• Microbiology and Biotechnology Letters
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• v.51 no.3
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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.

• Korean Journal of Microbiology
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• v.39 no.2
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• pp.76-82
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• 2003

To prepare evolved PHB depolymerase with increased activity for PHB or P(3HB-co-3HV) compared to the activity of the original PHB depolymerase from Alcaligenes faecalis T1, random mutation of the cloned PHB depolymerase gene was performed by using a DNA shuffling method. A library of mutated PHB depolymerase genes from A. faecalis T1 was fused to the ice nucleation protein (INP) gene from Pseudomonas syringae in pJHCl 1 and approximately 7,000 transformants were isolated. Using M9 minimal medium containing PHB or P(3HB-co-3HV) as the carbon source, mutants showing alteration in PHB depolymerase activity were selected from the transformants. The PHB depolymease activity of the transformants was confirmed by the formation of halo around colony and the turbidity decrease tests using culture supermatants. The catalytic activity of PHB depolymerase of the best mutant II-4 for PHB or P(3HB-co-13 mol% 3HV) was approximately 1.8-fold and 3.2-fold, respectively, higher than that of the original PHB depolymerase. DNA sequence analysis revealed that three amino acid residues (Ala209Val, Leu258Phe, and Asp263Thr) were substituted in II-4. From the mutational analysis, it was presumed that the substitution of amino acids near catalytic triad to more hydrophobic amino acids enhance the catalytic activity of PHB depolymerase from A. faecalis T1.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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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.

• Proceedings of the Polymer Society of Korea Conference
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• 2006.10a
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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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• v.4 no.2
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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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• v.11 no.2
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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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• v.17 no.12
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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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• v.22 no.6
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• pp.371-377
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• 2007

Polyhydroxyalkanoates (PHAs) are a family of microbial polyesters that can be produced by fermentation from renewable resources. PHAs can be used as completely biodegradable plastics or elastomers. In this paper, novel applications of PHAs in biosensor are described. A general platform technology was developed by using the substrate binding domain (SBD) of PHA depolymerase as a fusion partner to immobilize proteins of interest on PHA surface. It could be shown that the proteins fused to the SBD of PHA depolymerase could be specifically immobilized onto PHA film, PHA microbead, and microcontact printed PHA surface. We review the results obtained for monitoring the specific interaction between the SBO and PHA by using enhanced green fluorescent protein, red fluorescent protein, single chain antibody against hepatitis B virus preS2 surface protein and severe acute respiratory syndrome coronavirus surface antigen as model proteins. Thus, this system can be efficiently used for studying protein-protein and possibly protein-biomolecule interactions for various biotechnological applications.