الفهرس 
Biotechnological Studies on Microbial Levansucrase
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Abstract
The present work focused on production, optimization and characterization of microbial levansucrase enzyme as well as improvement of enzyme properties to be more suitable for industrial applications. In addition, Levansucrase was used in enzymatic synthesis of several fructose-based saccharides and investigated for their potential biological activities.
Sixty-six microbial isolates were collected from different sources and investigated for their ability to produce LS. The most potent producer isolate was Aspergillus niger that was isolated from soil and identified by ITS technique then submitted in the gene bank database under accession number MK788296. The LS production by Aspergillus niger isolate was optimized by physiological and statistical optimization techniques. In physiological optimization, it was observed that LS production in submerged fermentation was higher than solid state fermentation by 5.3 folds. Potato peels was the main carbon source with the highest LS production followed by molokhya stalks, lemon peels, orange peels, sugarcane bagasse, wheat straw and finally banana peels. While, utilizing corn cobs was not suitable for LS production. In statistical optimization, Plackett-Burman design (PBD) and Central composite design (CCD) were established. The PBD studied the effect of eleven different factors on LS production. The CCD investigated the optimum levels for the significant factors determined by PBD. The methods used for optimization of LS production resulted in a massive increase of LS activity from 317.49 U/ml under the basic production medium conditions to 18870.3 U/ml under optimized conditions. In other words, an overall 59.4-fold increase in LS production was achieved after application of the optimization approaches. The optimum conditions for LS production were achieved through using 5.7 g potato peels in 50 ml statistically optimized medium (pH 7) for 12 days at 150 rpm at 30 °C. The statistically optimized medium was composed of (g/l): yeast extract, 15; sucrose, 5; fructose, 5; KCl, 1.5; MgSO4.7H2O, 1.5; MnCl2.4H2O, 0.001; FeSO4.7H2O, 0.001.
The crude LS produced after the optimization approaches was used for synthesis of FBS. Different enzymatic synthesis conditions were investigated by a CCD design and resulted in synthesis of fifteen different FBS. The synthesized FBS had different molecular weights ranging from 1278 to 41038 Da. The synthesized FBS showed DPPH scavenging activities with different degrees. Fructose-based saccharides no.11 and no.19 showed antitumor activities against breast adenocarcinoma, hepatocellular and colorectal cell lines. All the synthesized FBS exhibited prebiotic activities as they enhanced the growth of Bifidobacterium lactis and Lactobacillus plantarum probiotics. Most of the synthesized FBS had antimicrobial activities against E.coli and Staphylococcus aureus. Fructose-based saccharides no.1, 3, 13 and 20 showed fibrinolytic activities.