• Journal of Microbiology and Biotechnology
• /
• v.6 no.5
• /
• pp.309-314
• /
• 1996

Alcaligenes sp. SH-69 can synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from a single carbon source such as glucose. To clone the phaC gene from Alcaligenes sp. SH-69, a polymerase chain reaction was performed using the oligomers synthesized based on the conserved regions of the phaC genes from other bacteria. A PCR product (550 bp) was partially sequenced and the deduced amino acid sequence was found to be homologous to that of the phaC gene from Alcaligenes eutrophus. Using the PCR fragment Southern blotting of Alcaligenes sp. SH-69 genomic DNA digested with several restriction enzymes was carried out. To prepare a partial genomic library, about 5-Kb genomic DNA fragments digested with EcoRI, which showed a positive signal in the Southern blotting, were eluted from an agarose gel, ligated with pUC19 cleaved with EcoRI, and transformed into Escherichia coli. The partial library was screened using the PCR fragment as a probe and a plasmid, named pPHA11, showing a strong hybridization signal was selected. Restriction mapping of the insert DNA in pPHA11 was performed. Cotransformation into E. coli of the plasmid pPHA11 and the plasmid pPHA21 which has phaA and phaB from A. eutrophus resulted in turbid E. coli colonies which are indicative of PHA accumulation. This result tells us that the Alcaligenes sp. SH-69 phaC gene in the pPHA11 is functionally active in E. coli and can synthesize PHA in the presence of the A. eutrophus phaA and phaB genes.

• Korean Journal of Microbiology
• /
• v.36 no.2
• /
• pp.84-90
• /
• 2000

Biodegradation of vanous medium-chain-length polyhydroxyalkanoates (MCL-PHAs) by an extracellular depolymerase system from Pseudomonas sp. RY-1 was investigated under laboratoly conditions. The degradation rate of the polymers was determined by quantitative clem zone technique, enzyme (turbidity) assay, and respirometry assay. Although the enzyme system secreted by Pscudomor~as sp. RY-1 was capable of degrading all MCL-PHAs tested. its secretion was influenced by the availability of secondary carbon sources. The rate of enzymatic degradation of MCL-PHAs was dependent upou the monomeric composition of the polyesters and reduced as the chain lengths of the monomer m t s in the polyesters increased. MCL-PHAs containing C-even monomer units showed faster degradation rate than MCL-PHAs containing C-odd monomer units. Respiration rates of MCL-PHAs with C-even monomer uuts were also much faster than those of MCL-PHAs with C-odd monomer units. The degmdation rate of MCL-PHAs bearing unsaturated substituents was faster than that of mcl-PHAs without functional substituents, which is suggesting the correlation between the degradation rate and the crystallinity of MCL-PHAs.