• 자연과학논문집
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• 제9권1호
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• pp.53-56
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• 1997

Cyanobacteria Chlorogloea fritschii를 여러 생장조건(인산 충분 혹은 결핍)에서 배양한 후 poly-$\beta$-hydroxybutyrate(PHB)를 추출하여 그의 구조를 적외선 분광법에 의해 분석하였다. 이들은 공통적으로 C=O의 신축 진동에 의한 1700-1800 $cm^-1$영역에서 대단히 강한 흡수 피이크를 나타내고 또한 2900$cm^-1$에서 C-H의 비대칭 및 대칭 신축 진동흡수 피이크를 나타냄으로써 PHB의 특징을 잘 나타내고 있었다. 그러나 생장조건에 따라 C-H의 신축 진동흡수 피이크 세기에 변화를 관측할 수 있었으며 인산 결핍 생장조건에서 C-H의 신축 진동흡수 피이크의 세기는 나머지 피이크에 비해 증가된 양상을 나타냄으로 인산의 공급여하에 따른 PHB구조의 변화를 시사하였다.

• Applied Biological Chemistry
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• 제34권3호
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• pp.273-278
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• 1991

Methanol 자화성 세균 Pseudomonas sp. ILS-003 균주를 이용하여 methanol로부터 PHB생산의 최적조건을 검토하였다. PHB 생산에 있어서 초기 pH 6.4, 온도 $30^{\circ}C$ 및 methanol 농도가 1.0(v/v)일 때 최적이었으며, 질소원으로는 $(NH_4)_2SO_4$가 최적이었으며 농도는 0.8g/l로서 C/N비가 17.4이었다. 또한 2가 금속이온의 결핍은 PHB축적효과를 나타내었다. Fed-batch culture에서 methanol 첨가의 효과는 0.25%(v/v)씩 첨가했을 때 가장 좋았다. 상기의 최적조건하에서 96시간 배양시 균체량은 2.78g/l였고 PHB의 양은 1.94g/l로서 건조균체량의 69.8%이었다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권5호
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• pp.281-288
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• 2002

Recombinant Escherichia coli strain harboring the ${\lambda}$pR-pL promotor and heterologus poly-${\beta}$-hydroxybutyrate (PHB) biosynthesis genes was used to investigate the effect of culture conditions on the efficient PHB production. The expression of phb genes was induced by a temperature upshift from $33^{\circ}C\;to\;38^{\circ}C$. The protein expression levels were measured by using two-dimensional electrophoresis, and the enzyme activities were also measured to understand the effect of culture temperature, carbon sources, and the dissolved oxygen (DO) concentration on the metabolic regulations. AcetylCoA is an important branch point for PHB production. The decrease in DO concentration lowers the citrate synthase activity, thus limit the flux toward the TCA cycle, and increase the flux for PHB production. Since NADPH is required for PHB production, the PHB production does not continue leading the overproduction of acetate and lac-tate. Based on these observations, a new operation was considered where DO concentration was changed periodically, and it was verified its usefulness for the efficient PHB production by experiments.

• Journal of Microbiology and Biotechnology
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• 제7권4호
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• pp.215-222
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• 1997

The regulatory mechanism of the flux of acetyl-CoA in Alcaligenes eutrophus in unbalanced growth conditions was investigated using a PHB-negative mutant and transformants reintroduced PHB-biosynthesis enzymes through the transformation of cloned phbCAB genes. The PHB-negative mutant was defected absolutly in PHB synthase but partially in ${\beta}$-ketothiolase and acetoacetyl-CoA reductase, and excreted substantial amount of pyruvate to culture broth at late growth phase. The excretion was due to the inhibitory effect of acetyl-CoA on the activity of pyruvate dehydrogenase. The cloned phbC and phbCAB genes were transformed to the PHB-negative mutant strain to reintroduce PHB biosythesis enzymes. Pyruvate excretion could be decreased substantially but not completely by transformation of PHB synthase alone, while pyruvate excretion was ceased by transformation of all three PHB biosynthesis enzymes. To identify the most critical PHB biosynthesis enzyme influencing on the flux of acetyl-CoA, the effect of the variation of PHB biosynthesis enzymes on pyruvate dehydrogenase was investigated. ${\beta}$-Ketothiolase influenced the activity of pyruvate dehydrogenase more sensitively than PHB synthase. ${\beta}$-Ketothiolase, the first step enzyme of PHB biosynthesis that condense acetyl-CoA to acetoacetyl-CoA, seems to be the major enzyme determining the flux of acetyl-CoA to PHB biosynthesis or TCA cycle, and the rate of PHB biosynthesis in A. eutrophus.

• Journal of Microbiology and Biotechnology
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• 제4권2호
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• pp.146-150
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• 1994

Production of poly-$\beta$-hydroxybutyrate (PHB) in fed-batch fermentation was studied. Utilization of carbon for PHB biosynthesis was investigated by using feeding solutions with different ratios of carbon to nitrogen (C/N). It was observed that at a high C/N ratio carbon source was more preferably utilized for PHB accumulation while its consumption for cellular metabolism appeared to be more favored at a low C/N value. A high cell concentration (184 g/l) was achieved when ammonium hydroxide solution was fed to control the pH, which was also utilized as the sole nitrogen source. For the mass production of PHB, two-stage fed-batch operations were carried out where PHB accumulation was observed to be stimulated by switching the ammonium feeding mode to the nitrogen limiting condition. A large amount of PHB (108 g/l) was obtained with cellular content of 80% within 50 hrs of operation.

• KSBB Journal
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• 제6권1호
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• pp.35-43
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• 1991

The production of poly-$\beta$-hydroxybutyrate(PHB) from methanol by batch and fed-batch cultivations of Methylobacterium sp. GL-10 was studied. PHB accumulation was stimulated by the nutrients deficiency including, NH4+, SO42-, and K+. The nitrogen deficiency was the most critical factor for PHB accumulation. In batch cultivation, the maximum cell concentration and PHB content were 1.86g/l and 0.62g/l, respectively, with 1.0%(v/v) of methanol and 0.5g/1 of ammonium sulfate. The mass doubling time of Methylobacterum sp. GL-10 was in the range of 4-5 hrs. The cell growth and PHB accumulation were severely inhibited at the methanol concentration over than 2% (v/v). To overcome methanol Inhibition, constant feeding and intermittent feedillg fed-batch cultivations were adopted, using C/N molar ratio as a control factor. In constant feeding fed-batch process, cell concentration was increased up to 2.67g/1, and PHB yield was enhanced from 0.33 of batch culture to 0.53. The relatively low cell concentration was caused by methanol accumulated in culture broth at late growth phase. To prevent methanol accumulation and to maximize PHB production, DO-state intermittent fed-batch cultivation was attempted. The cell and PHB concentration was reached up to 4.55g/1 and 1.80g/1, respectively. It was possible to maintain methanol concentration low and also to feed nutrient of desired C/N molar ratio.

• 폴리머
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• 제31권3호
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• pp.228-232
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• 2007

본 연구에서는 의료용 흡수성 재료 및 환경적합성 재료의 개발을 목적으로 생체흡수성의 poly(glycolic acid)(PGA)를 심선(芯線)에 제작하고, 환경분해성의 poly(butylene succinate-co-L-lactate)(PBSL) 및 poly [(R)-3-hydroxybutylate] (PHB)를 외층(外層)에 지닌 복합섬유를 제작하여, PGA의 가수분해에 의해 발생되는 glycolic acid에 의한 PBSL 및 PHB의 가수분해성의 향상을 도모하고자 하였다. 그 결과 PBSL/PGA 복합섬유에 대한 연신은 $65^{\circ}C$에서 실시함에 의해 결정배향이 잘 배열된 섬유를 얻었다. 그러나 PHB/PGA 복합섬유는 $50^{\circ}C$에서는 결정배향이 양호한 섬유를 얻을 수 없었기 때문에 $50^{\circ}C$ 이상의 온도에서 연신을 실시해야 양호한 복합섬유를 얻을 수 있음을 알았다. 또한 in-line 연신에서는 섬유표면에 요철이 발생되기 때문에 on-line 연신을 실시하는 것이 매끄러운 표면을 얻을 수 있음을 알았다.

• 한국염색가공학회지
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• 제30권2호
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• pp.130-140
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• 2018

In the bio-synthesis of poly(3-hydroxybutyrate)(PHB), which is a kind of poly(3-hydroxyalkanoate)(PHA), aimed to control the low molecular weight of PHB and obtain a telechelic PHB. As a result of incubation of R. eutropha at $30^{\circ}C$ with ethylene glycol added as a chain transfer agent, PHB content on the dry cell weight increased up to 24h, however, it decreased after that, and the molecular weight of PHB increased from 9h to 12h, and then, decreased up to 72h. The decrease of the content and the molecular weight of PHB indicates that PHB was decomposed as an energy source in bacterial cells and was incorporated into metabolic pathways. $^1H-NMR$ of the obtained PHB after incubation for 72h was measured to determine the terminal groups of the PHB during incubation. As the results of $^1H-NMR$ measurement, the peaks derived from ethylene glycol in both terminals of PHB were observed. Which indicate that the terminal reaction was caused by the addition of ethylene glycol, and that telechelic PHB having hydroxyl group at the both terminals where molecular weight was controlled was successfully synthesized.

• Journal of Microbiology and Biotechnology
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• 제20권1호
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• pp.71-77
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• 2010

The time, yield, and related genes expression of PHB accumulation of Acidiphilium cryptum DX1-1 were investigated under four different initial C/N ratios, 1.2, 2.4, 7.5, and 24. The results of time and yield of poly-$\beta$-hydroxybutyrate (PHB) accumulation show that the initial C/N ratio of 2.4 was optimum for strain DX1-1 to accumulate PHB, but both higher and lower initial C/N ratios did not favor that process. Based on the genome of Acidiphilium cryptum JF-5, 13 PHB accumulation related genes in strain JF-5 were chosen and successfully cloned from strain DX1-1. The differential expressions of the 13 functional genes, in different C/N ratios as cited above, were then studied by real-time PCR. The results show that all the 13 genes were most upregulated when the initial C/N ratio was 2.4, and among which the gene Acry_3030 encoding poly-$\beta$-hydroxybutyrate polymerase and Aery_0626 encoding acetyl-CoA synthetase were much more upregulated than the other genes, which proved that they play the most important role for PHB accumulation, and acetate is the main initial substance for PHB accumulation for strain DX1-1. Potential regulatory motifs analysis showed that the genes related to PHB accumulation are regulated by different promoters and that the motif had weak similarity to the model promoters, suggesting that PHB metabolism in Acidiphilium cryptum may be mediated by a different mechanism.

• KSBB Journal
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• 제10권5호
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• pp.533-539
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• 1995

재조합 대장균에 의해 생합성된 PHB를 분리정제하기 위하여 박테리오파아지의 세포파괴작용을 이용하는 방법의 가능성에 대해 알아 보았다. 먼저, $cI_{857}$ 유전자를 지난 박테리오파아지 A를 대장균에 감염시킨 후 lysogen, XLl-Blue($\lambda$HL1)를 선별하고, 이 균주에 PHA 생합성 플라스미드를 도입시켜 원하는 균주인 XLI-Blue($\lambda$HL1, pSYLl05)를 만들였다. 숙주인 XLl-Blue, 열적유도에 의해 세포파괴가 가능한 XLl-Blue($\lambda$HL1), 그리고 세포파괴와 PHB 생합성이 모두 가능한 XLl-Blue(AHL1, pSYL105) 에 대하여 여러 가지 조건에서의 실험결과를 비교.검토하였다. XLI-Blue($\lambda$IL1, pSYLl05)의 경우 대수기에서는 열적유도만으로 세포파괴를 효과적으로 야기할 수 있었으나 PHB가 축적되는 정지기에서는 열척유도만으로 세포파괴를 일으킬 수 없었다. 세포 파괴를 보다 용이하게 하기 위하여 열적유도 빛 2% (v/v)의 chloroform을 사용하는 화학적용균을 병행 하였는데, 이 경우 세포파괴가 효과적으로 일어남을 관찰할 수 있었다.