Abstract
A novel strategy to enhance the yield of levan-type fructooligosaccharide (LFOS) was recently introduced, whereby levansucrase and inulosucrase reactions were coupled together in one pot. In order to simplify the process, in the present study we report the first example of a recombinant levansucrase-inulosucrase fusion protein and investigate its impact on LFOS production. Sequences for levansucrase from Bacillus amyloliquefaciens KK9 and inulosucrase from Lactobacillus reuteri 121 were fused genetically with a flexible eighteen residue glycine-serine peptide linker. SDS-PAGE analysis showed that the molecular weight of obtained fusion protein is approximately of 120 kDa, corresponding to the expected fusion protein molecular weight. Kinetic analysis revealed that after protein combination, the kinetic parameters of both enzymes are slightly changed. Biochemical characterization revealed that fusion did not affect the optimum pH and temperature for catalysis, but significantly change the stability of the enzyme. HPAEC-PAD analysis and enzymatic hydrolysis assays demonstrated that the fusion enzyme produced the desired higher yield of LFOS compared to those of individual levansucrases.
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