J Ind Microbiol Biotechnol (2013) 40:1–10 DOI 10.1007/s10295-012-1198-6 123 BIOCATALYSIS Production of organic acids by periplasmic enzymes present in free and immobilized cells of Zymomonas mobilis Eloane Malvessi · Sabrina Carra · Flávia Cristina Pasquali · Denise Bizarro Kern · Mauricio Moura da Silveira · Marco Antônio Záchia Ayub Received: 23 May 2012 / Accepted: 3 September 2012 / Published online: 30 September 2012  Society for Industrial Microbiology and Biotechnology 2012 Abstract In this work the periplasmic enzymatic com- plex glucose-fructose oxidoreductase (GFOR)/glucono- -lactonase (GL) of permeabilized free or immobilized cells of Zymomonas mobilis was evaluated for the bioconversion of mixtures of fructose and diVerent aldoses into organic acids. For all tested pairs of substrates with permeabilized free-cells, the best enzymatic activities were obtained in reactions with pH around 6.4 and temperatures ranging from 39 to 45 °C. Decreasing enzyme/substrate aYnities were observed when fructose was in the mixture with glu- cose, maltose, galactose, and lactose, in this order. In bio- conversion runs with 0.7 mol l ¡1 of fructose and with aldose, with permeabilized free-cells of Z. mobilis, maxi- mal concentrations of the respective aldonic acids of 0.64, 0.57, 0.51, and 0.51 mol l ¡1 were achieved, with conver- sion yields of 95, 88, 78, and 78 %, respectively. Due to the important applications of lactobionic acid, the formation of this substance by the enzymatic GFOR/GL complex in Ca-alginate-immobilized cells was assessed. The highest GFOR/GL activities were found at pH 7.0–8.0 and temper- atures of 47–50 °C. However, when a 24 h bioconversion run was carried out, it was observed that a combination of pH 6.4 and temperature of 47 °C led to the best results. In this case, despite the fact that Ca-alginate acts as a barrier for the diVusion of substrates and products, maximal lac- tobionic acid concentration, conversion yields and speciWc productivity similar to those obtained with permeabilized free-cells were achieved. Keywords Zymomonas mobilis · Glucose-fructose oxidoreductase/glucono--lactonase · Organic acids · Immobilized cells Introduction In the last two decades the ethanol-producing bacterium Zymomonas mobilis has been the subject of considerable research eVorts due to its ability to produce sorbitol and gluconic acid from a mixture of fructose and glucose, in reactions catalyzed by its own periplasmic enzymatic com- plex of glucose-fructose oxidoreductase (GFOR; EC 1.1.1.99), and glucono--lactonase (GL; EC 3.1.1.17) [3, 4, 20, 24, 31]. Research has been particularly focused on the biotechnological production of sorbitol, because this polyol has several important applications in the pharmaceutical and food industries [11, 23, 28]. However, since sorbitol and gluconic acid are formed in equimolar amounts when mediated by the GFOR/GL complex, the industrial applica- tion of this enzymatic system has been hindered by the fact that the commercial demand for sorbitol is signiWcantly higher than that for gluconic acid and its salts. In order to avert this problem, some technical alterna- tives have been tried. For instance, it has been reported that puriWed GFOR from Z. mobilis oxidizes diVerent aldoses other than glucose, to their respective lactones, which are then hydrolyzed to form linear aldonic acids by the action of GL [21, 31]. One of these products is lactobionic acid, E. Malvessi (&) · S. Carra · F. C. Pasquali · D. B. Kern · M. M. da Silveira (&) Biotechnology Institute, University of Caxias do Sul, PO Box 1352, Caxias do Sul, RS 95001-970, Brazil e-mail: emalvess@ucs.br M. M. da Silveira e-mail: mmsilvei@ucs.br E. Malvessi · M. A. Z. Ayub (&) Biotechnology and Biochemical Engineering Laboratory (BiotecLab), Federal University of Rio Grande do Sul State, PO Box 15090, Porto Alegre, RS 91501-970, Brazil e-mail: mazayub@ufrgs.br