An extracellular ␤-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70°C) and thermostability (with a T 50 in the range 66 to 71°C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-␤-(231)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the k cat /K m ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial ␤-fructofuranosidases, the enzyme from X. dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Using a 41% (wt/vol) sucrose solution, the maximum fructooligosaccharide concentration reached was 65.9 g liter ؊1. In addition, we isolated and sequenced the X. dendrorhous ␤-fructofuranosidase gene (Xd-INV), showing that it encodes a putative mature polypeptide of 595 amino acids and that it shares significant identity with other fungal, yeast, and plant ␤-fructofuranosidases, all members of family 32 of the glycosyl-hydrolases. We demonstrate that the Xd-INV could functionally complement the suc2 mutation of Saccharomyces cerevisiae and, finally, a structural model of the new enzyme based on the homologous invertase from Arabidopsis thaliana has also been obtained.

Schwanniomyces occidentalis invertase is an extracellular enzyme that hydrolizes sucrose and releases ␤-fructose from various oligosaccharides and essential storage fructan polymers such as inulin. We report here the three-dimensional structure of Sw. occidentalis invertase at 2.9 Å resolution and its complex with fructose at 1.9 Å resolution. The monomer presents a bimodular arrangement common to other GH32 enzymes, with an N-terminal 5-fold ␤-propeller catalytic domain and a C-terminal ␤-sandwich domain for which the function has been unknown until now. However, the dimeric nature of Sw. occidentalis invertase reveals a unique active site cleft shaped by both subunits that may be representative of other yeast enzymes reported to be multimeric. Binding of the tetrasaccharide nystose and the polymer inulin was explored by docking analysis, which suggested that medium size and long substrates are recognized by residues from both subunits. The identified residues were mutated, and the enzymatic activity of the mutants against sucrose, nystose, and inulin were investigated by kinetic analysis. The replacements that showed the largest effect on catalytic efficiency were Q228V, a residue putatively involved in nystose and inulin binding, and S281I, involved in a polar link at the dimer interface. Moreover, a significant decrease in catalytic efficiency against inulin was observed in the mutants Q435A and Y462A, both located in the ␤-sandwich domain of the second monomer. This highlights the essential function that oligomerization plays in substrate specificity and assigns, for the first time, a direct catalytic role to the supplementary domain of a GH32 enzyme.Fructans, the fructose-rich polymers derived biosynthetically from sucrose, are important storage oligosaccharides and polysaccharides in many bacteria and fungi and numerous plant species. Furthermore, sucrose is one of the most widespread disaccharides in nature and is especially ubiquitous in higher plants as the first free sugar resulting from photosynthesis. It is the major transport compound to bring energy and carbon skeletons from source to sink tissues. Carbohydrate partitioning and sugar sensing are intimately connected to sucrose metabolism; these processes are vital throughout plant development. Therefore, the enzymes involved in fructans and sucrose processing are essential to plant cell metabolism.The enzymes that hydrolyze sucrose are referred to collectively as invertases or ␤-fructofuranosidases (EC 3.2.1.26) and catalyze the release of ␤-fructose from the nonreducing end of various ␤-D-fructofuranoside substrates (Fig. 1). The cleavage of the ␤-glycosidic bond is carried out by a double displacement catalytic mechanism that retains the configuration of the fructose anomeric carbon, two conserved residues, an aspartic and a glutamic acid, being the nucleophile and the general acid-base catalyst, respectively. On the basis of the amino acid sequences (1) they are classified into family 32 of the glycosylhydrolases (GH32), 3 which are included in...

beta-Fructofuranosidases are powerful tools in industrial biotechnology. We have characterized an extracellular beta-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 (1)H, (13)C and 2D-NMR techniques shows that the major product is the prebiotic trisaccharide 6-kestose. The 6-kestose yield obtained with this beta-fructofuranosidase is, to our concern, higher than those reported with other 6-kestose-producing enzymes, both at the kinetic maximum (76gl(-1)) and at reaction equilibrium (44gl(-1)). The total FOS production in the kinetic maximum was 101gl(-1), which corresponded to 16.4% (w/w) referred to the total carbohydrates in the reaction mixture.

The transgalactosylation activity of Kluyveromyces lactis cells was studied in detail. Cells were permeabilized with ethanol and further lyophilized to facilitate the transit of substrates and products. The resulting biocatalyst was assayed for the synthesis of galacto-oligosaccharides (GOS) and compared with two soluble β-galactosidases from K. lactis (Lactozym 3000 L HP G and Maxilact LGX 5000). Using 400 g/L lactose, the maximum GOS yield, measured by HPAEC-PAD analysis, was 177 g/L (44% w/w of total carbohydrates). The major products synthesized were the disaccharides 6-galactobiose [Gal-β(1→6)-Gal] and allolactose [Gal-β(1→6)-Glc], as well as the trisaccharide 6-galactosyl-lactose [Gal-β(1→6)-Gal-β(1→4)-Glc], which was characterized by MS and 2D NMR. Structural characterization of another synthesized disaccharide, Gal-β(1→3)-Glc, was carried out. GOS yield obtained with soluble β-galactosidases was slightly lower (160 g/L for Lactozym 3000 L HP G and 154 g/L for Maxilact LGX 5000); however, the typical profile with a maximum GOS concentration followed by partial hydrolysis of the newly formed oligosaccharides was not observed with the soluble enzymes. Results were correlated with the higher stability of β-galactosidase when permeabilized whole cells were used.

BackgroundChitinases are ubiquitous enzymes that have gained a recent biotechnological attention due to their ability to transform biological waste from chitin into valued chito-oligomers with wide agricultural, industrial or medical applications. The biological activity of these molecules is related to their size and acetylation degree. Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-d-glucosamine (GlcNAc) units. Gene chit42 was previously characterized, and according to its sequence, the encoded protein included in the structural Glycoside Hydrolase family GH18.ResultsChit42 was expressed in Pichia pastoris using fed-batch fermentation to about 3 g/L. Protein heterologously expressed showed similar biochemical properties to those expressed by the natural producer (42 kDa, optima pH 5.5–6.5 and 30–40 °C). In addition to hydrolyse colloidal chitin, this enzyme released reducing sugars from commercial chitosan of different sizes and acetylation degrees. Chit42 hydrolysed colloidal chitin at least 10-times more efficiently (defined by the kcat/Km ratio) than any of the assayed chitosan. Production of partially acetylated chitooligosaccharides was confirmed in reaction mixtures using HPAEC-PAD chromatography and mass spectrometry. Masses corresponding to (d-glucosamine)1–8-GlcNAc were identified from the hydrolysis of different substrates. Crystals from Chit42 were grown and the 3D structure determined at 1.8 Å resolution, showing the expected folding described for other GH18 chitinases, and a characteristic groove shaped substrate-binding site, able to accommodate at least six sugar units. Detailed structural analysis allows depicting the features of the Chit42 specificity, and explains the chemical nature of the partially acetylated molecules obtained from analysed substrates.ConclusionsChitinase Chit42 was expressed in a heterologous system to levels never before achieved. The enzyme produced small partially acetylated chitooligosaccharides, which have enormous biotechnological potential in medicine and food. Chit42 3D structure was characterized and analysed. Production and understanding of how the enzymes generating bioactive chito-oligomers work is essential for their biotechnological application, and paves the way for future work to take advantage of chitinolytic activities.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0895-x) contains supplementary material, which is available to authorized users.

Background: Schwanniomyces occidentalis ␤-fructofuranosidase synthesizes 6-kestose and 1-kestose, prebiotics that stimulate beneficial bacteria. Results:The ␤-sandwich domain is involved in substrate binding, and the Gln-228/Asn-254 pair plays a crucial role in acceptor/ donor substrate binding and product specificity. Conclusion: First evidence of ␤-sandwich domain functionality. Variants with new specificities, synthesizing neokestose, were obtained. Significance: Understanding the molecular mechanism that regulates product specificity is crucial for designing improved new enzymes.

Schwanniomyces occidentalis ␤-fructofuranosidase (Ffase) releases ␤-fructose from the nonreducing ends of ␤-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (k cat /K m ) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes' specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme.

Among several commercial enzymes screened for chitosanolytic activity, Neutrase 0.8L (a protease from Bacillus amyloliquefaciens) was selected in order to obtain a product enriched in deacetylated chitooligosaccharides (COS). The hydrolysis of different chitosans with this enzyme was followed by size exclusion chromatography (SEC-ELSD), mass spectrometry (ESI-Q-TOF), and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Neutrase 0.8L converted 10 g/L of various chitosans into mostly deacetylated oligosaccharides, yielding approximately 2.5 g/L of chitobiose, 4.5 g/L of chitotriose and 3 g/L of chitotetraose. We found out that the neutral protease was not responsible of the chitosanolytic activity in the extract, whilst it could participate in the deacetylating process. The synthesized COS were tested in vitro for their neuroprotective (towards human SH-S5Y5 neurons) and anti-inflammatory (in RAW macrophages) activities, and compared with other functional ingredients, namely fructooligosaccharides.