Serratia marcescens 로 부터 5 가지 Chitinase Isozymes 의 동정

황재령,갈상완,이경애,신용철,조무제,이상열 ( Jae Ryoung Hwang,Sang Wan Gal,Kyeong Ai Lee,Yong Chul Shin,Moo Je Cho,Sang Yeol Lee ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.3

S. marcescens KCTC2172 produced five extracellular chitinases that were involved in chitin degradation. These enzymes were identified as chitinase isozymes by substrate activity staining in SDS-PAGE gel after removal of SDS, because chitinases were resistant to SDS and existed as monomeric proteins. Their apparent molecular weights obtained by SDS-PAGE were 58, 47, 43, 37, and 21 kd. The chitinase activities were induced about seven folds by the addition of 1.5% chitin to LB-medium compared with those obtained without chitin, and the induction was dependent on the various forms of chitin. Chitinases were purified using the regenerated chitin column by successive pH changes of elution buffer. When the column was eluted with enough volume of pH 3.3 buffer, five chitinase isozymes were eluted. However, three of them whose molecular weights were 58, 47 and 43 kd were further eluted by the pH changes of elution buffer from pH 3.3 to 2.0. Considering the pH dependent stability of chitinases, it was known that S. marcescens produced two groups of chitinase isozymes; One is resistant and the other is very sensitive to acidic conditions, at pH below 3.3.

Klebsiella pneumoniae 로 부터 nif A Gene 의 크로닝 및 Azospirillum 에서의 발현

조무제,정명철,강규영,갈상완 ( Moo Je Cho,Myung Chul Chung,Kyu Young Kang,Sang Wan Gal ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.3

The nifA gene in the 3.0 kb SalI fragment from the nifA gene cluster of Klebsiella pneumoniae was cloned into BglII site of pRK290 and XhoI site of pVK100, a derivative of a wide host range plasmid RK2 (Inc P-1 group) and named pMK23 and pMK26, respectively. The constructed pMK23 and pMK26 carring nifA gene could be introduced into Azospirillum by transformation and conjugation with the helper plasmid pRK2013 and maintained stably in the host in constrast to some instability of RSF1010 (Inc Q group) or pSa (Inc W group)-derived vector. The nifA gene in the former recombinant nifA plasmid was expressed under the Tc^r gene promoter of pRK290 and the latter under the Km^r gene promoter of pVK100 in the Azospirillum host. The degree of derepression of the nitrogenase activity by the nifA expression from K. pneumoniae in the presence of 16 mM ammonia was about 18% in A. lipoferum (pMK23) and 10% in A. lipoferum (pMK26) compared to the nitrogenase activity in the absence of fixed nitrogen. Different level of MoFe-protein (nitrogenase component I) and Fe-protein (nitrogenase component II) synthesis between nifA plasmid introduced Azospirillum transconjugant and wild type were observed in the cells grown with the fixed nitrogen.

Cloning of nitA Gene from Klebsiella pneumoniae and Expression in Azospirillum

조무제,정명철,강규영,갈상완,Cho, Moo-Je,Chung, Myung-Chul,Kang, Kyu-Young,Gal, Sang-Wan 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.3

Klebsiella pneumoniae로부터 nifA 유전자를 크로닝하여 Azospirillum에 도입 발현시킴으로서 암모니아 비제어 질소고정을 유도하고자 하였다. Azospirillum 숙주에서 안정하게 복제 유지될 수 있는 vector로서 RK2 (IncP-1 group plasmid) 변형 vector인 pRK290과 pVK100를 선별하였으며 pKT230 (RSF1010 변형 vector, IncQ group) 및 pSa151(pSa 변형 vector, IncW group)는 불안정하였다. nifA 유전자 크로닝은 nifA 3.0 kb Sal1 절편을 pRK290의 Bgl II 위치에 그리고 pVK100의 Xho1 위치에 각각 크로닝하고 pMK23 및 pMK26으로 명명하였다. pMK23에서 nifA의 발현은 $Tc^r$ 유전자 promoter pMK 26에서는 $Km^r$ 유전자 promoter에 의하여 constitutive expression을 유도하였다. A lipoferum KY 6 에서는 16mM의 암모니아 첨가로 질소고정효소의 발현이 완전히 억제되었으나 A.lipoferum KY6 (pMK23) 및 A.lipoferum KY6 (pMK26)에서는 암모니아를 첨가하지 않았을 경우의 질소고정력의 18% 및 10%를 나타내므로써 K. pneumoniae nifA 유전자의 Azospirillum에서 발현이 확인되었다. 한편 암모니아 존재하에서의 질소고정 효소 합성도 전기영동 결과 확인되었다. The nifA gene in the 3.0 kb SalI fragment from the nifA gene cluster of Klebsiella pneumoniae was cloned into BglII site of pRK290 and XhoI site of pVK100, a derivative of a wide host range plasmid RK2(Inc P-1 group) and named pMK23 and pMK26, respectively. The constructed pMK23 and pMK26 carring nifA gene could be introduced into Azospirillum by transformation and conjugation with the helper plasmid pRK2013 and maintained stably in the host in constrast to some instability of RSF1010 (Inc Q group) or pSa (Inc W group)-derived vector. The nifA gene in the former recombinant nifA plasmid was expressed under the $Tc^r$ gene promoter of pRK290 and the latter under the $Km^r$ gene promoter of pVK100 in the Azospirillum host. The degree of derepression of the nitrogenase activity by the nifA expression from K. pneumoniae in the presence of 16mM ammonia was about 18% in A. lipoferum (pMK23) and 10% in A. lipoferum (pMK26) compared to the nitrogenase activity in the absence of fixed nitrogen. Different level of MoFe-protein (nitrogenase component I) and Fe-protein (nitrogenase component II) synthesis between nifA plasmid introduced Azospirillum transconjugant and wild type were observed in the cells grown with the fixed nitrogen.

Over-Expression of E. coli pro BA Genes in Azospirillum and Enhanced Osmotolerance

CHO, MOO JE,YANG, MIN SUK,CHOI, YOUNG JU,GAL, SANG WAN 경상대학교 유전공학연구소 1988 遺傳工學硏究所報 Vol.7 No.-

The proBA(pro-74) mutant genes which synthesize the less sensitive γ-glutamyl kinase, the first enzyme in proline proline biosynthesis, to the proline feedback inhibition than the wildtype was cloned into Bgl II and EcoR I site of pRK290, a derivative of a wide host range plasmid RK2(Inc. P-1 group), and named pCMK25a and pCMK30a, respectively. The former contains 5.0kb proBA(pro-74) Bgl II fragment and the latter contains 10.6kb proBA(pro-74) EcoR I fragment in pRK290 and each recombinant plasmid has two different types due to orientation of the fragment. The recombinant plasmid containing pro genes were introduced into A. lipoferum by conjugation with E.coil SM10 harboring the plasmid. The A. lipoferum KY6(pCMK25a) transconjugant produced about 10 folds of proline in the absence of NaCl and 25 folds in the presence of 0.4M NaCl than the A. lipoferum KY6 wildtype did. The inhibition of nitrogenase activity by NaCl was partially overcomed by the proline overproduction with the introduced proBA genes in A. lipoferum KY6.

Antifungal Activity of Serratia marcescens Chitinases against Phytopathogenic Fungi

CHO, MOO JE,GAL, SANG WAN,HWANG, JAE RYOUNG,SHIN, YONG CHUL,LEE, SANG YEOL 경상대학교 유전공학연구소 1990 遺傳工學硏究所報 Vol.9 No.-

The extracellular chitinase activities of Serratia marcescens were induced by the addition of 1.5% swollen chitin to LB-broth about seven folds compared to that obtained from the culture medium without chitin. S. marcescens was co-cultured with various phytopathogenic fungi, including Phytophtora parasitica, Botryosphaeria oxyaporium in an LB-agar medium containing 1.5% swollen chitin. Fungal hyphae grew rapidly outward from the center, however the hyphal extension of pathogenic fungi was significantly inhibited in a perimetric contract area with S. marcescens. Each of the culture supernatant of S. marcescens, E. coli, or the chitinase of Streptomyces griceus purchased from Sigma was incubated with the mycelium of F. oxysporium as a sole carbon source in a slide glass with single depressiom under the inverted microscope at 30℃. It was observed that the mycelium of F. oxysporium was gradually bursted as the cultivation time increased and completely lysed after incubation for 24 hrs by the chitinolytic activities of S. marcescens. From these data, it was concluded that the chitinolytic activites of S. marcescens play an important role in biocontrol of phytopathogenic fungi.

Expression of Proline Biosynthetic Genes(proBA) From E. coli In Tomato(Lycopersicon esculentum L.cv Ailsa Craig) Cells

CHO, MOO JE,GAL, SNAG WAN,KIM, JAE IM,CHOI, YOUNG JU 경상대학교 유전공학연구소 1989 遺傳工學硏究所報 Vol.8 No.-

Proline was observed to be a major osmoprotectant in such plants as rice soybean, tomato, tobacco and radish with 5 to 15 folds increase of proline accumulation in callus state under 300mM salt stress. Therefore the proBA(pro-74) mutant genes from E. coli which synthesize the less sensitive γ-glutamyl kinase, the first enzyme in proline biosynthetic pathway, to proline feed-back inhibition were attempted to introduce into plant cell in order to express the bacterial pro genes and construct salt tolerant plants. The 4.1 kb BgI II fragment of proBA(pro-74) genes were cloned into BgI II site of pGA580, a binary vector for plant cell transformation derived from Ti-plasmid and the recombinant plasmid containing proBA (pro-74) gene was named pGAP16. The proBA (pro-74) gene in pGA16 was expressed in E. coli CSH26 and Agrobacterium tumefaciens A281 under the control of nos promotor. Tomato protoplasts were transformed with A. tumefaciens A281 (pGAP16) or by electroporation with naked pGAP16 DNA. The transformed plant cells were screened with the kanamycin resistance first which originated from pGA580. The kanamycin sensitive wildtype calli of the tomato(Lycopersicon esculentum, L. ev. Ailsa Craig) could be survived in the media containing up to 1.4% NaCl but the kanamycin resistant transformants could be survived in the media containing up to 2.0% NaCl.

Identification of Five Chitinase Isozymes from S. marcescens

Hwang, Jae Ryoung,Gal, Sang Wan,Lee, Kyeong Ai,Shin, Yong Chul,Cho, Moo Je,Lee, Sang Yeol 慶尙大學校 기초과학연구소 1991 基礎科學硏究所報 Vol.7 No.-

SDS 존재하에서 전기영동을 한 후, substrate activity staining을 한 결과, S. marcescens는 5가지의 chitinase isozyme을 생성함을 알아내었다. 이 효소는 inducible enzyme으로서, 30℃에서 7일간 진탕배양하였을 때 1.5% chitin을 첨가한 LB-배지에서 얻은 효소활성이 chitin을 첨가하지 않은 배지에서 얻은 것보다 약 7배의 효소활성의 증가를 보였다. Chitinase를 최대로 생성시키는 조건에서 S. marcescens를 배양한 후 regenerated chitin을 이용한 친화성 chromatography를 행하여, 이 배양상등액으로부터 5가지의 chitinase isozyme를 분리하였으며 이들의 분자량이 58, 47, 43, 37 및 21 kd임을 확인하였다. 충분히 세척한 chitin column으로부터 chitinase를 elution시키는 방법으로, pH3.3인 완충액을 사용하였을 때, 5가지 chitinases가 모두 용출되었으며, pH를 2.0인 완충액으로 바꾸어 용출시켰더니 5가지 중 분자량이 58, 47, 43 kd인 3가지 chitinase가 더 회수되었다. 또한 Chitinases 의 pH에 대한 안정성을 실험한 결과 S. marcescens가 분비하는 chitinases는 산성조건에서 매우 불안전한 chitinases와 비교적 안정한 chitinase의 두가지 group이 존재함을 알 수 있었다. S. marcescescens KCTC2172 produced five extracellular chitinases that were involved in chitin degradation. These enzymes were identified as chitinase isozymes by substrate activity staining in SDS-PAGE gel after removal of SDS, because chitinases were resistant to SDS and existed as monomeric proteins. Their apparent molecular weights obtained by SDS-PAGE were 58, 47, 43, 37, and 21kd. The chitinase activities were induced about seven folds by the addition of 1.5% chitin to LB-medium compared with those obtained without chitin, and the induction was dependent on the various forms of chitin. Chitinases were purified using the regenerated chitin column by successive pH changes of elution buffer. When the column was eluted with enough volume of pH 3.3 buffer, five chitinase isozymes were eluted. However, three of them whose molecular weights were 58,47 and 43 kd were further eluted by the pH changes of elution buffer from pH 3.3 to 2.0 Considering the pH dependent stability of chitinases, it was known that S. marcescens produced two groups of chitinase isozymes; One is resistant and the other is very sensitive to acidic conditions, at pH below 3.3.