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

Abstract

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.

Genes, Structural, and Biochemical Characterization of Four Chlorophyllases from Solanum lycopersicum

“…Previous studies have demonstrated the importance of the catalytic triad (Ser-Asp-His) for CaCLH activity [ 29 ]. In one of the studies, one catalytic residue (Ser-Asp-His) was replaced with other residues, and the activity of CLH was either reduced or completely lost [ 10 , 29 , 30 ]. In this study, introduction of three mutations into the putative catalytic triad of SlCLH1 (S135, D162, and H239) showed the complete loss of CLH activity ( Figure 6 C).…”

“…Previous studies have shown that the N-terminal peptide processing of CLHs plays a contradictory role in their Chl dephytylation activity. For example, the chlorophyllases from Pachira macrocarpa and Cyanobacterium Oscillatoria cuminata PCC6304 were functionally inactive when the N-terminal peptide was removed [ 8 , 10 ]. However, CLH lacking the N-terminal 21 amino acids from Citrus sinensis showed more enzymatic activity in vivo [ 35 ].…”

“…The mutant enzymes Ser135Ala, Asp162Asn, and His239Ala of SlCLH1 were constructed to identify the role of these putative catalytic residues in the activity of SlCLH1. The desired mutations were introduced into the Mal-p2x vector with site-directed mutagenesis using the PCR overlap extension method [ 10 ]. The primers listed in Table S1 were designed to introduce the desired mutations.…”

“…Since it was first reported more than a century ago, the physiological and biochemical characteristics of CLHs from various plants and algal species, including Chenopodium album [ 5 ], Citrus limon [ 6 , 7 ], Pachira macrocarpa [ 8 ], Chlamydomonas reinhardtii [ 9 ], Oscillatoria acuminata PCC6304 [ 10 ], and Arabidopsis thaliana [ 11 ], have been intensively studied. CLH was considered to localize in chloroplasts and the thylakoid membrane because endogenous Chl hydrolyzation only takes place when chloroplast membranes are disrupted [ 12 , 13 , 14 ].…”

Chlorophyllides: Preparation, Purification, and Application

“…The results indicated that recombinant CyanoCLH preferred hydrolyzing bacteriochlorophyll a from R. sphaeroides. The characterization of a chlorophyllase (OaCLH) from the cyanobacterium Oscillatoria acuminata PCC 6304 was reported [130]. This study demonstrated that the substrates of recombinant OaCLH were chlorophyll a, chlorophyll b, bacteriochlorophyll a, and pheophytin a, and chlorophyll b and chlorophyll a were especially preferred.…”