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

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 Km, Vmax, Kcat, and Kcat/Km 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.

Production, characterization, and bio-ethanologenic potential of a novel tripartite raw starch-digesting amylase from Priestia flexa UCCM 00132.

The biological conversion of agro-waste biomass into value-added metabolites is one of the trendy biotechnological research areas in recent times. One of the major drawbacks of the bioprocess is the saccharification potential of the amylolytic enzyme that releases reducing sugar from complex biomass to serve as substrate for fermentation. The present study reports the production of a novel tripartite raw starch-digesting amylase (RSDA) by an indigenous Priestia flexa strain with α-, ß-, and gluco-amylolytic activities and its potential for bioethanol production. Response surface statistics was employed to develop a suitable medium for improved production of the tripartite enzyme by submerged fermentation. The bioprocess selected raw starch (4.36%) Ca2+(2.71 g/L) and Zn2+ (0.0177 g/L) as significant variables which demonstrated a total RSDA activity of 7208.23 U/mL in a 5-L batch bioreactor. SDS/Native-PAGE determined the molecular weights of the 27-fold purified product as 25.2 kDa, 57.3 kDa, and 90.1 kDa for α-, ß-, and gluco-amylases, respectively. Optimum temperature and pH for enzyme activity were respectively broad at 30-70 °C and 4-11. The enzyme mixture demonstrated digestibility above 90% against a variety of raw starches and simultaneous fermentation of digestate with Saccharomyces cerevisiae generated 71.69 g/L of bioethanol within 24 h suggesting great potential for bioethanologenesis.

Strain improvement, artificial intelligence optimization, and sensitivity analysis of asparaginase-mediated acrylamide reduction in sweet potato chips.

In recent times, L-asparaginase has emerged as a potential anti-carcinogen through hydrolysis of L-asparagine in the blood for anti-leukemic application, and in carbohydrate-based foods, for acrylamide reduction applications. In this study, Aspergillus sydowii strain UCCM 00124 produced an L-asparaginase with a baseline acrylamide reduction potential of 64.5% in sweet potato chips. Plasma mutagenesis at atmospheric pressure and room temperature (ARTP) was employed to improve L-asparaginase production while artificial neural network embedded with genetic algorithm (ANN-GA) and global sensitivity analysis were used to identify and optimize process conditions for improved acrylamide reduction in sweet potato chips. The ARTP mutagenesis generated a valine-deficient mutant, Val-Asp-S-180-L with 2.5-fold L-asparaginase improvement. The ANN-GA hybrid evolutionary intelligence significantly improved process efficiency to 98.18% under optimized conditions set as 118.6 °C, 726.37 g/L asparagine content, 9.92 µg/mL L-asparaginase, 4.54% NaCl, and soaking time of 15 h without significant changes in sensory properties. The sensitivity index revealed initial asparagine content as the most sensitive parameter to the bioprocess. The enzyme demonstrated significant thermo-stability with Arrhenius deactivation rate constant, Kd, of 0.00562 min-1 and half-life, t1/2, of 123.35 min at 338 K. These conditions are recommended for sustainable healthier, and safer sweet potato chips processing in the food industry. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05757-5.