Comparison of novel GH 68 levansucrases of levan-overproducing Gluconobacter species | Acetic Acid Bacteria

Gluconobacter species are capable of incomplete oxidations which are exploited in food biotechnology. Levans isolated from exopolysaccharide (EPS)-overproducing Gluconobacter species are promising functional compounds for food applications. Fructan production strongly depends on the corresponding fructosyltransferases (Ftfs), which catalyze the formation of these polymers from sucrose. Therefore, we characterized novel Ftfs from three EPS-overproducing food-grade strains, i.e. Gluconobacter sp. TMW 2.767 and Gluconobacter sp. TMW 2.1191 isolated from water kefir, and Gluconobacter cerinus DSM 9533T isolated from cherries. Several PCR techniques, including degenerate gradient temperature PCR, modified and standard inverse PCR, modified site-finding PCR and modified single primer PCR, were used to finally detect complete open reading frames coding for Ftfs. The prospective ftf-gene sequences were heterologously expressed in Escherichia coli Top 10. E. coli transformants harboring one of the three different ftf-genes produced polysaccharides from sucrose in contrast to the E. coli wildtype. Each of the heterologously expressed proteins encoded a levansucrase, catalyzing the formation of b-(2→6)-linked fructose polymers, which corresponded to our previous analyses about the chemical nature of the isolated polymers formed by these Gluconobacter strains. Structurally, these enzymes belong to the glycoside hydrolase 68 family (GH 68), sharing the typical modular topology of levansucrases from gram-negative bacteria. In conclusion, we could identify novel active levansucrases, which can be used for ex situ (enzymatic catalyses) or in situ (fermentation) production of functional fructan polymers by Gluconobacter strains in food and other applications