The effects of selected reaction parameters, including solvent hydrophobicity, initial water activity, agitation speed, temperature and enzyme concentration, on the biocatalytic efficiency of a chlorophyllase enzymatic extract from Phaeodactylum tricornutum in neat organic solvent media were investigated. The highest chlorophyllase specific activity of 322 nmol hydrolyzed chlorophyll per gram of protein per minute and bioconversion yield of 91% were obtained in the reaction mixture of hexane/2-octanone (98.3:1.7, v/v), at a controlled initial water activity of 0.90. RO/A value, which is the ratio of the specific activity in the organic solvent to that in the aqueous/miscible organic solvent medium, was 1.5 x 10(-3). To reduce the substrate diffusional limitations, the appropriate agitation speed and enzyme concentration were determined. The optimum temperature for maximal enzymatic activity and activation energy were 35 C and 105.0 kJ/mol, respectively. Although the catalytic efficiency of chlorphyllase in the neat organic solvent mixture was lower than that in the aqueous medium, its half-life time in the first environment at temperature ranging from 35 to 50 C was increased by 5.0 to 15.0 times.