Molecular, structural and biochemical characterization of a novel recombinant chlorophyllase from cyanobacterium Oscillatoria acuminata PCC 6304

BACKGROUND: Chlorophyllase catalyzes the hydrolysis of chlorophyll and produces chlorophyllide and phytol. Cyanobacterial chlorophyllases are likely to be more highly heterologously expressed than plant chlorophyllases. A novel recombinant chlorophyllase from the cyanobacterium Oscillatoria acuminata PCC 6304 was successfully expressed in Escherichia coli BL21(DE3). RESULTS: The putative N-terminal 28-amino-acid signal peptide sequence of O. acuminata chlorophyllase (OaCLH) is essential for its activity, but may confer poor solubility on OaCLH. The C-terminal fusion of a 6 × His tag caused a partial loss of activity in recombinant OaCLH, but an N-terminal 6 × His tag did not destroy its activity. The optimal pH and temperature for recombinant OaCLH activity are 7.0 and 40 °C, respectively. Recombinant OaCLH has hydrolysis activities against chlorophyll a, chlorophyll b, bacteriochlorophyll a, and pheophytin a, but prefers chlorophyll b and chlorophyll a as substrates. The results of site-directed mutagenesis experiments indicated that the catalytic triad of OaCLH consists of Ser159, Asp226, and His258. CONCLUSIONS: The high-level expression and broad substrate specificity of recombinant OaCLH make it suitable for genetically engineering and a promising biocatalyst for industrial production, with applications in vegetable oil refining and laundry detergents.