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Two Pantoea anthophila bacterial strains were isolated from "tejuino", a traditional Mexican beverage, and studied as β-galactosidase producers for galactooligosaccharides synthesis. Using 400 g/L of lactose, 50 °C, and 15 U/mL of β-galactosidase activity with ethanol-permeabilized cells, the maximum galactooligosaccharides (GOS) yield determined by High performance anion exchange chromatography with pulse amperometric detection (HPAEC-PAD) was 136 g/L (34% w/w of total sugars) at 96% of lactose conversion for Bac 55.2 and 145 g/L (36% w/w of total sugars) at 94% of lactose conversion for Bac 69.1. The main synthesized products were the disaccharides allolactose [Gal-β(1 → 6)-Glc] and 6-galactobiose [Gal-β(1 → 6)-Gal], as well as the trisaccharides 3'-galactosyl-lactose [Gal-β(1 → 3)-Gal-β(1 → 4)-Glc], 6-galactotriose [Gal-β(1 → 6)-Gal-β(1 → 6)-Gal], 3'-galactosyl-allolactose [Gal-β(1 → 3)-Gal-β(1 → 6)-Glc], and 6'-galactosyl-lactose [Gal-β(1 → 6)-Gal-β(1 → 4)-Glc]. The β-galactosidases present in both strains showed a high transgalactosylation activity and formed principally β(1 → 3) and β(1 → 6) linkages. Considering the stability and bifidogenic properties of GOS containing such types of bonds, P. anthophila strains Bac 55.2 and Bac 69.1 possess a high potential as novel biocatalysts for prebiotic industrial production. [ABSTRACT FROM AUTHOR]

The β-galactosidase from Bacillus circulans was covalently attached to aldehyde-activated (glyoxal) agarose beads and assayed for the continuous production of galactooligosaccharides (GOS) in a packed-bed reactor (PBR). The immobilization was fast (1 h) and the activity of the resulting biocatalyst was 97.4 U/g measured with o-nitrophenyl-β-D-galactopyranoside (ONPG). The biocatalyst showed excellent operational stability in 14 successive 20 min reaction cycles at 45˚Cin a batch reactor. A continuous process for GOS synthesis was operated for 213 h at 0.2 mL/min and45˚C using 100 g/L of lactose as a feed solution. The efficiency of the PBR slightly decreased withtime; however, the maximum GOS concentration (24.2 g/L) was obtained after 48 h of operation, which corresponded to 48.6% lactose conversion and thus to maximum transgalactosylation activity. HPAEC-PAD analysis showed that the two major GOS were the trisaccharideGal-β (1!4)-Gal-β (1!4)-Glc and the tetrasaccharide Gal-β(1!4)-Gal-β(1!4)-Gal-β(1!4)-Glc.The PBR was also assessed in the production of GOS from milk as a feed solution. The stability of the bioreactor was satisfactory during the first 8 h of operation; after that, a decrease in the flow rate was observed, probably due to partial clogging of the column. This work represents a step forward in the continuous production of GOS employing fixed-bed reactors with immobilized β-galactosidases. [ABSTRACT FROM AUTHOR]

During directed evolution to functionally express the high redox potential laccase from the PM1 basidiomycete in Saccharomyces cerevisiae, the characteristic maximum absorption at the T1 copper site (Abs610T1Cu) was quenched, switching the typical blue colour of the enzyme to yellow. To determine the molecular basis of this colour change, we characterized the original wild-type laccase and its evolved mutant. Peptide printing and MALDI-TOF analysis confirmed the absence of contaminating protein traces that could mask the Abs610T1Cu, while conservation of the redox potential at the T1 site was demonstrated by spectroelectrochemical redox titrations. Both wild-type and evolved laccases were capable of oxidizing a broad range of substrates (ABTS, guaiacol, DMP, synapic acid) and they displayed similar catalytic efficiencies. The laccase mutant could only oxidize high redox potential dyes (Poly R-478, Reactive Black 5, Azure B) in the presence of exogenous mediators, indicating that the yellow enzyme behaves like a blue laccase. The main consequence of over-expressing the mutant laccase was the generation of a six-residue N-terminal acidic extension, which was associated with the failure of the STE13 protease in the Golgi compartment giving rise to alternative processing. Removal of the N-terminal tail had a negative effect on laccase stability, secretion and its kinetics, although the truncated mutant remained yellow. The results of CD spectra analysis suggested that polyproline helixes were formed during the directed evolution altering spectral properties. Moreover, introducing the A461T and S426N mutations in the T1 environment during the first cycles of laboratory evolution appeared to mediate the alterations to Abs610T1Cu by affecting its coordinating sphere. This laccase mutant is a valuable departure point for further protein engineering towards different fates. [ABSTRACT FROM AUTHOR]

The regioselective α-glucosylation of hesperetin was achieved by a transglycosylation reaction catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. using soluble starch as glucosyl donor. By combining mass spectrometry (ESI-TOF) and 2D-NMR analysis, the main monoglucosylated derivative was fully characterized (hesperetin 7-O-α-d-glucopyranoside). In order to increase the yield of monoglucoside, several reaction parameters were optimized: Nature and percentage of cosolvent, composition of the aqueous phase, glucosyl donor, temperature, and the concentrations of hesperetin and soluble starch. Under the optimal conditions, which included the presence of 30% of bis(2-methoxyethyl) ether as cosolvent, the maximum concentration of monoglucoside was approximately 2 mM, obtained after 24 h of reaction. To our knowledge, this is the first report of direct glucosylation of hesperetin employing free enzymes instead of whole cells. [ABSTRACT FROM AUTHOR]

The synthesis of a novel α-glucosylated derivative of pterostilbene was performed by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. The reaction was carried out in a buffer containing 20% (v/v) DMSO to enhance the solubility of pterostilbene. Due to the formation of several polyglucosylated products with CGTase, the yield of monoglucoside was increased by the treatment with a recombinant amyloglucosidase (STA1) from Saccharomyces cerevisiae (var. diastaticus). This enzyme was not able to hydrolyze the linkage between the glucose and pterostilbene. The monoglucoside was isolated and characterized by combining ESI-MS and 2D-NMR methods. Pterostilbene α-d-glucopyranoside is a novel compound. The α-glucosylation of pterostilbene enhanced its solubility in water to approximately 0.1 g/L. The α-glucosylation caused a slight loss of antioxidant activity towards ABTS˙+ radicals. Pterostilbene α-d-glucopyranoside was less toxic than pterostilbene for human SH-S5Y5 neurons, MRC5 fibroblasts and HT-29 colon cancer cells, and similar for RAW 264.7 macrophages. [ABSTRACT FROM AUTHOR]

The β-fructofuranosidase (Xd-INV) from the basidiomycota yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) is unique in its ability to synthesize neo- fructooligosaccharides (neo-FOS). In order to facilitate its industrial application, the recombinant enzyme expressed in Pichia pastoris (pXd-INV) was immobilized by entrapment in polyvinyl alcohol (PVA) hydrogels. The encapsulation efficiency exceeded 80%. The PVA lenticular particles of immobilized pXd-INV were stable up to approximately 40 °C. Using 600 g/L sucrose, the immobilized biocatalyst synthesized 18.9% (w/w) FOS (59.1 g/L of neokestose, 30.2 g/L of 1-kestose, 11.6 g/L of neonystose and 12.6 g/L of blastose). The operational stability of PVA-immobilized biocatalyst was assayed in a batch reactor at 30 °C. The enzyme preserved its initial activity during at least 7 cycles of 26 h. [ABSTRACT FROM AUTHOR]