الفهرس 
A study on possible fructosyltransferase production by bacterial isolates from honey and its application in prebiotic synthesis
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Abstract
Levan is a β (2,6)-linked polymer of repeating five-memberfructofuranosyl rings; its fundamental chain is branched by β-(2 →1). Levan can be used as prebiotic, antiviral and anticancer agent. Biosynthesis of microbiallevan occurs by the action of levansucrase enzyme (E.C.2.4.1.10). Therefore, the aim of this study was to isolate levansucrase-producer bacteria from honey with optimization of the conditions for levansucrase production. The produced levan was evaluated for different microbial activities.
A total of eighteen microbial isolates were recovered from honey. The isolate with the highest levansucrase activity was identified asBacillus subtilis MT453867. Effect of agitation speed and addition of agricultural wastes on levansucrase activity were evaluated. Medium composition was optimized usingOne-factor-at-a-time technique andPlackett–Burman design. Theidentification of the produced levan was confirmed by molecular weight determination, thin-layer chromatography, Fourier-Transform Infrared spectrometric analysis, 13C- and 1H-nuclear-magnetic resonance. Both crude and dialyzed levan were tested for their cytotoxicity against normal retinal cell and five cancer cell lines. The effect of dialyzed levan (DL) on gene expression and DNA fragmentation in pancreatic and skin cancer cells was evaluated. Acute oral toxicity and LD50 for DL were determined. The partialpurification of levansucrase was carried out by fractional precipitation.The purified enzyme was used for levan and fructo-oligosaccharides synthesis.The produced crude levan, DL and oligosaccharides were tested for theirantioxidant activity.
The optimum agitation speed for levansucrase activity was 150 rpm. Addition of banana peel recorded the highest levansucrase activity. Levansucrase activity increased by 60% when MgSO4 was eliminated. Optimization of production conditions and medium components by Plackett–Burman design led to 60-fold increase in levansucraseactivity. Both crude and dialyzed levan completely inhibited the pancreatic cancer cell line and skin cancer cell line at 100 ppm with no cytotoxicity on the normal retinal cell line. DL significantly reduced theexpression of CXCR4 and MCM7 genes in pancreatic cancer cells and HBD-1 and HBD-2 in skin cancer cells with significant DNA fragmentation. Mice did not show any illness signs during 14 days after oral administration of crude levan. The LD50of crude levan was4833 mg/kg body weight.The produced Levan had strong antioxidant activity. Acetone (20%) was the best solvent for partial purification of levasucrase. Oligosaccharides synthesized by levansucrase action had low antioxidant activity.
In conclusion, Bacillus subtilis MT453867 levan had promising anticancer activity against pancreatic and skin cancer with strong antioxidant activity.