• 한국미생물·생명공학회지
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• 제24권3호
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• pp.336-343
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• 1996

Maximum amount of the aminopeptidase M inhibitors produced by Streptomyces griseoplanus SL20209 in 500 ml-Erlenmeyer flask was accumulated after cultivation for 3 days at 28$\circ$C, thereafter the amount of inhibitors decreased slowly with a pH change to alkaline. Arabinose, xylose, mannose and soluble starch were good carbon sources for the production of the inhibitors. On the other hand, glucose was only good for the cell growth but potently inhibited the production of inhibitors. Natural organic nitrogen sources such as soybean meal, fish meal, gluten meal and peanut powder were good for the production of inhibitors, however, soytone, peptone and inorganic nitrogens such as NH$_{4}$C1 and NH$_{4}$NO$_{3}$ were poor. Inclusion of yeast extract (0.5%, w/v) or K$_{2}$HPO$_{4}$ (0.05%) into the production medium increased the production of inhibitors by accelerating cell growth. The production of inhibitors was slightly increased on the medium containing CaCO$_{3}$ (0.3%) and zeolite (0.5%), respectively. Optimal temperature and initial pH range for the production ot inhibitors were determined to be 28$\circ$C and 6.0-7.0, respectively. Employing two improved production media consisting of 3% arabinose or soluble starch, 2.5% soybean meal, 0.5% yeast extract, 0.05% K$_{2}$HP0$_{4}$, 0.1% CaCO$_{3}$ and 0.3% zeolite (pH 6.8), 1.8-fold increase in the amount of inhibitors was achieved, comparing with the basal medium used.

• 한국미생물·생명공학회지
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• 제23권1호
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• pp.47-52
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• 1995

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 $\mu$g/ml) than that of the wild type(6.7 $\mu$g/ml). This mutant was resistant to 3,4-dehydro-DL-proline, an antimetabolite of proline, with 25 $\mu$g/ml 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 nhibitor 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-phenylbutanoic acid. IC$_{50}$ value (89.1 $\MU$g/ml) 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.

• Journal of Microbiology and Biotechnology
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• 제6권1호
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• pp.7-11
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• 1996

MR-387Al (ARPA-Val-Pro) and A2 (AHPA-Val-Hyp) were prepared as aminopeptidase N inhibitors through the synthesis of peptide MR-387A and B analogues which contained 3-amino-2-hydroxy-4-phenyl butanoic acid (ARPA) as a zinc-chelating moiety. They are competitive inhibitors of aminopeptidase N with inhibition constants(Ki) of 4.1 $\times 10^{-7}\;and 1.1 \times 10^{-6}$ M, respectively. MR-387Al also strongly inhibited aminopeptidase B of human myelogenous leukemia K-562 cell with $IC_50$ of 0.35 $\mu$ M. Inhibitions of aminopeptidase N activity by ARPA-bearing inhibitors of various peptide chain lengths also have been studied. $IC_ 50$ values of AHPA-Val (bestatin), ARPA-Val-Pro (MR-387Al) and ARPA-Val-Pro-Leu (MR-387C) compared against porcine kidney aminopeptidase N were 20.1, 0.60 and 0.08 $\mu$ M, respectively. These results support that a multiple interaction between the $S_1\to S'_3$ sites of aminopeptidase N and the $P_1\to P'_3$ of the inhibitor plays a crucial role in stabilizing strongly the enzyme-inhibitor complex.

• 한국미생물·생명공학회지
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• 제24권1호
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• pp.1-8
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• 1996

Characterization and numerical identification were carried out for an actinomycetes SL20209. Morphological, cultural and physiological perperties of SL20209 which porduced valistatin and des-$asp^4$-amastatin as inhibitors of aminopeptidase M were evaluated. The isolate was identified to be the genus of Streptomyces. Fourty-three taxonomic units were analysed by using a TAXON program. The isolate was classified into the major cluster 29 of Streptomyces and best-matched to Streptomyces griseoplanus.

• BMB Reports
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• 제28권1호
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• pp.83-86
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• 1995

In the course of screening for new aminopeptidase M inhibitors which were expected to be analgesic, immunopotentiating, or anti-metastatic agents, the novel synthetic substance MR-387C[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl-L-prolyl-L-leucine] (M.W. 504 daltons) was obtained. It was competitive with the substrate and had an $IC_{50}$ value of $0.04\;{\mu}m/ml$ ($7.9{\times}10^{-8}\;M$) and an inhibition constant ($K_i$) of $3.8{\times}10^{-8}\;M$. This novel MR-387C was compared with various known inhibitors of aminopeptidase M. It inhibited the enzyme more strongly than any other microorganism-originated inhibitor, except probestin.

• 한국미생물·생명공학회지
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• 제22권5호
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• pp.447-452
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• 1994

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.

• Applied Biological Chemistry
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• 제38권2호
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• pp.100-105
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• 1995

토양으로부터 분리한 Streptomyces sp. SL-387 균주에 의하여 신규 aminopeptidase M 저해제 MR-387A 및 B를 생산하기 위한 최적 배지 조건을 검토하였다. 최적 배지 조건으로 glucose 1%, soybean meal 3%, yeast extract 0.2%, beef extract 0.1%, NaCl 0.3%, $K_2HPO_4$ 0.01%, $CaCO_3$ 0.03%, $MnCl_2{\cdot}4H_2O$ 0.001%, $ZnCl_2{\cdot}7H_2O$ 0.001%, $MgSO_4{\cdot}7H_2O$ 0.0005%, pH 7.0가 적당한 것으로 나타났다. 이 배지를 발효용 배지로 사용하였을때 저해제의 생산은 배양 120시간에 최대에 도달하였으며, 그때의 최대 생산성(total productivity)은 909.1 U/ml이었다.

• Applied Biological Chemistry
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• 제38권3호
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• pp.196-201
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• 1995

토양으로부터 분리한 신규의 aminopeptidase M 저해제 MR-387A 및 B를 생산하는 균주 SL-387의 화학동정 및 수리동정을 하였다. 배양학적 및 형태학적 특성과 화학지표에 의하여 분리균주는 Streptomyces에 속하는 것으로 판단되었다. 종의 동정을 위하여 41개 동정 단위형질에 대하여 조사한 후 TAXON 프로그램을 사용하여 수리동정을 하였다. 분리균주는 Streptomyces의 주군집 18에 속하는 균주로 Streptomyces eyagawaensis와 공유도계수($S_{SM}$) 75.6%로 가장 유사 하였다. 이상의 화학동정 및 TAXON 분석에 의하여 분리균주 SL-387은 Streptomyces neyagawaensis이거나 그 유연 균주인 것으로 동정 되었다.

• Journal of Microbiology and Biotechnology
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• 제5권4호
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• pp.213-217
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• 1995

The effect of inorganic phosphate on the fermentative production of aminopeptidase M inhibitors MR-387A and B by Streptomyces sp. SL-387 has been studied. With inorganic phosphate concentrations higher than 0.78 mM, an inverse correlation was found between the maximum inhibitor production and the initial phosphate concentration added. Growth sensitivity of this actinomycete to arsenate, a phosphate analogue, and the use of magnesium carbonate, a phosphate-trapping agent, suggested that the inhibitor formation was under phosphate repression. Exogenous ATP further increased the degree of phosphate interference in both phosphate-repressed and non repressed culture conditions. The use of a phosphate analogue and a protein synthesis inhibitor also suggested that the phosphate itself repressed inhibitor formation.

• Journal of Microbiology and Biotechnology
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• 제5권3호
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• pp.158-162
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• 1995

The effect of carbon and nitrogen sources on the production of novel aminopeptidase M inhibitors MR-387A and B by Streptomyces sp. SL-387 has been studied. High D-glucose and ammonia concentrations (5$\%$ and 1$\%$, respectively) exerted a negative influence on the inhibitor formation. The suppressive effect of glucose on the inhibitor formation is probably caused by an effect of medium pH rather than that of cyclic AMP. To establish the optimum conditions for inhibitor overproduction, various nitrogen sources and ammonium ion-trapping agents were examined. The use of ammonia slow-releasing nitrogen sources such as soybean meal and fish meal, or ammonium ion-trapping agents such as kaoline, celite, and natural zeolite achieved the enhancement of inhibitor production. These results also indicate that inhibitor formation is affected by ammonium ion repression.