Characterization and optimization of glutaminase enzyme from Bacillus albus isolated from agriculture waste

Glutaminase is an enzyme that catalyzes the hydrolysis of glutamine to ammonia and glutamate. Although it can be isolated from bacteria, fungi, plants, and animals, microorganisms remain the preferred source due to their biochemical diversity and ease of cultivation. In this study, 34 soil samples were collected from agricultural waste areas in district Kohat to isolate glutaminase-producing bacteria. Primary screening using phenol red indicator and subsequent biochemical characterization identified 10 positive isolates (30% positivity rate). Among these, four representative strains (A1, G2, P3, and 1R) were selected for detailed analysis, with the majority belonging to the Bacillus genus. Optimal enzyme activity was observed at neutral pH (7.0) and 37°C. SDS-PAGE (12%) analysis was used to estimate the molecular weight of the purified glutaminase. 16S rRNA gene sequencing identified isolate A1 as Bacillus albus, isolates G2 and P3 as closely related to Bacillus anthracis, and isolate 1R as Alkalibacillus bogoriensis. Phylogenetic analysis revealed that Alkalibacillus bogoriensis (1R) clustered distinctly from other Bacillus species. Among all isolates, Bacillus albus demonstrated the highest glutaminase activity, showing good pH and temperature stability. These findings suggest that the isolated strains, particularly Bacillus albus, have potential for future pilot-scale studies and industrial applications in glutaminase production.