• KSBB Journal
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• v.11 no.5
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• pp.586-592
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• 1996

Poly(3-hydroxybutyric-co-3-hydroxyvaleric) acid(PHB/HV) copolymer synthesis by Pseudomonas sp. HJ from glucose and cosubstrate was investigated. Taxonomic analysis suggested that Pseudomonas sp. HJ was best marched to Pseudomonas picketti having 78.8% similarity. Pseudomonas sp. HJ produced PHB/HV copolymer containing 60.8 mol% HV and 44.9 mol% HV when supplied with hexadecane and propionic acid as a cosubstrate, respectively. The HV composition in PHB/HV copolymer was controlled by varying the concentration of hexadecane and propionic acid. Propionic acid added after 24 hours of incubation was incorporated as the HV monomer in the PHB/HV copolymer up to 49.6 mol%.

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.333-336
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• 2001

An isolated phbC gene from Alcaligenes latus was reintroduced into the parent A. latus through the transformation process, and the effect of the amplified phbC gene on the biosynthesis of P(3-hydroxybutyrate-3-hydroxyvalerate) [P(3HB-3HV)] and P(3-hydroxybutyrate-4-hydroxybutyrate) [P(3HB-4HB)] in the transformant A. latus was investigated. The biosynthesis rate and content of the above copolymers increased up to 1.3-fold after enforcing its own phbC gene, and the molar fractions of 3HV and 4HB in P(3HB-3HV) and P(3HB-4HB) also changed remarkably from 35.0 to 48.0% and from 34.0 to 56.0%, respectively, showing a critical role of PHB synthase which catalyzes the polymerizing reactions between eiher 3HV or 4HB from precursor compounds and 3HB.

• Microbiology and Biotechnology Letters
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• v.26 no.6
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• pp.537-544
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• 1998

The cultivation conditions of transformant Alcaligenes eutrophus AER5 harboring cloned phbC gene for mass production of poly (3-hydroxybutyrate-3-hydroxyvalerate)[P(3HB-3HV)] containing high molar fraction of 3-hydroxyvalerate (3-HV) were investigated. In two-stage batch cultivation, transformant accumulated P(3HB-3HV) containing 52.2 mol% of 3HV compared to 30 mol% of parent strain A. eutrophus H16. The increased 3-HV molar fraction was due to the amplified activity of PHB synthase participating in condensation of 3-HB and 3-HV. To increase efficiency of P(3HB-3HV) accumulation, fructose was added along with precursor compound valerate, and total cell mass and P(3HB-3HV) concentrations remarkably increased, but not 3-HV molar fraction. The effect of magnesium ion showed that P(3HB-3HV) concentration and 3-HV molar fraction were significantly increased upto 6.1 g/L and 71.3 mol% at 0.01 g/L of MgSO$_4$, respectively. The efficiency of several pH adjuster, NaOH, NaOH and (NH$_4$)$_2$SO$_4$, and NH$_4$OH, on total cell mass, p(3HB-3HV) concentration, and 3-HV molar fraction was also compared. To overcome the disadvantage of two-stage cultivation, one-stage intermittent fed-batch cultivation was attempted, such a way 10.0 g/L of fructose was supplied for cell growth at initial 36 hr and then 10.0 g/L of valerate and 5.0 g/L of fructose were applied to induce the accumulation of P(3HB-3HV), consequently, 10.4 g/L of P(3HB-3HV) with 38 mol% of 3-HV fraction could be obtained after 72 hr. These results can be used for elucidating cultivation strategy for mass production of P(3HB-3HV) containing high 3-HV molar fraction using transformant A. eutrophus AER5 harboring cloned phbC gene.

• KSBB Journal
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• v.10 no.4
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• pp.349-356
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• 1995

To produce PHA(polyhydroxyalkanoic acid) from microbr, dozens of microorganism have been screened from sewage sludge. Selected a strain HJ out of 50 strains of PHA producing bacteria has a capability of accumulating large amounts of PHB/HV copolymer when grown in batch culture with a single carbon source (glucose) that was not generally considered as precursor of hydroxyvalerate monomer unit. The strain HJ was identified as the genus Pseudomonas with respect to morphological, cultural, and biochemical characteristics. The optimal temperature and pH for cell growth were $37^{\circ}C$ and 7.0. The optimal medium compositions for cell growth were glucose 1% as a carbon source, (NH4) 2SO4 0.2% as a nitrogen source, K2HPO4 0.3%, and KH2PO4 0.45%. TO investigate she optimal condition for PHA production two-step cultivation method was employed. PHA production was inducted by deficiency of NH4+, SO4-2, Mg+2. Besides carbon source, deficiency of all nutrients stimulated PHA productivity but deficiency of NH4+ stimulated the most HV monomer content. The highest PHA production was C/N molar ratio 95.2. Pseudomonas sp. HJ was also able to pyoduc PHB/HV copolymer when cultivated on alkane, alkanoate, alcohol as carbon sources. The contents of PHA and she proportions of hydroxyvalerate monomer units varied depending on the carbon sources. Especially Pseudomonas sp. HJ was able to incorporate hydroxyvalerate into PHB/HV to level as high as from 49 to 74 mol% when grown in a medium containing hexadecane and propionate. The purified PHA was identified PHB/HV copolymer by HNMR analysis.

• 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.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.220-228
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• 1995

For polyhydroxyalkanoic acid (PHA) production, several microorganisms were isolated from sewage sludge. One of them, GB-77 strain, was chosen from its PHB/HV copolymer production on only fructose without cosubstrate. The isolated strain GB-77 was identified as the genus Alcaligenes. Optimal temperature and pH for cell growth were 36C and 6.8. Optimal medium composition was 10 g/l of fructose and 5 g/l of polypeptone, 1 $\times$ 10$^{-2}$M Na$^{2}$HP0$^{4}$, 1.3 $\times$ 10$^{-2}$M KH$^{2}$PO$^{4}$. To investigate the optimal condition for polyhydroxyalkanoic acid production two-stage culture technique was used; first stage for cell growth and second stage for PHA production on unbalanced growth conditions. Optimal conditions for high PHA production were C/N ratio 50, temperature 36$\circ$C and pH 6.8. To overcome fructose inhibition on cell growth, intermittent feeding fed-batch culture technique was used. Total cell concentration was 17.4 g/l with 9.1 g/l of PHA. The purified PHA was identified PHB/HV copolymer by NMR analysis.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.771-774
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• 2001

NADPH has been known as a regulating factor the biosynthesis of polyhydroxyalkanote(PHA), and the flux of NADPH for PHA biosynthesis could be enforced by the amplification of zwf gene encoding glucose 6-phosphate dehydrogenase. The recombinant plasmid pCZWF harboring PHA synthase, phbC from R. eutropha and zwf from E. coli were constructed, and were transformed to R. eutropha by electroporation. The biosynthesis of P(3HB-3HV) copolymer were carried out in transformant R. eutropha through the two-stage cultivation method using valerate as a precursor. The biosynthesis rate and PHA content of transformant R. eutropha harboring pCZWF were increased compared with transformant R. eutropha harboring only phbC. Especially, the molar fraction of 3HV was increased from 68% to 74% due to amplification of zwf gene. And the biosynthesis P(3HB-3HV) and P(3HB-4HB) carried out using propionate and ${\gamma}-butyrolactone$ as a precursor, respectively. But the rate, content, and molar fraction of biosynthesis copolymers were not influenced appreciably. This may be due to the reduced availability of NADPH.

• Journal of Environmental Science International
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• v.6 no.4
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• pp.379-384
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• 1997

The biodegradable characteristics of poly-$\beta$-hydroxybutyrate(PHB) film by fun맥 and soil burial are Investigated. As the results of the American Standards for Testing and Materials(ASTM) method, the you of Aspergillus niger was apparent on the PHB containing plate. This suggests that PHB was utilized as the sole carbon source by Aspergillus niger and ASTM method may have applications as measuring means of biome gradability of polyhydroxyalkanoic acid(PHA). PHB film was studied by monitoring the time-dependant changes in weight loss of PHB film under 30% and relative humidity 80 % during pot-test. As the results of pot-test, PHB film was decomposed about 87 % in 30 days by soul microorganisms. PHB film was more slowly degraded than PHB/HV film.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.175-178
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• 2000

Biodegradability of the $poly({\beta}-hydroxybutyrate-co-3 -hydroxyvalerate)$ [PHB/V] containing 0, 10 and 15mol% hydroxyvalerate [HV] was studied. Bioderadability of PHB/V was evaluated at $30^{\circ}C$ for 58 days and $55^{\circ}C$ for 33 days by monitoring the time-dependent changes in weight loss(erosion) of aerobic conditions in a temperature-controlled microcosms containing the earthworm cast($30^{\circ}C$) and compost ($55^{\circ}C$). It was found that PHB/V biodegradability occurred with increasing HV monomer concentration from 0 mol% to 15 mol%.

• Journal of Life Science
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• v.9 no.6
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• pp.737-742
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• 1999

The extremely halophilic archaebacterium Haloarcular sp. EH-1 was isolated from solar salts. Haloarcular sp. EH-1 accumulated poly(3-hydroxyalkanoate) (PHA) as intracellular granules. PHA production in batch culture have been studied. The PHA was identified as poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHB/HV) of 3-hydroxybutyric acid and 3-hydroxyvaleric acid by the analysis of GC, IR and NMR. The melting temperature of PHB/HV was 152.46$^{\circ}C$, viscosity was 1.25 ㎗/g, and molecular weight was $1.44 X 10^5.$