• 미생물학회지
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• 제36권2호
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• pp.84-90
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• 2000

Psudomonas sp. RY-1이 생성하는 extracellular depolymerase system을 이용하여 단위체의 결가지에 서로 다른 탄소 길이와 불포와기를 함유하는 medium-chain-length polyhdroxyalkanoates (MCL-PHAs)의 생분해도를 시럼실 조건에서 조사하였다. 생분애도는 평파내지에서의 clear zone 형성, 효소 처리에 의한 고분자 현탁액의 탁도 감소 및 호흡량의 경시적 변화로 측정하였다. Pseudomonas sp. RY-1은 MCL-PHA depolymerase의 생성을 통하여 조사된 모든 종류의 MCl-PHAs를 분해할 수 있었으나, 이 효소의 생성은 쉽게 이용될수 있는 이차기질에 의해 저해받는 것으로 나타났다. MCl-PHAs의 분해율이 단위체의 탄소수가 홀수개로 구성된 고분자에 비하여 보다 높았다. 곁가지에 분포화기를 함유한 MCl-PHAs는 불포화기를 갖지 아니하는 고분자에 비하여 분해가 빠르게 이루어졌으며, 이들의 분해는 고분자의 결정화도와 밀접한 관련이 있는 것으로 나타났다.

• KSBB Journal
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• 제29권6호
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• pp.443-447
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• 2014

Biosynthesis of lactate-containing polyhydroxyalkanoates (PHAs) was examined in recombinant Escherichia coli W strain from sucrose. The Pseudomonas sp. MBEL 6-19 phaC1437 gene and the Clostridium propionicum pct540 gene, which encode engineered Pseudomonas sp. MBEL 6-19 PHA synthase 1 ($PhaC1_{Ps6-19}$) and engineered C. propionicum propionyl-CoA transferase ($Pct_{Cp}$), respectively, were expressed in E. coli W to construct key metabolic pathway to produce poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)]. The recombinant E. coli W expressing the phaC1437 gene and the pct540 gene could synthesize P(3HB-co-13mol%LA) up to the polymer content of 31.3 wt% when it was cultured in chemically defined MR medium containing 20 g/L of sucrose and 2 g/L of sodium 3-hydroxybutyrate. When Ralstonia eutropha phaAB genes were additionally expressed to provide 3-hydroxybutyrate-CoA (3HB-CoA) from sucrose, P(3HB-co-16mol%LA) could be synthesized from sucrose as a sole carbon source without supplement of sodium 3-hydroxybutyrate in culture medium, but the PHA content was decreased to 12.2 wt%. The molecular weight of P(3HB-co-16 mol%LA) synthesized in E. coli W using sucrose as carbon source were $1.53{\times}10^4$ ($M_n$) and $2.78{\times}10^4$ ($M_w$), respectively, which are not different from those that have previously been reported by other recombinant E. coli strains. Engineered E. coli strains developed in this study should be useful for the production of lactate-containing PHAs from sucrose, one of the most abundant and least expensive carbon sources.

• KSBB Journal
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• 제10권2호
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• pp.137-142
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• 1995

Sucrose를 탄소원으로 이용할 수 있는 대장균 W 를 A. eutrophus PHA 합성 유전자가 cloning된 높 은 복제수의 plasmid pSYL105로 형질전환하여 단 순배지에서 배양시 $0.21g/\ell$ 의 낮은 PHB 농도를 얻었다. 이에 107}지의 복합질소원을 소량 첨가하여 PHB 합성에 미치는 영향을 살펴 보았다. Tryptone, casamino acids, casein hydrolysate, 혹은 soy bean hydrolysate의 첨가시 PHB 합성 이 10배 이 상 증대되었다. 또한 이때 sucrose로부터의 PHB 수율도 10배 이상 증가하여 이 네 가지 복합질소원 을 척정 농도로 첨가함으로써 sucrose로부터 효과적인 PHB 생산이 가능함을 알았다.

• Journal of Microbiology and Biotechnology
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• 제9권6호
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• pp.847-853
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• 1999

A Pseudomonas sp. strain that is capable of utilizing dicarboxylic acids as a sole carbon source was isolated from activated sludge by using the enrichment culture technique. This organism accumulated polyhydroxyalkanoates (PHAs) with an unusual pattern of monomer units that depends on the carbon sources used. Polyhydroxybutyrate (PHB) homopolyester was synthesized from glucose or small $C_{-even}$ alkanoic acids, such as butyric acid and hexanoic acid. Accumulation of PHB homopolyester was also observed in the cells grown on $C_{-odd}$ dicarboxylic acids, such as heptanedioic acid and nonanedioic acid as the sole carbon sources. In contrast, a copolyester consisting of 6 mol% 3-hydroxybutyrate (3HB) and 94 mol% 3-hydroxyvalerate (3HV) was produced with a PHA content of as much as 36% of the cellular dry matter. This strain produced PHAs consisting both of the short-chain-length (SCL) and the medium-chain-length (MCL) 3-hydroxyacid units when heptanoic acid to undecanoic acid were fed as the sole carbon sources. Most interestingly, polyester consisting of significant amount of relevant fractions, 3HB, 3HV, and 3-hydroxyheptanoate (3HHp), was accumulated from heptanoic acid. According to solvent fractionation experiments, the polymer produced from heptanoic acid was a blend of poly(3HHp) and of a copolyester of 3HB, 3HV, and 3HHp units. The hexane soluble fractions contained only 3HHp units while the hexane-insoluble fractions contained 3HB and 3HV units with a small amount of 3HHp unit. The copolyester was an elastomer with unusual mechanical properties. The maximum elongation ratio of the copolyester was 460% with an ultimate strength of 10 MPa, which was very different from those of poly(3HB-co-3HV) copolyesters having similar compositions produced from other microorganisms.

• 한국미생물·생명공학회지
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• 제52권1호
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• pp.102-104
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• 2024

제주도 매립지에서 분리된 Massilia strain KACC 81254BP는 생분해성플라스틱 폴리하이드록시알카노에이트(PHA)를 분해할 수 있다. 이 균주의 유전체는 5,028,452 bp의 원형 염색체로 구성되어 있으며, G+C 함량은 64.6%이다. 이 유전체는 PHB depolymerase를 포함한 4,513개 유전자가 확인되었다. 이 유전체는 신호전달과 아미노산 수송 등 대사와 관련한 다양한 유전자를 포함하고 있다. 이 연구는 Massilia sp. KACC 81254BP의 폴리에스터 플라스틱 분해 효소와 관련된 유전학적 정보를 제공한다.

• 미생물학회지
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• 제34권4호
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• pp.219-224
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• 1998

Alcaligenes sp. SH-69에서 탄소원의 고갈 또는 새로운 탄소원의 첨가시 포도당을 단일탄소원으로 하여 생합성된 poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)] 공중합체의 물질대사 변화 양상을 조사하였다. 회분배양 과정 중 탄소원이 고갈된 후에는 세포 내에 축척되었던 poly(3HB-co-3HV) 공중합체의 분해가 일어나 고분자 함량의 감소와 함께 무게평균 분자량도 감소하였다. 분해 과정 중에는 저분자량의 PHA에 비하여 상대적으로 고분자량의 PHA양이 크게 감소하여 평균 분자량 분포가 보다 낮은 방향으로 이동되는 양상을 보였다. 이에 반하여 1차 탄소원(기질)으로 사용된 포도당의 고갈 직후 2차 기질로서 포도당과 함께 3HV의 전구물질인 levulinic acid를 혼합기질로 첨가해 주었을 경우, 세포 건체량의 지속적인 증가와 아울러 3HV 함량이 높은 poly(3HB-co-3HV) 공중합체의 합성이 이루어졌다. 그러나 아세톤을 이용한 고분자의 분획 실험 결과, 2차 기질로부터의 poly(3HB-co-3HV) 공중합체의 생합성과 1차 기질로부터 생합성된 공중합체의 분해가 동시에 일어나며, 또한 각 기질로부터 생합성된 고분자가 단지 3HV의 함량이 다른 poly(3HB-co-3HV) 공중합체임에도 불구하고 혼합물 형태로 존재하고 있음을 확인하였다.

• KSBB Journal
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• 제13권2호
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• pp.187-194
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• 1998

For mass production of polyhydroxyalkanoates (PHA), high cell density cultures of Alcaligenes eutrophus by fed-batch culture under phosphate-limitation condition has been investigated. PHA accumulation by the regulation by the regulation of initial phosphate concentration could be automatically induced, and high density cell culture above 200 g/L also could be successfully produced. The production of Poly-$\beta$-hydroxybutyrate (PHB) and dry cell weight increased with increasing the initial phosphate concentration. When the initial concentrations of phosphate were in the ranges of 1.5~4.5 g-PO$_4$/L, PHB and dry cell weight obtained were 83~266 g/L and 61~216 g/L, respectively, and PHB productivity was in the ranges of 1.35~3.10 g/L.h. When a mixture of glucose and propionic acid is used as carbon sources, poly(3-hydroxybutyrate-co-poly-3-hydroxyvalerate), P(3HB-co-3HV), could be also successfully produced under phosphate limitation condition. When the mole ratio of propionic acid to glucose in the feeding solution is 0.22, a final dry cell weight of 150 g/L and a P(3-HB-co-3HV) of 90 g/L were produced. Morphological changes and size distribution of PHB granules synthesized in A. eutrophus under phosphate-limitation condition are determined by TEM during the course of fed-batch. Mean granule diameters of PHB produced are in the range of 0.36~0.39 $\mu$m, and mean cell size was elongated from 0.54~0.59 $\mu$m$\times$ 1.3~1.5 $\mu$m to 0.83~0.89 $\mu$m $\times$2.0~2.3 $\mu$m. Phosphate concentration in media did not affect size distribution of PHB granule and cell.

• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.382-391
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• 2019

Many poultry eggs are discarded worldwide because of infection (i.e., avian flu) or presence of high levels of pesticides. The possibility of adopting egg yolk as a source material to produce polyhydroxyalkanoate (PHA) biopolymer was examined in this study. Cupriavidus necator Re2133/pCB81 was used for the production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) or poly(3HHx), a polymer that would normally require long-chain fatty acids as carbon feedstocks for the incorporation of 3HHx monomers. The optimal medium contained 5% egg yolk oil and ammonium nitrate as a nitrogen source, with a carbon/nitrogen (C/N) ratio of 20. Time course monitoring using the optimized medium was conducted for 5 days. Biomass production was 13.1 g/l, with 43.7% co-polymer content. Comparison with other studies using plant oils and the current study using egg yolk oil revealed similar polymer yields. Thus, discarded egg yolks could be a potential source of PHA.

• Journal of Microbiology and Biotechnology
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• 제29권5호
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• pp.776-784
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• 2019

Polyhydroxybutyrate (PHB), the most well-known polyhydroxyalkanoate, is a bio-based, biodegradable polymer that has the potential to replace petroleum-based plastics. Lignocellulose hydrolysate, a non-edible resource, is a promising substrate for the sustainable, fermentative production of PHB. However, its application is limited by the generation of inhibitors during the pretreatment processes. In this study, we investigated the feasibility of PHB production in E. coli in the presence of inhibitors found in lignocellulose hydrolysates. Our results show that the introduction of PHB synthetic genes (bktB, phaB, and phaC from Ralstonia eutropha H16) improved cell growth in the presence of the inhibitors such as furfural, 4-hydroxybenzaldehyde, and vanillin, suggesting that PHB synthetic genes confer resistance to these inhibitors. In addition, increased PHB production was observed in the presence of furfural as opposed to the absence of furfural, suggesting that this compound could be used to stimulate PHB production. Our findings indicate that PHB production using lignocellulose hydrolysates in recombinant E. coli could be an innovative strategy for cost-effective PHB production, and PHB could be a good target product from lignocellulose hydrolysates, especially glucose.

• Korean Chemical Engineering Research
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• 제54권5호
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• pp.665-670
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• 2016

본 연구에서는 톨루엔 분해 균주인 Pseudomonas putida와 아세트산 분해 균주인 Cupriavidus necator에 무세포 효소 시스템(cell-free enzyme system)을 적용하여 톨루엔과 아세트산에 대한 분해 가능성을 확인하는 실험을 수행하였다. P. putida는 톨루엔 존재 하에서만 toluene dioxygenase를 생성하여 톨루엔을 cis-toluene dihydrodiol로 분해하며, C. necator는 acetyl coenzyme A synthetase-1을 생성하여 아세트산을 acetyl CoA로 전환시켜 생존에 필요한 ATP나 생분해성(biodegradable) 고분자인 Polyhydroxyalkanoate (PHA)를 합성한다. P. putida의 톨루엔 분해 효소인 toluene dioxygenase는 유도효소이기 때문에 toluene dioxygenase 생성 전과 후로 나누어 실험을 진행하였다. P. putida의 톨루엔 분해능력 확인을 위한 gas chromatography (GC) 분석 결과, 대조군과 toluene dioxygenase 생성 전인 실험군 1에서는 검출된 톨루엔의 양이 거의 유사하였으나, toluene dioxygenase 생성 후인 실험군 2에서는 검출된 톨루엔의 양이 대조군 및 실험군 1에 비해 감소하였다. 또한 C. necator의 아세트산 분해능력 확인을 위한 gas chromatography-mass spectrometer (GC-MS) 분석 결과, 무세포 효소 시스템을 적용한 실험군에서는 아세트산에 대한 피크가 검출되지 않았다. 따라서 P. putida와 C. necator는 무세포 효소 시스템 적용 후에도 톨루엔 및 아세트산 분해 능력이 유지되었으나, P. putida는 무세포 효소 시스템을 적용하기 전에 유도 효소를 생성하는 과정이 필요하다.