Abstract: Recombinant E. coli cells were disrupted by combined non-mechanical methods, such as chemical permeation, repeated freeze-thaw and enzymatic lysis for the extraction of recombinant lipoxygenase (LOX). Based on one-factor-at-a-time experiments, an L25(56) orthogonal array design was employed to optimize five process parameters. The best results for bacterial cell disruption were achieved through enzymatic lysis with 1.5 mg/mL of lysozyme for 40 min in the presence of 2.0 mmoL/L EDTA-2Na and 2% Tween-60 followed by 3 repeated freeze-thaw cycles, yielding an LOX activity of 6840 U/mL in crude enzyme solution, which was 1.44-fold higher than before the optimization. Compared with spectrophotometry and xylenol orange method, potassium iodide-starch method was more simple, sensitive and rapid and the reaction system showed characteristic color visible to the naked eye. Thus, this method is suitable for high throughput screening of LOX activity.

Key words: lipoxygenase, chemical permeation, freeze-thaw, enzymatic lysis, enzyme activity assay