Characterization On Astaxanthin Esters And Fatty Acids Of Haematococcus Pluvialis And Analysis Of Differentially Expressed Genes

The astaxanthin ester composition of Haematococcus pluvialis were characterized by LC-(APCI)MS, and a HPLC method was developed to determine the astaxanthin content. The astaxanthin ester composition and the fatty acid composition in four different cell types of H. pluvailis were characterized by HPLC and GC-MS. The stability of astaxanthin and astaxanthin esters was studied. The content of free astaxanthin obtained by saponification and enzymatic hydrolysis were examined. A subtractive cDNA library of genes related to cyst formation and astaxanthin synthesis was contracted by SSH.1. Four free carotenoids, fifteen astaxanthin monoesters, twelve astaxanthin diesters and three astacin monoesters in H. pluvialis were identified by LC-(APCI)MS. In addition, a set of compounds with maximum absorbance at 400 nm, detected by HPLC-DAD, had strong characteristic fragment ions at m/z 871 and m/z 593 in the positive ion mode MS₂. They were presumed to be linolenic acid or an isomer ofω-6-γ-linolenic acid esters of astacin. The presence of these compounds has not been reported ever in H. pluvialis cells.2. The astaxanthin ester contents in H. pluvialis were quantified by the external standard calibration curve method through HPLC. The linear calibration curves were y = 1027.3x+245.55 (R²=0.9997) when the astaxanthin content ranging from 0.1 to 2.0μg. The average recovery was 100.277 and the RSD (%) was 2.14. This method, with better sensitivity and better reproducibility, could identify more astaxanthin esters than those ever reported3. The astaxanthin ester types were identical in red mobile cells and red cysts, and the fatty acid types were identical in four types of H. pluvialis cells. The major fatty acid contents in astaxanthin esters had the same tendency with the contents of the total fatty acids in red motile cells and red cysts. The oleic acid (C18:1) and linoleic acid (C18:2) were dominant in both red mobile cells and red cysts, and red mobile cells and red cysts contained much more oleic acid and linoleic acid than green motile cells and green cysts. It indicated that the active synthesis of oleic acid and linoleic acid was necessary to astaxanthin ester synthesis. The HPLC chromatogram of green cysts was similar to that of green motile cells, and neither free astaxanthin nor astaxanthin esters were detected in these two cells. Furthermore, the fatty acid composition was the same in green cysts and green motile cells. It suggested that astaxanthin ester accumulation coordinated with the synthesis of fatty acids from another point of view. The synthesis and accumulation of fatty acids was independent of the formation of cysts, but was highly correlated with the synthesis and accumulation of astaxanthin. The red cysts contained more than 30% of fatty acids, in which the unsaturated fatty acids accounted for about 81%. Therefore, H. pluvialis was not only a good resource of precious astaxanthin, but also would become a good resource of valuable fatty acids in future.4. Both oxygen and temperature had strong effects astaxanthin stability. Both vitamin C and vitamin E, the common antioxidant, decreased the astaxanthin stability. There were no significant changes in the astaxanthin content or the relative content of each astaxanthin ester after the dry algal meal with broken cell wall stored under vacuum at -20℃for 1.5 years or the algal meal stored at -20℃for 1.5 years. After stored for 2 years, the astaxanthin content of each treatment decreased about 10%, and the relative content of each astaxanthin ester was stabile in algal meal, but decreased significantly in dry algal meal.5. 72.04% free astaxanthin was obtained after the extraction mixture was treated at 37℃for 75 min with the presence of 1.02 units cholesterol. And it was the optimal treatment for hydrolysis by enzyme. However, only 51.48% free astaxanthin was obtained after the reaction mixture was treated at 22℃for 40 min with the presence of 1.07 M NaOH. And it was the optimal treatment for hydrolysis by alkali. Therefore, it was not accurate to determine astaxanthin content by HPLC after hydrolysis especially after saponification, for hydrolysis by enzyme or by alkali would destroy astaxanthin in some degree.6. H. pluvialis formed red cysts with astaxanthin accumulation in cells under stresses. In order to analyze the change of gene expressions and isolate these related genes inducing astaxanthin synthesis, a subtractive cDNA library of cyst formation and astaxanthin synthesis was constructed by using the Suppression Subtractive Hybridization (SSH) techniques. In this paper, the quality of cellular RNAs and adaptor ligation efficiency were analyzed, and the housekeeping gene,α-tubulin, was used to estimate the efficiency of subtractive cDNA. In this subtractive cDNA library,α-tubulin was subtracted very efficiently at appropriate 2¹⁰-2¹⁵ fold, indicating that some differentially expressed genes including the genes related to the cyst formation and astaxanthin synthesis were also enriched at the same folds. The construction of the subtractive cDNA library would be essential for rapid isolation and clone of the genes related to cyst formation and astaxanthin synthesis, and would be helpful to find out the molecular mechanism of the astaxanthin accumulation in H. pluvialis.