Amylase activities from various cultivars of pearl millet and a sorghum cultivar and their values in cold water and hot water extracts were studied. The α-amylase and β-amylase activities and values in hot water and cold water extracts were determined at intervals of 24 h for a period of 96 h. The correlation between amylase activities and values in hot water and cold water extracts was also determined. Activities of α-amylase and β-amylase increased progressively during germination with a concomitant increase in values in hot water and cold water extracts. The amylase activities and values in hot water and cold water extracts of the pearl millet cultivars were found to be comparable to those of sorghum. There was positive correlation (p<0.05) between amylase activity and values in hot water and cold water extracts. This study has shown that pearl millet could be used for producing various malt beverages and malt-based products.
Microbial conversion of lactose to lactobionic acid (β-O-D-galactosyl D-gluconic acid) was carried out with the resting cells of a mutant strain of Burkholderia cepacia that showed tolerance to high lactose concentrations, high oxidizing activity, and no β-galactosidase activity. To obtain the cells carrying high lactose-oxidizing activities, the strain was cultivated at 28°C for 216 h in a medium (pH 7.0) consisting of 10% (w/v) lactose, 1.5% (w/v) CaCO3 (1/2 molar equivalent of lactose), 3.0% (w/v) corn steep liquor, 0.02% (w/v) yeast extracts, and other mineral salts. The oxidizing activity of the resulting cells was most active at around pH 6 and at 55°C and the activity remained almost stable between pH 5 and 9 and below 40°C. Under the optimized conditions, the washed cells of 2.0 U/mL of the oxidase activity converted 15% (w/v) lactose and 2.2% (w/v) CaCO3 almost completely to calcium lactobionate within 15 h at 40°C. The cells were recovered from the reaction mixtures and used for the repeated bath operations five times. The initial conversion velocity depended on the reaction temperature, and the degree of conversions at 30, 35, and 40°C in the first cycle reached nearly 100% after 27, 18 and 15 h, respectively. In the fifth cycle of the reactions, however, incubation times for the complete conversion should be extended to 54, 36 and 30 h, respectively. Low content of impurities and high conversion efficiency enabled simple refining of calcium lactobionate in a high yield (about 99.3%), which was achieved by the treatment with activated carbon.
An expression plasmid containing the aglA gene encoding Aspergillus niger GN-3 α-glucosidase was constructed and inserted into Emericella nidulans JCM10259. The transformant secreted about 61 mg/L of the recombinant α-glucosidase into its culture medium. The recombinant enzyme was purified from the culture filtrate through ammonium sulfate precipitation and three chromatographic steps. It was confirmed that, like wild-type A. niger GN-3 α-glucosidase, the purified recombinant enzyme consisted of two subunits. Although the molecular mass of the recombinant enzyme was slightly smaller than that of wild-type A. niger α-glucosidase (attributed to differences in glycosylation), the pH optima and substrate specificities of the wild-type and recombinant enzymes were comparable.
A Gram-negative bacterium, strain IM944, belonging to the family Oxalobacteraceae which produced a new water-absorbing polysaccharide (WAP) was isolated from soil. The water-absorbing capacity of the WAP purified by the cetyltrimethylammonium bromide treatment in a potassium hydroxide solution was 120 times as much as its own weight without stickiness. The differential scanning calorimetry (DSC) of WAP showed the reversible heat transition at a peak temperature of 60.5°C, suggesting that this polysaccharide sample is homogeneous and forms a highly ordered structure in aqueous solutions.
Eight fractions of polished flours were prepared by gradually polishing soft-type whole-wheat grains using a rice-polisher. The gluten matrix of doughs and breads made from polished flours was broken by some pericarp layers and the appearances were not sufficient for breadmaking. Polished flours contained water-soluble pentosan (WSP) with a significantly larger amount of xylose as a main chain, while water-insoluble pentosan (WISP) had a smaller amount of xylose than those from N61 and commercial flour (Hermes). The addition of WSP obtained from polished flours of the innermost fraction 30-0% to Hermes significantly improved the dough and baking properties, as compared with that from N61. The improvement of polished flours for breadmaking was due to the characteristics of WSP, namely its high content, high ratio of xylose to arabinose, large amounts of ferulic acid and excellent foaming stability.
Brown rice is commonly considered to have an effect on various diseases including life-style related diseases. Pre-germinated brown rice is characterized by its easier cooking properties and better taste after cooking when compared with normal brown rice. Because of the rich content of gamma-aminobutyric acid (GABA) in brown rice, which can prevent the increase of blood pressure, the market for brown rice is now growing. However, the taste of the cooked pre-germinated brown rice is still unsatisfactory because of the peculiar smell. We performed a study aimed at establishing a processing method for obtaining a brown rice product with more GABA accumulation than in the commercially available brown rice products by introducing a high-pressure treatment. The result was that the content of GABA in the obtained brown rice is higher than that in the commercially available brown rice products and the functional components such as ferulic acids and oryzanol are also retained. Further, such brown rice with increased GABA accumulation was found to be digested more quickly than the commercially available brown rice products when those cooked rice products were evaluated by the artificial digestion method. The GABA-increased brown rice was also found to compare favorably with commercially available normal brown rice in terms of taste after cooking.
Cyclodextrin-hydrolyzing enzymes (CDases) such as cyclomaltodextrinase (CDase), neopullulanase (NPase), Thermoactinomyces vulgaris amylase II (TVA II), and maltogenic amylase (MAase) are multisubstrate enzymes, belonging to a subfamily of the Glycoside Hydrolase family 13, and act on cyclodextrins, various maltooligosaccharides, pullulan and starch. In terms of quaternary structure, many CDases are unique since they act not only as monomers, but also as oligomers by forming dimers, tetramers or even higher oligomers. The N-terminal domain of approximately 130 residues absent in ordinary α-amylases contributes to the formation of the oligomeric state in this group of enzymes. Dimerization and oligomerization can provide enzymes with a number of functional advantages such as high stability and efficacy in accessibility and specificity of active sites. CDase from Thermus sp. exists as a 3D domain-swapped dimer which exhibits different binding preferences for various substrates due to increased specificity via dimerization. Three-dimensional domain swapping is a basic unit of the oligomer. CDase from alkalophilic Bacillus sp. I-5 exists as a dodecamer by forming an assembly of six 3D domain-swapped dimeric subunits. Oligomerization of the CDase also affects the catalytic activity of transglycosylation, thereby preferentially forming an α-1,6-glycosidic linkage in the transfer product. We demonstrated that Glu 332 at the interdomain interface played an important role in the binding of the acceptor molecules. The association/dissociation process of CDase examined in various oligomeric states is of great interest to identify the mechanism and forces that contribute to the supramolecular assembly and function of the enzyme. In this paper we discuss the physiochemical properties of CDase in light of the consequences of oligomerization: 1) three-dimensional structure, 2) multisubstrate specificity/catalytic efficiency, 3) transglycosylation activity at the interface of the dimer, 4) dissociation/association of supramolecular assembly and 5) a possible physiological role in microorganisms.
The term “extremophiles” is used for organisms that thrive under extreme conditions, and are designated the enzymes that are active and tolerant under extreme reaction conditions “extremozymes”. We have long been engaged in the screening, gene cloning, and industrial applications of alkaline enzymes from alkaliphilic bacilli. Alkaliphilic bacilli have made a great impact on the detergent industry, because they often produce alkaline enzymes that improve the detergency of detergents. We isolated a number of alkaline enzymes from alkaliphiles, such as cellulase, α-amylase, protease, mannanase, and pectate lyase. We incorporated an alkaline cellulase into super-compact detergents for the first time in the world. Some of the alkaline enzymes were crystallized, and their tertiary structures were determined. The alkaline adaptation mechanism of these enzymes was analyzed by determination of the amino acid substitutions that and deletions occur during the alkaline adaptation process. The alkaline adaptation appeared to be a remodeling of ion pairs so that the charge balance is kept in the high alkaline pH range.
In germinating plant seeds, α-amylases degrade starch accumulated in seeds, and that requires two functions: catalysis itself and starch granule binding ability. All plant α-amylases belong to the α-amylase family and share the same catalytic machinery as other members, but are different in extended subsite structure accommodating the non-reducing end side of substrate even with high affinity, particularly in subsite -6, shown in α-amylases of kidney bean as well as barley. Barley α-amylase isozyme 1 (AMY1) mutants introduced site-directed mutagenesis along the predicted substrate binding site and the recent crystal structure solved in complex with a substrate occupying subsite -1 to -7 revealed that amino acid residues situated in a shallow cleft extending between domain A and B were involved in the subsite formation. Although plant α-amylases possess no additional starch-binding domain as seen in several α-amylases from microorganisms, plant α-amylases examined acted on starch granules. The residue corresponding to “sugar tongs” Tyr380AMY1 was proven to be involved in starch granule binding in adzuki bean α-amylase.
Trehalose (α-Glcp-(1↔1)-α-Glcp) is widely distributed in nature such as microorganisms, insects, plants, and invertebrates. This sugar exists not only as an energy source but also as an important functionality-material that protects the organization from damage by various stresses such as drying, freezing, and osmotic pressure. Therefore, organisms have various trehalose-related enzymes that participate in degradation or synthesis of trehalose to adjust the concentration in response to the environment. In this study, we obtained trehalase, trehalose synthase or trehalose phosphorylase producing bacterium from soil or an already identified bacterium. The trehalose-related enzymes are classified from the catalyst style into three groups named the degradation, the intramolecular transglucosylation, and the intermolecular transglucosylation. Three enzymes we screened were different from other kinds of trehalose-related enzymes. In addition, we clarified some properties of these enzymes, and examined the synthesis of useful oligosaccharides. Trehalase, which hydrolyzes trehalose to glucose, was purified from the Bacillus sp. T3 cultures. Trehalose synthase, which catalyzes the interconversion of maltose and trehalose by intramolecular transglucosylation, was purified from cell-free extracts of Pimelobacter sp. R48 and the thermophilic bacterium Thermus aquaticus ATCC33923. Trehalose phosphorylase, which catalyzes the reversible phosphorolytic cleavage of trehalose, was purified from a cell-free extract of thermopholic anaerobe, Thermoanaerobacter brockii ATCC 35047. Trehalose synthase was useful for not only the synthesis of trehalose but also the production of trehalulose (1-O-α-D-glucopyranosyl-D-fructose) from sucrose. Moreover, a non-reducing disaccharide, α-galactosyl α-glucoside, was synthesized for the first time by trehalose phosphorylase using galactose as an acceptor.
In 1988 we recognized glycosylated components in pyrodextrin and started a study to obtain an amylase-resistant ingredient. We succeeded in establishing a series of processes for industrial-scale separation of the indigestible component with superior appearance and taste by roasting starch (pyrolysis), enzymatic hydrolysis, purification, chromatographic fractionation and spray drying. The component was named indigestible dextrin (ID). In order to utilize ID as a source of dietary fiber, a low-calorie ingredient, and a physiologically active ingredient, we first confirmed that ID is a highly safe ingredient by conducting an acute toxicity study, a mutagenicity study, long-term administration studies in both rats and humans, and a study of diarrhea caused by long-term consumption. A novel determination method using the enzyme-gravimetric method in combination with high performance liquid chromatography (enzyme-HPLC method) was proposed to the Association of Official Analytical Chemists (AOAC) and approved as Final Action Method AOAC 2001. 03 in January 2005. Animal and human studies showed the energy value of 1 kcal/g dietary fiber fraction. Based on these results, ID has been approved in many countries. Moreover, it has been confirmed by both animal and clinical studies that ID has physiological functions such as intestinal regularity, moderating postprandial blood glucose level, lowering serum lipid, and reducing body fat. As for the physicochemical properties of ID, it is similar to DE10 maltodextrin in sweetness and browning property. The properties of not being fermented easily by yeast or lactobacillus impart an interesting characteristic to beer and lactic acid drinks. At present ID is commercially available for use in a wide range of food products not only in Japan but also in many countries around the world.