米の品種間における食味の違いが何に起因するかを知る目的で,白飯および桜飯の炊飯特性について糊化度や物性ならびに官能評価の面から実験を行った.また低温保存や再加熱が飯の食味に及ぼす影響についても検討した.試料は食味が劣るといわれる北海道産米トモヒカリをコシヒカリ・日本晴と比較し次のような結果を得た.　1)β-アミラーゼ・プルラナーゼ法による糊化度の測定からトモヒカリは炊飯直後の白飯の糊化度が低く,また桜飯は白飯に比べて低温保存による糊化度の低下が大であった.　2)破断試験から食味が劣るといわれる米はコシヒカリ・日本晴に比べて白飯の破断応力,破断歪および破断エネルギーが大きく,また炊飯後の時間経過に伴う破断特性値の変化が大であった.　3)テンシプレッサーによる白飯の硬さは5.0～6.5kgを示し,桜飯は4.1～5.4kgと白飯に比べて軟らかい飯となり付着性の低下が認められた.再加熱飯の付着性は一噛み目が小さく現れこの付着性の出方が食味に関与することが示唆された.　多重バイト試験のパラメータは食味が劣るといわれる米の値が小さくコシヒカリは大であり米の種類による物性の違いが明らかであった.とくに桜飯は白飯に比べて米の品種間の違いが明らかに示され,パラメータは飯の物性測定の重要な項目としてあげることができた.　4)白飯の官能評価から硬さがあり粘り・光沢の少ないトモヒカリの嗜好性は低く,また低温保存による食味の違いが顕著に認められコシヒカリとの差が明らかであった.　物性と官能評価との相関性から炊飯直後の白飯では硬さが食味に大きく関与し,低温保存後再加熱した飯では付着性が硬さよりもより大きく食味に関与する因子であった.　本報告においては白飯の物性を桜飯と比較し,また低温保存後の再加熱飯について炊飯直後と比較することにより,米の品種間の違いをより明らかにとらえることができた.　本報告は日本澱粉学会創立40周年記念平成4年度大会(1992)において発表した.

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Using three rice varieties with different tastes and newly developed rice varieties as samples, the relation between the texture of cooked rice (white rice) and starch structure was examined. The amylose contents of the rice starches ranged from 6.3 to 27.3% on gel filtration. The chain length distribution of amylopectins indicated that Fr.II ranged from 17.3 to 25.3% and Fr.III/Fr.IIratios ranged from 2.7 to 3.2. Rice starch with a lower amylose content showed a lower Fr.III/Fr.II ratio. The chain length distribution of amylopectins proved that amylopectin from favorable white rice showed a small amount of Fr. I and a large amount of Fr.II as well as a low Fr.III/Fr.II ratio. The average chain length of amylopectins, as determined by modified SMITH's degradation method, ranged from 18.2 to 23.1. These values agreed well with those determined by the enzymatic method. Rice starch having a low amylose content showed a shorter average chain length. White rice significantly differed in physicochemical properties and sensory evaluation, especially in firmness and adhesiveness by rice variety. Thus, the taste of white rice appeared to be closely related to the structure of the rice starch.

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　熱帯産澱粉4種すなわち食用カンナ,アロールート,キャッサバ,サゴの澱粉と対照として馬鈴薯とトウモロコシの澱粉を選び,2,3,4%の澱粉糊液につき,ずり速度流動化流動,チキソトロピー,降伏値,流動の見かけの活性化エネルギーを測定した.　各澱粉糊液は非ニュートン流動を示した.流動曲線につき,10s^-1から300s^-1の範囲で直線が得られたのでベキ則を適応し,流動方程式τ=Kγⁿを求め,粘性定数(K)および流動性指数(n)を求めた.　キャッサバとサゴの各濃度の澱粉糊液および4%アロールート澱粉糊液の流動性指数(n)は,20～60℃に比べて10℃ の値が極端に小さく馬鈴薯澱粉糊液に近い性質を示し,食用カンナと2,3%アロールート澱粉糊液は,温度依存性が小さく,トウモロコシ澱粉糊液に近い性質を示した.流動性指数(n)と分子特性の関係は,10℃ で値が小さく20～60℃ で大きくなる馬鈴薯に代表されるタイプのnはアミロースとアミロペクチンの分子量が両方とも大きい傾向であり,10～60℃ まで変化が少ないトウモロコシに代表されるnを示すものは,アミロースとアミロペクチンの分子量が両方とも小さい傾向がみられた.サゴはこの傾向に一致しなかった.　チキソトロピー性はキャッサバ,食用カンナ,サゴ,4%アロールート糊液に10～60℃ まで認められ,キャッサバ,サゴが大きく,食用カンナは中位,アロールートは最小であった.10℃ でチキソトロピー性が大きい馬鈴薯とキャッサバはアミロースとアミロペクチンの分子量が両方とも大きい傾向で,食用カンナ,アロールート,トウモロコシは,チキソトロピー性は中位から最小であるが,これらはアミロースとアミロペクチンの分子量は両方とも中位から最小の傾向が認められた.サゴはこの傾向と一致しなかった.　降伏応力は4%アロールート澱粉糊液の10,20℃と3,4%トウモロコシ澱粉糊液の10～60℃ に認められた.　流動の見かけの活性化エネルギーは,6種の澱粉の2,3,4%糊液について,約8.5～17kJmol^-1の範囲であった.

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　トラマメ(Phaseolus vulgaris L.)登熟種子より調製した粗抽出液を50倍に濃縮後,可溶性澱粉,β-リミットデキストリンおよびマルトースに作用させたところ,いずれの基質にも水解作用を示し,アミラーゼの存在が示唆された.このような粗抽出液をネイティブ電気泳動後,pH5.6で可溶性澱粉を基質に活性染色すると3本のバンドが,β-リミットデキストリンでは2本の活性バンドが認められた.長径12mmの粗抽出液を70℃ で処理すると,いずれを基質とした場合にも2本のバンドが認められた.精製α-アミラーゼから調製した抗体を用いたウエスタンブロット法でも幾つかのバンドが検出された.したがって,登熟種子にもα-アミラーゼが存在するものと考えられた.一方,熱処理およびβ-リミットデキストリンゲルで消失する活性バンドはβ-アミラーゼないしは枝つけ酵素と想定されるため,電気泳動後,アミロースを含むゲルに転写し,pH7.0で活性バンドの検出を行った.その結果,想定したバンドは本条件では出現せず,精製枝つけ酵素と同位置の2本を含む数本のバンドが新たに認められた.登熟期におけるα-アミラーゼの存在をより確実なものとするため,粗抽出液をβ-リミットデキストリンに作用させ,生成物を薄層クロマトグラフィーで分析した.無処理の酵素液では主としてグルコースとマルトースを,熱処理酵素液では上記の2種の他に,マルトトリオース,マルトテトラオースなどのオリゴ糖も生じた.この反応様式は発芽種子より精製したα-アミラーゼのものによく類似していた.

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From tea leaves (Camellia sinensis var. sinensis cvs. Shuixian and Maoxie) to be processed to oolong tea, we have isolated and identified alcoholic aroma precursors of geraniol, linalool, etc., which are known to contribute to the floral aroma of oolong tea and black tea, as β-primeverosides (6-O-β-Dxylopyranosyl-β-D-glucopyranosides), guided by an enzymatic hydrolysis followed by GC analysis. Aroma precursors of linalool oxides III and IV were found to be exceptionally present as 6-O-β-D-apiofuranosyl-β-D-glucopyranosides. We have also purified β-primeverosidases from fresh leaves of cv. Yabukita for Japanese green tea, cv. Shuixian for oolong tea and a cultivar of C. s. var. assamica. The enzymatic characteristics were very similar to each other (molecular weight, 60.2-60.5 kDa; optimum temperature, 45°C; stable temp., 40-45°C; optimum pH, 4; pH stability, pH 3-5). The enzyme was confirmed to effectively hydrolyze the aroma precursors, β-primeverosides as well as 6-O-β-Dapiofuranosyl-β-D-glucopyranoside, into disaccharides and each aglycon (alcoholic aroma) without further hydrolysis. We were also interested in the molecular mechanism of the floral aroma emission in flowering. We applied the same experimental procedures as used for the tea to the study of Gardenia jasminoides, Jasminum sumback, and Rosa damascene to find that disaccharide glycosides such as β-primeverosides, β-vicianosides, etc. are also important alcoholic aroma precursors in flowers. In Gardenia jasminoides, glycosidic activities in floral buds reach a maximum at flower opening stage. Purification of the enzyme concerned with the floral aroma formation is now in progress. The enzyme has been characterized as a glycosidase which hydrolyzes the aroma precursors, the disaccharide glycosides, into aglycons and disaccharides.

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Erythritol (1, 2, 3, 4-butanetetrol, molecular weight: 122.12) is a tetra-carbon sugar alcohol contained in fermented foods, animal bodies and plants, and humans have consumed it naturally from ancient times. Erythritol possesses such characteristics as 75% of the sweetness level of sucrose, low hygroscopicity, high endothermic reaction, and easy crystallization. Physiological aspects include low energy value (0.2 kcal/g), low laxative effect and no affect to blood glucose level or secretion of insulin. The National Food Research Institute of the Ministry of Agriculture, Forestry and Fisheries in Japan and Nikken Chemicals Co., Ltd. have done research and development concerning erythritol production by fermentation, and have discovered micro-organisms which can produce erythritol at a high yield and not generate much foam. As the consequence, we succeeded in applying manufacturing technology for highly efficient production of erythritol from glucose by fermentation. Nikken launched the commercial production of erythritol from 1990 and plans to introduce industrial uses in the future.

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Xylanase C (XynC) from Aspergillus kawachii is stable between pH 1-9 and the optimum pH is 2.0. In order to obtain the structural basis of its extreme acid stability and low pH optimum, we conducted X-ray cystallography of the enzyme. Good crystals were grown by using sodium sulfate as a precipitant, and the space group of the crystal was P4₃2₁2 with unit cell dimensions a=b=62 .1 Å, c=113.3 Å. The structure of the enzyme was solved by molecular replacement using the coordinates of Trichoderma reesei XYN I. The final R-factor was 0.194 for data between 6.0 and 2.0 A resolution with F>3σ(F). A structural feature around Asp37 was clearly observed, which is thought to be characteristic for xylanases with a low pH optimum. A mutational analysis confirmed that the residue is important for the low pH optimum of the enzyme. The “Ser/Thr surface” of the enzyme was covered by acidic residues, and thought to be a cause of its extreme acid stability.

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Alkaliphilic Bacillus sp. strain 41M-1 was isolated from soil. The strain secreted a xylanase (xylanase J) that had an alkaline pH optimum. The gene encoding xylanase J was cloned and sequenced. The putative xylanase J gene contained an open reading frame of 1062 by and encoded a 27-amino-acid (aa) leader peptide followed by a 327-aa mature enzyme. The deduced amino acid sequence of xylanase J was compared with those of other bacterial xylanases. The potential catalytic domain belonging to family G was located at the N-terminus of xylanase J. A linker-like sequence occurred between the catalytic domain and an additional functionally-unknown domain at the Cterminus. A mutational analysis of xylanase J indicated that G1u93 and G1u183 should act as catalytic residues, and that Trpl8, Trp86, Tyr84 and Tyr95 might play important roles in substrate binding. Furthermore, the substitution of Asp20 by Asn or Trp144 by Phe caused an acidophilic shift in the optimum pH of xylanase J. A deletion derivative of xylanase J lacking the C-terminal region retained xylanase activity, suggesting that the C-terminal region was not essential for catalytic activity. Wild type xylanase J bound to insoluble xylan specifically, while the C-terminal truncated enzyme completely lost binding ability. On the other hand, the fusion protein between glutathione S-transferase and the C-terminal region of xylanase J showed xylan-binding activity. These results suggested that the C-terminal region of xylanase J is a novel xylan-binding domain. Wild-type xylanase J hydrolyzed insoluble xylan more efficiently than the C-terminal truncated enzyme. Thus, the xylan-binding domain enhances hydrolyzing activity toward the insoluble xylan of the catalytic domain.

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We have been investigating the mechanism of cellulose degradation by various cellulases . Cellulases were classified into three groups such as R-glucosidase, endo-R-1, 4-glucanase and exo-β-1, 4-glucanase, and the differences of reactivities on Avicel and CMC as substrates discriminated endo-β-1, 4-glucanase from exo-β-1, 4-glucanase. However, results which deviate from the above classification were obtained when we investigated the reactivities of some cellulase components on other cellulosic substrates, such as bacterial cellulose, with a different type of structure . From these results, we propose that bacterial celluloses with different crystalline structures are convenient substrates for characterizing the mode of action of cellulases produced from various microorganisms.

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The β-glucosidase (EC 3.2.1.21, βGA) preparation purified from Aspergillus niger (Novozym 188DCN0003) was confirmed to be a monomer protein of Mw 137, 000. With this preparation, binding of glucono-l:5-lactone (GLN) to βGA was studied using fluorescence-spectrophotometric and stopped flow kinetic experiments on the basis of Trp-residue (s), and an inhibition kinetic experiment for the cellobiose substrate. The dissociation constant (K_d) and inhibitor constant (K_i) (competitive inhibition) for the GLN-βGA complex were evaluated to be 10μM and 15μM, respectively. The subsite structure of βGA was analyzed with the steady-state kinetic method. It was confirmed that the active site was composed of six subsites, affinity at subsite 2 (A₂) was the largest and subsites 4-6 had negative affinity. We tried to interpret the binding of GLN on the basis of the subsite structure, where subsite 1 carried an indispensable role in the productive binding mode. Determining the kinetic parameters for several β-glucosides as substrates, it was shown that the molar activity (k₀) and Michaelis constant (K_m) were very characteristic of the substituent species in aglycone phenyl-residue. Furthermore, the value of k₀/K_m, which reflects the productive-binding mode, strongly depends on substituent constant n (degree of hydrophobicity), suggesting that subsite 2, in which the aglycone residue is bound, is intimately related to the "hydrophobic-driven" mechanism for ES-complex formation. Further, the effect of acetonitrile (0-32.3%) on the hydrolytic reaction was examined for the cellobiose substrate. The results support the hydrophobic-driven mechanism.

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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 undertaken. A biochemical analysis of the purified potato D-enzyme suggested that high molecular weight starch (amylose and amylopectin) can serve as donor and acceptor, and very long α-1, 4-glucans or even highly branched glucans can be transferred by the enzyme. 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. From these results, the possible role of Denzyme in starch metabolism is discussed.

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Cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. #1011 and its mutant enzymes are cocrystallized with acarbose at pH 4.5. A triclinic unit cell contains two independent molecules, MOLL and MOL2, related by pseudo two-fold symmetry . In both MOLL and MOL2 of wild-type . CGTase, acarbose occupies subsites 2 to 2' of the enzyme and the catalytic triad Asp229, Glu257 and Asp328 are in hydrogen-bonding contact with B and C residues which are connected by a scissile bond (residue A is at the non-reducing end of acarbose). The corresponding catalytic residues in a mutant enzyme, F183L/F259L, constituting subsite 2' form hydrogen bonds with C and D residues in MOLL and only with the D residue in MOL2. In addition, the cleavage sites of 3-ketobutylidene-2-chloro-4-nitrophenyl f3-maltopentaoside (3KB-G5CNP) are observed to be shifted in the direction of the reducing end in the catalytic reaction of the mutant. This mutant shows a 10, 000-fold increase of IC50 of acarbose on starch degrading activity as compared to that of wild-type CGTase. From these findings, we discuss the effects of the stacking interaction of Phe183 and Phe259 on substrate binding, cyclization reaction and the acarbose-inhibition mechanism of CGTase . The replacement of Tyr100 with leucine causes a drastic reduction in catalytic activity as well as an increase in IC50 of more than 170, 000-fold. Interestingly, in MOL1 and MOL2 of Y100L, acarbose forms fewer hydrogen bonds with the active site than that bound to wild-type CGTase and the O₄ atom of each scissile bond moves in the direction of Tyr195, away from G1u257. As a result, Phe183 and Phe259 can no longer stack on the D residue. Since the significant changes in the protein backbone conformation between Y100L-acarbose and wild-type acarbose have not been found, the drastic reduction of catalytic activity of the mutant Y100L is ascribed to decreased interaction between enzymes and substrates. This again points to an essential role for the residue constituting subsite 1 in the catalysis of CGTase.

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　好熱性細菌Bacillussp.SAM1606の生産するα-グルコシダーゼは,マルトース,イソマルトース,スクロースのようなさまざまな1-O-α-D-グルコシドを加水分解できる幅広い基質特異性を示す.本酵素はトレハロースを分解できる唯一のα-グルコシダーゼでもある.本酵素のアミノ酸配列中には,α-アミラーゼ・ファミリー酵素に見いだされる保存領域(CR)が存在し,CR中にある三つの触媒残基も保存されていることから,本酵素も同ファミリーに属すると考えられる.Bacillus属細菌の生産するoligo-1,6-glucosidases(016G)が全領域で60%以上,CR領域では80%の配列同一性を有することがわかったが,016G酵素の基質特異性はα-1,6-結合に限定され,トレハロースを分解できない.本酵素のユニークな基質特異性を特徴づけるアミノ酸残基を明らかにするために,CR中で016G酵素と異なる5残基のアミノ酸について016G型に置換した変異体酵素,およびこれらを組み合わせた計12種類を作成し,速度論解析を行った.いずれの変異体酵素でも各基質に対するV_maxの顕著な低下はなく,野生型酵素のV_maxの20%以上の値を保持していた.また,マルトース,スクロース,イソマルトースに対する著しいV_max値の変動も認められなかった.しかしながら,Gly273Proの単一変異はトレハロースに対する親和性の著しい低下をもたらした.Gly273Pro変異を含む多重変異体酵素の解析の結果,本酵素のトレハロースに対する親和性には,Gly273が最も重要,Thr342はGly273の効果をさらに増強する働きがあることがわかった.最近発表されたブタ膵臓α-アミラーゼと阻害剤との複合体の立体構造解析の結果から,本酵素のGly273に相当するアミノ酸残基は基質と非常に近接した位置にあることが示唆された.

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The domain structure of Aspergillus niger glucoamylase was studied by calorimetry and steady-state kinetics. Two forms of the enzyme were used; G1 is the whole protein with two main domains of the enzyme (the catalytic domain and the starch-binding domain), and G2 is an isozyme that lacks the starch-binding domain of Gi. Adiabatic differential scanning calorimetry (DSC) revealed that the catalytic domain and starch-binding domain were independent of each other concerning thermally induced unfolding, and that the former domain unfolded irreversibly and the latter reversibly . DSC and isothermal titration calorimetry also showed that there was no detectable interaction for the binding of ligands to those domains. Steady-state kinetic studies showed that the starch-binding domain did not change the kinetic parameters for the hydrolysis of p-nitrophenyl glucoside or the binding modes of inhibitors. These results lead us to the following conclusion; the essential role of the starch-binding domain seems simply to increase the local concentration of starch around the enzyme molecule .

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Soybean β-amylase was mutated by site-directed mutagenesis at residues (His93, Cys95, Asp101, Glu186, Cys208, Cys343, Glu345, Asp348, Glu380 and Leu383) conserved in highly similar regions of the α-amylase family found in plants and bacteria. The substitutions of Asp101, Glu186 and Glu380 completely eliminated the activity without inducing any significant change in the binding affinity for α-cyclodextrin (α-CD). These data and X-ray crystallographic works confirm Glu186 and Glu380 as catalytic residues of the enzyme. On the other hand, substitutions of Leu383 led to remarkable decreases in the k_cat/K_m values, and those mutants also showed marked reductions in their binding affinity to α-CD. Leu383, therefore, may be important for both substrate penetration and subsequent retention at the active site. Based on the foregoing, we propose an action mechanism of soybean β-amylase involving the interaction of three essential amino acid residues (Asp101, Glu186 and Glu380) in concert with leu383.

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