No abstract

The beta-galactosidases (beta-Gals) of Lactobacillus reuteri L103 and L461 proved to be suitable biocatalysts for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. Maximum GOS yields were 38% when using an initial lactose concentration of 205 g/L and at approximately 80% lactose conversion. The product mixtures were analyzed by capillary electrophoresis (CE) and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Disaccharides other than lactose and trisaccharides made up the vast majority of GOS formed. The main products were identified as beta-d-Galp-(1-->6)-d-Glc (allolactose), beta-d-Galp-(1-->6)-d-Gal, beta-d-Galp-(1-->3)-d-Gal, beta-d-Galp-(1-->6)-Lac, and beta-d-Galp-(1-->3)-Lac. There were no major products with beta1-->4 linkages formed. Both intermolecular and intramolecular transgalactosylation were observed. d-Galactose proved to be a very efficient galactosyl acceptor; thus, a relatively large amount of galactobioses was formed. Monosaccharides could be conveniently separated from the mixture by chromatography using a strong cation-exchange resin.

The intracellular beta-galactosidase (beta-gal) enzymes from two strains of Lactobacillus reuteri, L103 and L461, were purified by ammonium sulfate fractionation, hydrophobic interaction, and affinity chromatography. Both enzymes are heterodimers with a molecular mass of 105 kDa, consisting of a 35 kDa subunit and a 72 kDa subunit. Active staining of L. reuteri L103 and L461 beta-gal with 4-methylumbelliferyl beta-d-galactoside showed that the intact enzymes as well as the larger subunits possess beta-galactosidase activity. The isoelectric points of L. reuteri L461 and L103 beta-gal were found to be in the range of 3.8-4.0 and 4.6-4.8, respectively. Both enzymes are most active in the pH range of 6-8; however, they are not stable at pH 8. The L. reuteri beta-galactosidases are activated by various mono- and divalent cations, including Na(+), K(+), and Mn(2+), and are moderately inhibited by their reaction products d-glucose and d-galactose. Because of their origin from beneficial and potentially probiotic lactobacilli, these enzymes could be of interest for the synthesis of prebiotic galacto-oligosaccharides.

To characterize catalysis by NAD-dependent long-chain mannitol 2-dehydrogenases (MDHs), the recombinant wild-type MDH from Pseudomonas fluorescens was overexpressed in Escherichia coli and purified. The enzyme is a functional monomer of 54 kDa, which does not contain Zn(2+) and has B-type stereospecificity with respect to hydride transfer from NADH. Analysis of initial velocity patterns together with product and substrate inhibition patterns and comparison of primary deuterium isotope effects on the apparent kinetic parameters, (D)k(cat), (D)(k(cat)/K(NADH)), and (D)(k(cat)/K(fructose)), show that MDH has an ordered kinetic mechanism at pH 8.2 in which NADH adds before D-fructose, and D-mannitol and NAD are released in that order. Isomerization of E-NAD to a form which interacts with D-mannitol nonproductively or dissociation of NAD from the binary complex after isomerization is the slowest step (>/=110 s(-)(1)) in D-fructose reduction at pH 8.2. Release of NADH from E-NADH (32 s(-)(1)) is the major rate-limiting step in mannitol oxidation at this pH. At the pH optimum for D-fructose reduction (pH 7.0), the rate of hydride transfer contributes significantly to rate limitation of the catalytic cascade and the overall reaction. (D)(k(cat)/K(fructose)) decreases from 2.57 at pH 7.0 to a value of

During growth on d-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76%. Aldose reductase consists ofa single 43 kDa polypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last. The catalytic efficiency (kcat/Km) in d-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants. The enzyme prefers NADP(H) approx. 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 microM), whereas NAD+ is not. Unlike mammalian aldose reductase, the enzyme from C. tenuis is not subject to oxidation-induced activation. Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5'-phosphate. The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase.