Characterization of a class III peroxidase from Artemisia annua: relevance to artemisinin metabolism and beyond.

KEY MESSAGE: A class III peroxidase from Artemisia annua has been shown to indicate the possibility of cellular localization-based role diversity, which may have implications in artemisinin catabolism as well as lignification. Artemisia annua derives its importance from the antimalarial artemisinin. The -O-O- linkage in artemisinin makes peroxidases relevant to its metabolism. Earlier, we identified three peroxidase-coding genes from A. annua, whereby Aa547 showed higher expression in the low-artemisinin plant stage whereas Aa528 and Aa540 showed higher expression in the artemisinin-rich plant stage. Here we carried out tertiary structure homology modelling of the peroxidases for docking studies. Maximum binding affinity for artemisinin was shown by Aa547. Further, Aa547 showed greater binding affinity for post-artemisinin metabolite, deoxyartemisinin, as compared to pre-artemisinin metabolites (dihydroartemisinic hydroperoxide, artemisinic acid, dihydroartemisinic acid). It also showed significant binding affinity for the monolignol, coniferyl alcohol. Moreover, Aa547 expression was related inversely to artemisinin content and directly to total lignin content as indicated by its transient silencing and overexpression in A. annua. Artemisinin reduction assay also indicated inverse relationship between Aa547 expression and artemisinin content. Subcellular localization using GFP fusion suggested that Aa547 is peroxisomal. Nevertheless, dual localization (intracellular/extracellular) of Aa547 could not be ruled out due to its effect on both, artemisinin and lignin. Taken together, this indicates possibility of localization-based role diversity for Aa547, which may have implications in artemisinin catabolism as well as lignification in A. annua.

Supercritical CO2 fractionation of bio-oil produced from wheat-hemlock biomass.

The biomass i.e. wheat-hemlock used in this study was first characterized for its composition. The physical and chemical characterization of biomass was estimated using proximate analysis, calorific value, crystallinity, devolatilization behaviour, ultimate analysis, ICP-MS of ash, FT-IR, XRD, CHNS, and HPLC analysis. For commercial purpose the same biomass was used for conversion to bio-oil by fast pyrolysis process. Therefore, in order to investigate its composition, the bio-oil was also characterized using proximate analysis, calorific value, whereas the chemical composition of the bio-oil was estimated using CHNS, (1)H NMR, GC-FID and GC/MS. The bio-oil obtained from wheat-hemlock biomass was supplied by Advanced Biorefinery Co. and after the analysis, its composition has been determined. It contains a mixture of hydrocarbons, pyranoids, furanoids, benzenoids and fatty acids/alcohols with 45% of water, which forms azeotrope with organic polar compounds. The supercritical CO(2) (SC-CO(2)) is an advanced method for selective extraction of valuable chemicals from bio-oil without solvent residue. The organic fraction of the bio-oil was isolated by SC-CO(2). It was observed that SC-CO(2) fractions collected at 10 and 25 MPa pressure were enriched with furanoids, pyranoids and bezenoids. Similarly the bio-oil was also fractionated by conventional column chromatographic method and the yields and chemical compositions were compared with fractionated bio-oil obtained using SC-CO(2).