Poly-3-hydroxybutyrate (P(3HB)), a member of the polyhydroxyalkanoate (PHA) family, is a biodegradable polyester with diverse industrial applications. NADPH-dependent acetoacetyl-CoA reductase (phaB) is the enzyme which plays an essential role in P(3HB) synthesis by catalyzing the conversion of the intermediates. The expression of phaB enzyme using the recombinant Escherichia coli BL-21(DE3) and the purification of the synthesized enzyme were studied. The pET-B3 plasmid harbouring the phaB gene derived from Ralstonia eutropha H16, was driven by the lac promoter in E. coli BL-21(DE3). The enzyme was expressed with different induction time, temperatures and cell age. Results showed that the cell age of 4 h, induction time of 12 h at 37°C were identified as the optimal conditions for the enzyme reductase expression. A specific activity of 0.151 U mg-1 protein and total protein concentration of 0.518 mg mg-1 of dry cell weight (DCW) were attained. Affinity chromatography was performed to purify the His-tagged phaB enzyme, in which enhanced the specific activity (14.44 U mg-1) and purification fold (38-fold), despite relative low yield (44.6%) of the enzyme was obtained. The purified phaB showed an optimal enzyme activity at 30°C and pH 8.0. The findings provide an alternative for the synthesis of the reductase enzyme which can be used in the industrial-scale production of the biodegradable polymers.
Vivipary with precocious seedlings in mangrove plants was thought to be a hindrance to long-range dispersal. To examine the extent of seedling dispersal across oceans, we investigated the phylogeny and genetic structure among East Asiatic populations of Kandelia candel based on organelle DNAs. In total, three, 28 and seven haplotypes of the chloroplast DNA (cpDNA) atpB-rbcL spacer, cpDNA trnL-trnF spacer, and mitochondrial DNA (mtDNA) internal transcribed spacer (ITS) were identified, respectively, from 202 individuals. Three data sets suggested consistent phylogenies recovering two differentiated lineages corresponding to geographical regions, i.e. northern South-China-Sea + East-China-Sea region and southern South-China-Sea region (Sarawak). Phylogenetically, the Sarawak population was closely related to the Ranong population of western Peninsula Malaysia instead of other South-China-Sea populations, indicating its possible origin from the Indian Ocean Rim. No geographical subdivision was detected within the northern geographical region. An analysis of molecular variance (AMOVA) revealed low levels of genetic differentiation between and within mainland and island populations (phiCT = 0.015, phiSC = 0.037), indicating conspicuous long-distance seedling dispersal across oceans. Significant linkage disequilibrium excluded the possibility of recurrent homoplasious mutations as the major force causing phylogenetic discrepancy between mtDNA and the trnL-trnF spacer within the northern region. Instead, relative ages of alleles contributed to non-random chlorotype-mitotype associations and tree inconsistency. Widespread distribution and random associations (chi2 = 0.822, P = 0.189) of eight hypothetical ancestral cytotypes indicated the panmixis of populations of the northern geographical region as a whole. In contrast, rare and recently evolved alleles were restricted to marginal populations, revealing some preferential directional migration.
Nipa (Nypa fruticans Wurmb.) is an important mangrove palm species, but it is understudied due to lack of information on genetic patterns within its distribution range. In this study, we identified 18 informative microsatellite markers to assess genetic variations among local populations in the Indo-West Pacific (IWP). Results showed population stratification based on high genetic differentiation (FST = 0.22131) with the Mantel test indicating significance to isolation-by-distance. We found a pronounced differentiation between the west populations in Sri Lanka and east populations in Southeast Asia. The east populations around the South China Sea were more genetically similar than those along the Malacca Strait and Java Sea. These genetic clines were shaped by ocean circulations and seasonal monsoon reversals as plausible factors. The Malacca Strait was confirmed as both a genetic and a geographic barrier rather than a corridor according to the Monmonier plot. Simulations of directional migration indicated a statistically strong contemporary genetic connectivity from west to east where Sri Lankan immigrants were detected as far as central Philippines via long-distance dispersal. This is the first report on the recent migration patterns of nipa using microsatellites. Assignment of first-generation (F0) immigrants suggested Mainland Southeast Asia as a melting pot due to the admixture associated with excess of homozygosity. The western populations were recent expansions that emerged in rapid succession based on a phylogram as supported by footprints of genetic drift based on bottleneck tests.