Over-expression of the ß-fructofuranosidase xd-inv from Xanthophyllomyces dendrorhous, an enzyme producer of Prebiotics

Trabajo presentado en el ANQUE.ICCE.BIOTEC - Congress on Chemistry, Chemical Engineering and Biotechnology, celebrado en Madrid del 01 al 04 de julio de 2014.The β-fructofuranosidase Xd-INV from Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) is an extracellular glycoprotein with a content of 59-67% N-linked carbohydrates and an estimated molecular mass of 160-200 kDa. This protein is included in family 32 of glycosylhydrolase (GH 32) on the basis of its amino acid sequence. Xd-INV, independently of the glycoforms, catalyzes the synthesis of neofructooligosaccharides (neoFOS), mainly neokestose and neonystose, sugars with enhanced prebiotic properties and higher chemical stability compared to traditional fructooligosaccharides (FOS). To the best our knowledge, this enzyme is the largest neoFOS microbial producer reported up to now [1]. Functionality of Xd-INV gene in Saccharomyces cerevisiae was previously reported [2], but only weak Xd-INV invertase activity was quantified in the cellular fraction of the transformant strain. In order to improve Xd-INV activity level, different heterologous expression systems, based in S. cerevisiae and Pichia pastoris, were evaluated. The DNA fragment encoding Xd-INV was fused to the S. cerevisiae MFα1 secretion signal and included in different expression vectors. The best expression level was obtained using P. pastoris and the produced enzyme was enzymatically characterized as well as analyzed on polyacrylamide gels using native or denaturing conditions and mass spectrometry. This study may provide the basis for a further mutational analysis of this enzyme that should shed additional light on the determinants responsible for fructosyltransferase activity within GH 32 enzyme family and the improvement of neoFOS production.Peer Reviewe