Interspecific protoplast fusion of atmospheric and room-temperature plasma mutants of Aspergillus generates an L-asparaginase hyper-producing hybrid with techno-economic benefits

Abstract

The axenic culture of Aspergillus candidus (Asp-C) produced an anti-leukemic L-asparaginase while Aspergillus sydowii (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 Ile^-Thr^-Asp-C-180-K and Val^-Asp-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 K_m, V_max, K_cat, and K_cat/K_m as 1.667 × 10⁻³ M, 1666.67 µmol min⁻¹ mg⁻¹ protein, 645.99 s⁻¹ and 3.88 × 10⁵ M⁻¹ s⁻¹ respectively. In-vitro cytotoxicity of HL-60 cell lines by fusant-09 L-asparaginase improved 3.00 and 18.71-folds from mutants Ile^-Thr^-Asp-C-180-K and Val^-Asp-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.

Keywords:

• Acrylamide mitigation
• anti-leukemic activity
• ARTP mutagenesis
• protoplast fusion
• L-asparaginase
• techno-economics

Acknowledgements

The assistance of technical aids at the Research Laboratory, Calabar, Environmental and Biotechnology Unit of Microbiology Department, University of Calabar, Hematology Department of University of Nigeria Teaching Hospital (UNTH) and the Faculty of Technology Laboratory at the University of Ibadan, Nigeria are highly appreciated.

Data availability statement

Data associated with this study have been included in the manuscript and supplementary file. More data may be provided by the corresponding author on reasonable request.