• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.356-365
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• 2004

In analyzing the region of the Amycolatopsis mediterranei S699 chromosome responsible for the biosynthesis of the ansamycin antibiotic rifamycin, we identified a gene, designated orj0, which is located immediately upstream of the rifamycin polyketide synthase (PKS). Orj0 encodes a protein, on the basis of sequence-comparative analysis, that is similar to several cytochrome P450 monooxygenases from different sources. The rifamycin producer, A. mediterranei, predominantly produces rifamycin B from its macrocyclic intermediate, proansamycin X, through dehydrogenation and hydroxylation steps. However, an A. mediterranei strain, deleted in orj0 by gene replacement, no longer produced rifamycin B. Furthermore, a versatile replicative vector in A. mediterranei was constructed and rifamycin B production was restored in a complementation experiment of orj0 using this novel vector. These consecutive results verified that the arf0 protein, which is a P450 hydroxylase, is required for the production of rifamycin B in A. mediterranei.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.613-619
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• 2003

The polyketide backbone of rifamycin B is assembled by the type I rifamycin polyketide synthase (PKS) encoded by the rifA-rifE genes. In order to produce novel analogs of rifamycin via engineering of the PKS genes, inactivation of the ${\beta}-ketoacyl:acyl$ carrier protein reductase (KR) domain in module 8 of rifD, by site-specific mutagenesis of the NADPH binding site, was attempted. Module 8 contains a nonfunctional dehydratase (DH) domain and a functional KR domain that is involved in the reduction of the ${\beta}-carbonyl$ group, resulting in the C-21 hydroxyl of rifamycin B. This mutant strain produced linear polyketides, from tetraketide to octaketide, which were also produced by a rifD-disruption mutant as a consequence of premature termination of the polyketide assembly. Another attempt to replace the DH domain of module 7, which has been considered nonfunctional, with a functional homolog derived from module 7 of rapamycin-producing PKS also resulted in the production of linear polyketides, including the heptaketide intermediate and its precursors. Premature release of the carbon chain assembly intermediates is an unusual property of the rifamycin PKS. that is not seen in other PKSs such as the erythromycin PKS.

• Journal of Microbiology and Biotechnology
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• 제1권1호
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• pp.70-74
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• 1991

The rheological properties of rifamycin B fermentation broth using Nocardia mediterranei were characterized in concomitance with the chemical changes of the fermentation broth. The data were interpreted with various rheological models. As results, it was found that the rheological behaviour at the early growth phases of the culture was the Newtonian fluid, but it was changed to the non-Newtonian fluid (Casson plastic behaviour) at the later phases of the culture. Rheological parameters viz., apparent viscosity ($\mu_a$), yield shear stress ($\tau_o$), and Casson constant ($K_c$) were changed through the fermentation where cell concentrations were changed and mycelia were greatly branched. Those results indicated that cell concentration and cell morphology played important roles to determine the rheological characters.

• 한국미생물·생명공학회지
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• 제9권4호
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• pp.225-230
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• 1981

The kinetic study of rifamycin B production in batch culture of Nocardia mediterranei was undertaken in part of our endeavor to optimize the fermentation condition. The growth parameters such as $\mu$$_{m}$ and Ks values for nitrogen source were evaluated by employing Monod equation. From the experiments, $\mu$$_{m}$ and Ks were 0.15hr$^{-1}$ and 8.35g/1, respectively. The growth kinetics in batch culture was found successfully interpreted by logistic law, i.e., the initial specific growth rate and the maximum cell mass concentration were determined as function of pH and both found to have maxima. For the production of rifamycin B, a non-growth associated production kinetics was employed and the specific productivity as a function of pH was found to have two maximum points. The yield coefficient and the specific productivity were calculated as mean values in production phase. Utilizing these experimental data as a function of pH, the optimal condition for the rifamycin B production was discussed with regad to the pH effect on the cell growth and production of the antibiotic. As a result, growth phase at pH 6.5 and production phase at pH 7.0 were found to be recommended.ded.

• 한국미생물·생명공학회지
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• 제13권2호
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• pp.115-118
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• 1985

리파마이신 B 산화효소의 활성을 갖고 있는 Humicola spp. (ATCC 20620)를 아세톤 처리 한 후 cellulose acetate로 고정화하여 그 특성을 조사하였다. 고정화 효소에 있어서 pH는 7.2온도는 50-55%에서 최대 활성을 보였으며, 비고정화 효소에 비하여 pH와 온도변화에 덜 민감하였다. 외경 3mm bead size에서 20%의 활성회수율을 보였으며, storage stability는 4$0^{\circ}C$ 이하에서는 아주 좋았으며 5$0^{\circ}C$ 이상에서는 급격히 활성이 감소하였다. 고정화 담체로서 cellulose acetate는 물리적으로 견고하여 삼상반응기의 packing material로써 적당하였다.

• 한국미생물·생명공학회지
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• 제20권4호
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• pp.470-476
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• 1992

Nocardia mediterranei NCK-4를 동결건조시 생존도에 미치는 영향을 검토하기 위해 보호물질의 종류와 농도, cell 농도 및 건조시간에 대해 실험한 data를 반응표면분석법을 활용하여 분석한 결과, 추정된 최대치의 생존도는 39.3이었으며, 이 최적 조건은 sucrose 11.6(v/v), cell 농도 $1.16{\times}10^{11}$(CFU/ml), 건조 시간 6.18hrs로 나타났다. N.mdeiterranei NCK-4를 동결건조하여 rehydration한 후 계대배양없이 starter cell로서 발효생산에 사용가능성을 검토한 결과, 전배양시 대조군인 FVM(frozen vegetative mycelium)과 거의 비슷하게 cell의 농도를 맞추어 발효함으로써 가능하다. 따라서 이 starter cell은 lyophilization 후 약 18개월간 저장하여 안정하게 사용할 수 있었다.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2002년도 학술발표대회
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• pp.18-25
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• 2002

The pikromycin biosynthetic system in Streptomyces venezuleae is unique for its ability to produce two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolides narbomycin and pikromycin. The metabolic pathway also contains two post polyketide-modification enzymes, a glycosyltransferase and P450 hydroxylase that have unusually broad substrate specificities. In order to explore further the substrate flexibility of these enzymes a series of hybrid polyketide synthases were constructed and their metabolic products characterized. The plasmid-based replacement of the multifunctional protein subunits of the pikromycin PKS in S. venezuelae by the corresponding subunits from heterologous modular PKSs resulted in recombinant strains that produce both 12- and 14-membered ring macrolactones with predicted structural alterations. In all cases, novel macrolactones were produced and further modified by the DesVII glycosyltransferase and PikC hydroxylase leading to biologically active macrolide structures. These results demonstrate that hybrid PKSs in S. venezuelae can produce a multiplicity of new macrolactones that are modified further by the highly flexible DesVII glycosyltransferase and PikC hydroxylase tailoring enzymes. This work demonstrates the unique capacity of the S. venezuelae pikromycin pathway to expand the toolbox of combinatorial biosynthesis and to accelerate the creation of novel biologically active natural products. The polyketide backbone of rifamycin B is assembled through successive condensation and ${\beta}$-carbonyl processing of the extender units by the modular rifamycin PKS. The eighth module, in the RifD protein, contains nonfunctional DH domain and functional KR domain, which specify the reduction of the ${\beta}$-carbonyl group resulting in the C-21 bydroxyl of rifamycin B. A four amino acid substitution and one amino acid deletion were introduced in the putative NADPH binding motif in the proposed KR domain encoded by rifD. This strategy of mutation was based on the amino acid sequences of the corresponding motif of the KR domain of module 3 in the RifA protein, which is believed dysfunctional, so as to introduce a minimum alteration and retain the reading frame intact, yet ensure loss of function. The resulting strain produces linear polyketides, from tetraketide to octaketide, which are also produced by a rifD disrupted mutant as a consequence of premature termination of polyketide assembly. Much of the structural diversity within the polyketide superfamily of natural products is due to the ability of PKSs to vary the reduction level of every other alternate carbon atom in the backbone. Thus, the ability to introduce heterologous reductive segments such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) into modules that naturally lack these activities would increase the power of the combinatorial biosynthetic toolbox. The dehydratase domain of module 7 of the rifamycin PKS, which is predicted to be nonfunctional in view of the sequence of the apparent active site, was replaced with its functional homolog from module 7 of rapamycin-producing polyketide synthase. The resulting mutant strain behaved like a rifC disrupted mutant, i.e., it accumulated the heptaketide intermediate and its precursors. This result points out a major difficulty we have encountered with all the Amycolatopsis mediterranei strain containing hybrid polyketide synthases: all the engineered strains prepared so far accumulate a plethora of products derived from the polyketide chain assembly intermediates as major products instead of just analogs of rifamycin B or its ansamycin precursors.

• Archives of Pharmacal Research
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• 제24권2호
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• pp.100-104
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• 2001

Differential pulse polarographic(DPP) analytical procedure for the rifampicin antibiotic, which can be applied to monitor its synthetic process from the starting antibiotic of rifamycin B or rifamycin SV has been developed based on the electrochemical reduction of an azomethine group. Rifampicin exhibited a cathodic peak due to the azomethine group in the side chain of 3-[(4-methyl-1-piperazinyl)imino]methyl moiety and another cathodic peak due to the carbonyl group in rifamycin SV by DPP. The experimental peak potential shift of an azomethine reduction was -73 mV/pH in the pH range between 3.0 and 7.5, agreeing with involvement of 4 e-and 5 $H^5$ in its reduction. By the cyclic voltammetric(CV) studies, the azomethine and the carbonyl reductions in rifampicin were processed irreversibly on the mercury electrode. The plot of peak currents vs. concentrations of rifampicin ranging $1.0{\times}10^{-7} M~$1.0{\times}10^{-5} M yielded a straight line with a correlation coefficient of 0.9996. The detection limit was $1.0{\times}10^{-8} M with a modulation amplitude of 50 mV DPP has been successfully applied for the determination of rifampicin in the pharmaceutical preparations.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.623-624
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• 2005

• 한국식품과학회지
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• 제34권1호
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• pp.73-78
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• 2002

본 연구에서는 식품용 프로바이토틱 생균인 비스루트균의 인공위액과 인공담즙산에 대한 내성, 효소활성, 항생물질과 알콜에 대한 내성, 그리고 병원성 세균에 대한 항균기작 등 주요 생균제 특성에 대하여 검토하였다. 인공위액 내성 실험에서는 pH 2.5로 조성된 인공위액을 통과한 생존율이 매우 높았으며 균수가 $2.4{\times}10^7\;CFU/mL$의 수준이었다. 인공담즙산에 대한 내성에서는 pH 2.5의 인공위액을 통과한 비스루트균의 인공담즙산의 내성은 oxgall이 함유되지 않은 대조구$(1.6{\times}10^7\;CFU/mL)$에 비해 오히려 균수가 증가되었으며, 최종적인 생존수가 $3.3{\times}10^7\;CFU/mL$ 으로 확인됨으로써 프로바이오틱 생균으로 매우 우수한 특성을 가지고 있었다. 효소활성을 측정한 결과, 비스루트균은 발암효소인 ${\beta}-glucuronidase$ 및 ${\beta}-glucosidase$ 의 활성이 나타나지 않았다. 또한 항생물질에 대한 내성은 rifamycin $10\;{\mu}g/mL$ 이상의 농도에 대해서는 생육 저해를 보였으나, $nisin(100\;{\mu}g/mL)$, $streptomycin(20\;{\mu}g/mL)$, $tetracycline(20\;{\mu}g/mL)$에 대해 내성을 보였다. 그리고 비스루트균의 알콜에 대한 내성은 4%의 농도에서 저해를 받았으며, 8%의 농도에서 더욱 저해를 받으나, $8{\sim}32%$ 범위의 농도에서는 균수가 더욱 크게 감소를 하지는 않았다. Listeria에 대한 향균기작을 측정한 결과, 24시간이내에 L. monocytogenes ATCC 19111을 완전히 저해를 하였으며, bactericidal한 양상을 나타내었다. 본 연구를 통해 밝혀진 비스루트균의 프로바이오틱 특성은 기능성 식품의 개발 등 산업적으로 매우 유용하다고 판단된다.