Characterization Of A Highly Active Polyhydroxyalkanoate Synthase
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Date
2012-06
Authors
Chuah, Jo-Ann
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Polyhydroxyalkanoate (PHA) synthase from a locally isolated
Chromobacterium sp. USM2 (PhaCCs) exhibited superior polymerizing ability and
broad in vivo substrate specificity with preferences for short chain length (SCL) [3-
hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV)] and medium chain length
(MCL) [3-hydroxyhexanoate (3HHx)] monomers. For further characterization of the
synthase, a Strep2-tagged PhaCCs for expression in and purification from
Escherichia coli, was constructed in this study. In vitro enzymatic assay revealed an
activity of 253 ± 13 U/mg for polymerization of 3-hydroxybutyryl-coenzyme A
(3HB-CoA), which was approximately fivefold higher than that of model PHAproducing
strain Cupriavidus necator (39 ± 5 U/mg). Its activity for polymerization
of 3-hydroxyvaleryl-coenzyme A was twice as great as that for 3HB-CoA, while
corresponding activity for 3-hexanoyl-coenzyme A polymerization rivaled that of
another SCL-MCL synthase, from Aeromonas caviae. This discovery prompted
further characterization studies and application of the highly active synthase for in
vivo PHA production. Here, the gene encoding the PHA synthase from
Chromobacterium sp. USM2 (phaCCs) was used to replace the native PHA synthase
gene in wild type C. necator by homologous recombination. The resultant strain
showed improved productivity of flexible poly(3-hydroxybutyrate-co-3-
hydroxyhexanoate) from crude palm kernel oil with concomitant good growth.
Various approaches such as alteration of culture parameters, addition of precursor or
inhibitor compounds and manipulation of biosynthetic pathway successfully
increased the 3HHx monomer fraction, further enhancing flexibility of the
copolymer.
Description
Keywords
Characterization of a highly active , Polyhydroxyalkanoate synthase