Lk2 and Lk3 were thermostable recombinant lipase and highly expressed in Escherichia coli BL21 (DE3). However, Lk2 and Lk3 accumulated as an inclusion body. To further characterize both recombinant lipases, the soluble enzyme must be obtained first. This study aimed to optimize the disruption of the cell membrane in order to obtain soluble and active lipases. The effects of temperature lysis, pH, and SDS concentration on lipolytic activity Lk2 and Lk3 were investigated using a three-factor Box-Behnken design response surface methods. The optimum condition for the temperature variables at 50°C, pH 8, and 0.34% SDS which gave a lipolytic activity of 0.9 U for Lk2. Meanwhile, Lk3 lipolytic activity of 0.9 U obtained at the temperature of 50°C, pH 8, and 0.1% SDS. This result showed efficient one-step membrane disruption methods using thermolysis with addition of a low concentration of detergent at pH 8. The methods used were effective and applicable in the production of active and soluble thermostable recombinant lipase.