الفهرس 
AIM OF THE WORKOnly 14 pages are availabe for public view
 |  | from | 121 |  |  |
| | | from | 121 | | |
Abstract
Screening for levansucrase producing bacteria was carried out using static culture techniques. A survey of 18 bacterial cultures (isolated from honey and honey bees) for the production of levansucrase was carried out of the tested bacteria, Bacillus subtilisNRC16, showed the highest production value ( 45.6 U/ml) of levansucrase under static conditions. Therefore, it was selected as the most potent isolate to pursue the work. In preliminary experiments of the present work, different incubation periods were tested. The highest levansucrase yield (45.6 U/ml) was produced by the experimental bacteria after 24 hrs incubation in static cultures. Effect of different carbon sources was investigated. Also some physical conditions affecting the yield of levansucrase by Bacillus subtiis NRC16 were studied. Sucrose proved to be the best substrate for levansucrase production.Yield of levansucrase in the presence of sucrose was approximately (45.6 U/ml) higher than of cultures supplemented with molasses. Highest yield of levansucrase was at temperature 50oC. Enzyme activity was increased by increasing the time of incubation to reach and the maximum value was obtaind (50.8U/ml) after 28 h. A gradual decrease was observed by increasing incubation periods from (32 to 48 h). Summary 82 The addition of sucrose at 120 g/l in the medium exhibited a high levansucrase productivity (55 U/ml) and specific enzyme activity (39.1 U/mg). Different concentrations of sodium chloride revealed that the most favorable concentration for enzyme production was (5g/L). The highest enzyme productivity (56.4U/ml) and specific enzyme activity (49.5 U/mg). Different carbon sources were tested. It was noticed that starch (5g/l) gave the maximum levansucrase activity (70.5 U/ml) and specific enzyme activity (137 U/mg). Different nitrogen sources were substituted by equivalent amounts of nitrogen added to the fermentation media to test there effects on levansucrase activity. The maximum activity was obtained when (12.5g/l) baker yeast was used (166 U/ml) and specific enzyme activity (86 U/mg). Some salts were individually added to the production medium. AlCl3 (5mM) was the best salt increased the levansucrase and specific enzyme activities (224 U/mland127U/mg, respectively). Partial purification of the crude levansucrase produced by Bacillus subtilisNRC16 was carried out by fractionation with acetone and ethanol. The salting out with ammonium sulfate was tried. The molecular weight of the partially purified enzyme was 16KDa. The fraction 30% acetone showed the highest specific enzyme activity and purification fold (228.7U/ml and 1.98 fold, respectively). The study of the properties of the partially purified levansucrase enzyme was carried out. The optimum pH value was 8.2. The optimum temperature was at 40oC. The enzyme retained full activity (100%) at 45 oC Summary 83 for 90 min. The enzyme showed complete pH stability on alkaline side from (5.2- 9.2). At pH 4.2 the activity decreased gradually. The metal salts had adverse effect on the partially purified levansucrase. All salts, (0.02M) were accompanied by drastic decrease in levansucrase relative activity. The calculated values of Km and Vmax using different concentrations of sucrose as a substrate were found to be (0.05 and 15.9 mg/ml, respectively). Carbohydrate content of the partially purified enzyme was 8%. A lso, the biological activities like anticoagulation and fibrinolytic activity were tested on the levan samples and their sulfated levan. The results of anticoagulation activity showed no activity comparable to that of standard preparation of heparin sodium. On the other hand, the fibrinolytic activity showed that the levan14 exhibited fibrinolytic activities equivalent to double of the same amount of standard ”Hemoclar” preparation.