Production and purification of protease from mutant of locally isolated Aeromonas hydrophila

In this study, a total of 89 samples were collected for isolation of Aeromonas hydrophila. These include 34 water samples taken from pools of fish farms and 55 samples of gills pieces of fresh fishes collected from different locations in Baghdad city. From these samples, a total of 91 bacterial isolates were obtained and subjected to the identification according to there morphological, cultural and biochemical characteristics. Results showed that only seven of these isolates were identified as A. hydrophila. The ability of these isolates for protease production was examined. Results showed that all seven isolates of A. hydrophila were protease producers with variable degrees. Among them, the isolate symbold A. hydrophila A4 was the most efficient in protease production. Its specific activity of protease in crude filtrate was 31.01U/mg protein. Upon this, the isolate was selected to enhance its ability in protease production by random mutagenesis.
Isolate A. hydrophila A4 was subjected to physical mutagenesis by UV irradiation and chemical mutagenesis by using Mitomycin C. Results showed that an over protease producing mutant symboled A. hydrophila A4-78 arose after mutagenesis with UV irradiation when the enzyme specific activity in its crude filtrate was 46.6 U/mg protein in comparison to the wild type (30.01U/mg protein). On the other hand another overproducer mutant symboled A. hydrophila A4-127 was obtained after chemical mutagenesis with Mitomycin C characterized with its higher ability in protease production. The enzyme specific activity in its crude filtrate was 38.4 U/mg protein in comparison with the wild type. According to these results, mutant A4-78 was selected to determine the optimum conditions for protease production. Results showed that maximum protease production was achieved after supplementation of the production medium (pH7) with 0.5% sucrose, 1.5% tryptone and 0.5% K2HPo4 before inoculated with a 106 cells/1ml of fresh bacterial culture and incubated at 35oC in shaker incubator (150 rpm) for 24h. Under these conditions, the specific activity of protease produced in culture medium was sharply increased to 90 U/mg protein. Protease produced under the optimum conditions was purified in four purification steps, first by precipitation with 60% saturation of ammonium sulfate, second by the dialysis, third by ion exchange chromatography using DEAE-Cellulose column, while the gel filtration chromatography throughout Sephacryl S-200 column was the fourth step. Specific activity of the purified enzyme was 1200 U/mg with 13.3 folds of purification and 40% enzyme recovery. Some biochemical characteristics of the purified enzyme were studied. Results showed that the molecular weight of protease produced by A. hydrophila A4 was about 68000 Dalton, the optimum pH for enzyme activity was 8 while that for stability was 8.5. Optimum temperature for enzyme activity was 35oC, hence the enzyme was stable with full activity at a range of temperatures between 20-40oC.
The effect of chelating and reducing agents and heavy-metal ions on purified protease activity was investigated. Results showed that protease produced by the A. hydrophila A4 was a metalloenzyme because its enzyme activity was inhibited after incubation with 5mM EDTA.