Purification and characterization of amylase from local isolate Pseudomonas sp.SPH4

The amylase produced from local isolate pseudomonas sp. sph4 was purified by precipitation with 30% saturation ammonium sulphate, followed by ion-exchange chromotography using deae- cellulose column, and gel filtration using sephacryl s-300 column.the two iso-enzymes (a, b) were purified to (2.83, 3.47) times in the last step with an enzymes yields of (32.36, 76.34)% respectively. enzyme characterization of the two iso-enzymes indicated that the optimum ph for the two iso- enzymes a and b were (7, 7.5) respectively, while the optimum ph for the iso-enzymes stability were (6.5, 7) respectively. the maximum activity for iso-enzymes (a, b) appeared at 45ºc and stable for 15 min at 30-50ºc and lost approximately 50% of it's activity at rang above 75ºc. enzyme characterization results showed that the chlorides of silver and mercury had inhibitory effect on enzyme activity, the remaining enzyme activity for the iso-enzymes (a, b) were (46.66, 36.36)% for s ilver ions and (41.33, 33.63)% for mercury ions at 5 mm respectively, and (28, 28.18)% for silver ions and (25.33, 19.09)% for mercury ions at 10 mm respectively. the iso-enzymes a and b were affected by chelating agent ethylene diamine tetra acetic acid (edta) at concentration 2mm the remaining activity (45.33, 43.63)% respectively, and 5mm the remaining activity (28, 28.18)% respectivily, and these iso-enzymes (a, b) refered to metalloenzymes. the iso-enzymes (a, b) were kept their activity when treated by reducing agent (2-mercaptoethanol) at 2 mm the remaining activity (92, 92.72)% respectively, and 5 mm the remaining activity (85.3, 89.09)% respectivily. the iso-enzymes (a, b) were kept their activity when treated by phenyl methyl sulphonyl fluoride (pmsf) at concentration 1mm the remaining activity (93.33, 90.90)% respectivily,and 5 mm the remaining activity (90.66, 87.27)% respectivily, and these indicated that these iso-enzymes didnot referred to serine amylases group.