Statistical Improvement of Batch Culture with Immobilized Pichia pastoris Cells for rPOXA 1B Laccase Production

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.