Interspecific Protoplast Fusion of Atmospheric and Room-temperature Plasma Mutants of Generates an L-asparaginase Hyper-producing Hybrid with Techno-economic Benefits

The axenic culture of (Asp-C) produced an anti-leukemic L-asparaginase while (Asp-S) produced the acrylamide-reduction type. Upon mutagenesis by atmospheric and room-temperature plasma (ARTP), their individual L-asparaginase activities improved 2.3-folds in each of IleThrAsp-C-180-K and ValAsp-S-180-E stable mutants. Protoplast fusion of selected stable mutants generated fusant-09 with improved anti-leukemic activity, acrylamide reduction, higher temperature optimum and superior kinetic parameters. Submerged (SmF) and solid-state fermentation (SSF) types were compared; likewise batch, fed-batch and continuous fermentation modes; and fed-batch submerged fermentation was selected on the basis of impressive techno-economics. Fusant L-asparaginase was purified by PEG/Na citrate aqueous two-phase system and molecular exclusion chromatography to 69.96 and 146.21-fold, respectively, and characterized by molecular weight, specificity, activity and stability to chemical and physical agents. Michaelis-Menten kinetics, evaluated under optimum conditions gave , , , and / as 1.667 × 10 M, 1666.67 µmol min mg protein, 645.99 s and 3.88 × 10 M s respectively. In-vitro cytotoxicity of HL-60 cell lines by fusant-09 L-asparaginase improved 3.00 and 18.71-folds from mutants IleThrAsp-C-180-K and ValAsp-S-180-E, and from 5.73 and 32.55 from respective original strains. Free-radical scavenging and acrylamide reduction improvements were intermediate. Fusant-09 L-asparaginase is strongly recommended for sustainable economic anti-leukemic and food industry applications.