Sweet potato dietary fiber (SPDF) was prepared from sweet potato pulp on an industrial scale. Its dietary fiber content was 83.8%. Male Wistar Hannover GALAS rats, 4 wk old, were fed diets containing cellulose or SPDF for 27 d. The cellulose and SPDF level was adjusted to 10%. The effects of SPDF on the cecal fermentation products and microflora in rats were investigated. The body weight gain of the rats fed SPDF-containing diet (SPDF rats) was lower than that of the rats fed the cellulose-containing diet (control rats) because of a significant decrease in food intake. There was no significant difference in the pH value of the cecum content. In the SPDF rats, the cecum weights of content and tissue were heavier, the weight of wet feces was lower, and the moisture content of feces was significantly higher than in the control rats. The numbers of intestinal bacteria of the SPDF rats were higher than those of the control rats, and Bifidobacterium sp. was found only in the SPDF rats. The ratio of the total anaerobes to the total aerobes in the SPDF rats was higher than that in the control rats. The propionate concentration significantly increased in the cecum content of the SPDF rats.
Thermus maltogenic amylase (ThMA), one of the cyclodextrin (CD)-degrading enzymes, is expected to have a calcium-binding site (Ca2 site) based on multiple sequence alignments. In spite of the presumption of the Ca2 site in ThMA, however, its thermostability is independent of calcium ions. In order to investigate the effect of calcium ions on thermostability, two mutations (Ile152Asn and Ser153Asn) were introduced into Ca2 sites of a thermostabilized ThMA mutant (ThMA-DM2) using site-directed mutagenesis. The resultant mutant (ThMA-DM2-Ca) showed highly improved thermostability in the presence of calcium ions. The relative hydrolysis activity of ThMA-DM2-Ca also increased in comparison with that of ThMA-DM2 whereas transglycosylation activity was not affected by substitutions. The result confirmed that the residues located in the Ca2 site (Ile152 and Ser153) played critical role in stabilizing CD-degrading enzymes.
The wheat dough and baking properties of various strong-type wheat grains cultivated in Japan—Kitanokaori (strong), Glenlea and Bluesky (extra-strong), and Haruyutaka (semi-strong)—were evaluated by comparing them with commercial wheat flour, Cameria (strong). Protein contents of Glenlea and Bluesky (15.8 and 15.6%, respectively) were significantly higher than that of Cameria (12.4%), whereas Kitanokaori and Haruyutaka had significantly lower protein contents (11.4 and 9.7%, respectively). The amylose contents of starches of Kitanokaori and Haruyutaka were significantly lower than those of the others. Kitanokaori also had the lowest lipid and ash contents among the wheat flours used. The doughs made from Kitanokaori, Glenlea and Bluesky had significantly higher water absorption, and increased rapidly the resistance to stretch during proofing. The bread baked from Kitanokaori had bigger loaf volume and lower firmness than the others after baking and during storage, whereas Glenlea and Bluesky breads had lower loaf volumes and higher firmness than did Cameria and Kitanokaori though they had higher protein contents. In addition, Haruyutaka bread had the lowest loaf volume and increased in firmness rapidly during storage.
A cDNA (cel2) coding for an exo-type cellobiohydrolase was isolated from the basidiomycete Irpex lacteus strain MC-2. The cel2 cDNA was expressed in a heterologous host Aspergillus oryzae by using an expression vector pNAN-8142. A recombinant cellobiohydrolase was secreted in culture fluid of A. oryzae transformant cells with the aid of a signal sequence of the cel2 gene. The recombinant cellobiohydrolase purified from the culture fluid exhibited similar enzymatic properties to those of a major cellobiohydrolase (Ex-1) previously purified and characterized from Driselase (a commercial enzyme produced by strain MC-2).
Acetobacter xylinum ATCC23769 produces not only cellulose but also various oligosaccharides during cell growth. These oligosaccharides accumulated and increased gradually up to about half the amount of cellulose with the increase of endo-1,4-α-glucanases activity in culture broth. These oligosaccharides were identified cello-oligosaccharides, gentiobiose and rhamnose, which were constituent sugars of acetan. It is suggested that they are the degradation products from acetan, as enzymes prepared from culture broth hydrolyzed acetan rather than cellulose.
Studies have been made of the effects of sodium dodecyl sulfate (SDS) on the pasting properties of wheat, corn, potato and waxy corn starches with the Rapid Visco-Analyzer. The peak viscosity and break down of wheat, corn and waxy corn starches during heating were markedly increased with the increasing concentration of SDS. Those of potato starch were markedly decreased at the lower concentrations of SDS and then increased at the higher concentrations. The maximum setback viscosity during cooling was markedly increased for wheat, corn and potato starches and slightly increased for waxy corn starch with the increasing concentration of SDS. From the results it was assumed that the striking increase in the peak viscosity during heating might be ascribed to the complex formation of amylopectin inside the swollen granules with SDS and the increase in the viscosity during cooling might be ascribed to the complex formation of the amylose leached with SDS followed by the gelation.
About three centuries have already passed since Antoni van Leeuwenhoek observed the gelatinization of starch granule under a microscope in 1719. Now, many people know not only the word “starch,” but also the reaction in which starch is digested by α-amylases contained in our saliva. However nobody can correctly address the question how plant starch is made. The final aims of our research were to elucidate the mechanism of plant starch biosynthesis and then to take advantage of the knowledge in our lives. For that purpose, we tried to understand the characteristics of various enzymes related to starch biosynthesis. We have isolated cDNA clones for ADPglucose pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (DBE) from kidney bean (Phaseolus vulgaris L.) plants. Northern blot analyses showed that transcripts for all AGPases and SBEs isolated in this study accumulate in both leaves and seeds, whereas the profiles of organ-specific expression for SSs and DBEs differ with each isozyme. To investigate enzymatic properties, several recombinant enzymes were purified from Escherichia coli cells. Three SS isozymes (designated rPvSSI, rPvSSIIb and rPvGBSSIa) showed distinct chain-length specificities for the extension of glucan chains. rPvGBSSIa acts on chains of approximately DP=15 of amylopectin and elongates them processively to synthesize ultimate long chains. The elongation properties of chains by PvGBSSIa isozyme must have a pivotal role in amylose biosynthesis. Two SBE isozymes (designated rPvSBE1 and rPvSBE2) were also purified from E. coli cells and their enzymatic properties were determined. Western blot analysis with antisera raised against rPvSBE1 and rPvSBE2 showed that these two SBEs were located in different amyloplast fractions of developing seeds of kidney bean: PvSBE2 was present in the soluble fraction, whereas PvSBE1 was associated with starch granule fraction. These differences in location suggest that these two SBE isozymes have different roles in amylopectin synthesis in kidney bean seeds. Moreover, we showed that a larger form of PvSBE2, LF-PvSBE2, exists which contains an extended N-terminal region. Unlike the soluble location of PvSBE2, LF-PvSBE2 is observed in both the soluble and starch-granule fractions. We also demonstrated that the extended N-terminal region in LF-PvSBE2 alters not only its subcellular location but also its kinetic properties as well. The two isoforms are encoded by the same gene, which produces two distinct transcripts generated by alternative splicing of the first two exons. Our results shed new insights on our understanding of the regulatory mechanism of amylopectin biosynthesis.
To study starch biosynthesis in terms of molecular structures of organ-specific starches within one plant species is interesting. The structural properties of starches from tuberous roots of various sweetpotato varieties and breeding lines, and also starches from leaf and callus of sweetpotato cv. Koganesengan were investigated in this study. Examination of the structure and pasting properties of the 16 kinds of root starches suggested that, statistically, the variation in pasting properties of the starches was more accurately reflected by amylopectin structures than by amylose. During the course of examination on starch structures of the latest breeding lines, we found two lines having root starches with unique characteristics. One had low amylose starch and the other had low temperature gelatinizing starch. The physicochemical properties of these starches were likewise characterized. In leaves where the starch structures and their diurnal-nocturnal changes were examined from leaves harvested at different times of a day. Gel-permeation chromatography of the leaf starches and amylopectins showed that, compared to root starch, the leaf starches had higher amount of materials eluted at the low molecular weight fraction which contained both amylose and a large amount of small amylopectin molecules. It was found that the amylose content of the leaf starches dropped during the daytime whereas the branch aspect of the leaf amylopectins remained relatively constant throughout the whole day. Finally, the structural properties of starch formed in the sweetpotato callus were studied. The starch content increased from nearly zero to the maximum content in the first week of culture, and then decreased afterward. Based on the chain-length distributions of the callus starches at different culture periods, it was demonstrated that the structural change from waxy to normal feature of starch granule occurred during the culture of the callus. Thus, the structural characteristics of starches from tuberous root, leaf and callus of sweetpotato have been revealed.
We found a novel α-amylase (AmyK38) in a culture of a novel, alkaliphilic Bacillus sp. strain KSM-K38. The enzyme was an alkaline, liquefying α-amylase, having a pH optimum of 8.0-9.5, and exhibiting strong resistance to chemical oxidants and chelating reagents. Therefore, the enzymatic properties of AmyK 38 fulfill the essential requirements for enzymes that can be used as effective additives in detergents. To further characterize and understand the unique features of AmyK38, we cloned and sequenced the gene for the enzyme. The amino acid sequence of the mature enzyme showed moderate homology with those of liquefying α-amylases from the genus Bacillus. By building a molecular model, we concluded that the high oxidative stability of AmyK38 was because the amino acid residue corresponding to Met197 in BLA is replaced by non-oxidizable Leu. We also suggested that the loss of coordination geometries of the Ca in AmyK 38 reflects its high resistance to chelating reagents. The previously reported α-amylases all contain one or more calcium per protein molecule. Surprisingly, AmyK38 was found to contain no Ca. Thus, this is the first report of calcium-free α-amylase. Additionally, AmyK38 required monovalent cations for manifestation of activity. Furthermore, we have determined the crystal structure of AmyK38, which revealed that sodium ions, instead of calcium ions, are used to retain the structure and function of this α-amylase. To make AmyK38 industrially useful, we improved the thermostability of this enzyme by protein engineering without any changes in the enzymatic properties.
Gentiooligosaccharide-containing syrup and powder (Gentose®) were industrially produced by the condensation and transglucosylation reactions of a fungal β-glucosidase from a high concentration of glucose. Gentose® is strongly characterized by its bitter taste derived from gentiooligosaccharide; consequently Gentose® #45 had both a bitter taste and the sweet taste of glucose. The other physicochemical properties of Gentose® solution such as viscosity, osmotic pressure, water activity, etc. were closely related to those of sucrose solution. Incidentally, the hydrolysis rate of β-glucobioses by rat small intestinal enzymes was much lower than that of lactose. In particular, the hydrolysis rate of gentiobiose was only 4% compared to that of lactose. These results suggested that β-glucooligosaccharides would be low-digestible sugars. Low-digestible sugars in general show the effect of the mineral absorption, and it has also been revealed that these β-glucooligosaccharides developed have a physiologically important function. Furthermore, β-glucooligosaccharides were selectively utilized in vitro, by Bifidobacterium and Lactobacillus, and the administration of β-glucooligosaccharides promoted the growth of Bifidobacteria and lowered fecal pH, in vivo. The applications of Gentose® to food processing were intensively tested. The unique bitter taste of Gentose® matched exceedingly well the bitter taste of various foods such as chocolate, coffee, beer, cocoa, tea, etc. The slight addition of Gentose® to food processing exhibited an unexpected improvement in food tastes. In the case of fruit juices, the addition of a small amount of Gentose® enhanced the original flavor. Moreover, the addition of Gentose® to vegetable drinks improved exceedingly the acridity and salient tastes of vegetables. This amazing improvement effects for food tastes pushed up the sales volume of Gentose®. The surprising usefulness of gentiooligosaccharides will expand the advanced uses of Gentose® in food processing and additives.
Mammalian milks usually contain a few percent of carbohydrate. The milk of most of the Eutheria, with a few exceptions, contains lactose (Gal(β 1-4)Glc) as the dominant saccharide; lactose accounts for more than 80% in the carbohydrate fractions. The milk or colostrum also contains other free oligosaccharides, called milk oligosaccharides, which have N-acetylglucosamine, galactose, fucose and/or sialic acid residues as well as a lactose unit in reducing end. Lactose serves as an energy source for the young, while it is thought that milk oligosaccharides are anti-infection factors against pathogenic microorganisms. Why did mammals select lactose as an energy source for their youngs? This issue should be discussed from a phylogenetic aspect as well as from nutritional and physiological aspects. The answers are as follows from the latter aspects: the half osmolarity as two equivalent monosaccharides, the growth stimulation for beneficial colonic bacteria such as Bifidobacterium and provision of galactose as a material for brain development. From the phylogenetic aspect, one should understand how lactation was acquired during the evolution from Amniotes to extant mammals. Free lactose is synthesized within lactating mammary glands from UDP-galactose (donor) and glucose (acceptor) by a transgalactosylation catalysed by lactose synthase. This enzyme is a complex of a β4 galactosyltransferase I and α-lactalbumin, one of the milk proteins. The acquisition of α-lactalbumin is a key for the presence of lactose and oligosaccharides containing the reducing lactose unit in milk. It is believed that α-lactalbumin evolved from lysozyme, which is an enzyme which cleaves the bond in peptidoglycans of bacterial cell walls. When α-lactalbumin first appeared, its concentration would have been very low. As it is assumed that the primitive glands contained most of the glycosyltransferases which are found in the mammary glands of mammals today, these enzymes would have catalyzed the synthesis of oligosaccharides from lactose. Lactose would have unable to accumulate; thus the early secretions would have continued much higher concentrations of oligosaccharides than of lactose. The milk oligosaccharides would have served to inhibit the attachment of pathogenic microorganisms in the infant colon. The molecular evolution from lysozyme, an antipathogen, to α-lactalbumin, a milk protein, should have had an advantage to provide milk oligosaccharides, the antipathogen, to the infants, and thus this gene expression survived and defeated the natural selection. The high content of lactose would be caused by the significant increase of α-lactalbumin expression within lactating mammary glands of eutherians. On the other hand, the acquisition of intestinal neutral lactase in eutherians provided an efficient mechanism for the digestion of lactose. Lactose therefore became a significant energy source for most eutherian young while milk oligosaccharides continued to serve mainly as anti-microbial agents for them.