Characterisation & Kinetics of Glutaminases Produced by Certain Pseudomannas Species & Studies on Mycolases produced by Certain Microorganims and Their Combined Effect with Antibiotics

A total of 298 microbial isolates were qualitatively and quantit¬atively screened for their ability to produce L-glutaminases. The isolate No. 18C.1 showed the maximum activity and so was chosen for further studies.The isolate No. 180 showed maximum glutaminases activity when cultivated on plate count agar and lactose yeast extract medium. The glutaminases activity in intact cells had an optimum pH of 8, and showed linearity with time up to 60 minutes. The optimum temperature for the enzyme activity was 37o c and the optimum substrate concentration was 6.25 x 10-5 M.The enzyme of the isolate was specific only for the D and L isomers of glutamine and asparagine, and was totally intracellular. Celis of the isolate were disrupted by sonification and the crude enzyme extract-was concentrated and purified with manganese chloride, ammonium sulphate, DEAE Servacell 32 chromatography and gel filtration on Sephadex G 200, to a total apparent purification factors of 19.6 and 11.3 for glutaminase and. asparacitnase respectively. .The kinetics of the glutaminase-asparaginase after .purification were studied. The reaction progress with time exhibited a linear response up to 7.5 minutes for both glutaminase and asparaginase activities. The glutaminase activity was linear between concentrations of 14 and 28 mU/ml, and the asparaginase activity was linear between concentrations of 2.5 and 7.5 mU/mlThe pH optimum for the glutaminases- asparaginase activities lies in 7.5 and 9, with the maximum activity around 8 for glutaminaee and9 for asparaginase. The substrate affinity of the glutaminase-asparaginase as measured by Km values was 0.9 x 10-5 and 0,34 x 10-5 M for L-glutamine and L- asparagine respectively. This enzyme proved to had a molecular weigh of 170,000 as measured by gel fillration on Sephadex G 200 .