Extraction, partial purification and Characterization of Isoamylase produced by Locally isolated Psuedomonas

Eighty soil and sewage samples were collected from different environmental locations to isolate isoamylase producing Pseudomonas. From these samples, 100 isolates were obtained, among them 40 were identified as Pseudomonas sp. according to the morphological characteristics and biochemical tests. These isolates were screened to examine their ability in isoamylase production. Results showed that all these isolates were producers of isoamylase. An isolate (Pseudomonas sp. H3) was the most efficient in producing isoamylase when the activity of the crude enzyme in the culture was 0.394 U/ml.  Optimum conditions for isoamylase production by Pseudomonas sp. H3 were studied. These conditions include the optimum; pH, growth temperature, type and concentration of carbon source, type and concentration of nitrogen source type and concentration of phosphate source.Results showed that the maximum activity for crude enzyme was (0.70 U/mg protein) which gained when the bacterial isolate was grown in a production medium containing ; 1 % maltose, 0.4 % peptone, 0.4% K2HPO4, pH 6.0, in a shaker incubator at 150 rpm at and incubation at 35ºC for 24 hr.  Enzyme produced under the optimum conditions was purified by precipitation with ammonium sulphate (60% saturation), followed by dialysis
and purification by gel filtration using Sephacryl G-300. Enzyme activity of the partially purified enzyme was 1.853 U/ml. Results of enzyme characterization showed that optimum pH for the activity and stability were 5.0 and 5.5, respectively, while optimum temperature for the activity and stability was 35ºC.