Annual Research & Review in Biology

Protease-producing bacteria from insect gut microbiota may provide useful enzyme-producing strains for biotechnological applications. This study isolated and characterised a proteolytic bacterium, Bacillus siamensis D4i (GenBank accession PQ481981.1), from the larval gut of Helicoverpa armigera and evaluated culture optimisation and low-frequency ultrasonication. The isolate was selected after screening on skim-milk agar and was confirmed taxonomically through phenotypic and molecular characterisation. Physical and nutritional fermentation variables were screened sequentially using a one-factor-at-a-time approach. Maximum protease production under the tested physical conditions occurred at 37 °C, pH 8.0, 120 rpm, and a 4.5% (v/v) inoculum. Nutritional optimisation identified soybean meal at 1.5% (w/v) and glucose or starch at 1.0% (w/v), together with defined mineral supplementation, as favourable conditions. Under optimised conditions, protease activity reached approximately 400–425 U mL⁻¹, while the time-course experiment recorded 415.92 ± 2.80 U mL⁻¹ at 72 h. Mid-exponential-phase cultures were subsequently exposed to low-frequency ultrasonication at 33 ± 2 kHz and 3.26 W cm⁻² for 0–28 min. Exposure for 2–8 min significantly enhanced biomass and protease production, with the strongest response at 8 min, whereas prolonged exposure for 24–28 min reduced both responses. These findings identify B. siamensis D4i as an insect-gut-derived protease producer and demonstrate a defined, dose-dependent window in which low-frequency ultrasonication can stimulate growth and enzyme production under the tested conditions.