Co-Cultivation Enhances Extracellular Endoglucanase Activity in Two Insect-Gut-Derived Bacillus Isolates

Insect guts harbor microorganisms with carbohydrate-active enzymes, yet their potential as enzyme-production strains and the benefits of co-cultivation remain insufficiently characterized. In this study, 180 bacterial isolates from nine insect species were screened for carboxymethyl cellulose (CMC)-hydrolyzing activity. Two non-hemolytic isolates, H11 and Q5-3, were assigned to the genus Bacillus based on morphological characteristics and 16S rRNA gene analysis. Their initial extracellular endoglucanase activities were 156.67 ± 4.17 and 190.36 ± 4.29 U/mL, respectively, and increased to 228.01 ± 2.73 and 283.28 ± 2.06 U/mL after orthogonal array optimization. Central composite design predicted a maximum volumetric activity of 340.81 U/mL at approximately 16% (v/v) total inoculum and a Q5-3/H11 inoculum ratio of 2.08; independent validation yielded 339.17 ± 3.97 U/mL. Additional H11 and Q5-3 monoculture controls evaluated at the same total inoculum proportion showed lower mean volumetric activities than the validated co-culture. PCR screening produced expected-size amplicons for five putative endoglucanase-related targets in both isolates. These results indicate that H11 and Q5-3 are promising insect-gut-derived candidates for extracellular endoglucanase production and that co-cultivation can enhance volumetric enzyme activity on CMC without genetic modification. This study establishes candidate strains and a validated co-cultivation process in a controlled soluble-CMC model system, providing a process-development basis for subsequent evaluation of strain interactions and performance on insoluble cellulose and representative lignocellulosic substrates.