Application of The Polyhydroxyalkanoate Synthase of Chromobacterium Sp. USM2 for the Production of Biodegradable Plastics

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dc.contributor.authorBhubalan, Kesaven
dc.date.accessioned2016-01-12T03:36:42Z
dc.date.available2016-01-12T03:36:42Z
dc.date.issued2010-05
dc.description.abstractA local isolate, Chromobacterium sp. USM2 was found to synthesize polyhydroxyalkanoate (PHA) with very high molar fractions of 3-hydroxyvalerate (3HV) monomer under certain culture conditions. The PHA synthase gene of this isolate was cloned and expressed in a mutant Cupriavidus necator PHB-4. Interestingly, the synthase was able to incorporate 3-hydroxyhexanoate (3HHx) monomer when this transformant was cultured with palm oil as the sole carbon source. In this study, the PHA synthase of Chromobacterium sp. USM2 (PhaCcs) was characterized via in vivo and in vitro assays using Escherichia coli JM109 to better understand its ability to synthesize PHA. Results from the in vitro assays showed that the expression level of PhaCcs was higher compared to the PHA synthase of C. necator HI6 (PhaCcn) which was used as a positive control. This phenomenon was clearly exhibited in the SDSPAGE and Western blot analyses. The total enzyme activity of PhaCcs (2132 U/g) was nearly 8-fold higher compared to that of PhaCcn (253 U/g). These findings were further supported by the results obtained from in vivo assay whereby the E. coli transformant was able to produce 76 ± 2 wt% of poly(3-hydroxybutyrate) homopolymer within 24 h of cultivation with glucose. Positive results from the in vitro and in vivo assays demonstrated the superior ability of PhaCcs to efficiently synthesize PHA. As it was previously proven that PhaCcs could incorporate both 3HV and 3HHx monomers, effort was taken to produce a terpolymer consisting of 3-hydroxybutyrate, 3-hydroxyvalerate XXV and 3-hydroxyhexanoate [P(3HB-co-3HV-co-3HHx)]. This terpolymer was believed to possess improved physical and mechanical properties compared to its respective copolymers. In this study, P(3HB-co-3HV -co-3HHx) terpolymers were synthesized using PHB-4 transformant harboring the PHA synthase gene of Chromobacterium sp. USM2 and the results were compared with that containing the PHA synthase gene of Aeromonas caviae. Terpolymers with various monomer compositions were synthesized from mixtures of crude palm kernel oil and 3HV precursors namely sodium valerate and sodium propionate. The 3HV and 3HHx monomers were regulated by varying the feeding techniques of precursors which resulted in their compositions ranging from 1 - 91 mol% and 1 -20 mol%, respectively. Subsequently, the P(3HB-co-3HV-co- 3HHx) terpolymer synthesized by both strains were characterized and evaluated. Terpolymers with relatively high Mw at around 8 x 105 - 25 x 105 Da were produced. In general, terpolymers exhibited greater flexibility and mechanical strength compared to the copolymers. Interestingly, terpolymer containing 69 mol% 3HB, 24 mol% 3HV and 7 mol% 3HHx exhibited mechanical properties similar to that of common low-density polyethylene.en_US
dc.identifier.urihttp://hdl.handle.net/123456789/1414
dc.subjectPolyhydroxyalkanoateen_US
dc.titleApplication of The Polyhydroxyalkanoate Synthase of Chromobacterium Sp. USM2 for the Production of Biodegradable Plasticsen_US
dc.typeThesisen_US
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