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

A strain of , 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 Mn suggesting a manganese-dependent serine‑protease. The 45.7 kDa thermo-halo-stable alkaline protease demonstrated keratinolytic and blood-stain removal potentials showing prospects in textile and detergent industries, respectively.