Optimization of aqueous two-phase micellar system for partial purification of L-asparaginase from Penicillium sp. grown in wheat bran as agro-industrial residue

L-asparaginase has been used in the remission of malignant neoplasms such as acute lymphoblastic leukemia. The search for new sources of this enzyme has become attractive for therapeutics. Traditional methods for biomolecule purification involve several steps. A two-phase system may be a good strategy to anticipate one of these stages. This study aimed to produce and purify a fungal L-asparaginase through an aqueous two-phase micellar system (ATPMS) using Triton X-114. The fungus Penicillium sp.-encoded 2DSST1 was isolated from Cerrado soil. Plackett-Burman design followed by a 2⁴ full factorial design was used to determine the best conditions to produce L-asparaginase. The evaluated variables were L-asparagine, L-proline, wheat bran, potato dextrose broth, ammonium sulfate, yeast extract, sucrose and glucose concentrations, incubation temperature, incubation period, and initial pH of the culture medium. L-asparaginase quantification was valued by the formation of β-aspartyl hydroxamate. The significant positive variables, L-asparagine, L-proline, potato dextrose broth, and sucrose concentrations, were evaluated at 2 levels (+ 1 and - 1) with triplicate of the central point. After 34 runs, maximum activity (2.33 IU/mL) was achieved at the factorial design central point. A central composite design was performed in ATPMS at two levels (+ 1 and - 1) varying Triton X-114 concentration (w/v), separation phase temperature, and crude extract concentration (w/v). The L-asparaginase partition coefficient (K) was considered the experimental design response. Out of the 16 systems that were examined, the most promising presented a purification factor of 1.4 and a yield of 100%.

Keywords: ATPMS; Agro-industrial residue; L-Asparaginase; Plackett-Burman design; Purification.