Immobilized Pichia pastoris X33/pGAPZαA-LaccPost-Stop in Ca2+ alginate beads was employed for Pleurotus ostreatus rPOXA 1B laccase batch production. Sequential statistical improvement was achieved through Plackett-Burman (PBED), (PBED-T11, 29.5±0.8 UL-1), which allowed to increase activity by 2.36-fold (12.5±2.6 UL-1) obtained in a preliminary study. Following, Box-Behnken Experimental Design (BBED) was implemented and obtained enzymatic activity in PBED-T11 was further increased by 33.5-fold (BBED-T12 989.31±187.45 UL-1). After BBED-T12 extrapolation to column, cell release remained high. To demonstrate laccase was not acting on Ca2+ alginate polymer, it was shown that both untransformed P. pastoris and S. cervisiae were able to be released from the alginate matrix and proliferate. Molecular docking evaluating interaction between rPOXA 1B and Ca2+ alginate, exhibited weak interactions between the active center and Ca2+ alginate polymer. Moreover, the active center conformation was not appropriate for ligand transformation. Immobilization conditions decreasing cell release (17.01±0.12 gL-1) allowed for high enzymatic activity (1,453.93±0.43 UL-1) with greater specific activity (18.33 Umg-1). These conditions were: 4% Na2+ alginate (w/v) and 0.3 M CaCl2, suggesting that Na2+ alginate and CaCl2 concentrations can control cell release from this matrix.