Secretion of Bacillus amyloliquefaciens Levansucrase and Its Mutants from L. lactis NZ9000 and Their Applications in the Synthesis of Levan

Abstract

Levan, a kind of fructan connected by β-(2,6) and β-(2,1) glycosidic bonds, is synthesized by levansucrase and has attracted extensive attention because of its excellent physiochemical properties. In order to improve the productivity and enzymatic activity of levansucrase, we cloned the gene of Bacillus amyloliquefaciens levansucrase (Ba-SacB) and its three mutants (E313V, E313L, and E313F) into the plasmid pNZ8048, respectively, and transformed them into Lactococcus lactis for levansucrase heterologous expression. After purification by immobilized metal affinity chromatography, wild-type Ba-SacB showed the highest yield of 225.2 ± 1.2 mg/L, and the mutant E313F showed the highest enzyme activity of 298.89 ± 0.6 U/mg. Moreover, free E313F was immobilized on magnetic porous Fe3O4 nanoparticles (Fe3O4@NPs). The generated immobilized E313F (E313F@NPs) retained up to ∼61.93% of the initial enzyme activity even after 10 uses and was able to catalyze levan formation with a yield of ∼41.65%.