Kazuhiro Yoshinaga

Characterization of exo-(1,4)-alpha glucan lyase from red alga Gracilaria chorda. Activation, inactivation and the kinetic properties of the enzyme

Exo-(1,4)-alpha glucan lyase (GLase) was purified from a red alga Gracilaria chorda. The enzyme was activated 1.3-fold in the presence of Ca(2+) and Cl(-) ions. The ions also stabilized the enzyme increasing the temperature of its maximum activity from 45 degrees C to 50 degrees C. GLase was inactivated by chemical modification with carbodiimide and a carboxyl group of the enzyme was shown essential to the lyase activity. A tryptophanyl residue(s) was also shown to be important for the activity and was probably involved in substrate binding. K(m) values of the enzyme were 2.3 mM for maltose, 0.4 mM for maltotriose and 0.1 mM for maltooligosaccharides of degree of polymerization (dp) 4-7, and the k(0) values for the oligosaccharides were similar (42-53 s(-1)). The analysis of these kinetic parameters showed that the enzyme has four subsites to accommodate oligosaccharides. The subsite map of GLase was unique, since subsite 1 and subsite 2 have large positive and small negative affinities, respectively. The subsite map of this type has not been found in other enzymes with exo-action on alpha-1,4-glucan. The K(m) and k(0) values for the polysaccharides were lower (0.03 mM) and higher (60-100 s(-1)), respectively, suggesting the presence of another affinity site specific to the polysaccharides.

Preparation and reactivity of a novel disaccharide, glucosyl 1,5-anhydro-d-fructose (1,5-anhydro-3-O-α-glucopyranosyl-d-fructose)

A novel disaccharide, glucosyl 1,5-anhydro-D-fructose (1,5-anhydro-3-O-alpha-glucopyranosyl-D-fructose, GAF) was enzymatically prepared from 1,5-anhydro-D-fructose (1,5-AF) and cyclomaltoheptaose (beta-cyclodextrin). Cyclodextrin glucanotransferase transferred various sizes of maltooligosaccharide to 1,5-AF. Glucoamylase digested the maltooligosyl chain of the products to a glucosyl residue giving a final product, GAF. An NMR analysis of GAF elucidated that the glucose residue was linked to C-3 of the 1,5-AF residue with an ether linkage. Reactivity on the aminocarbonyl reaction of GAF with bovine serum albumin was lower than that of 1,5-AF, but was higher than that of glucose.

Determination of kinetic parameters for maltotriose and higher malto-oligosaccharides in the reactions catalyzed by α-d-glucan phosphorylase from potato

Abstract For kinetic studies on its synthetic and phosphorolytic reactions, α- d -glucan phosphorylase from potatoes was purified chromatographically until free of D-enzyme. Purified maltotriose (G 3 ) is a poor primer in the phosphorylase-catalyzed synthetic reaction, showing an anomalous time course and making previous attempts to determine its kinetic parameters unsuccessful. In the present work the true rate of the G 3 -primed reaction was obtained from linear plots obtained by incorporating a sufficient quantity of β-amylase in the digest to eliminate the more rapidly reacting G 4 formed from the G 3 . A K m value of 9.4 ± 0.8 m m for G 3 was calculated from the data by a nonlinear least-squares method. Kinetic parameters for a series of higher malto-oligosaccharides (G 4 –G 8 ) were also determined in both the synthetic and the phosphorolytic directions. A large change in the values of K m and V/e was seen on going from G 3 to G 4 for the synthetic reaction, and from G 4 to G 5 for the phosphorolytic. For the higher saccharides the V/e values do not vary strongly with increasing d.p., while the K m values tend to decrease, as has seen in the reactions of other plant phosphorylases.

1,5-anhydro-D-fructose and its derivatives: biosynthesis, preparation and potential medical applications.

1,5-Anhydro-D-fructose (AF) was first found in fungi and red algae. It is produced by the degradation of glycogen, starch and maltosaccharides with α-1,4-glucan lyase (EC 4.2.2.13). In vivo, AF is metabolized to 1,5-anhydro-D-glucitol (AG), ascopyrone P (APP), microthecin and other derivatives via the anhydrofructose pathway. The genes coding for the enzymes in this pathway have been cloned, enabling the large-scale production of AF and related products in a cell-free reactor. The possible applications of these products in medicine have been evaluated using both in vitro and in vivo systems. Thus AF is a useful anticariogenic agent as it inhibits the growth of the oral pathogen Streptococcus mutans, impairing the production of plaque-forming polysaccharides and lactic acid. AF also shows anti-inflammatory and anticancer effects. AG is used as a diabetic marker for glycemic control. AG also stimulates insulin secretion in insulinoma cell lines. in vivo, APP has been shown to lengthen the life span of cancer-afflicted mice. It interferes with tumor growth and metastasis by its cidal effects on fast multiplying cells. Microthecin inhibits the growth of the human pathogen Pseudomonas aeruginosa PAO1, particularly under anaerobic conditions. The pharmaceutical usefulness of the other AF metabolites 1,5-anhydro-D-mannitol,1-deoxymannojirimycin, haliclonol, 5-epipentenomycin I, bissetone, palythazine, isopalythazine, and clavulazine remains to be investigated. In this review AF and its metabolites as the bioactive natural products for their pharmaceutical potentials are discussed.