Leveraging mutagenesis for enhanced enzymatic output: Detergent-stable α-amylase and lipase co-production by Bacillus...

The sustainable bioproduction of industrial enzymes from low-cost substrates is a primary objective for circular bioeconomy strategies. This study reports the development of a strain of Bacillus subtilis via strain improvement that is capable of producing both α-amylase and lipase simultaneously from kitchen waste. The bacterium was subjected to EMS mutagenesis to improve its natural secretory potential, and a mutant named Bacillus subtilis 250 was selected based on its concurrent overproduction of both enzymes in a medium containing kitchen waste. Leftover fries and used vegetable oil were identified as major drivers for α-amylase and lipase, respectively. Fermentation conditions were sequentially optimized using One-Factor-At-a-Time (OFAT) and Response Surface Methodology (RSM), resulting in 1.53- and 1.45-fold increases in α-amylase and lipase activities, reaching final yields of 89.4 IU/ml and 74.1 IU/ml, respectively. The partially purified enzyme preparations exhibited apparent optimal activity at pH 7 and 50 °C for α-amylase, and pH 7 and 60 °C for lipase, with considerable activity under alkaline and high-temperature conditions. Both enzymes retain prominent residual activities when incubated with commercial detergents, surfactants, metal ions and additives showcasing their compatibility and industrial stability. This work establishes Bacillus subtilis as a versatile biocatalytic platform for the simultaneous valorization of food waste into industrially relevant amylase and lipase.