Low-cost media statistical design for laccase rPOXA 1B production in P. pastoris

Laccases (E.C. 1.10.3.2) are multicopper oxidases of great importance in the industry due to their non-specificity and high oxidative potential. Laccases are useful to bleach synthetic dyes, oxidize phenolic compounds and degrade pesticides, among others. Hence, the objective of this work was to optimize low cost culture media for recombinant (rPOXA 1B) laccase production from Pleurotus ostreatus in Pichia pastoris. To this end, low cost nitrogen sources were studied, such as malt extract, isolated soy protein and milk serum. Following, two central composite designs (CCD) were performed. In CCD-1 different concentrations of glucose USP (0-13.35 gL^-1), protein isolated soy protein (5-25 gL^-1), malt extract (3.5-17.5 gL^-1) and (NH₄)₂SO₄ (1.3-6.5 gL^-1) were evaluated. In CCD-2 only different concentrations of glucose USP (7.9-22 gL^-1) and isolated soy protein (15.9-44.9 gL^-1) were evaluated. CCD-2 results led to a One Factor Experimental design (OFED) to evaluate higher isolated soy protein (20-80 gL^-1) concentrations. In all designs, (CCD-1, CCD-2 and OFED) CuSO₄ (0.16 gL^-1) and chloramphenicol (0.1 gL^-1) concentrations remained unchanged. For the OFED after sequential statistical optimization, an enzyme activity of 12,877.3 ± 481.2 UL^-1 at 168 h was observed. rPOXA 1B activity increased 30.54 % in comparison with CCD-2 results. Final composition of optimized media was: 20 gL^-1 glucose USP, 50 gL^-1 isolated soy protein 90 % (w/w), 11.74 gL^-1 malt extract, and 4.91 gL^-1 (NH₄)₂SO₄. With this culture media, it was possible to reduce culture media costs by 89.84 % in comparison with improved culture media previously described by our group.