PCR法を用いて,Clostridium thermocellum YM4株由来セロデキストリンホスホリラーゼ遺伝子のクローニングを行った.セロデキストリンホスホリラーゼ遺伝子は,984アミノ酸残基のポリペプチド鎖をコードする2952bpのオープンリーディングフレームを含んでいた.得られたアミノ酸配列は,C.thermocellum ATCC27405株由来セロデキストリンボスホリラーゼと高い相同性(92%)を示したが,c.stercorarium由来のセロデキストリンホスホリラーゼとは相同性が低かった(20%).組替セロデキストリンホスホリラーゼはセロトリオース以上のセロオリゴ糖を加リン酸分解し,セロビオース以上のセロオリゴ糖をアクセプターとして認識した.温度安定性は60℃以下であり,至適pHは7.5であった.

抄録全体を表示

　Irpex lacteus由来のエキソセルラーゼ(Ex-1),エンドセルラーゼ(En-1),β-グルコシダーゼおよびセロビオース脱水素酵素(CDH)の4種によるバクテリアセルロース(BC),アビセルおよびリン酸膨潤アビセル(HA)の分解に対する相乗効果を検討した.CDHはEx-1存在下およびEx-1とEn-1の両者の存在下においてセルロース分解を効果的に促進したが,En-1存在下のみでは相乗効果を示さなかった.セルラーゼは主生成物であるセロビオースによって生成物阻害を受けるが,これの除去反応の早いCDHはβ-グルコシダーゼよりもEx-1のセルロース分解を促進することが速度論的に明らかにされた.セルラーゼ系による相乗作用は結晶化度の違いにかかわらずその効果が観察されるが,CDH存在下ではさらにその作用が促進された.しかしながら,低結晶化度の基質を用いたときに確認されたEx-1とEn-1の相乗効果は,CDHを加えてもこの効果は観察されなかった.電子受容体が存在するとき,CDHにより多くのセロビオースが消化されるが,本菌のCDHはセルラーゼの生成物阻害の解除だけでなく,種々のセルロースの構造に対し効果的に働き,生成したグルコースを菌体に供給するものと考えられる.

抄録全体を表示

　

抄録全体を表示

　加水比の異なる米飯を冷蔵保存した試料を用いて,米飯の初期老化を評価する方法について比較検討し,次のような結果を得た.1)色彩色差計で測定したL*値で初期の老化程度を判断することは可能であったが,精確さに問題があった.2)DSC測定はX線回折に比べ,保存時間のより短い試料の老化を捉えていた.3)BAP法による糊化度では保存12時間以降に,DSC測定では9時間後に,水分量の異なる試料間の老化程度の差が現れた.4)DSC曲線から算出されたエンタルピー変化量には,米飯試料における8%の水分差の影響が現れなかった.一方,米飯試料中の4%の水分減少は吸熱ピークを有意に高温側に移動させた.以上より,冷蔵のより早期に試料の差を検出でき,直接測定が可能である簡便さから考えて,米飯の初期老化の評価にはDSC測定が適していると考察された.

抄録全体を表示

Amylases of Streptomyces griseus, Pseudomonas stutzeri and Klebsiella pneumoniae produced mainly G₃, G₄ and G₆ at the initial stage of the reaction. The amylase of Bacillus licheniformis had a dual product-specificity for the formation of G₅ and G₃. Amylases of Pseudomonas stutzeri and Bacillus licheniformis catalyzed the degradation of water-insoluble, cross-linked blue starch . All four amylases also hydrolyzed partially-oxidized potato amylose and the degree of hydrolysis increased gradually. The action patterns of four amylases were investigated by two-dimensional paper chromatography by using ¹⁴C-reducing-end-labeled maltooligosaccharides. Three amylases of S. griseus, P. stutzeri, and K pneumoniae were characterized as exo-amylases, and that of B. licheniformis was an endo-amylase. Three such exo-amylases, namely maltotriohydrolase, maltotetraohydrolase, and maltohexaohydrolase formed products having α-configuration . I propose to classify this new group of amylases as “exo-α-amylase” with high product-specificity . Maltohexa ose was also formed from maltotetraose by a transfer reaction of the exo-maltohexaohydrolase, with an action pattern dependent on the substrate concentration. In addition, continuous production of maltotetraose using a dual immobilized enzyme system of maltotetraohydrolase and pullulanase was studied. The effects of operating conditions on the maltotetraose production were examined to confirm that the maltotetraose content of the products could be analyzed using the specific space velocity, SSV. The effectiveness of using immobilized pullulanase along with the maltotetraohydro lase was confirmed from constant-conversion operations in which the maltotetraose content in the product was kept at 50% (w/w) for 60 days in laboratory and bench scale experiments. Furthermore, industrial production and utilization of brand-new starch-related functional oligosaccharides will be described in this paper.

抄録全体を表示

Using possible monodeoxy derivatives of p-nitrophenyl (pNP) α-D-glucopyranoside, -mannopyranoside, and -galactopyranoside as probe substrate, glycon specificities of α-glucosidases, -mannosidases, and -galactosidases from various sources were investigated, through hydrolysis of them. α-Glucosidases of Saccharomyces cerevisiae, Bacillus stearothermophilus, and honeybee hydrolyzed no deoxy derivatives, while the enzymes of rice, sugar beet, flint corn, and Aspergillus (A.) niger hydrolyzed the 2-deoxy derivative with substantially high activities. Moreover, flint corn and A. niger enzymes showed, although low, activities against the 3-deoxy derivative. Jack bean and almond α-mannosidases both showed sufficient activities toward 6-deoxy derivative. A. niger α-galactosidase acted on only 2-deoxy derivative with substantially high activity, while the enzymes of green coffee bean and Mortierella vinacea hydrolyzed not only the 2-deoxy derivative but also 6-deoxy one with low activities. Oligosaccharides that contain 2- or 3-deoxygenated glucose were synthesized by the transesterification reaction of A. niger α-glucosidase. α- and β-Anomers of methyl 6-ο-(p-tolylsulfonyl)-D-glucopyranoside, -mannopyranoside, and -galactopyranoside were acetylated partially by lipase-catalyzed transesterification with vinyl acetate. A lipase from Pseudomonas cepacia (lipase PS) reveaed high activity and regioselectivity for the esterification of them.α-Glycopyranosides were acetylated preferentially at the C-2 hydroxyl group, while corresponding β-anomers were acetylated preferentially at the C-3 hydroxyl group. The best selectivity was observed in the reaction of the glucopyranosides. Using methyl 3-ο -acetyl-6-ο -(p-tolylsulfonyl)-β-Dglucopyranoside, which was prepared in quantitative yield by the lipase PS-catalyzed regioselective acetylation, as a common starting material, highly deoxygenated monosaccharides, namely 2, 6-dideoxy-D-arabino-hexopyranose, 2, 4-dideoxy-D-threo-hexopyranose, and 2, 4, 6-trideoxy-D-threo-hexopyranose, respectively, were chemically synthesized in good yields.

抄録全体を表示

Isomalto-dextranase (EC 3.2.1.94) was purified from the culture of a soil bacterium, Arthrobacter globiformis T 6 by successive chromatographies on CM-cellulose and CM-sepharose to a homoge neous state as confirmed by PAGE. The molecular weight of the enzyme was estimated to be about 69 kDa by SDS-PAGE. The enzyme hydrolyzed α-1, 6-glucosidic linkages of dextran or isomalto oligosaccharides to release exolytically α-isomaltose from the non-reducing ends . The optimum pH and temperature of the enzyme were pH 5.3 and 65°C, respectively . The enzyme showed a weak isopullulanase activity, an endo-type attack on pullulan to produce isopanose. The isomalto dextranase expressed by the recombinant E. coli cells also produced isopanose from pullulan . The enzyme hydrolyzed α-1, 4-glucosidic linkage of panose as well as α-1, 6-glucosidic linkage of isomaltotriose. The kinetic features of the experiments with the mixed substrates of isomaltotriose and panose were in good agreement with those expected for a single catalytic site mechanism. The ionization constants, pKel and pKe2, of the essential ionizable groups 1 and 2 of the enzyme were 3.3 and 6.3 for dextran T2000 and 3.5 and 6.1 for isomaltotriose. The heats of ionization for groups 1 and 2 were 0 kcal/mol or less with both the substrates. These kinetic results suggested that the ionizable groups essential for the enzyme activity were carboxyl and carboxylate . Modification experiments with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), modifying carboxyl residues specifically, also indicated that the carboxyl groups were essential to the enzyme activity . The subsite affinities of the enzyme were culculated to be >7.3, <-7.2, 6.7, 0.74 and 0.18 kcal/mol for subsites 1, 2, 3, 4 and 5, respectively, from the rate parameters (Km and 10) for the hydrolysis of isomaltooligosaccharides. Subsites 1 and 3, showing large affinity values, were thought to attract the substrates and form the productive bindings. A new method for preparation of isomaltose was developed by using the enzyme and an acid-treated dextran. The branch points of dextran were selectively hydrolyzed by a mild acid pretreatment. When the acid-treated dextran was acted on by the enzyme, the maximal degree of hydrolysis went up to over 90%.

抄録全体を表示

4^G-Galactosylsucrose (β-D-fructofuranosyl 4-ο -β-D-galactopyranosyl-α-D-glucopyranoside or lactosucrose; LS) is selectively utilized by bifidobacteria in the human intestinal canal. This saccharide is found in the fermentation of yogurt containing sucrose as a sweetener. In 1957, Avigad and co-workers reported that LS was synthesized from sucrose and lactose by transfructosylation of levan sucrase. Production of LS was then proposed, using transfructosylation of levan sucrase or transgalactosylation of β-galactosidase from sucrose and lactose. However, industrial production of LS was not undertaken at that time. We have therefore attempted to establish production of LS, and to develop utilization of LS as an ingredient in health foods. Arthrobacter sp. K-1 isolated from soil produces β-fructofuranosidase. The enzyme catalyzes both transfructosylation and hydrolysis when incubated with only sucrose. However, in the presence of a suitable acceptor such as lactose, the enzyme predominantly catalyzes transfructosylation and transfers the fructosyl residue preferentially to an acceptor molecule rather than to a sucrose molecule. LS is commercially produced as follows. The mixture of sucrose and lactose (45-55: 55-45, w/w) is solubilized in water at 40% (w/w), and incubated with Arthrobacter sp. β-fructofuranosidase and invertase-deficient yeast at a temperature of 30-35°C for 24 h. The yeast is added to remove residual products of glucose derived from sucrose by assimilation, as these materials inhibit LS production. Utilizing this method, LS production is increased more than 65%. After heating is used to terminate the reaction, the reaction mixture is purified by decoloration, carbonation, filtration, desalination, ultra filtration and concentration. LS syrup containing over 55% LS can then be obtained. Three kinds of products, Nyuka-oligo LS-40 L, LS-55 L, and LS-55 P are commercially available, with LS contents of 40, 55, and 55%, respectively. The sweetness of LS, LS-40 L, LS-55 L, and LS-55 P is about 30%, 79%, 50-55% that of sucrose, respectively. These products have a high quality taste similar to sucrose. LS is not digestible in the human small intestine, but human intestinal microorganisms, particularly bifidobacteria, ferment it. The minimum effective dose of LS to improve intestinal microflora fecal conditions and defecation is 2 g/day. The saccharide is less likely to result in watery stool compared to other low-caloric sweetening agents. LS is used in soda, soft drinks, frozen yogurt, candy, biscuits, cookies, powdered soft drinks, sweet pastries (e.g., croissants), and table

抄録全体を表示

It is well known that the carbohydrate moieties of glycoproteins or glycolipids, which exist on mammalian cell surfaces, are modified by sulfate or phosphate, etc, in a few cases. These modifiers are recognised as possible receptors in intercellular communications. On the other hand, mammalian milk or colostrum contains many kinds of free oligosaccharides other than lactose in common. These oligosaccharides are also suggested to have biological roles such as inhibition of the adhesion of pathogenic microorganisms to the intestinal tract of infant or brain development - stimulation in infants. Recently, a few milk oligosaccharides have also been shown to be modified and the biological significance of these saccharides are assumed as follows: 1) Sialyllactose lactone: Some proportion of Neu5Gc(α2-3)Gal(β1-4)Glc was found to occur as free lactones between the carboxyl group of Neu5Gc and OH of Gal or Neu5Gc itself (sialyllactose lactone) in ovine colostrum. The sialyl oligosaccharides in milk or colostrum may attach to bacterial toxin and viruses, such as influenza virus in the intestine, thus protecting the infant. The lactone can be assumed to be resistant to virus sialidase. When the sialic acid is liberated from sialyl oligosaccharides by virus sialidase, they would lose their protective effect but the lactone should retain this effect because of its resistance to the action of virus sialidase. 2) 4-O-acetylated sialyllactose: Neu5Ac(α2-3)Gal(β1-4) Glc, whose Neu5Ac was O -acetylated at OH-4, was identified in the milk of echidna, one of the monotremes. Although this saccharide was assumed to be a possible inhibitor for the adhesion of viruses or bacteria whose receptors are O -acetylated Neu5Ac, the exact function of this saccharide is still unknown. 3) sulfated oligosaccharides: Gal(β1-4)Glc-6'-O-sulphate (lactose 6'-O -sulfate) and Neu5Ac(α2-3)Gal(β1-4)Glc-6'-O-sulfate (N-acetylneuraminyllactose 6'-O-sulfate) were identified in rat milk, whereas Gal(β1-4)Glc-3'-O-sulfate (lactose 3'-O-sulfate) was found in dog milk. The presence of N-acetylneuraminyllactose 6'-O -sulfate is suggested in human milk, too, and, in addition, this milk contained oligosaccharides whose GlcNAc residues were replaced by sulfate at OH-6 position. These compounds may permit the simultaneous delivery of two essential nutrients, sulfate and calcium, in early life, avoiding the precipitation of insoluble calcium sulfate in milk. 4) phosphorylated oligosaccharides: Bovine or mare colostrum contained phosphorylated oligosaccha-rides including Neu5Ac(α2-6)Gal(β1-4)GlcNAc-a1-phosphate (cow), Neu5Ac(α2-6)Gal(β1-4)GlcNAc-6-phosphate (cow) and Gal(β1-4)GlcNAc-α1-phophate (horse). These oligosaccharides may also permit the simultaneous delivery of phosphate and calcium, avoiding the precipitation of calcium phosphate. The existence of these modified oligosaccharides in milk may offer valuable in-formation for manufacturing bio-functional materials in the food industry.

抄録全体を表示