Application of The Polyhydroxyalkanoate Synthase of Chromobacterium Sp. USM2 for the Production of Biodegradable Plastics
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Date
2010-05
Authors
Bhubalan, Kesaven
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Abstract
A 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.
Description
Keywords
Polyhydroxyalkanoate