Tomonori Nagahama

Release of bound lipids in cereal starches upon hydrolysis by glucoamylase

ABSTRACT The raw starch granules from corn, rice, and wheat were hydrolyzed by practically pure glucoamylase (Rhizopus niveus). The bound lipids remaining in the residual starches were investigated, of which the major components of the lipids, free fatty acids (FFA) in corn starch, FFA and phospholipids (PL) in rice starch, and PL in wheat starch were determined. In each case, the bound FFA and PL were decreased to some extent during the initial stage of hydrolysis. During the later stages, the FFA continued to gradually decrease, while the level of PL stabilized. It was interesting that some of the bound lipids were released from the granules upon glucoamylase hydrolysis, differing from the model amylose-lipid complexes. Furthermore, the structures of the residual starches were investigated. The blue value and λmax of the starches were increased by partial hydrolysis of the starch granules using practically pure glucoamylase. Two gel-permeation chromatography analyses revealed that the relative amount of...

Elucidation of the subsite structure of bacterial saccharifying alpha-amylase and its mode of degradation of maltose

The subsite structure of bacterial saccharifying alpha-amylase (BSAm) was elucidated by two methods using a series of maltooligosaccharides labeled with [14C]D-glucose at the reducing end. The rate parameter k0/Km and the cleavage frequency were obtained using the labeled substrates at sufficiently low concentrations to eliminate transglycosylation and condensation. This evaluation showed that the active center is composed of five subsites, with the catalytic site located between the 3rd and the 4th subsites from the nonreducing end. The evaluated affinity values of a subsite varied with the set of data used, which suggests some stimulation factor resulting from the chain length effect. The appearance of a time lag during the digestion of the poor substrate, maltose, was studied using radioactively labeled maltose (81.6 mM). Radioactive oligosaccharides larger than maltose were found at a significant level of more than 2% of the initial substrate in the digests, including a product peculiar to condensation, G-G*-G, as 8-10% of the maltotriose in the digests. This indicates that transglycosylation is a main side reaction (ca. 90%). A degradation pathway for maltose via maltosyl transfer was proposed, in which G3 behaves as a kind of catalyst.

Study of the action of human salivary alpha-amylase on 2-chloro-4-nitrophenyl α-maltotrioside in the presence of potassium thiocyanate

The degradation mechanism of a synthetic substrate, 2-chloro-4-nitrophenyl alpha-maltotrioside (CNP-G3), by human salivary alpha-amylase (HSA) was investigated by kinetic and product analyses. It was observed that the enzyme attacked the various CNP-maltooligosaccharides (CNP-G3 to CNP-G6) releasing free CNP. Addition of 500 mM potassium thiocyanate (KSCN) was also found to greatly increase the rates of CNP-release. It was the fastest with CNP-G3, and, in the presence of KSCN, was almost comparable to that of degradation of maltopentaose (G5). On the other hand, addition of KSCN decreased the rate of cleavage between glucan-glucan bonds in maltopentaose. Product analysis showed that KSCN addition altered the cleavage distribution which occurred 100% at the bond between CNP and G3, and that product distribution of free CNP was largely dependent on substrate concentration. Formation of CNP-G6, a larger product than the original substrate CNP-G3, was found to be present in the digest at high concentrations of substrate and in the presence of KSCN. Based on these results, a degradation pathway for CNP-G3 involving transglycosylation besides direct hydrolysis is proposed. The increase of the CNP-release by the addition of KSCN would result from a corresponding increase in the interaction between the CNP moiety and the corresponding subsite near the catalytic site, as well as the enhancement of the catalytic efficiency.

Analysis of the characteristic action of D-enzyme from sweet potato in terms of subsite theory

Abstract Disproportionating enzyme (D-enzyme, EC 2.4.1.25) was purified chromatographically from a β-amylase-deficient variety of sweet potatoes, and obtained free of amylases and glucosidases. Some heavy metal ions, particularly Hg2+ and Ag+, inactivated the enzyme. The Km value for maltrotriose was found to be 7.3m m . Analysis by h.p.l.c. of digests of malto-oligosaccharides (G3–G7) showed that, as with other D-enzymes, maltose is not formed in any case. The products from all substrates except G4 are those resulting from maltosyl transfer as the predominant reaction, wherein Gn−2 and Gn+2 are produced from Gn. The various malto-oligosaccharides undergo reaction at almost the same rates. The frequency of cleavage of the various bonds in malto-oligosaccharides was estimated from the observed molar ratios of products smaller than the substrates. A possible subsite structure for the D-enzyme is proposed on the basis of computer modelling of data describing the cleavage pattern etc.

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.

Some properties of branched and linear dextrins from Nägeli amylodextrin

Abstract Branched dextrin (BD) and linear dextrin (LD) were prepared from Nageli amylodextrin, and their molecular structure and some properties (the interaction with 2-p-toluidinylnaphthalene-6-sulfonate (TNS) in solution and the aggregation into precipitate) were investigated. BD and LD were singly branched dextrin (d.p. = 25.7) with the branched point near the reducing end, and practically linear dextrin (d.p. = 12.4), respectively. The fluorescence intensity of TNS increased with increasing concentrations of α-cyclodextrin, BD and LD, indicating their interactions with each other, in which the values of the dissociation constant were estimated to be 20mM, 3.4mM and 7.8 mM, respectively. In aqueous 16 M methanol, the aggregation of BD was much faster than that of LD, and both dextrins were precipitated into a type A crystallite. It is considered that the branch point in the BD which is a connection of two linear dextrins may trigger the association of the unit chains. On the other hand, the aggregation of dextrins was retarded in the presence of lauric acid. These results may be applicable to the local phenomenon of the whole amylopectin.

Studies on Cycasin, a New Toxic Glycoside, of Cycas revoluta Thunb: Part V. Quantitative Determination of Cycasin in Cycad Seeds

Quantitative paper chromatography of cycasin is described, where the cycasin and sugars separated on the paper are eluted out and determined, according to the colorimetric micro analysis of sugars by Plumel. The contents of cycasin and free sugars contained in both premature and matured cycad seeds determined by this method are presented.

Interconversion between the Azoxyglycosides by Cycad Emulsin:Studies on Some New Azoxyglycosides of Cycas revoluta Thunb. Part III

glycoside, neocycasin C, is also produced. In this case, it is noteworthy that neocycasin A, containing a ƒÀ-1.3g lucosyl linkage, is predominantly produced from cycasin as substrate, although in the hitherto reported transglucosylation 4, 5) a ƒÀ-1.6 or 1.4 linkage was mainly formed by emulsins obtained from various origins. The cycad emulsin employed here is an acetone-dried preparation obtained from the cycad seeds by the tannic acid precipitation methods 6)

Studies on Some New Azoxy Glycosides of Cycas revoluta Thunb.: Part I. On Neocycasin A, β-Laminaribiosyloxyazoxymethane

The isolation of a new glycoside, named here as neocycasin A, with use of carbon chromatography, is described. It is one of a series of aliphatic azoxy glycosides, found in the seeds of Japanese cycad together with cycasin which is β-glucosyloxyazoxymethane as reported previously. The glycoside monohydrate gives m.p. 162° ~ 163° (decomp.), − 35.1°; its heptaacetylate, m.p. 142° ~ 143°, − 55.5°, from which octaacetyl-β-laminaribiose is isolated. On the basis of examination of the products obtained from partial or complete hydrolysis, and spectroscopic measurements, neocycasin A is concluded to be β-laminaribiosyloxyazoxymethane, i.e. 3-O-β-d-glucopyranosylcycasin.

Studies on Cycasin, a New Toxic Glycoside, of Cycas revoluta Thunb:Part VI. Polarography of Cycasin

The polarographic behaviors and the determination of cycasin, glucosyloxyazoxymethane, are described here. In the whole pH range, cycasin shows a reduction wave, which is considered to be due to the reduction of its aliphatic azoxy group. The polarograms from pH 4 to 7 run in two steps, and two electrode reaction mechanisms may be infered. The following facts are observed on the limiting current at pH 1 or 7: It is controlled by the diffusion process, and its temperature coefficient has a value resembling that of the usual diffusion current. The wave height shows a linear proportionality to the concentration of cycasin. The contents of cycasin in cycad seeds, determined polarographically, are presented, and compared with the results obtained by the paper chromatographic method.