The microalgal cell wall breakage has been identified as complex phenomena and highly dependent upon the nature and composition of cell wall. Detailed analysis of plastids and their function requires the breaking open of cell without any damage to cellular components. To develop a rapid and universal methodology for cell wall breakage; liquid nitrogen crushing, sonication, enzymatic lysis and homogenization procedures were applied to various microalgal species. Homogenization based procedure for isolation of intact chloroplast was found to have wide application. The crucial key parameters affecting the intactness of isolated chloroplast were optimized. The isolated chloroplasts were subjected to chloroplast integrity analysis. The intact chloroplast exhibited positive maximum quantum yield, Fv/Fm values ranging from 0.1 to 0.4 as measured by pulse amplitude modulation fluorimetry and was found to be suitable for further downstream applications like isolation of protein-pigment complexes involved in photosynthetic O2 evolution. The developed methodology is quick and efficient technique for isolation of intact chloroplasts across different genera of microalgae by employing minor changes in base protocol as a species specific characteristic.
Key words: Microalgae, Chloroplast isolation, Cell wall lysis
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