A moderate yield of a purified enzyme can be achieved by using the simple technique of reverse micellar extraction (RME). RME is a liquid-liquid extraction method that uses a surfactant and an organic solvent to extract biomolecules. Instead of traditional chromatographic purification methods, which are tedious and expensive, RME using the nonionic surfactant Triton X-100 and toluene is used as an alternative purification technique to purify a recombinant cold-adapted lipase, AMS8. Various process parameters were optimized to maximize the activity recovery of the AMS8 lipase. The optimal conditions were found to be 50 mM sodium phosphate buffer, pH 7, 0.125 M NaCl, and 0.07 M Triton X-100 in toluene at 10 • C. Approximately 56% of the lipase activity was successfully recovered. Structural analysis of the lipase in a reverse micelle (RM) was performed using an in silico approach. The predicted model of AMS8 lipase was simulated in the Triton X-100/toluene reverse micelles from 5 to 40 • C. The lid 2 was slightly opened at 10 • C. However, the secondary structure of AMS8 was most affected in the non-catalytic domain compared to the catalytic domain, with an increased coil conformation. These results suggest that an AMS8 lipase can be extracted using Triton X-100/water/toluene micelles at low temperature. This RME approach will be an important tool for the downstream processing of recombinant cold-adapted lipases.2 of 15 absence of co-factors [6][7][8][9]. Cold-active lipases cover a broad spectrum of biotechnological applications, such as additives in detergents (cold washing), an additive in food industries (fermentation, cheese manufacture, bakery, meat tenderizing), bioremediations (Digesters, composting, oil, or xenobiotic biology applications), and molecular biology applications [10][11][12].Several conventional purification methods for cold-adapted lipases have been applied for many years in industrial and laboratory procedures [13,14]. For example, the lipase KE38 from Pseudomonas fluorescens KE38 was successfully purified using (NH 4 ) 2 SO 4 precipitation, Sephadex G-100 and, lastly, ultrafiltration with a recovery of 54.99% and a 41.13-fold purification [15]. Likewise, three purification steps were used to purify extracellular LipP from Pseudomonas sp. strain BII-I by using 20-65% (NH 4 ) 2 SO 4 precipitation followed by a diethylaminoethyl (DEAE)-cellulose and graphite column with 38-fold purification and a recovery of 17% of the lipase activity [13]. Although a good yield of purified enzyme was obtained from these chromatographic techniques, the production and purification of large amounts of enzyme are often laborious and expensive, which hinders applications for these enzymes [16]. Therefore, a standard method that enables the purification of large amounts of highly pure, active, and stable enzymes is urgently needed. Reverse micellar extraction is one of the alternative purification methods under consideration for continuous downstream processing of bulk enzymes.A reverse micelle (RM) is a surfactant...