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.

A novel strain of Stenotrophomonas acidaminiphila produces thermostable alkaline peptidase on agro-industrial wastes: process optimization, kinetic modeling and scale-up.

Bacterial alkaline peptidases, especially from Bacillus species, occupy the frontline in global enzyme market, albeit with poor production economics. Here, we report the deployment of response surface methodology approximations to optimize fermentation parameters for enhanced yield of alkaline peptidase by the non-Bacillus bacterium; Stenotrophomonas acidaminiphila. Shake flask production under optimized conditions was scaled up in a 5-L bench-scale bioreactor. Logistic and modified Gompertz models revealed significant fits for biomass formation, total protein, and substrate consumption models. Maximum specific growth rate (µmax = 0.362 h-1) of the bacterium in the optimized medium did not differ significantly from those in Luria-Bertani and trypticase soy broths. The aqueous two-phase system-purified 45.7 kDa alkaline protease retained 83% activity which improved with increasing sodium dodecyl sulfate concentration thus highlighting potential laundry application. Maximum enzyme activity occurred at 75ºC and pH 10.5 but was inhibited by 5 mM phenyl-methyl-sulfonyl fluoride suggesting a serine-protease nature.

Kinetic modeling and quasi-economic analysis of fermentative glycolipopeptide biosurfactant production in a medium co-optimized by statistical and neural network approaches.

This study presents the kinetics of production of a glycolipopeptide biosurfactant in a medium previously co-optimized by response surface and neural network methods to gain some insight into its volumetric and specific productivities for possible scale-up towards industrial production. Significant kinetic parameters including maximum specific growth rate, µmax, specific substrate consumption rate, qs and specific biosurfactant yield, Yp/x were determined from logistic model parameters after comparison with other kinetic models. Results showed that bio-catalytic rates of lipase and urease reached exponential values within the first 12 h of fermentation leading to high specific rates of substrate consumption and bacterial growth. Volumetric biosurfactant production reached significantly high levels during prolonged stationary growth and specific urease activity. This suggests that glycolipopeptide biosynthesis may proceed through stationary phase transpeptidation of the glycolipid base. A high cross-correlation coefficient of 0.950 confirmed that substrate consumption and glycolipopeptide production occurred contemporaneously during the 66-h fermentation. The maximum biosurfactant concentration of 132.52 g/L, µmax of 0.292 h-1, qp of 1.674 g/gDCW/h, rp of 2.008 g/(Lh) and Yp/x of 4.413 g/g predicted by the selected logistic model and a unit cost of €0.57/g glycolipopeptide in the optimized medium may lead to technical and economic benefits.

Production, characterization and techno-economic evaluation of Aspergillus fusant L-asparaginase.

Protoplast fusion is one of the most reliable methods of introducing desirable traits into industrially-promising fungal strains. It harnesses the entire genomic repertoire of fusing microorganisms by routing the natural barrier and genetic incompatibility between them. In the present study, the axenic culture of a thermo-halotolerant strain of Aspergillus candidus (Asp-C) produced an anti-leukemic L-asparaginase (L-ASNase) while a xylan-degrading strain of Aspergillus sydowii (Asp-S) produced the acrylamide-reduction type. Protoplast fusion of the wild strains generated Fusant-06 with improved anti-leukemic and acrylamide reduction potentials. Submerged fed-batch fermentation was preferred to batch and continuous modes on the basis of impressive techno-economics. Fusant-06 L-ASNase was purified by PEG/Na+ citrate aqueous two-phase system (ATPS) to 146.21-fold and global sensitivity analysis report revealed polymer molecular weight and citrate concentration as major determinants of yield and purification factor, respectively. The enzyme was characterized by molecular weight, amino acid profile, activity and stability to chemical agents. Michaelis-Menten kinetics, evaluated under optimum conditions gave Km, Vmax, Kcat, and Kcat/Km as 6.67 × 10-5 M, 1666.67 µmolmin-1 mg-1 protein, 3.88 × 104 min-1 and 5.81 × 108 M-1.min-1 respectively. In-vitro cytotoxicity of HL-60 cell lines by Fusant-06 L-ASNase improved significantly from their respective wild strains. Stability of Fusant-06 L-ASNase over a wide range of pH, temperature and NaCl concentration, coupled with its micromolar Km value, confers commercial and therapeutic value on the product. Free-radical scavenging and acrylamide reduction activities were intermediate and the conferred thermo-halo-stability could be exploited for sustainable clinical and food industry applications.

Overproduction of a thermo-stable halo-alkaline protease on agro-waste-based optimized medium through alternate combinatorial random mutagenesis of Stenotrophomonas acidaminiphila.

A strain of Stenotrophomonas acidaminiphila, isolated from fermenting bean-processing wastewater, produced alkaline protease in pretreated cassava waste-stream, but with low yield. Strain improvement by alternate combinatorial random mutagenesis and bioprocess optimization using comparative statistical and neural network methods enhanced yield by 17.8-fold in mutant kGy-04-UV-25. Kinetics of production by selected mutant modeled by logistic and modified Gompertz functions revealed higher specific growth rate in mutant than in the parent strain, likewise volumetric and specific productivities. Purification by PEG/Na+ citrate aqueous two-phase system recovered 73.87% yield and 52.55-fold of protease. Its activity was stable at 5-35% NaCl, 45-75°C, and was significantly enhanced by 1-15 mM sodium dodecyl sulfate (SDS). The protease was inhibited by low concentrations of phenyl-methyl-sulfonyl fluoride but was activated by 1-5 mM Mn2+ suggesting a manganese-dependent serine­protease. The 45.7 kDa thermo-halo-stable alkaline protease demonstrated keratinolytic and blood-stain removal potentials showing prospects in textile and detergent industries, respectively.