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.
Subject(s)
Bacterial Proteins/metabolism , Carboxylic Ester Hydrolases/metabolism , Chlorophyll A/metabolism , Chlorophyll/metabolism , Oscillatoria/enzymology , Recombinant Proteins/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Biocatalysis , Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/genetics , Cloning, Molecular , Escherichia coli/genetics , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Models, Molecular , Oscillatoria/genetics , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid , Substrate Specificity , TemperatureABSTRACT
DHA production by Schizochytrium sp. S31 was studied in batch cultures on glycerol with stepwise dissolved oxygen strategy. Three growth stages were identified as cell growth, lipid accumulation and lipid turnover. It was revealed that fatty acid (FA) shifts during the three growth stages involved the activity changes of glycerol kinase (GK), FAD(+)-dependent glycerol-3-phosphate dehydrogenase (FAD(+)-G-3-PDH), malic enzyme (ME), ATP citrate lyase (ACL) and NAD(+)-dependent isocitrate dehydrogenase (NAD(+)-ICDH). Glycerol dissimilation in Schizochytrium sp. S31 was suggested via a phosphorylation by GK and a following oxidation by FAD(+)-G-3-PDH. Lipid accumulation of this strain was a growth-associated process, but the assimilable nitrogen depletion enhanced the accumulation of lipids. The exhaustion of glycerol induced the lipid turnover stage, where the short chain fatty acids were preferentially degraded and converted into lipid-free biomass (Xf) which was correlated to the increase of DHA content in biomass.
