Investigating and optimizing the immobilization of levansucrase for increased transfructosylation activity and thermal stability

- Optimal immobilization conditions for LS (levansucrase) on glyoxyl agarose-IDA/Cu/ polyethylenimine (PEI) were determined.
- Interactions between immobilization parameters were explored and modulated.
- PEI allows the stabilization of LS immobilized onto glyoxyl agarose-IDA/Cu without compromising the retained activity.
- The thermal stability of LS was increased 4.7 times compared to the native enzyme.

Levansucrase (LS) represents a key enzyme in glycoside synthesis of novel prebiotics and β-2,6-levan. The study of the effects of immobilization parameters of LS, produced from Bacillus amyloliquefaciens, onto glyoxyl agarose-iminodiacetic acid/Cu (glyoxyl agarose-IDA/Cu) by response surface methodology revealed the significance of their interactive effects. Retention of activity was altered by interactive effects from buffer molarity/time and buffer pH/buffer molarity. The optimized immobilization conditions were identified to be a protein loading of 9.09 mg protein/g support, a buffer concentration of 608 mM at pH 6.8 and an incubation time of 49 h. Normally a reducing agent is applied to the immobilized enzyme in order to promote the formation of covalent bonds. This step was replaced with the addition of the ionic polymer polyethylenimine (PEI), which provided a better compromise between retained activity and thermal stability of the immobilized LS. Indeed, LS immobilized onto glyoxyl agarose-IDA/Cu/PEI had a retention of activity of 70.91% with a protein yield of 44.73% and an activity yield of 54.69%, while exhibiting a half-life 4.7 times higher than that of the free LS at 50 °C.

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