respective pure culture. When the mixture of these bacteria were subcultured on the rich medium or other enrichment medium except PVA and sodium acetate as a carbon source, their PVA utilizing activity was lost. Thus, it had been found that carbon sources of medium effect maintenance of PVA utilizing activity. In the case of Pyrroloquinoline quinone(PQQ) addition to PVA mdium, Pseudomonas sp. G5Y strain was able to degrade PVA in the pure culture. PQQ was effective as growth factorf or PVA utilization of Pseudomonas sp. G5Y strain.