Characterization of a β-fructofuranosidase from Schwanniomyces occidentalis with transfructosylating activity yielding the prebiotic 6-kestose

Abstract

β-Fructofuranosidases are powerful tools in industrial biotechnology. We have characterized an extracellular β-fructofuranosidase from the yeast Schwanniomyces occidentalis. The enzyme shows broad substrate specificity, hydrolyzing sucrose, 1-kestose, nystose and raffinose, with different catalytic efficiencies (k_cat/K_m). Although the main reaction catalysed by this enzyme is sucrose hydrolysis, it also produces two fructooligosaccharides (FOS) by transfructosylation. A combination of ¹H, ¹³C and 2D-NMR techniques shows that the major product is the prebiotic trisaccharide 6-kestose. The 6-kestose yield obtained with this β-fructofuranosidase is, to our concern, higher than those reported with other 6-kestose-producing enzymes, both at the kinetic maximum (76 g l⁻¹) and at reaction equilibrium (44 g l⁻¹). The total FOS production in the kinetic maximum was 101 g l⁻¹, which corresponded to 16.4% (w/w) referred to the total carbohydrates in the reaction mixture.

Introduction

Enzymes that hydrolyse sucrose are collectively referred to as invertases or β-fructofuranosidases (EC 3.2.1.26), and they catalyse the release of β-fructose from non-reducing termini of various β-d-fructofuranoside substrates. Yeast β-fructofuranosidases have been widely studied in Saccharomyces cerevisiae (Taussig and Carlson, 1983, Reddy and Maley, 1990, Reddy and Maley, 1996), Schizosaccharomyces pombe (Moreno et al., 1990), Pichia anomala (Rodríguez et al., 1995, Pérez et al., 1996), Candida utilis (Chávez et al., 1998) and Arxula adeninivorans (Boer et al., 2004). In general, these enzymes exhibit a high degree of sequence homology, and based on their amino acid sequences, they fall into family 32 of the glycosyl-hydrolases (GH) (Coutinho and Henrissat, 1999). They share two significant located acidic residues, which are necessary for the cleavage of glycosidic bonds. The reaction proceeds by a double-displacement mechanism in which a covalent glycosyl-enzyme intermediate is formed. Furthermore, the three-dimensional structures of a β-fructofuranosidase from Thermotoga maritima (Alberto et al., 2004) and an exoinulinase from Aspergillus niger (var. awamori) (Nagem et al., 2004) have been also reported.

In addition to releasing d-glucose and d-fructose from sucrose, microbial β-fructofuranosidases may catalyse the synthesis of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked to the sucrose by different glycosidic bonds depending on the enzyme source (Sangeetha et al., 2005). The extent to which the transglycosylation takes place varies among the different β-fructofuranosidases (Antosova and Polakovic, 2001). FOS containing β-(2 → 1)-bonds (1-kestose, nystose and 1^F-fructofuranosylnystose) are produced commercially in vast quantities as their prebiotic properties, and exert a beneficial effect on human health because they are selectively fermented by colonic flora (Ghazi et al., 2005).

There is great interest in the development novel FOS with improved prebiotic and physiological properties. In this context, β-(2 → 6)-linked FOS (6-kestose being first in the series) were metabolized by different Bifidobacteria strains when supplied as the sole carbon source (Marx et al., 2000). The FOS were synthesized by acid hydrolysis of β-(2 → 6)-linked polymers containing a glucose at one terminus (levans), these being produced by several microorganisms growing in sucrose-based medium (Bekers et al., 2002). The discovery of novel enzymes that synthesize β-(2 → 6)-linked FOS from sucrose may, however, provide a non-pollutant alternative to acid hydrolysis of levans.

Schwanniomyces occidentalis efficiently utilizes a wide variety of inexpensive carbon compounds by employing a number of extracellular enzymes. It has been extensively used in biotechnology, where it has a high potential for enzyme production. Several of its amylolytic enzymes (glucoamylase and amylases) have been well characterized (Abarca et al., 1989, Abarca et al., 1991, Dohmen et al., 1990, Yáñez et al., 1998) and invertase, one of the predominant secreted proteins when lactose is used as the carbon source (Klein et al., 1989a, Costagliogli et al., 1997), purified (Klein et al., 1989a). The invertase gene encodes a 533 amino acids protein, which shares similar sequence similarity with other invertases (Klein et al., 1989b). In this work, we have studied the biochemical properties of Sw. occidentalis β-fructofuranosidase, and kinetic studies of the hydrolase activity were developed using different substrates. The fructosyltransferase ability of this enzyme was investigated in detail.