Glycoside hydrolase family 32 enzymes from Bombella spp. catalyze the formation of high molecular weight fructans from sucrose

Abstract Aims Acetic acid bacteria of the genus Bombella have not been reported to produce exopolysaccharides. In this study the formation of fructans by B. apis TMW 2.1884 and B. mellum TMW 2.1889 was investigated. Methods and results Out of eight strains from four different Bombella species, only B. apis TMW 2.1884 and B. mellum TMW 2.1889 showed exopolysaccharide (EPS) formation with 50 g L−1 sucrose as substrate. Both EPS were identified as high molecular weight (HMW) polymers (106-107 Da) by AF4-MALLS/UV and HPSEC-MALLS/RI analyses. Monosaccharide analysis via TFA hydrolysis showed that both EPS are fructans. Determination of glycosidic linkages by methylation analysis revealed mainly 2,6-linked fructofuranose (Fruf) units with additional 1,2-linked Fruf units (10%) and 1,2,6-Fruf branched units (7%). No glycoside hydrolase (GH) 68 family genes that are typically associated with the formation of high molecular weight fructans in bacteria could be identified in the genomes. Through heterologous expression in E. coli Top10, an enzyme of the GH 32 family could be assigned to the catalysis of fructan formation. The identified fructosyltransferases could be clearly differentiated phylogenetically and structurally from other previously described bacterial fructosyltransferases. Conclusions The formation of HMW fructans by individual strains of the genus Bombella is catalyzed by enzymes of the GH 32 family. Analysis of the fructan revealed an atypical structure consisting of 2,6-linked Fruf units as well as 1,2-linked Fruf units and 1,2,6-Fruf units.