L-Glutaminase (EC 3.5.1.2) is an amidohydrolase enzyme of considerable therapeutic, industrial and ecological significance that catalyzes the hydrolytic deamidation of L-glutamine to L-glutamic acid and ammonia. The present study was conducted to isolate, screen, characterize and produce crude L-glutaminase enzyme from soil bacteria collected from garden and forest sites of Government Institute of Science, Chhatrapati Sambhajinagar, Maharashtra, India. Serial dilution and spread plate technique on M9 minimal salt medium containing L-glutamine as the sole nitrogen source yielded six distinct bacterial isolates designated L1 to L6. Primary screening using phenol red indicator confirmed L-glutaminase activity in all six isolates through the development of pink or red halo zones. Morphological characterization by gram staining revealed that L2, L3 and L6 were gram positive while L1, L4 and L5 were gram negative. Negative staining confirmed diverse cell morphologies including cocci and rod-shaped cells. Biochemical characterization through IMViC, catalase and urease tests provided a comprehensive metabolic profile of each isolate. Secondary screening by Nessler's quantitative assay identified isolate L6 as the most potent L-glutaminase producer with the highest ammonia concentration of 0.58 mg/L per 5ml (OD 0.3870 at 456nm), followed by L3 (0.55 mg/L) and L2 (0.50 mg/L). Crude enzyme extract was successfully prepared by centrifugation of fermented broth at 10,000 rpm for 15 minutes at 4°C. These results establish that local soil environments of Chhatrapati Sambhajinagar harbour potent L-glutaminase producing bacteria with significant biotechnological and agricultural potential.
Gujar et al. (Fri,) studied this question.