The characteristic aroma of mushrooms is one of their attractive elements as food materials. The major aroma compound in most mushrooms is 1-octen-3-ol. The biosynthesis of 1-octen-3-ol starts with the oxidation of linoleic acid by lipoxygenase (LOX). The resultant intermediate hydroperoxide is then cleaved by hydroperoxide lyase. LOX is a non-heme iron-containing dioxygenase widely found in plants, animals, fungi, and bacteria. It catalyzes the insertion of molecular oxygen into polyunsaturated fatty acids containing Z,Z-1,4-pentadiene moieties, such as linoleic acid, linolenic acid, and arachidonic acid, yielding the corresponding hydroperoxides. Two LOX genes, Polox1 and Polox2, have been isolated from P. ostreatus, which has higher LOX activity than other edible mushrooms. Polox1 and Polox2 were found to show different expression patterns during the development of the fruiting body. However, the biochemical properties of PoLOX1 and PoLOX2, encoded by Polox1 and Polox2, respectively, have been not fully elucidated. In this study, we engineered these two LOX genes of P. ostreatus into a heterologous host, Escherichia coli, and characterized the recombinant proteins. The coding regions of Polox1 and Polox2 were amplified by RT-PCR from the total RNA of P. ostreatus PC15 mycelia. The RT-PCR products were digested with appropriate restriction enzymes and ligated into an expression vector (pET-16b). The resultant plasmids were introduced into E. coli BL21 (DE3) via transformation. Polox1 and Polox2 were then expressed by induction at 15°C with 0.4 mM IPTG for 18 h. The cells were harvested by centrifugation and resuspended in 10 mM potassium phosphate buffer (pH 7.0). The cell suspension was sonicated and again centrifuged at 15,000 ×g for 20 min at 4 °C. The resultant cell-free extract was used for subsequent experiments. Recombinant PoLOX1 and PoLOX2 were confirmed by SDS-PAGE analysis of the cell-free extract. PoLOX1 and PoLOX2 were estimated to have molecular weights of approximately 76,000 Da and 78,000 Da, respectively. The LOX activity was determined with linoleic acid as a substrate by a spectrophotometric procedure based on the formation of conjugated dienes. To characterize the biochemical properties of PoLOX1 and PoLOX2, in vitro enzymatic assays were performed using the total cell protein from E. coli expressing the two Polox genes, with linoleic acid as a substrate. The optimum pH of recombinant PoLOX1 and PoLOX2 was 7.5 and 5.5, respectively; the optimum temperatures of recombinant PoLOX1 and PoLOX2 were 55 °C and 30 °C, respectively. Recombinant PoLOX1 and PoLOX2 were stable at pH 5.0-9.0 and 6.0-8.0, respectively; recombinant PoLOX1 and PoLOX2 were relatively stable below 50 °C and 40 °C, respectively. Thus, PoLOX1 had higher thermal and pH stability than PoLOX2. The calculated Km values of PoLOX1 and PoLOX2 were 121 μM and 249 μM, respectively. The calculated Vmax values of PoLOX1 and PoLOX2 were 17.2 μmol/mg･min and 17.5 μmol/mg･min, respectively. These results indicated that PoLOX1 had a higher affinity for linoleic acid than PoLOX2. Collectively, our findings suggested that there were some differences between the biochemical properties of PoLOX1 and PoLOX2.