Ecophysiological significance and antisolvent micronization of zeaxanthin synthesized by marine Flavobacteria

• Main Authors: Asif Hameed, 哈錫夫
• Other Authors: Dr. Chiu-Chung Young
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/d3xe7u

博士 === 國立中興大學 === 土壤環境科學系所 === 100 === Marine Flavobacteria affiliated to the phylum Bacteroidetes are ecologically and phylogenetically diverse, playing significant role in the global oceanic carbon cycle. Previous culture-based and in silico analysis have demonstrated potential carotenoid producing ability of the marine Flavobacteria. However, predominant carotenoid produced and its ecophysiological relevance in native marine Flavobacteria remains elusive. The present study clarifies this uncertainty, besides exploring the role played by zeaxanthin in ATP synthesis and carbon fixation in marine Flavobacteria for the first time. Comparative genomic data (15 genera, 17 strains) and in vitro carotenoid screening via UV–visible spectroscopy and tandem mass spectrometry of randomly selected isolates (Muricauda lutaonensis CC-HSB-11T, Lutaonella thermophila CC-MHSW-2T, Siansivirga zeaxanthinifaciens CC-SAMT-1T, Leeuwenhoekiella blandensis MED217T, Zeaxanthinibacter enoshimensis TD-ZE3T, Mesoflavibacter zeaxanthinifaciens TD-ZX30T, Polaribacter dokdonensis DSW-5T, Dokdonia donghaensis DSW-1T and Robiginitalea biformata HTCC2501T) suggested zeaxanthin as a predominant carotenoid widespread in phylogentically distant marine Flavobacteria. In addition, expression analyses of a gene encoding β-carotene hydroxylase (CrtZ) of MED217T provided primary experimental evidence for the putative photoreceptor ability of zeaxanthin particularly to the green light in native marine Flavobacteria. Proteomic data of a hot spring isolate CC-HSB-11T showed succinyl-CoA synthetase (SCS) as a putative zeaxanthin-binding protein. Moreover, binding study via circular dichroism spectroscopy using a recombinant prokaryotic SCS revealed bathochromic shift in the chirality of zeaxanthin (~7–8 nm) suggesting protein-binding, confirming the preliminary proteomic data. All together, these results provided foremost evidence for the novel linkage between zeaxanthin and SCS. Green light illumination induced expression of α and β subunits of SCS of MED217T revealed a distinct green light-dependent ATP synthesis. Based on the genomic, transcriptomic and proteomic data, a hypothetical model representing green light-driven, zeaxanthin-mediated ATP synthesis in marine Flavobacteria was constructed. Furthermore, genomic screening detected genes encoding proteins of inorganic carbon uptake and fixation for the first time in marine Flavobacteria regardless of proteorhodopsin-photophysiology. The expression analysis of genes encoding bicarbonate transporter (BicA), carbonic anhydrase (CA) and pyruvate carboxylase (PC) of MED217T provided primary experimental evidence for the previously unknown, green light-driven anaplerotic inorganic bicarbonate uptake and fixation existing in marine Flavobacteria irrespective of the lack of proteorhodopsin-photophysiology. The study provided initial experimental proofs which imply marine Flavobacteria could reduce ocean acidification particularly during day time. Based on the comparative genomics and gene expression data analysis, a hypothetical model was derived to explain observed anaplerotic inorganic carbon fixation mechanism with response to light. Taken together, the present study discovers a novel aerobic, proteorhodopsin-independent, zeaxanthin-mediated, anoxygenic photoautotrophy existing in marine Flavobacteria which are being characterized as chemoorganotrophs before. Moreover, the study identified green light as a potential energy source driving inorganic carbon sequestration in the oceanic surface waters worldwide. These finding will have significant impact on our current understanding of the biogeochemistry of marine Flavobacteria which are ecologically diverse. In addition, these findings may prove truly valuable for developing strategies to enhance carbon sequestration and minimize global warming. The bioactive compounds in micronized forms attain much importance in pharmaceutical industry due to their enhanced efficacy, and tendency to accumulate at the target site. In the present work, zeaxanthin was isolated from strain CC-HSB-11T and subsequently subjected to microparticle generation using SC-CO2 ASP technology. The results provided baseline information for the industrial utilization of zeaxanthin produced by marine Flavobacteria for the generation of microparticles using SC-CO2 ASP technology.