الفهرس Only 14 pages are availabe for public view
 |  | from | 222 |  |  |
| | | from | 222 | | |
Abstract
A survey of 7 bacterial cultures for the production of fructosyltransferase was carried out. Among the tested bacteria Bacillus cereus, a local isolate, showed the highest fructosyltransferase activity. The composition and concentration of different nutrients forming the production medium were investigated and optimized. Of the different carbon sources used in the culture medium, sucrose proved to be the best substrate at a concentration of 160 g/l. The most suitable nitrogen source for highest fructosyltransferase activity was baker,s yeast at a concentration of 1.5 g nitrogen/L. The utilization of different agro-industrial wastes in solid state cultures revealed that wheat straw was the most suitable substrate for production of relatively high yield of fructosyltransferase activity (29.1 U/ml). The crude B. cereus fructosyltransferase was partially purified by fractional precipitation with ethanol, acetone and ammonium sulphate. Salting out with ammonium sulphate at 80-90% saturation proved to be the best way for partial purification of B. cereus fructosyltransferase (1.29-fold purification). This fraction was used for immobilization by 4 different methods namely physical adsorption, covalent binding, entrapment and ionic binding. The partially purified FTFase immobilized by ionic binding on DEAE-cellulose 53 gave the highest immobilization yield (76.29%). Comparison between some properties of free and immobilized B. cereus fructosyltransferase on DEAE- cellulose 53 was carried out. The activation energy for immobilized enzyme (5.09 K cal/ mol) was lower than that of free one (7.15 K cal/ mol) while the calculated half life values at 50, 55 and 60 ˚C for the immobilized enzyme were (207.90, 148.50 and 124.64 min) higher than those for the free enzyme (100.00, 83.16, 69.30 min, in the same order), indicating that the enzyme immobilization process increased enzyme stability. The partially purified B. cereus FTFase was purified by chromatography on DEAE-cellulose 53 column (5.70- fold purification), followed by further purification on Sephadex G-150 filtration. The purity of fructosyltransferase was confirmed by SDS-polyacrylamide gel electrophoresis and its molecular weight was determined to be 35 KDa. Characterization and some properties of purified fructosyltransferase was optimally active at pH 5.2 and 30 ˚C. The Michael’s constant (Km) and maximum velocity (Vmax) for the pure fructosyltransferas were calculated to be 90.9 mM and 420.16 U/mg protein respectively. The carbohydrate content of pure fructosyltransferase was determined to be 20% and the protein moiety of enzyme was found to be composed of 17 amino acid.