Enzymes serve as remarkably efficient biological catalysts synthesized by living organisms, playing a crucial role in various metabolic processes both within and outside the cell. They accelerate the rates of a diverse array of chemical reactions essential for sustaining life. L-glutaminase(EC.3.5.1.2), a member of the amidohydrolase family, is responsible for catalyzing the deamination of L-glutamine which results in the generation of L-glutamic acid and ammonia. Notably, L-glutaminase exhibits wide distribution across the biological spectrum, being found in microorganisms to mammals. The purification of L-glutaminase from Pseudomonas sp. GPB-06 was achieved through a two-step process. Initially, cell disruption was carried out using a Bead-Beater in five cycles. Subsequently, the membrane-free enzyme extract underwent an initial precipitation step at 30% ammonium sulphate saturation to eliminate contaminating proteins and a final cut at 80% saturation resulting in the precipitation of the target protein. Dialysed enzyme was further purified using DEAE ion exchange chromatography which resulted in 1.66-fold purification, with a respectable 41.15% yield. The purified protein was identified as a monomer, comprised of subunits with a molecular weight of 41 kDa each. Characterization of the purified L-glutaminase revealed its optimal activity in a 100 mM potassium phosphate buffer at pH 7.0 and a temperature of 30oC. The enzymatic properties of the purified L-glutaminase from Pseudomonas sp. GPB-06 included a Vmax of 144.92 µmole/min/mg protein. Furthermore, the Km values for L-glutamine were determined to be 0.6 mg/ml.

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