Thermostability and chelator resistance of the liquefying alkaline α-amylase (AmyK) from alkaliphilic Bacillus sp. strain KSM-1378 were examined by deletion of either Argl81-Glyl82 or Thr 183-G1y184 on a loop in domain B. In the tertiary structure of Bacillus stearothermophilus α-amylase (BSA), Ilel81-Glyl82 (Thr183-G1y184 in AmyK) pushes away a spatially contacting region containing Ca2+-coordinating Asp207 (Asp209 in AmyK). Therefore, the deletion of Ilel81- G1y182 rather than Argl79-G1yl80 was predicted to result in a higher thermostability of BSA. However, our results with AmyK were clearly contrary to this prediction. The resistance to EDTA of both mutant enzymes from AmyK was essentially equal, and the Argl81-Glyl82-deleted mutant was more thermostable than the Thr183-G1y184-deleted one. It strongly implies that the microenvironmental topology around the loop containing these dipeptides in AmyK is different from that in BSA.
The substrate specificity and the active site of β-D-glucosidase F-1 (Mr, 50, 000; optimum pH, 5.5) purified from a Streptomyces sp. were kinetically investigated. The β-D-glucosidase showed a broad substrate specificity for synthetic glycosides and disaccharides having β-glycosidic linkage, but the former was more favorable substrate than the latter. The enzyme was characterized by the ability to hydrolyze rapidly not only ρ-nitrophenyl β-glucoside (Km, 0.72 mM; k0, 63 s-1) but also ρ-nitrophenyl β-fucoside (Km, 0.19 mM; k0, 44 s-1) and laminaribiose (Km, 1.6 mM; k0, 70 s-1). The kinetic study was made as to whether the hydrolyses of these synthetic glycosides were catalyzed at a single active site or at dual active sites in the β-D-glucosidase. In the experiments with the mixed substrates of p-nitrophenyl β-glucoside (PNPG) and ρ-nitrophenyl β-fucoside (PNPF) or ρ-nitrophenyl β-galactoside (PNPGal), the kinetic features, the linearity of Lineweaver-Burk plots and the dependence of the apparent maximal veloities and Km value on the mole fraction (f) of PNPG in the mixed substrate, f = [PNPG/([PNPG] + [PNPF or PNPGal]) agreed very closely with those theoretically predicted for a single catalytic site mechanism. The findings strongly support the notion that the β-D-glucosidase attacks the synthetic glycosides at a common active site.
Waxy corn starch (WCS) was treated with the Neurospora crassa glycogen branching enzyme (BE), and the BE-treated WCS was characterized. The chain-length distribution pattern of the BEtreated WCS was similar to that of glycogen by HPAEC analysis, and the average chain-length changed from 21.2 to 15.2. The molecular mass of the BE-treated WCS was estimated at 2000 kDa using a calibration curve of pullulan by gel filtration chromatography. The apparent viscosity of a 3% paste of the BE-treated WCS was about 1/10 that of the same paste concentration containing the untreated WCS. The BE-treated WCS was soluble in water. The solution remained clear and no precipitate was observed after being stored at 4°C for 1 week.
A novel α-amylase (AmyK38) from an alkaliphilic Bacillus designated KSM-K38 is strongly resistant to chelators and oxidative reagents and contains no calcium. However, thermostabilization of AmyK38 is essential if it is to have industrial applications. Several chimeric enzymes between AmyK38 and the thermostable Arg181-G1y182-deleted mutant (dRG) of an α-amylase AmyK were constructed. A chimeric enzyme containing the N-terminal 21 amino acid residues of dRG was found to have higher thermostability than the parental AmyK38. By site-directed mutagenesis, AmyK38 was successfully thermostabilized by the single substitution of Tyrl1 by Phe without any changes in the kinetic features.
A gene encoding an extracellular β-fructofuranosidase (β-FFase), designated bff A, was clonedfrom the chromosomal DNA of Arthrobacter sp. K-1, and expressed in Escherichia coli. The putativebff A consisted of 1734 by encoding β-FFase of 578 amino acids. Comparison of the DNA sequencedata with the N-terminal amino acid sequence of the purified enzyme indicated the presenceof a precursor protein with a signal sequence of 36 residues. The deduced amino acid sequence ofthe β-FFase demonstrated a high degree of homology with that of levansucrase from Acetobacterdiazotrophicus. The N-terminal amino acid sequence and some enzymatic properties of the recombinantenzyme were identical to those of the native enzyme.
Cooking and processing properties of four kinds of newly developed waxy rice, Oumochi 349, Odorokimochi, Kantomochi 171 and Komochi 360 were studied. Samples were tested for their physical properties and eating qualities as flour, rice cakes and steamed rice. To investigate the influenceof one-year storage on cooking properties, waxy rice samples harvested in 1994 and 1995 were compared. The pasting properties of flour were determined with a Rapid Visco Analyser (RVA) and the textural properties of gel with a Tensipresser. Rice cakes were prepared in a rice cooker, and the textural change with storage time was studied. Sensory evaluation of rice cakes and steamed rice was done by a scoring method. RVA curves differed among the samples. Pasting viscosity of Odorokimochi was as high as that of Himenomochi, and that of Mochiminori was as low as that of Hiyokumochi. Firmness and stickiness of the gel showed a positive relationship with viscosity. As a rice cake, Odorokimochi was highest in firmness, followed by Himenomochi, and the firmness increased drastically with time. The rice sample harvested in 1994 was higher in pasting viscosity as flour and firmness as rice cake than that form 1995. Hence, there was a one-year storage influence on the cooking properties of waxy rice. Sensory evaluation of rice cake and steamed rice showed the same tendency. Hiyokumochi was preferred in all profiles and the sensory score for Odorokimochi was between those for Hiyokumochi and Oumochi 349.
The retrogradation of starch gels from 5 kinds of sweet potato cultivars (Shiroyutaka, Satumastarch, Joywhite, Kyushu No. 123 and Kyushu No. 126) were compared, and the relationship between properties of starch and molecular structure of amylopectin was also investigated. Considerable differences in the retrogradation of sweet potato starch gels were found in each varietiy . In Joywhite, Kyushu No. 123 and Satumastarch, retrogradation for starch gels was slow. The varieties of starches with slow rate of retrogradation showed common characteristics: the pasting temperature is low, and both the phosphorus content and amylose content are low. In addition, the degree of gelatinization determined by the beta-amylase-pullulanase method was correlated with the tendency of retrogradation of sweet potato starch gels. The chain length distribution of isoamylasedebranched amylopectin by gel permeation chromatography (GPC) showed that the content of long chains was low in varieties with slow rate of retrogradation. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of isoamylase-debranched amylopectin showed that the content of short chains with DP 6-10 was high and the content of chains with DP 11-17 was low in varieties with a slow rate of retrogradation. The GPC analysis of the phosphorylated chains revealed that the phosphorus of sweet potato starch was attached to the long chains of amylopectin. Thus, these results suggest that the molecular structure of amylopectin is related to the phosphorus content, which also influences the retrogradation properties.
The common method for treating industrial wastewater is the activated-sludge process in which specific carriers of bacteria capable of forming active sludge are put in contact with treated water . As a raw material for the carriers, cellulose sponges (native cellulose) are advantageous after use, but one disadvantage is their weakness in physical contact with wastewater as it is being treated. Because of this disadvantage, epoxydated-resin coated carriers (coated cellulose) with hardened surface are used in the treatment plants. It is often observed that the carriers are shortly dissolved, leaving fragments of resins possibly resulting from cellulase. In order to overcome this disadvantage, the cellulose is pretreated with ethyleneglycol-diglycidylether, then crosslinked with polyethyleneimine to ether on the surface of cellulose-sponges (crosslinked cellulose). It has been found that the crosslinked cellulose has an especially superior ability to resist cellulase action, while its adhesion capacity for bacteria is equivalent or greater, compared with that of coated cellulose.
Dynamic viscoelastic measurements and functional tests were carried out for diets of paients with dysphagia. Both storage and loss shear moduli G' and G" for diets of paients with dysphagia were strongly frequency dependent. Frequency was changed from 0.01 to 2 Hz. It is believed at present that a dominant factor governing the mechanics of gelation of polysaccharide gels is the balance between the hydrophilic and hydrophobic properties of the molecule of polysaccharides, or the bal ance between the affinity to water and the tendency toward formation of microcystalline structure in polysaccharide molecules.
Proteoglycans (PGs) are complex glycoconjugates that are composed of a core protein and glycosaminoglycan (GAG) chains. The GAG chains are covalently bound to the serine residue of the core protein via a common core tetrasaccharide (glucuronic acid-galactose-galactose-xylose) as the linkage region. The endo-type glycosidases were investigated with the aim of performing enzymatic synthesis of PG. It is known that many glycosidases catalyze a transglycosylation reaction as a re-verse reaction in addition to their main hydrolysis reaction. Therefore, the transglycosylation mechanism of testicular hyaluronidase, which is an endo-β-N-acetylhexosaminidase, was investi-gated. It was found that disaccharides are successively released from the nonreducing terminal of a donor hyaluronic acid (HA) and rapidly transferred to the nonreducing terminal of an acceptor HA. It was also found that testicular hyaluronidase also acted on chondroitin (Ch), chondroitin 4-sulfate (Ch4S), chondroitin 6-sulfate (Ch6S), and other GAGs as well as HA. Therefore, by repeating the transglycosylation using suitable combinations of Ch, Ch4S, Ch6S, and other GAGs as acceptors and donors, it was possible to custom synthesize GAGs. It is likely that application of this system would facilitate artificial reconstruction of GAG moieties of PG. Subsequently, we found that an endo-β-xylosidase activity was present in rabbit liver. This enzyme specifically hydrolyzed the xylose-serine linkage between the core protein and GAG chains of PG, thereby allowing intact GAGs to be obtained. At present, we are studying the enzymatic transfer of the reconstructed GAG chains to the core protein using the transglycosylation activity of this enzyme.
The prevalence of lifestyle-related disease has been continuously on the increase with the western ization of diet in Japan. Recently, attention has been focused on the development of “Nutraceuti cals, ” which are beneficial foods for human health. The development and the effect of some nu traceuticals, based on clinical evidence, are reviewed in this report. 1) Dietary Fiber Drink: This beverage contains 7 g of water-soluble dietary fiber (Polydextrose) which improves the bowel movement of females with constipation. 2) Oligosaccharide Drink: This beverage contains 5 g of indigestible oligosacchareide (Lactosucrose) which increases the amount of intestinal bifidobacteria and leads to the improvement of the intestinal environment. 3) New Sports Drink: This beverage contains fructose an as energy source and antioxidant nutrient. We demonstrated that fructose did not suppress the oxidation of fat during exercise and that ingestion of β-carotene before/during ex ercise decreased the urinary 8-OHdG excretion which is a marker of oxidative DNA-base damage. 4) Protein Supplement: We found that it is more effective to administer amino acids immediately after exercise than later for protein synthesis in muscle tissue. We propose the effective timing of protein intake to develop the skeletal muscle tissue of athletes.
At first, the author would like to state a philosophy for the highly utilization of carbohydrates. Carbohydrates synthesized from the combination of carbon dioxide and water by photosynthesis, are converted to aminosugar by amination, consecutively to a special kind of δ-amino acids having several hydroxyl groups, presumably D type from D-aminosugar, L type from L-aminosugar by the oxidation on C l position, α-amino acid by the oxidation on C6 position. The composition for living activity are consecutively formed to act in the living body essentially such as carbohydrate, aminosugar, nucleic acid, the polymer such as DNA, protein, polysaccharides and so on. Various and important thesis on the biofunctional activity and food related properties of carbohydrates and carbohydrate complex are existed. As for complex carbohydrates (glycosides), many examples were reported. Taste properties such as sweetness were of course able to be changed by the change of the structure of aglycon itself. Sweetness and biofunctional activity were considerably changed by the change of carbohydrate portion of the carbohydrate complexes, and even the change of linkage type of the carbohydrate portion were drastically resulted in the change of the properties. In case of some glycosides, only aglycon shows sweetness. There are numerous number of organic compounds which are formed to carbohydrate complexes. It is interested to know how and how much the change of the properties of the complexes which are formed by the addition of carbohydrate are. The higher-order structure of these complexes may give some important activity on lives, the addition of time factor to the complexes, some unknown property might be emerged.
Trehalose is a non-reducing disaccharide consisting of two glucose residues. Industrial mass-production of trehalose from starch has been achieved by using enzymatic reactions of maltooligo-syltrehalose synthase and maltooligosyltrehalose trehalohydrolase. This saccharide is available for various foods and has many useful functions; sweetening, low-damage to nutrients, and prevention of degeneration of starch and proteins. In this review, we present several functions of trehalose revealed by recent studies. Degradation of fatty acids and the formation of volatile aldehydes from fatty acids are remarkably suppressed by trehalose. This saccharide is preventive of the body odor (volatile aldehydes) of aged persons. In addition, trehalose has inhibitive effects on DNA-strand scission and protein modification by lipid oxidative products. Trehalose is effective in stabilizing vitamin C and SOD-like activity. Trehalose has shown ability to interact with minerals, including calcium chloride. Because of the interaction between trehalose and minerals, trehalose can be applied to improve of the property of mineral-containing products. From these results, it has been seen that trehalose has the benefits of lipid, minerals and vitamins other than starch and protein, including its function as a malti-functional saccharide. Trehalose has wide applications in foods, cosmetics and phamaceuticals.