• 한국미생물·생명공학회지
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• 제51권3호
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• pp.257-267
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• 2023

The potential polyhydroxyalkanoates (PHA)-producing bacteria, Bacillus megaterium PP-10, was successfully isolated and studied its feasibility for utilization of pineapple peel waste (PPW) as a cheap carbon substrate. The PPW was pretreated with 1% (v/v) H₂SO₄ under steam sterilization and about 26.4 g/l of total reducing sugar (TRS) in pineapple peel hydrolysate (PPH) was generated and main fermentable sugars were glucose and fructose. A maximum cell growth and PHA concentration of 3.63 ± 0.07 g/l and 1.98 ± 0.09 g/l (about 54.58 ± 2.39%DCW) were received in only 12 h when grown in PPH. Interestingly, PHA productivity and biomass yield (Y_x/s) in PPH was about 4 times and 1.5 times higher than in glucose. To achieve the highest DCW and PHA production, the optimal culture conditions e.g. carbon to nitrogen ratios of 40 mole/mole, incubation temperature at 35℃ and shaking speed of 200 rpm were performed and a maximum DCW up to 4.24 ± 0.04 g/l and PHA concentration of 2.68 ± 0.02 g/l (61% DCW) were obtained. The produced PHA was further examined its monomer composition and found to contain only 3-hydroxybutyrate (3HB). This finding corresponded with the presence of class IV PHA synthase gene. Finally, certain thermal properties of the produced PHA i.e. the melting temperature (T_m) and the glass transition temperature (T_g) were about 176℃ and -4℃, respectively whereas the M_w was about 1.07 KDa ; therefore, the newly isolated B. megaterium PP-10 is a promising bacterial candidate for the efficient conversion of low-cost PPH to PHA.

• 미생물학회지
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• 제39권2호
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• pp.76-82
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• 2003

Alcaligenes faecalis T1의 Poly(3-hydroxybutyrate)(PHB) depolymerase활성 증진을 위해 DNA shuffling방법을 이용하였다. 제조된 A. faecalis T1의 PHB depolymerase 돌연변이 유전자의 library를 Pseudomonas syringae의 icenucleation protein유전자를 포함하는 발현벡터 pJHCll에 클로닝하여 약 7,000개의 형질전환체를 얻었다. 탄소원으로 PHB또는 poly(3-hydroxybutyrate-co-3-hydroxyvalerate)를 포함하는 M9최소배지를 이용하여 형질전환체들로부터 활성이 서로 다른 돌연변이주들을 선별하였다. 이들의 PHB depolymease 활성은 평판배지에서의 halo형성 및 배양 상등액을 이용한 탁도 감소 실험으로 확인하였으며,형질전환체들 중에서 shuffling전의 대조군에 비하여 사용된 기질에 따라 효소활성이 1.8-3.2배 증진된 II-4 돌연변이주를 얻었다. DNA 염기서열의 분석을 통하여 II-4의 PHB depolymease에는 3개의 아미노산 치환(A1a209Va1, Leu258Phe, Asp263Thr)이 이루어졌음을 확인하였다. 여러 가지 돌연변이주의 아미노산 서열의 변화를 분석한 결과, PHB depolymerase의 catalytictriad주위에 기존 아미노산에 비하여 보다 소수성인 아미노산으로의 치환이 소수성 기질인 PHB에 대한분해 활성 중진에 기여하는 것으로 추정되었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.735-738
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• 2001

Metabolically engineered Escherichia coli strains harboring a plasmid containing a novel artificial polyhydroxyalkanoate (PHA) operon consisting of the Aeromonas PHA biosynthesis related genes and Ralstonia eutropha reductase gene were developed for the production of poly(3-hydroxybutyrate-co-hydroxyhexanoate) [P(3HB-co-3HHx)] from dodecanoic acid. By applying stepwise reduction of dissolved oxygen concentration (DOC) during the fermentation, the final dry cell weight, PHA concentration, and PHA content of 79 g/L, 21.5 g/L, and 27.2 wt%, respectively, were obtained in 40.8 h, which resulted in the PHA productivity of 0.53 g/L/h. The 3HHx fraction slowly increased during the fed-batch culture to reach a final value of 10.8 mol%. The 3HHx fraction in the copolymer could be increased by three fold when the Aeromonas hydrophila orfl gene was co-expressed with the PHA biosynthesis genes.

• 한국고분자학회:학술대회논문집
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• 한국고분자학회 2006년도 IUPAC International Symposium on Advanced Polymers for Emerging Technologies
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• pp.351-351
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• 2006

The possible interactions between cyclodextrins and biodegradable polyesters were investigated. The hydrophobicity of cyclodextrin could be varied with the methyl substitution of host CD, and the possibility of IC formation and the types of interaction between respective CDs and polyesters were subsequently changed. Further, the effect of cyclodextrins on the morphological change of biodegradable polymer was shown to depend on the degree of IC formation between cyclodextrin and biodegradable polymer as well as on the type of interaction between respective CDs and polyesters. That is, the enhancement and/or the restriction of the crystallization of P(3HB) were observed by the incorporation of various kind of cyclodextrins with different cavity size and hydrophobicity.

• Journal of Microbiology and Biotechnology
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• 제4권2호
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• pp.126-133
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• 1994

A Pseudomonas putida strain able to accumulate high amount of polyesters of medium-chain-length 3-hydroxyalkanoic acids ($PHA_{MCL)$) was isolated from soil in a landfill site using an enrichment technique. Culture condition of the isolated strain for polyester production in a one-step culture was optimized in a mineral-salts medium against pH and concentrations of ammonium sulfate, carbon source(e.g., octanoate), and phosphate. The optimal values for maximal cell growth and PHA accumulation were: pH; 7$\sim$8, $(NH_4)_2SO_4$; 8 mM, octanoate; 40 mM. The optimum temperature was in the range of $20\sim30^{\circ}C$, which was rather broader than in other bacteria. Cell growth was strongly inhibited by the phosphate limitation to less than 1 mM. An increase of phosphate concentration above 1 mM showed little effect on cell growth and polyester accumulation. When the strain was grown on octanoate under this optimized condition it produced 3.4 g dry biomass per liter and yielded 1.7 g PHA per liter amounting to 53 wt% of dry cells. The monomer units composing the polyester synthesized from octanoate were 3-hydroxyoctanoate (3HO), 3-hydroxycaproate (3HC), and 3-hydroxybutyrate (3HB) (85:13:2, mole ratio). Other low linear $C_3\simC_{10}$ monocarboxylic acids were also tested for polyester production.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권2호
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• pp.173-177
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• 2006

In this study, a novel strategy was developed for the highly selective immobilization of proteins, using the polyhydroxyalkanoate (PHA) depolymerase substrate binding domain (SBD) as an active binding domain. In order to determine the appropriacy of this method for immunodiagnostic assays, the single-chain antibody (ScFv) against the hepatitis B virus (HBV) preS2 surface protein and the severe acute respiratory syndrome coronavirus (SARS-CoV) envelope protein (SCVe) were fused to the SBD, then directly immobilized on PH A-coated slides via microspotting. The fluorescence-labeled HBV antigen and the antibody against SCVe were then utilized to examine specific interactions on the PHA-coated surfaces. Fluorescence signals were detected only at the spotted positions, thereby indicating a high degree of affinity and selectivity for their corresponding antigens/antibodies. Furthermore, we detected small amounts of ScFv-SBD (2.7 ng/mL) and SCVe-SBD fusion proteins (0.6ng/mL). Therefore, this microarray platform technology, using PHA and SBD, appears generally appropriate for immunodiagnosis, with no special requirements with regard to synthetic or chemical modification of the biomolecules or the solid surface.

• Journal of Microbiology and Biotechnology
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• 제17권12호
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• pp.2018-2026
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• 2007

A bacterium, Pseudomonas aeruginosa BM114, capable of accumulating a blend of medium-chain-length (MCL)- and short-chain-length (SCL)-polyhydroxyalkanoic acid (PHA), was isolated. Salicylic acid (SA), without being metabolized, was found to specifically inhibit only the accumulation of MCL-PHA without affecting cell growth. An addition of 20 mM SA selectively inhibited the accumulation of MCL-PHA in decanoate-grown cells by 83% of the control content in one-step cultivation, where overall PHA accumulation was inhibited by only ${\sim}11%$. Typically, the molar monomer-unit ratio of the PHA for 25 mM decanoate-grown cells changed from 46:4:25:25 (=[3-hydroxybutyrate]:[3-hydroxycaproate]: [3-hydroxyoctanoate]:[3-hydroxydecanoate]) at 0 mM SA (dry cell wt, 1.97 g/l; PHA content, 48.6 wt%) to 91:1:4:4 at 20 mM SA (dry cell wt, 1.85 g/l; PHA content, 43.2 wt%). Thus, the stimulation of SCL-PHA accumulation was observed. Growth of P. aeruginosa BM114 on undecanoic acid also produced a PHA blend composed of 47.4% P(3HB-co-3-hydroxyvalerate) and 52.6% P(3-hydroxyheptanoate-co-3-hydroxynonanoate-co-3-hydroxyundecanoate). Similar to the case of even-carboxylic acids, SA inhibited the accumulation of only MCL-PHA, but stimulated the accumulation of SCL-PHA. For all medium-chain fatty acids tested, SA induced a stimulation of SCL-PHA accumulation in the BM114 strain. SA could thus be used to suppress only the formation of MCL-PHA in Pseudomonas spp. accumulating a blend of SCL-PHA and MCL-PHA.

• KSBB Journal
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• 제22권6호
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• pp.371-377
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• 2007

나노기술과 바이오기술의 융합연구에 의해 나노바이오기술이 발전되고 있다. 나노바이오기술의 중요한 응용연구 중의 하나로서, 진단이나 바이오센서 분야에서 단백질-단백질 및 단백질-바이오물질간의 상호작용을 연구하기 위한 단백질 센서 칩이 개발되어 왔다. 본 논문에서는 단백질의 선택적 고정화를 위한 새로운 생체고분자 기질로 PHA를 이용하는 첫 번째 예로서, 단백질-단백질 및 항원-항체 반응의 구현을 나타내고자 하였다. 본 시스템은 PHA 표면 위에서 PHA depolymerase의 SBD와의 선택적 결합에 기반한 것으로, PHA depolymerase의 SBD와 융합된 단백질이 PHA가 코팅된 표면 위에 spotting 될 수 있고 미세접촉인쇄방법에 의해 PHA 위에 미세패턴이 제조되어지는 것을 알 수 있었다(52, 53). 이러한 새로운 전략이 PHA depolymerase의 SBD와 다른 단백질을 융합함으로서 미세 spotting과 미세패터닝이 가능하게 되었고 항원-항체의 생물학적 반응을 통해 많은 바이오센서 칩 연구에 응용될 수 있음을 확인하였다. 또한, PHA 마이크로 비드에도 PHA depolymerase의 SBD와 융합된 단백질을 고정시킴으로서 항원-항체 반응을 유도할 수 있음을 확인하였다(54). PHA의 구조를 변경하여 PHA 기판, PHA 필름, PHA 미세패턴, PHA 마이크로 비드 등을 이용할 수 있으며 multiplex assay를 동시에 진행할 수 있는 다양한 융합 단백질을 사용할 수 있을 것이다. 생분해성 플라스틱으로서 성공적으로 개발된 PHA를 이용한 새로운 플랫폼 기술이 PHA depolymerase의 SBD를 이용함으로서 특이적이고 선택적인 단백질의 고정화에 이용될 수 있음을 확인하였다. 본 전략이 다양한 단백질-단백질 및 단백질-바이오물질 반응을 이용한 바이오칩 및 바이오센서의 응용연구에 유용하게 사용될 것이다.