• Journal of Microbiology and Biotechnology
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• 제6권2호
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• pp.120-127
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• 1996

The characteristics of cell growth and poly-$\beta$-hydroxybutyrate biosynthesis of Alcaligenes latus ATCC 29713 were investigated. The PHB accumulation pattern of A. latus followed a growth-associated type where the cell growth and PHB accumulation were carried out simultaneously. Various intermediate compounds such as metabolites involved in the TCA cycle, amino acids, and saturated and unsaturated fatty acids were added to examine their effect on cell growth and PHB accumulation. Citrate, tyrosine, and palmitic acid showed the most significant increase both on cell growth and PHB accumulation. Maximum PHB concentrations were noticeably increased about 1.4 to 1.6 times higher than that of control, corresponding to 5.54, 6.45, and 6.45 g/l for citrate, tyrosine, and palmitic acid, respectively. The stimulatory effects of the supplemented metabolites were analyzed in terms of the increment of enzyme activities related to sugar catabolism and PHB biosynthesis.

• 한국미생물·생명공학회지
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• 제24권3호
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• pp.344-351
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• 1996

Microorganisms growing alcoholic distillery wastes were isolated from soil. Of them, the selected strain EL-9 had a capability of accumulating poly-$\beta$-hydroxybutyrate (PHB) when grown in alcoholic distillery wastes. The strain EL-9 was identified as a genus Actinobacillus based on the morphological, cultural, and biochemical characteristics. The strain EL-9 was named temporarily as Actino- bacillus sp. EL-9. The optimal temperature and pH for cell growth of Actinobacillus sp. EL-9 were 30$\circ$C and 7.0, respectively. The optimal medium compositions for cell growth comprised glucose 2%, NH$_{4}$NO$_{3}$ 0.15%, Na$_{2}$HP0$_{4}$-12H$_{2}$O 0.25%, KH$_{2}$PO$_{4}$ 0.25%, MgSO$_{4}$4-7H$_{2}$O 0.15%, FeSO$_{4}$-7H$_{2}$O 0.005%, CaCl$_{2}$-2H$_{2}$O 0.003% and trace element solution 5m/l. To investigate the optimal conditions for PHB production, two-stage culture technique was used. The result showed that the optimal conditions for PHB production are identical those for cell growth. When propionic acid was added as a cosubstrate, PHB/HV copolymer was produced.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.425-431
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• 1996

The enzymatic characteristics of Alcaligenes latus were investigated by measuring the variations of various enzyme activities related to biosynthesis and degradation of poly-${\beta}$-hydroxybutyrate (PHB) during cultivation. All PHB biosynthetic enzymes, ${\beta}$-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase, were activated gradually at the PHB accumulation stage, and the PHB synthase showed the highest value among three enzymes. This indicates that the rate of PHB biosynthesis is mainly controlled by either ${\beta}$-ketothiolase or acetoacetyl-CoA reductase rather than PHB synthase. The enzymatic activities related to the degradation of PHB were also measured, and the degradation of PHB was controlled by the activity of PHB depolymerase. The effect of supplements of metabolic regulators, citrate and tyrosine, was also investigated, and the activity of glucose-6-phosphate dehydrogenase was increased by metabolic regulators, especially by tyrosine. The activities of ${\beta}$-ketothiolase and acetoacetyl-CoA reductase were also activated by citrate and tyrosine, while the activity of PHB depolymerase was depressed. The increased rate and yield of PHB biosynthesis by metabolic regulators may be due to the increment of acetyl-CoA concentration either by the repression of the TCA cycle by citrate through product inhibition or by the activation of sucrose metabolism by the supplemented tyrosine.

• Journal of Microbiology
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• 제40권1호
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• pp.20-25
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• 2002

An extracellular PHB depolymerase was purified from P. simplicissimum LAR13 cultural medium by Sepharose CL-6B chromatography. When the fungus was grown in a basal salt medium with poly(3-hydroxybutyrate) (PHB) as the sole carbon source, PHB depolymerase production reached maximum at its stationary phase. The mycelial growth rate was higher at 37$^{\circ}C$ than at 30$^{\circ}C$ and even higher than at 25$^{\circ}C$, However, the enzyme production was lower at 37$^{\circ}C$ than 30$^{\circ}C$ or 25$^{\circ}C$. The isolated enzyme is composed of a single polypeptide chain with a molecular mass of about 36 kDa as determined by SDS-PAGE. The optimum conditions for the enzyme activity are pH 5.0 and 45$^{\circ}C$. The enzyme was stable for 30 min at a temperature lower than 50$^{\circ}C$, and stable at pH higher than 2.0 but it was unstable at pH 1.0.1 mM Fe$\^$2+/ reduced the enzyme activity by 56% and the enzyme was inhibited almost completely by 4 mM Fe$\^$2+/ . The enzyme was partially inhibited by phenylmethylsulfonyl fluoride and was very sensitive to diazo-DL-norleucine methyl esters dithiothreitol and mercuric ion. However, N-p - tosyl - L - Iysinechloromethyl ketone, p -hydroxymercuricbenzoate and N- acetylimidazole had no influence upon its activity.

• Journal of Microbiology and Biotechnology
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• 제9권5호
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• pp.593-603
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• 1999

Poly(3-hydroxybutyrate) (PHB) production by fermentation was examined under both restricted- and ample-oxygen supply conditions in a single fed-batch fermentation. Recombinant Escherichia coli transformed with the PHB production plasmid pSYLl07 was grown to reach high cell density (227 g/l dry cell weight) with a high PHB content (78％ of dry cell weight), using a glucose-based minimal medium. A simple flux model containing 12 fluxes was developed and applied to the fermentation data. A superior closure (95％) of the carbon mass balance was achieved. When the data were put into use, the results demonstrated a surprisingly large excretion of formate and lactate. Even though periods of severe oxygen limitation coincided with rapid acetate and lactate excretion, PHB productivity and carbon utilization efficiency were not significantly impaired. These results are very positive in reducing oxygen demand in an industrial PHA fermentation without sacrificing its PHA productivity, thereby reducing overall production costs.

• 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.

• Bulletin of the Korean Chemical Society
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• 제22권8호
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• pp.872-876
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• 2001

Topological changes caused by the alkaline and enzymatic attacks of solution-grown, chain-folded lamellar crystals (SGCs) of poly[(R)-3-hydroxybutyrate] P(3HB) have been studied in order to investigate the chain-folding structure in P(3HB) crystal regions. NaOH and an extracellular PHB depolymerase purified from Alcaligenes faecalis T1 were used for alkaline and enzymatic hydrolysis, respectively. The measurements were performed on crystals attached to a substrate which is inactive to degradation mediums. Both alkaline and enzymatic attacks lead to a breakup of the lamellar crystals along the crystallographic b-axis during initial erosion. Since hydrolysis preferentially occurs in amorphous regions, this morphological result reflects relatively loosely packed chains in core parts of lamellar crystals. Additionally, it was supported by the ridge formation along the b-axis in the lamellar crystals after thermal treatment at a low temperature because of the thermally sensitive nature of the loosely packed chains in lamellar crystals. However, the alkaline hydrolysis accompanied the chain erosions or scissions in quasi-regular folded lamellar surfaces due to smaller size of alkaline ions in comparison to the enzyme, resulting in the decrease of molecular weight.

• 접착 및 계면
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• 제7권2호
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• pp.19-25
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• 2006

미생물 합성 고분자인 poly(hydroxylalkanoate)s (PHAs)의 초기효소분해는 표면침식의 메커니즘으로 진행하므로 이들의 분해거동은 표면특성을 개질로서 조절할 수 있다. 본 연구에서는 효소분해속도를 조절하기 위하여 플라즈마 기법을 PHAs 표면특성의 개질에 적용하였다. $CF_3H$와 $O_2$ 플라즈마를 사용하여 재료 표면에 각각 소수성 및 친수성을 부여하였다. 효소분해 실험은 pH 7.4의 0.1 M potassium phosphate 완충용액에서 Alcaligenes facalis T1에서 정제된 poly(hydroxybutyrate) 분해효소를 첨가하여 행하였다. $CF_3H$ 플라즈마 처리된 시편의 경우 표면 층의 불소화에 따른 소수성의 증가와 분해 효소에 대한 비활성으로 초기분해 속도가 상당히 지연됨을 관찰하였으나 $O_2$ 플라즈마 처리에 의한 표면 친수성은 분해속도의 촉진 등에 큰 영향을 미치지 않았다.

• Korean Chemical Engineering Research
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• 제55권3호
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• pp.363-368
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• 2017

메탄올자화균이란 일탄소 화합물인 메탄올을 주탄소원 및 에너지원으로 이용할 수 있는 미생물을 말한다. Methylobacterium extorquens AM1은 serine cycle을 탄소대사경로로 이용하는 메탄올자화균 중에서도 가장 많이 연구가 진행된 균주이다. M. extorquens AM1의 poly 3-hydroxybutyrate (PHB) cycle은 EMCP (ethylmalonyl-CoA pathway), glyoxylate regeneration cycle, TCA cycle과 연결되어 있으며 EMCP 유래 유기산 또는 TCA 유기산을 생산하기 위해서는 PHB cycle로 흐르는 carbon flux의 차단이 필요하다. 이를 위해서 PHB 합성과 acetyl-CoA flux의 조절유전자로 알려져 있는 PhaR 유전자를 markerless gene deletion 방법을 이용해서 M. extorquens AM1에서 knockout했다. 결과적으로, knockout 균주인 ${\Delta}phaR$에서 야생종 대비 확연히 PHB granule이 줄어든 것이 확인되었다. Lag phase가 약 12 h 늦어졌지만, ${\Delta}phaR$은 야생종과 비슷한 세포성장과 메탄올소비 경향을 보임을 확인하였다.

• 미생물학회지
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• 제37권1호
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• pp.92-99
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• 2001

Pi1ot plant (200 l)에서의 유가식 배양에 의한 Ralstonia eutopha KHB 8862의 고농도 배 양을 통하여 Poly(3-hydroxybutyrate) (PHB)의 대량생산을 모색하였다. 그 결과 배양 80시간 후 168 g/l의 건체량과 건체량의 74%에 달하는 PHB를 생산할 수 있었으며, 이때의 고과당 시럽으로부터의 PHB 전환수율 및 PHB 생산성은 각각 0.27 (w/w) 및 1.6 $gl^{-1}$ $h^{-1}$ /이었다. 이를 토대로 본 연구에서는 미생물 발효에 의한 PHB 생산 cost 및 그 경제성을 분석하였다. 신규설비투자를 고려하지 않은 경우의 PHB의 생산 cost는 US$2.41/kg으로 산출된 반면에 신규 설비투자를 고려한 경우에는 US$3.15/kg으로 상승되었다. 탄소원의 PHB로의 전환수율과 발효 생산성 모두 PHB 생산비를 결정하는 중요요인이지만 전체 생산비의 37%를 차지하는 탄소원 원료비의 비중이 설비투자의 감가상각비 비중 (17%)에 비해 높기 때문에 생산성을 높이는 노력보다는 전환수율을 개선하는 것이 PHB 생산비용 절감의 핵심이 되는 것으로 나타났다. PHB chip으로의 제조시 PHB 생산 cost는 US$4.0/kg의 수준으로 현재로서는 범용 합성플라스틱에 비하여 경쟁력을 확보하지 못한다. 따라서 생산비 절감을 통한 범용수지로써 경쟁력 제고와 함께 바이오의약 분야 등의 고부가가치 영역에서의 새로운 용도 개발 등이 적극 요구된다.