## Materials and Methods
### Isolation and Purification of LOX  
* Fractional precipitation using ammonium sulfate  
All steps were conducted below 4 \({}^{\circ}\)C following a previously described method [13]. An appropriate amount of crude enzyme solution was mixed with 114 g/L anhydrous ammonium sulfate solution in an ice water bath with stirring at a constant speed for 20 min. The mixture was passed through a double-layer filter paper or gauze to collect the filtrate. The supernatant was mixed with 243 g/L anhydrous ammonium sulfate solution in an ice water bath with stirring at a constant speed for 20 min. After the homogenate was centrifuged at 30,000\(\times\)\(g\) for 20 min, the supernatant was discarded and the residue collected and dissolved in an equal volume of phosphate buffer. Afterward, the mixture was recentrifuged at 90,000\(\times\)\(g\) for 20 min to obtain the supernatant.  
* DialysisThe enzyme solution was precipitated with 20-40% ammonium sulfate solution and then placed in dialysis bags and dialyzed with 50 mmol/L phosphate buffer (pH = 7.0) at 4 \({}^{\circ}\)C, during which the external solution was changed at an interval of 2-3 h. The supernatant was centrifuged and freeze-dried after overnight dialysis.
* DEAE Sepharose Fast Flow (FF) Anion Exchange Chromatography  
A DEAE Sepharose FF column was equilibrated with 50 mmol/L phosphate buffer (pH 7.0). The dialyzed enzyme solution was dissolved in an equal volume of phosphate buffer. The adsorbed protein was eluted from the column with a linear gradient of 0-0.5 mol/L NaCl at a rate of 0.8 mL/min. The absorption peak was measured at a wavelength of 280 nm and the tube with peak absorption was collected. Samples with LOX activity were collected, freeze-dried, and stored for further analysis.
* Electrophoresis  
Vertical plate sodium dodecyl sulfate\(-\)polyacrylamideamide gel electrophoresis (SDS\(-\)PAGE) was performed as previously described in the study by [PERSON], [PERSON], [PERSON], [PERSON], [PERSON], and [PERSON] [14]. After electrophoresis, the molecular weights of the samples were determined after staining with 0.25% CBB R-250 and decolorizing with glacial acetic acid-methanol.  
Enzymatic Characteristics (Storage Stability, Optimal Temperature, pH Value, and Substrate Specificity of LOX Extracted from Dry-Cured Mackerel)  
* Storage stability  
Enzymatic properties were measured as described in a previous study [15]. Briefly, the extracted LOX solution was stored at 25 \(\pm\) 3 \({}^{\circ}\)C for 7 days and its enzyme activity was measured at the same time every day.
* Optimal temperature and pH value for LOX activity in dry-cured mackerel  
The extracted LOX solution was dissolved in separate phosphate buffer solutions (50 mmol/L) with pH values of 6.6, 7.0, 7.4, 7.8, 8.2, and 8.6 and then heated in water baths set at 10, 20, 30, 40, 50, and 60 \({}^{\circ}\)C for 20 min. Afterward, the extracted LOX solution was mixed with separate solutions of linoleic acid, linoleic acid, and arachidonic acid to determine its activity.
* Substrate specificity  
The extracted LOX solution was reacted with linoleic acid, linoleic acid, and arachidonic acid, and enzyme activity was determined according to the method described in 2.3.