Characterization of a cell-associated inulosucrase from a novel source: A Leuconostoc citreum strain isolated from Pozol,a fermented corn beverage of Mayan origin V Olivares-Illana 1 , C Wacher-Rodarte 2 , S Le Borgne 1 and A Lo ´pez-Munguı ´a 1 1 Instituto de Biotecnologı ´a, Universidad Nacional Auto ´ noma de Me ´ xico, Apdo. Postal 510 - 3, Cuernavaca, Morelos, CP 62271, Mexico; 2 Facultad de Quı ´mica, Universidad Nacional Auto ´ noma de Me ´ xico, Cd. Universitaria, CP 04510, Me ´ xico, D.F., Mexico A cell - associated fructosyltransferase was extracted from a novel source, a strain of Leuconostoc citreum isolated from Pozol, a Mexican traditional fermented corn beverage, where lactic microflora are partially responsible for the transformation process. The enzyme is associated with the cell wall. It was characterized both in its cell - associated insoluble form and after separation by urea treatment. The fructosyltransferase has a molecular mass of 170 kDa, the highest reported for this type of enzyme, and in its insoluble form is highly specific for polymer synthesis, with low fructose transferred to maltose and lactose added to the reaction medium ( acceptor reactions ). The synthesized polymer has an inulin - like structure with  2 - 1 glycosidic linkages, as demonstrated by 13 C nuclear magnetic resonance ( NMR ). Bacterial inulosucrases have only been reported in Streptococcus mutans. Journal of Industrial Microbiology & Biotechnology (2002) 28, 112 – 117 DOI: 10.1038 / sj / jim / 7000224 Keywords: Leuconostoc citreum; inulosucrase; sucrose; levansucrase; dextransucrase Introduction Fructosyltransferases such as levansucrase (LS), as well as glucosyltransferases like dextransucrase ( DS ), are examples of glycosyltransferases that catalyze the transfer of a fructose or a glucose residue from sucrose to a growing polysaccharide chain, resulting in the synthesis of high molecular weight polymers of fructose ( fructans ) or glucose ( glucans ), respectively. The polymers produced by these enzymes have different sizes and structures, depending on the enzyme - producing strain. Dextrans are glucans containing a main linear chain with 1 - 6 glucosidic linkages [ 23 ], while mutans are distinguished by the 1-3 bonds in the main chain ‘‘alternate’’ 1-6 and 1 - 3 linkages in their structure. Branching may occur in different positions such as 1- 2, 1-3, 1-4 and 1 - 6, depending on the source of the enzyme [ 5 ]. On the other hand, levans are fructans containing 2-6 linkages in the main linear chain with 2 - 1 branch points, while the reverse is the case for inulin ( 2 - 1 in the main chain and 2-6 branch points ) [ 24 ]. The corresponding glycosyltransferases do not require any cofactors or high - energy phosphorylated inter- mediates, as they use the free energy liberated by the cleavage of sucrose for the synthesis [ 19 ]. All these polymers have found a variety of applications in the fields of cosmetics, foods, separation technology and medicine. An interesting feature of these enzymes is their ability to catalyze the synthesis of low molecular weight oligosaccharides from sucrose when efficient acceptor molecules like maltose are added to the reaction medium [ 13 ]. Some of these oligosacchar- ides are used as prebiotics in both cosmetic and food applications as they are highly resistant to attack by digestive enzymes, being substrates only for beneficial native probiotic flora [ 30 ]. The main producers of fructosyltransferases are Gram - positive bacteria such as several Bacillus species ( including Bacillus subtilis [4], Bacillus circulans [19], Bacillus polymyxa [3], Bacillus amyloliquefaciens [29]), Rahnella aquatilis [18], and Lactoba- cillus reuteri [ 32 ]. They are also found in some Gram - negative bacteria such as Zymomonas mobilis [14], Erwinia herbicola [5], Pseudomonas syringae pv. glycinea and P. syringae pv. phaseo- licola [9] and Acetobacter diazotrophicus [ 1,2 ]. All the fructosyl- transferases from these microorganisms are levansucrases ( LSs ), with levan as the main enzymic product. Inulosucrases have been isolated only from plants and fungi, with the exception of a similar activity reported in Streptococcus mutans by Shiroza et al [26]. Both Leuconostoc mesenteroides as well Streptococci from oral flora have been reported as dextran and levan producers [ 7,8 ]. Differences among bacterial fructosyltransferases are illustrated in Table 1. Dextransucrases (DSs) are extracellular enzymes, cell - associated or both, depending on the producing strain while LSs are generally extracellular with the exception of LS from Actinomyces viscosus, which produces extracellular and cell - associated forms [ 31 ] and that from Streptococcus salivarius, which is cell - associated in the absence of sucrose, but released from the cell and secreted into the culture medium in its presence [ 12 ]. DSs are large enzymes, with a reported molecular mass from 64 to 184 kDa [21], while LSs are smaller with a molecular mass between 45 and 64 kDa, with the exception of the S. salivarius and S. mutans enzymes, which have molecular masses of 140 ( fructosyltransferase ) and 87 kDa ( fructo- syltransferase with inulin- like activity ), respectively, as repor- ted in Table 1 [11,26 ]. There are few reports documenting LS activity in L. mesenteroides. Actually, due to very low expres- sion levels, LS activity in this source had previously been con- sidered as a minor contaminant of DS [ 17,20,33 ]. Up to now Correspondence: Dr A Lo ´ pez - Munguı ´a, Instituto de Biotecnologı ´a, Universidad Nacional Auto ´noma de Me ´xico, Apdo. Postal 510 - 3, Cuernavaca, Morelos, CP 62271, Me ´xico Received 25 September 2000; accepted 30 October 2001 Journal of Industrial Microbiology & Biotechnology (2002) 28, 112 – 117 D 2002 Nature Publishing Group All rights reserved 1367-5435/02 $25.00 www.nature.com/jim