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.

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休止菌体を用いた微生物変換法によるラクトビオン酸（β-O-D-galactosyl D-gluconic acid）の生産について検討を行った．酸化触媒の微生物菌株には，糖酸化活性と高濃度の乳糖に対する耐性をもち，かつβ-ガラクトシダーゼ活性をもたないBurkholderia cepacia変異株を用いた．高力価の菌体の調製は，乳糖10%，コーンスチープリカー3%，酵母エキス0.02%，炭酸カルシウム1.5%を含むpH7.0の培養基を用い，28℃で216時間振とう培養することにより行った．菌体の糖酸化活性は，pH 6.0，55℃で最も高く，pH 5-9の範囲，40℃以下で安定であった．反応最適条件は，洗浄した休止菌体2.0 U/mLを15%の乳糖と2.2%の炭酸カルシウムに40℃で作用させることで，このとき乳糖は約15時間でほぼ完全にラクトビオン酸カルシウムに変換された．触媒として反応に使用した菌体は，回収し数回繰り返し使用した．反応初速度は温度に依存し，30，35，40℃におけるおのおの27，18，15時間後の変換率はすべてほぼ100%であった．しかし5回目の繰り返し反応では，完全変換に必要な時間がおのおの54，36，30時間と遅延した．発酵と異なり反応液に培地成分を含まず不純物が少ないため，また変換率がほぼ100%と高いため，高い単離収率（99.3%）のラクトビオン酸カルシウムが得られた．

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Aspergillus niger GN-3由来のα-グルコシダーゼの遺伝子（aglA）を含む発現用プラスミドを構築し（Fig. 1），Emericella nidulans JCM10259に挿入して組換え酵素を発現させた（Fig. 2）．組換えα-グルコシダーゼは，培養液1リットル中に約61mg分泌されていると見積もられた。組換え酵素は，培養液より硫安塩析，2回のイオン交換カラムクロマトグラフィー，およびゲル濾過カラムクロマトグラフィーにより精製された．精製組換え酵素は，Aspergillus niger GN-3由来のもの（野生型酵素）と同様に，二つのサブユニットから形成されていることが確認された．組換え酵素は，野生型酵素よりも分子質量が若干小さいことがSDS-PAGEにより確認された（Fig. 3）．これは，酵素タンパク質に結合している糖鎖が，両者間で異なることに起因するものと推測される．酵素活性の最適pHや基質特異性については，組換え酵素と野生型酵素では同じであった（Table 1）．

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カルボキシメチルセルロースの部分架橋により多量の水を速やかに吸収し，かつ粘稠性を生じない性質が発現した知見に基づき，同様の性質をもつ多糖を菌体外に生産する細菌の検索を行った．そのような菌体外多糖は，特異的な化学構造，あるいは，水中での高次構造をもつことが，また，生分解性の吸水性物質としての応用が期待できる．ツァペック・ドックス培地を用いて探索した結果，土壌より，自重の200倍の吸水能をもつ粗多糖（粗WAP）を生産する1菌株（IM944株）を分離した．IM944株は，形態学的性質，生理・生化学的性質，16S rRNA遺伝子の全塩基配列解析の諸結果より，Oxalobacteraceae科に属する新種と推定された．また，粗WAPを水酸化カリウム水溶液中で，セチルトリメチルアンモニウムブロミドと処理することにより，マンノース，グルコース，ガラクトース（モル比1:3:3）を構成糖とする精製多糖（WAP）を得た．WAPは吸水倍率120倍，窒素含量0.3％の白色，繊維状の粉末で，ウロン酸，アミノ糖は含まれなかった．この多糖は構成糖およびそのモル比から，新規の多糖と推定された．WAPの示差走査熱量測定（DSC）を行った結果（Fig. 1）は，60.5℃付近に頂点のあるピークを示した．数回の昇降温をくり返しても，ピークの形状にほとんど変化がなかったことから，WAPは均質な試料であると判断された．また，WAPのDSC曲線が転移ピークを示したことは，実質的に水不溶性である吸水性多糖においても可溶性多糖と同様に，何らかの高次構造を形成していることを示唆した．DSC法は，均質性の証明が困難な水難溶性多糖の均質性の証明法として有用である可能性が示された．

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軟質小麦穀粒を酒米搗精機で段階的に削り製粉し，8分画の分級小麦粉を得た。分級粉で調製したドウやパンのグルテンはいくつかの外皮成分により切断され，製パンには不十分な形態であった。分級粉の水溶性ペントサン（WSP）は主鎖成分であるキシロースを通常粉（N61）や市販粉（ヘルメス）より有意に多く含み，一方不溶性ペントサン（WISP）中にはキシロース量は少なかった。中心部（30－0%）の分級粉から得られたWSPをヘルメスに添加すると，N61由来のWSPよりドウの物性と製パン性を有意に改善した。分級粉の製パン改善効果は，そのWSPの性質，すなわち含有量の高さ，アラビノースに対するキシロースの高い比率，多量のフェルラ酸，ならびに優れた泡沫安定性に起因すると考えられた。

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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.

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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.

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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.

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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” Tyr380_AMY1 was proven to be involved in starch granule binding in adzuki bean α-amylase.

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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.

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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.

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