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신규의 Aminopeptidase M 저해제 MR - 387A 및 B 생산균주의 수리동정
정명철(Myung Chul Chung),박동진(Dong Jin Park),김창진(Chang Jin Kim),김수일(Su Il Kim),고영희(Yung Hee Kho) 한국응용생명화학회 1995 Applied Biological Chemistry (Appl Biol Chem) Vol.38 No.3
Chemo- and numerical taxonomic studies on the isolate SL-387 producing novel aminopeptidase M inhibitors MR-387A and B were carried out. The genus of the isolate was determined as Streptomyces by cultural and morphological data and chemical indices. Forty one taxonomic unit characters were tested for determining the species of the isolate, and the data were analyzed numerically using a computer program as called TAXON. The isolate was best matched to Streptomyces neyagawaensis in the major cluster 18 of Streptomyces with S_(SM) value of 75.6%. On the base of chemotaxonomic data and TAXON analysis, the isolate SL387 was identified to be a member of Streptomyces neyagawaensis.
정명철,전효곤,이호재,고영희,Chung, Myung-Chul,Chun, Hyo-Kon,Lee, Ho-Jae,Kho, Yung-Hee 한국미생물 · 생명공학회 1994 한국미생물·생명공학회지 Vol.22 No.5
The strain SL-387 which produces new inhibitors of aminopeptidase M, MR-387A and B, was isolated from a soil sample. The strain has branched substrate mycelia, from which aerial hyphae develop in the form of open spirals. Spore surface is smooth. Melanoid and soluble pigme- nts were observed. The isolate contains LL-diaminopimelic acid in its cell wall hydrolysate, and has no pectinolytic activity. The strain SL-387 is closely related to Streptomyces griseoruber and S. naganishii, but is different from these strains in some cultural and physiological characteristics. This strain was, therefore, designated as Streptomyces sp. SL-387. The effects of several carbon and nitrogen sources on the production of the inhibitor were examined. Among them, glucose, galactose, mannose, and xylose were effective as a carbon source and soybean meal, soytone, fish meal, and gluten meal were effective as a nitrogen source. The maximum peak of the inhibitor production in jar fermentor was obtained on the fifth day of culture.
Streptomyces sp . SL - 387 에 의한 Aminopeptidase M 저해제 MR - 387A 및 B 의 생산 배지 최적화
정명철(Myung Chul Chung),전효곤(Hyo Kon Chun),이호재(Ho Jae Lee),이충환(Choong Hwan Lee),김수일(Su Il Kim),고영희(Yung Hee Kho) 한국응용생명화학회 1995 Applied Biological Chemistry (Appl Biol Chem) Vol.38 No.2
Media optimization for the production of MR-387A and B, novel aminopeptidase M inhibitors by Streptomyces sp. SL-387 isolated from a soil was studied. Optimized medium was consisted of 1% glucose, 3% soybean meal, 0.2% yeast extract, 0.1% beef extract, 0.3% NaCl, 0.01% K₂HPO₄, 0.3% CaCO₃. 0.001% MnCl₂·4H₂O, 0.001% ZnCl₂·7H₂O, and 0.0005% MgSO₄·7H₂O, and adjusted to pH 7.0 before autoclaving. When the optimized medium was used as a fermentation medium, maximum productivity of MR-387 was reached at 120 hours of fermentation, and total productivity was 909.1 U/㎖.
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.
신규의 Aminopeptidase M 저해제 MR-387A와 B를 생산하는 균주의 동정 및 저해제의 생산
정명철,전효곤,이호재,고영희 한국산업미생물학회 1994 한국미생물·생명공학회지 Vol.22 No.5
Aminopeptidase M에 대하여 강한 저해활성을 보이는 물질 MR-387A 및 MR-387B를 생산하는 균주 SL-387을 분리하고 동정하였다. 이 균주는 포자낭이 보이지 않고 기균사는 나선형이며 포자의 표면은 평활(smooth)형이었다. 멜라닌 색소 및 용해성 색소를 분비하였고, 단백질 및 전분 분해력이 강한 편이었다. 세포벽 성분중 diaminopimelic acid는 LL-형이었으며 펙틴 분해능은 없었다. 이 균주는 Streptomyces griseoruber 및 S. naganishi와 유사하였으나 몇몇 배양학적 특성 혹은 생리적 특성이 달라 Streptomyces sp. SL-387로 명명하고, 한국과학기술연구원 유전공학연구소 유전자원센타에 기탁하였다(KCTC0102BP). 저해제생산은 탄소원으로 glucose, galactose, mannose, xylose 등이, 질소원으로 soybean meal, soytone, fish meal, gluten meal 등에서 높게 생산되었으며, Jar fermentor에서 배양시 5일만에 가장 높은 저해 활성을 보였다. The strain SL-387 which produces new inhibitors of aminopeptidase M, MR-387A and B, was isolated from a soil sample. The strain has branched substrate mycelia, from which aerial hyphae develop in the form of open spirals. Spore surface is smooth. Melanoid and soluble pigments were observed. The isolate contains LL-diaminopimelic acid in its cell wall hydrolysate, and has no pectinolytic activity. The strain SL-387 is closely related to Streptomyces griseoruber and S. naganishii, but is different from these strains in some cultural and physiological characteristics. This strain was, therefore, designated as Streptomyces sp. SL-387. The effects of several carbon and nitrogen sources on the production of the inhibitor were examined. Among them, glucose, galactose, mannose, and xylose were effective as a carbon source and soybean meal, soytone, fish meal, and gluten meal were effective as a nitrogen source. The maximum peak of the inhibitor production in jar fermentor was obtained on the fifth day of culture.
Aminopeptidase M 저해제를 생산하는 Streptomyces sp. SL-387 (KCTC 0102BP) 변이주의 특성
정명철,전효곤,이호재,이충환,고영희 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.1
Streptomyces sp. SL-387 균주가 생산하는 신규의 aminopeptidase M 저해물질 MR-387A와 B의 생산성은 극히 적어, 이들 물질의 생산성 제고를 위하여 N-methyl-N'-nitro-N-nitrosoguanidine로 돌연변이를 유발시켜 N-3 균주를 선별하고 특성을 조사하였다. 변이주 N-3는 MR-387의 생산량이 wild type보다 6배 가량 높았으며, proline의 antimetabolite인 3,4-dehy-dro-DL-proline에 저항성을 나타냈다(MIC 25 ㎍/㎖). 특히 이 균주는 포자사슬이 나선형인 wild type과는 달리 직선형으로 형태적 변이가 일어났음을 관찰할수 있었다. 한편 N-3 균주의 배양액으로부터 aminopeptidase M 저해물질을 분리하고 구조분석을 실시하였다. 이 물질의 aminopeptidase M에 대한 저해활성은 IC_50 값이 89.1 ㎍/㎖로 다른 저해제들보다 낮았다. 이는 물질의 구조내에 3-amino-4-phenylbutanoic acid moiety가 있어 저해활성을 나타내는 2(S)-hydroxyl group이 구조 내에 없기 때문일 것으로 추론할 수 있었다. Since the original productivity of new aminopeptidase M inhibitors MR-387A and B by Streptomyces sp. SL-387 (KCTC 0102BP) was not enough for further chemical and biological evaluation, mutation of parent strain by the treatment of N-methyl-N'-nitro-N-nitrosoguanidine was performed in order to obtain a clone with greater inhibitory activity. Mutant N-3 was selected due to a 6-fold greater productivity (40 ㎍/㎖) than that of the wild type(6.7 ㎍/㎖). This mutant was resistant to 3,4-dehydro-DL-proline, an antimetabolite of proline, with 25 ㎍/㎖ of minimum inhibitory concentration. Furthermore, the characteristic morphological change from spiral spore chain in wild type to straight in mutant was observed. An aminopeptidase M inhibitor different from MR-387A and B was isolated from the culture broth of the mutant. This inhibitor was composed of 2 proline, 1 valine, and an unknown amino acid which is presumably 3-amino-4-phenylbuta-noic acid. IC_50 value (89.1 ㎍/㎖) of the purified inhibitor was lower than that of other inhibitors, which may be due to the absence of 2(S)-hydroxyl group within the structure of 3-amino-4-phenyl-butanoic acid.