Analysis of cultivation conditions and enzymatic efficiency enables high-yield levan production from Bacillus velezensis KKSB6.

Levan, a fructose-based biopolymer with significant industrial potential, was produced using the native strain Bacillus velezensis KKSB6. This study aimed to optimize levan production through different cultivation strategies and to characterize the enzyme responsible, levansucrase. Production was compared in batch, fed-batch and continuous systems, revealing a critical point between final yield and productivity. Batch cultivation achieved a high levan yield of 187 g/L (93.50% yield), while a fed-batch cultivation delivered high productivity at 3.46 g/L/h but a low levan yield (55.33%). Continuous cultivation demonstrated the highest levan production of 746 g/L (93.25% yield) and high productivity (7.77 g/L/h). In those systems, production was limited by the inhibitory effect of glucose accumulation. The purified levansucrase, a 42 kDa protein, exhibited optimal activity at 35 °C and pH 7.0. Significantly, in vitro synthesis using the purified enzyme produced primarily high-molecular-weight (HMW) levan, in contrast to the lower-molecular-weight product obtained from whole-cell fermentation. In conclusion, B. velezensis KKSB6 is an exceptionally potent strain, with batch cultivation or continuous cultivation producing levan yield and with continuous cultivation favoring productivity. The purified enzyme represents a promising biocatalyst for the targeted synthesis of HMW levan.