未熟モウソウチク(タケノコ)からレクチンをSephadexG-75を用いたアフィニティークロマトグラフィーによって単離,精製した.このレクチンはゲル濾過によって分子量48,000と推定され,数個の同一サブユニットから成っていた.赤血球凝集阻害反応によって,マルトースやα一メチルマンノシドに結合特異性を示すことが判明した.N末端配列はLys-Ser-Cys-Cys-Pro-Ser-Thr-Thr-Alaであり,これはヤドリギから得られているligatoxinAと同一であった

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　担子菌ベッコウタケ(Fomitopsis cytisina)の培養濾液から3種類のポリガラクツロナーゼをSDS.PAGE的に均一にまで精製した.これらは反応生成物の分析から,2種類がエンド型(EndoPG I,II),1種類がエキソ型(ExoPG)と決定された.EndoPG I,IIの分子量はSDS-PAGEでともに38kDa,ExoPGは50-60kDaと推定された.これらはいずれも酵素的脱糖鎖によりそれぞれの分子量は37kDaと44kDaに減少した.また,EndoPG I,IIおよびExoPGの等電点はそれぞれ4.3,4.2,4.0であった.いずれの酵素もアミノ酸組成はよく似ていた.また,EndoPG I,IIのN末端20アミノ酸配列には相同性がみられた.これらの至適pHはそれぞれ55,5.0,5.2であり,EndoPG I,IIのpH安定性は4.0から6.0,熱安定性は45℃までであり,ExoPGのpH安定性は3.5から65,熱安定性は45℃までであった.EndoPGIはヘプタ以下のオリゴガラクツロン酸には作用しづらくトリガラクツロン酸にはほとんど作用できなかった.一方,EndoPG IIはIよりオリゴガラクツロン酸によく作用した.本菌のEndoPGはこれまで報告されてきた真菌のものと似ていたが,同じAphyllophorales族のStereum purpureum endoPG IとはKm値,Vmax値,熱安定性が,かなり異なっていた.ExoPGは9GalUA付近の基質に対し最大活性を示し,平均重合度14から42までの基質には最大時の88%程度の活性で一定であった.また,ExoPGは植物起源で報告されていたタイプと同様,不飽和オリゴガラクツロン酸にはまったく作用できなかった.

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‘白くわい’,‘青くわい’および‘吹田くわい’の3品種と‘白くわい’ב青くわい’,‘ 白くわい’ב吹田くわい’,‘青くわい’ב白くわい’,‘青くわい’ב吹田くわい’,‘青くわい’ב青くわい’,‘吹田くわい’ב吹田くわい’の組合せで交配して得られた6系統の球茎から澱粉を調製し,その性質を検討した.　電流滴定による見かけのアミロース含量は28.2-29.9%,イソアミラーゼで枝切りした澱粉のゲル濾過分析よりアミロースに相当するFr.1は26.6-31.3%であった.HPAEC-PDA法によって,くわい澱粉・のアミロペクチンはモチトウモロコシ澱粉のアミロペクチンと比較して,重合度6および7の鎖長画分が増加し,重合度9の鎖長画分が減少するという特徴ある短鎖の鎖長分布を示した.X線回折図はいずれもA図形であった.‘ 白くわい’,‘ 青くわい’,‘ 吹田くわい’の3品種間では,澱粉の平均粒径およびDSCによる糊化温度は‘白くわい’>‘青くわい’>‘吹田くわい’,酵素分解性,70℃ における溶解度ならびに膨潤度は‘白くわい’<‘青くわい’<‘吹田くわい’の順であった.酵素による澱粉粒の分解性と60℃,70℃ における溶解度,膨潤度との間に正の相関が,酵素による澱粉粒の分解性とDSCによる糊化温度(ToおよびTp)との間に負の相関がみられた.

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　アミロペクチンの構造情報を直接取得する簡便な分析方法を確立する目的で,イソアミラーゼによるウルチトウモロコシ澱粉から切り出される短鎖アミロース鎖を調べた.アミロースとアミロペクチンを分離せずに得られる部分加水分解物を,Toyopearl HW-50Sのゲルクロマトグラフィーにより3区分(fr.1,fr.2,fr.3)に分けた.短い鎖長を含むfr.3の鎖長分布をHPAEC-PADで分析した.fr.3に含まれる短鎖アミロースはアミロペクチンのほかに分岐アミロース由来のものも含まれるが,後者由来のものはわずかであると推定した.このアミロペクチンの一般的な構造情報を直接得る分析法は,日本で栽培された代表的な米澱粉で応用性を調べた.

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　L-Arabitolの生理効果をしらべるために,まず, Sprague-Dawley系雄ラット15匹を3群(1%L-arabitol, L-arabitol,5%L-arabitol)に分けて4週間飼育し,高用量では下痢になることを観察した.その結果に基づいて,本実験は2.5%のL-arabitolを含む飼料で4週間飼育し,対照群と比較検討した.対照群の飼料はレarabitolの代りにcelluloseを用いた.両群ラットの諸臓器重量を定したところ,L-arabitol群の脂肪組織が有意に小さく,一方,腎臓ならびに盲腸が有意に大きかった.また,盲腸と結腸の内容物も対照群に比べて意に多かった.これらの所見はL-arabitolが水溶性食繊維様の生理効果を有することを示唆している.

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　トレハロースをグルコシル供与体,ガラクトースを受容体として,好熱嫌気性菌Thermoanaerobacter brockii由来トレハロースホスホリラーゼを用いて非還元性二糖を酵素的に合成した.本糖質をパン酵母処理によるグルコースの除去,トレハラーゼ消化,アルカリ処理,分取HPLCを用いて効率良く反応液から単離した.酸分解,メチル化分析,¹³C-NMRなどにより本二糖の構造をα-D-ガラクトピラノシルα-D-グルコピラノシドと決定した.本糖質はα-ガラクトシダーゼによりごくわずか加水分解されたが,α-またはβ-グルコシダーゼ,β-ガラクトシダーゼ,トレハラーゼでは分解をうけなかった.

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　1,5-アンヒドロ-D-フルクトース(1,5-AF)の紫外部領域の吸収スペクトルは215nmに最大吸収を示し,266nmにも小さなピークが認められた.215nmにおけるピークの分子吸光係数(34M^-1・cm^-1)は小さく,定量に用いるには適当でなかった.1,5-AFのフェノール一硫酸法での発色はグルコースの15%で,アンスロンー硫酸法ではまったく発色しなかったため,1,5-AFの定量にこれらの全糖量測定法は適さないことがわかった.一方,1,5-AFは比較的低温の35℃でもCu²⁺(ソモジイネルソン法,ネオクプロイン酸法)とFe³⁺(パークジョンソン法)を還元した.ソモジイネルソン法とネオクプロイン酸法では他のヘキソースやペントース存在下でも1,5-AFを特異的に定量できた.ネオクプロイン酸法はより高感度(0-50μg/mL)であり,マイクロタイタープレートを用いた小スケール(20μLの検液)の測定にも適用できた.

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Dextrin dextranase (DDase, EC 2.4.1.2) was extracellularly secreted in a culture medium of Acetobacter capsulatum ATCC 11894 containing both glucose and a small amount of dextrin as the essential carbon sources. The enzyme was simply purified by a high-speed refrigerated centrifugation, and immediately dialyzed against 50 mM acetate buffer (pH 4.5). The purified DDase gave a single protein band on both Native- and SDS-PAGE. The molecular mass of the purified enzyme was estimated to be about 152 kDa (SDS-PAGE). The optimum pH and temperature of the enzyme were 5.2 and 38°C, respectively. The K_m (mM) and V_max (mg dextran/mg protein/min) values for mal-tooligosaccharides (DP 3-7) and short-chain amylose (DP 17.3) were estimated to be about 10.2, 1.74; 6.41, 2.56; 3.34, 2.64; 2.59, 2.39; 1.66, 2.17; 0.12, 2.23, respectively. The conversion rate of maltodextrins into dextran increased with the increases in the DP number of donor sub-strates. The maximum yield of product dextran reached 73.9% by using short-chain amylose as a substrate. DDase showed a strong affinity on the sugars having non-reducing terminal linked with either α-1, 4- to α-1, 6-glucosidic bond. The affinity of enzyme on acceptor substrates increased with the increases in the DP number of sugars tested. Various oligosaccharides were formed, when DDase reacted on a maltose-dextran mixture. The ratio of α-1, 4- to α-1, 6-glucosidic linkages in a product dextran molecule was calculated to be about 1: 20. The average molecular mass of product dextran was estimated to be about 1270 kDa. The chemical structures of synthesized glucooligo-saccharides (DP =3-7) from a maltose-dextran mixture were 4-ο-α-isomaltosyl-D-glucose, 4-ο-α-isomaltotriosyl-D-glucose, 4-ο-α-isomaltotetraosyl-D-glucose, 4-ο-α-isomaltopentaosyl-D-glucose and 4-ο-α-isomaltohexaosyl-D-glucose. On the basis of many experimental data, we propose the possible transglucosylation actions of DDase which consist of five reaction routes.

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Malto-oligosyltrehalose synthase (EC 5.4.99.15, MTSase) is a glycosyltransferase catalyzing the conversion of maltodextrins to malto-oligosyltrehaloses by forming α, α-1, l-glucosidic linkage. The enzyme slightly catalyzes the release of glucose from maltodextrins by hydrolysis of the α-1, 4 linkage of maltodextrins on the reducing end. The amino acid sequence of MTSase from Sulfolobus acidocaldarius ATCC 33909 showed significant homologies to those of not only other bacterial MTSases but also α-amylase family enzymes. The S. acidocaldarius MTSase lost the enzyme ac tivity by site-directed mutation of Asp228, G1u255, or Asp443 of the enzyme, which corresponded to the catalytic residues of α-amylase family enzymes. The mutation of Lys390 or Lys445 brought about a decrease of the transfer activity and an increase of the hydrolysis activity of the enzyme. The two lysine residues are conserved among MTSases but not α-amylase family enzymes. The tertialy structure analysis revealed that MTSase as well as α-amylase family enzymes contained a (β/α)₈ barrel structure as the catalytic domain. The three carboxyl residues (Asp228, G1u255 and Asp443) proposed as the catalytic center of MTSase were found to be located in the active cleft and positioned similarly to those of a -amylase family enzymes. There were two loops and one he lix structures on one end of the active cleft, where a nearly closed space was formed. The lysine residues (Lys390 and Lys445) were found to be situated in the closed structure. It was suggested that several residues containing the two lysines in the closed structure were involved in catalysis of the α, α-1, 1 transfer activity of MTSase.

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Themoactinomyces vulgaris R-47 produces two a-amylases, TVA I and TVA II, both of which hydrolyze pullulan and cyclodextrins. Site-directed mutagenic and X-ray crystallographic studies ofboth enzymes were carried out. The putative catalytic residues, Asp325, G1u354, and Asp421 of TVA II were modified. The mutated enzymes retained a trace of activity, less than 0.01% of the wild-type enzyme, and the action pattern of D421N enzyme was different from wild-type and othermutated TVA II. The crystal structures of the mutated TVA II complexed with β-cyclodextrin were determined. TVA II has domains A, B and C, like other a-amylases, and also has domain N, which is not commonly found in α-amylase family enzymes. A β-cyclodextrin was bound to domain A, and was also located close to domain B. Crystals of TVA I were obtained by the vapor diffusion method in the presence of polyethylene glycol. Like TVA II, TVA Iwas composed of domain N, A, B, and C. Comparing the domains of TVA I and TVA II, the structure and position of domain N were the most different. To investigate the roles of domain N further, domains N of TVA I and TVA II were truncated, and the activities of these enzymes were drastically decreased.

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Thermococcus litoralis 4-α-glucanotransferase (GTase) belongs to family 57 of glycosyl hydro-lases and catalyzes the disproportionation and cycloamylose synthesis reactions. Using 3-ketobutylidene-β-2-chloro-4-nitrophenyl-maltopentaoside (3 KBG 5 CNP) as a donor and glucose as an acceptor, we showed that the disproportionation reaction of 4-α-glucanotransferase involves a Ping-Pong Bi Bi mechanism. Based on this reaction mechanism, we trapped the glycosyl-enzyme intermediate by treating the enzyme with 3 KBG 5 CNP in the absence of an acceptor. MALDITOFMS and PSD analysis of a peptic digest of the intermediate and great decrease in activity of the E 123 Q mutant enzyme indicated that completely conserved Glu 123 was the catalytic amino acid. Next, we have determined the structure of GTase, the first structure of the family 57 of glycosyl hydrolases, at 2.8 Å resolution. The structure of GTase is composed of three domains. N-terminal domain (domain A) contains two catalytic residues and has a novel pseudo-TIM barrel fold. C-terminal domain (domain C) is mainly made of β-strands arranged in two layered β-sheet sandwich. Domain B is located between domains A and C, and consists of α- and 3₁₀-helices. Active site cleft lies between domains A and B. The cleft is partially covered with aromatic residues to form a tunnel-like shape, which probably plays an important role in the formation of large cyclic glucans.

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The action of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) on amylose was reinvesti-gated by determining the product specificity of the cyclization reaction and the substrate specificity of the coupling and hydrolytic reactions of CGTases from alkalophilic Bacillus sp. A2-5a (A2-5a CGTase), Bacillus macerans CGTase (Bmac CGTase) and Bacillus stearothermophilus CGTase (Bste CGTase). The product specificity of the cyclization reaction of CGTase was determined by quantifying each cyclic α-1, 4-glucans with degree of polymerization (DP) from 6 to 31 (CD6 to CD31; CDn, cyclic α-1, 4-glucan with DP of n) produced at the initial reaction stage with HPAEC. All three CGTases produced CD6 to CD31 simultaneously. However, A2-5a CGTase produced more CD7, while Bmac CGTase produced more CD6 than other cyclic α-1, 4-glucans. The speci-ficity to produce CD7 or CD6 was very strict since the cyclic α-1, 4-glucan having a difference of only one glucose unit were not preferably produced by either enzyme. 4n the other hand, Bste CGTase produced more CD6 and CD7 than other cyclic α-1, 4-glucans, respectively. The substrate specificity of the coupling and hydrolytic reactions were determined by using CD6, CD7, CD8 and larger cyclic α-1, 4-glucans (mixture of CD22 to CD50). For the coupling and hydrolytic activities of all three CGTases, CD7 and CD8 were extremely poor substrate, while those activities on larger cyclic α-1, 4-glucans were higher than CD6, CD7 and CD8. The substrate specificity in the cou-pling and hydrolytic reaction could be attributed to the conversion of larger cyclic α-1, 4-glucans into smaller ones. The hydrolytic activity of CGTases on larger cyclic α-1, 4-glucans were 1/1.6 to 1/5 of the coupling activity, which indicated that the hydrolytic activity of CGTase was not so small as negligible. The hydrolytic activities of Bste CGTase were the highest among the three CGTases, while those of Bmac CGTase were the lowest, which would cause the difference in the conversion rate from larger cyclic a-1, 4-glucans into smaller ones. Based on these results, we discussed the action of CGTase on amylose.

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In Kagoshima, citric acid and shochu are produced on a commercial basis by the koji of Aspergillus groups. The black Aspergillus groups are known to produce two types of a-amylase, the acid-stable and the neutral, besides glucoamylase. In this study, we purified the acid-stable enzyme (UAA) from the acidic extract of the citric acid koji, and studied some enzymatic properties, especially the action pattern towards maltooligosaccharides, by comparison with the neutral enzyme (UNA) from the koji. Product analysis showed that UAA demonstrated a distinct cleavage pattern towards oligosaccharides more than G4, which suggests that UAA has a different subsite structure from the ordinary neutral a-amylase such as Taka-amylase A. The number of subsite is estimated to be 5, and subsite affinities of Al, A2, and A5 were evaluated to be 1.2 kcal/mol, 1.4 kcal/mol and 2.1 kcal/mol. Experiments with chemical modification suggested that UAA has a residue of Leu instead of Lys209 on Taka-amylase A, and a residue of Glu instead of His2l0. A new substrate Gal-G3-CNP was enzymatically synthesized from lactose and G3-CNP in order to discriminate between the two a -amylases, the acid-stable and the neutral.

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Endo-β-N-acetylglucosaminidase (endo-β-GlcNAc-ase) is a unique endoglycosidase that hydrolytically cleaves the N, N′-diacetylchitobiose moiety of asparagine-linked oligosaccharides of various glycoproteins. A novel endo-β-GlcNAc-ase in the culture fluid of Mucor hiemalis isolated from soil was found to have transglycosylation activity. This endo-β-GlcNAc-ase, Endo-M, couldtransfer the intact complex-type oligosaccharide from glycopeptide to suitable acceptors with an Nacetylglucosamine (GlcNAc) residue during hydrolysis of the glycopeptide. Using Endo-M, the chemo-enzymatic synthesis of a bioactive glycopeptide by chemical synthesis of N-acetylglucosaminyl peptide and enzymatic transfer of oligosaccharide was attempted. Peptide T can block HIV infection of human T cells. We added the sialo complex-type oligosaccharide to chemically synthesized N-acetylglucosaminyl Peptide T using the transglycosylation activity of Endo-M. The glycopeptide T thus produced showed a higher degree of resistance to protease digestion. We also prepared calcitonin glycopeptide by the chemo-enzymatic method described above. Calcitonin is a calcium-regulating hormone which is widely used in therapy for hyper calcemia. This glycopeptide demonstrated sufficient physiological activity. N-Acetylglucosaminyl glutamine was also a good glycoside acceptor of Endo-M. We were able to add oligosaccharides to the glutamine residues of Substance P neuropeptide and a-mating factor of yeast Saccharomyces cerevisiae using Endo-M. The glycosylated Substance P was biologically active and stable against peptidase digestion. On the other hand, the bioactivity of the glycosylated a-mating factor was lower than that of native one, although that of N-acetylglucosaminyl a-mating factor was higher. Using the transglycosylation activity of Endo-M, we prepared alkyl compound having oligosaccharide of glycoprotein. We used it as an antigen for preparing monoclonal antibody against oligosaccharide of glycoprotein.

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A recent trend has been to use commonly available foods in the prevention of disease. Such application is dependent on a food chemical and physical composition. We investigated functional ac tivity of substances derived from pentose when given in combination with various saccharides to rats. We observed that pentose-derivatives (fructosylxylosides, xylitol, some nucleosides) had a sup pressive effect on the elevation of blood glucose and insulin levels in rats given sucrose or soluble starch. The effect was due to the inhibition of disaccharide hydrolases. We also found that fructo sylxylosides and adenosine enhanced mineral (calcium and magnesium) absorption when given in combination with sucrose.

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Enzyme assay was carried out against wild, cultivate commercial stocks of strain and their variation stocks for analyzing their genetic differences and relations with the expression character. (1) Discrimination of various stocks of strain Flammulina velutipes : Zymograms of Esterase from F. velutipes showed many bands on the gel and variations among the different strains, and this method was very effective to distinguish the strains. The strains of F. velutipes in this experiment was largely classified to three groups (tentatively named A, B and C) according to the cluster analysis of esterase zymograms. All cultivate commercial strains belong to A group. The differ ences of physiological and morphological characteristic among the each groups were observed with mycelial growth, cap color and the size of basidiospore, and in addition these groups were different biological species each other from the crossbreeding test. (2) Screening of undesirable change in budding: The activity of peroxidase from F. velutipes was measured against normal stocks and variation stocks of strain for screening undesirable changes in budding. Also zymograms analysis of peroxidase was carried out concurrently. A specific band which is related with the germination abilities was observed on zymograms. Furthermore, a strong correlation (γ= 0.8590) between the peroxidase activities and the degree of germination abilities was found by statistical consideration. From this results it is suggested that peroxidase is one of the key enzyme in the process of germination of F. velutipes.

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Chitooligosaccharide and maltooligosaccharide were initially converted into useful oligosaccha rides, utilizing glycosidase-mediated transglycosylation. Hexa-N-acetylchitohexaose as a biologically active substance and p-nitrophenyl penta-N-acetyl β-chitopentaoside as a novel substrate of lysozyme assay were synthesized from chitooligosaccharides by hen egg-white lysozyme. p- Nitrophenyl α-maltopentaoside was synthesized from maltopentaose by maltotetraose-forming amylase. It was further converted into end-blocked p-nitropheny 4⁵-O-β-D-galactosyl-amaltopentaoside, which was designed as a substrate for diagnosis of human amylase in serum. Galactosylmaltooligonolactone was also designed and synthesized as substrate analogue inhibitor for mammalian α-amylase. Next an efficient method for synthesizing important di-, tri-, and tetrasaccharide units involved in glycoconjugate was developed. Beginning with β-galactosyl-disaccharide units library, a series of β-N-acetylglucosaminyl-, a-L-fucosyl- and β-mannnosyltrisaccharides were regioselectively synthesized by using some glycosidase-mediated transglycosylations. Consecutive synthesis of human milk tetrasaccharide and mucin type tisaccharide was carried out by enzymatic addition of two sugar residues. Such synthetic oligosaccharides were used to synthesize artificial glycopolymer. The polymers were shown to be useful as probes of carbohydrate recognition and modeling in the analysis of glycopolymer-lectin interaction.

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1. Starches were prepared from many kinds of sweet potatoes, which are mainly used for starch production, differing in variety, stage of development and tissue zone. Average granule size, amy lose content, gelatinization properties by differential scanning calorimetry and digestibility of raw starch by glucoamylase were compared. Digestibility by glucoamylase tended to be higher in the earlier stage of development and the cambium zone. Further, correlations among these starch properties were calculated to determine the factors relating to digestibility of raw starch by glucoamy lase. Negative correlations were observed between gelatinization heat and digestibility as well as average granule size and digestibility. 2. Starches were also prepared from many kinds of purple-and orange-fleshed sweet potatoes to investigate the effect of the cultivation conditions on starch properties. Earlier planting and harvesting dates apparently enhanced gelatinization temperature and tended to reduce the content of short chains of amylopectin. A major difference in starch properties, especially gelatinization temperature and amylopectin structure, occurs according to soil tem perature during the development of sweet potato tuberous roots. To elucidate the extensive variation in starch gelatinization properties within the same botanical origin for sweet potato, correlations were determined between gelatinization parameters and starch molecular characteristics. Starch with a lower gelatinization temperature had relatively abundant extremely shorter unit-chains of amylopectin.

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D-enzyme (EC 2.4.1.25) is believed to be involved in starch metabolism, but the function in vivo is not known. In order to investigate the role of D-enzyme, several approaches have been under taken. Transgenic potato plants with dramatically reduced D-enzyme activity were obtained by introducing sense and antisense D-enzyme cDNA sequences with the appropriate promoter sequence. The tubers from these plants sprouted later and the growth of sprouts was slower than the wild type. However, no significant difference was found in starch produced in tubers, either in its quantity or quality. These results seems to suggest the role of D-enzyme in starch degradation rather than starch synthesis. The analysis of the action of D-enzyme on amylose and amylopectin revealed that the enzyme can catalyze the cyclization reaction and produce cycloamylose with DP larger than 17. Cycloamylose produced are highly soluble in water and can incorporate various compounds in their helical cavities, thus expected to find exploitation in food and pharmaceutical industries.

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