Taka-amylase A (TAA) [EC 3.2.1.1 α-1, 4-glucan 4-glucanohydrolase, Aspergillus oryzae], which crystallized first by S. Akabori et al, in 1951, is a glycoprotein consisting of a single polypeptide chain of 478 amino acid residues with an amino-terminal alanine and a carboxylterminal serine. Crystalline TAA was further purified by ion-exchange chromatography on a DEAE-cellulose column. The homogeneous enzyme judged by polyacrylamide gel electrophoresis was reduced and carboxymethylated. The cyanogen bromide cleavage at methionine residues of the reduced and carboxymethylated TAA (RCM-TAA) was performed in 70% formic acid for 24 hr at room temperature. TAA contains nine methionine residues and the following ten CNBr fragments were isolated; CN 1(1-55), CN 2(56-112), CN3(113-115), CN4(116-123), CN5(124-246), CN6(247-269), CN 7(270-275), CN 8(276-395), CN 9(396-454) and CN 10(455-478). Their amino acid sequences were determined by automated Edman degradation or by the manual Edman method. Methionine-containing peptides were isolated from tryptic and chymotryptic digests of the maleylated RCM-TAA. The alignment of the CNBr fragments were performed based on the amino acid sequences of methionine-containing peptides and the entire amino acid sequence of TAA was established. The N-terminal sequences of α-amylases from various origins were compared. No sequence similarity among TAA, bacterial and animal α-amylases was found in their N-terminal regions. When the amino acid sequence of TAA was compared with those of animal (mouse, rat and hog) a-amylases, sequence homologies between TAA and animal α-amylases and among mouse, rat and hog were about 24% and 77%, respectively. Furthermore, on the view point of the structure and function relationship, some possibilities of a few residues as active sites in α-amylase were discussed.

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The crystal structure of Taka-amylase A has been determined by a combination of multiple isomorphous and molecular replacement methods to 3 .0 A resolution. A model of the protein has been completed with the aid of the amino acid sequence. The molecule consists of two globular domains, main and C-terminal ones which are connected by only one peptide chain. The main domain has an eight-fold α/β barrel and the C-terminal domain is made up of an eight -stranded β/β structure. The carbohydrate is located at the molecular surface on the opposite side of the active cleft. The essential Ca2+ was found to be buried in the molecule nearr the catalytic site.The binding mode of the substrate and the catalytic mechanism were examined briefly.

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The action patterns and their mechanisms of Taka-amylase A (TAA) and Bacillus subtilis saccharifying α-amylase (BSA) were investigated quantitatively and kinetically by product analysis, using a series of maltooligosaccharides (G₂*-G₇*) labeled at the reducing end with ¹⁴C-glucose. A better method for evaluating subsite affinities of amylase was devised . It is based on the combination of the kinetic parameter (k_o/K_m) and the bond-cleavage distribution at a sufficiently low substrate concentration, where transglycosylation and condensation can be ignored. This method was applied to evaluate the subsite affinities of TAA and BSA; TAA comprises as many as nine subsites and BSA has five subsites. On evaluating subsite affinities of BSA, accelerating factor (the dependency of k_int on the degree of polymerization of substrate) was strongly suggested . The product distribution dependent on substrate concentration was observed in the enzymic degradation of maltotriose by TAA. Time lag of glucose formation was observed in the early stage of the reaction of BSA with maltose as substrate. To discriminate condensation reaction from other reactions, the positional isomer (G*-G or G-G*-G) of specific product for condensation was detected in the enzymic digests. It was concluded that these anomalous behaviors were mainly due to glycosyl transfer activity of these enzymes at high substrate concentrations although condensation activity also was detected in the reactions of TAA and BSA to unnegligible extent. Based on a reaction scheme involving hydrolysis, transglycosylation and condensation, the time courses of various product formations from G₃* by TAA were simulated, using the Runge Kutta Gill method. Good agreement with the experimental results was obtained, and ascertained the above degradation mechanism where G₃ is degraded by TAA via G₅ and G₆ as transient products of transglycosylation at its high concentration.

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It has already been reported that α-amylase from Streptomyces hygroscopicus SF-1084 and Streptomyces praecox NA-273 hydrolyze starch to produce more than 75% maltose which rather higher than that produced by other α-amylases. There are two possible mechanisms for higher amount of maltose accumulation than would result from direct hydrolysis of maltotriose; one is condensation and the other is transglycosylation. To make clear the mechanism of higher amount of maltose accumulation, we studied the further conversion of maltotriose using cold and labelled maltotriose. The experiment showed the molar ratio of maltose was higher than glucose. From the results of our studies, it was seen that transglycosylation followed by hydrolysis played an important part in maltose accumulation.

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Two different works, inhibition study and chemical modification of histidine residue, concerning structure and function of Taka-amylase A [EC 3.2.1.1] were presented. 1. In inhibition study of Taka-amylase A catalyzed hydrolysis of phenyl α-maltoside (φM), most of about fifty substrate analogues showed competitive inhibition. However, the following analogues showed noncompetitive or mixed type inhibition: 2-deoxy D-glucose, D-glucosamine, methyl a-glucoside, etc. (noncompetitive); D-mannose etc. (mixed type). It was emphasized that even a subtle change in the structure of analogue could lead to the change in the inhibition type, as seen between D-glucose and 2-deoxy D-glucose. The location of the binding site for the three noncompetitive inhibitors in the specific region with about seven subsites of this enzyme was proposed. 2. Two out of six histidine residues of Taka-amylase A were modified with 4.6mM diethylpyrocarbonate (DEP) and three with 23 mM DEP. It was suggested that one histidine residue existed near the maltose binding site in the cleft. The modification of histidine residue of Taka-amylase A caused loss of the amylase activity and remarkable activation of the maltosidase activity (hydrolysis of φM). This alteration of enzyme activity by modification was not due to change in Michaelis constant K_m but change in molecular activity k_o. A role of the histidine residues in the enzyme was discussed.

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The α-amylase structural gene (ainyE⁺) maps near the arol locus on the Bacillus subtilis chromosome. The gene order around amyE⁺ is lin2-tmrA-amyR-amyE-tmrB-arol. A DNA fragment containing amyR2, amyE⁺, arol⁺ was cloned in 13. subtilis temperate phage P11 by the prophage transformation method. Partial diploids in the amyE⁺ gene were constructed by the transfer of another B. subtilis α-amylase gene, which showed different electrophoretic mobility in 7.5% polyacrylamide gel, into the chromosome of a strain lysogenic for the isolated specialized transducing phage (ρlld amyR2 afnyE⁺ arol⁺). The tmrA and amyR2 genes, which are regulatory genes forr the expression of amyE⁺, acted as cis-dominant on the hyperproduction of the enzyme. plld amyR2 amyE⁺ trrB arol⁺ DNA was partially digested by a restriction enzyme Sau 3A and was ligated with a plamid pUB 110 by T4 DNA ligase after pUB 110 DNA was cleaved by Barn-HI. The constructed plasmid, pTUB 4, have the 2.3 kilobase pairs insert containing the annyR 2 and amyE⁺ genes, α-Amylase expressed from pTUB4 was secreted into the culture medium and the enzyme activity secreted was neutralized by a rabbit anti-serum against B. subtilis α-amylase.

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The enzymatic reaction catalyzed by saccharifying α-amylase from Bacillus subtilis was studied kinetically by following the reaction using the polarimetric method and quantitative paper chromatography. The timecourse of the reaction with purified maltose as a substrate showed a distinct lag phase in the initial stage of the reaction, followed by a linear steady-state phase. The steady-state rate showed a strong sigmoidal dependency on the concentration of substrate maltose. The initial lag phase can be eliminated by the addition of a small amount of maltotriose. Quantitative paper chromatographic determination of the substrate and products during the course of the reaction clearly indicates that the reaction involves glycosyl transfer and/or condensation in addition to hydrolysis and that maltotriose and maltotetraose formed strongly accelerate the reaction. A reaction scheme involving hydrolysis, glycosyl transfer and condensation is presented. Computer simulation based on this scheme and the subsite structure of this enzyme reproduced the lag phase and strong sigmoidal concentration dependence of the rate, confirming the validity of the reaction mechanism proposed. Thus it was demonstrated that the reaction mechanism involved with glycosyl transfer and/or condensation in addition to hydrolysis by itself can account for the allosteric behavior of this enzyme, i.e., the appearance of lag phase in the time course of reaction and the sigmoidal dependency of reaction rate on substrate concentration, irrespective of ofrconmational change of the enzyme protein. One of the four tryptophan residues located on the surface of this enzyme was clearly distinguished by the reaction rate with NBS. This most reactive residue was considered not to be responsible for the enzyme activity. One of the other three residues was suggested to locate near the 5th subsite and be involved in the substrate binding in transglycosylation and/or condensation as well as hydrolysis with different contribution depending on the substrate.

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Recent results on the structure and catalytic mechanism of glycogen and starch phosphorylases [EC 2.4.1.1] are reviewed with special reference to the studies by the author's own group. The article contains the following three sections. 1) Primary and three-dimensional structures; 2) Glucan binding site; 3) Catalytic mechanism. The last section discusses the role of pyridoxal 5'-phosphate in the phosphorylase reaction.

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The action of Thermoactinomyces vulgaris α-amylase was examined in order to elucidate whether this α-amylase catalyzes the hydrolysis of α-1, 4-and α-1, 6-glucosidic linkages in some oligosaccharides at one catalytic site. Polyacrylamide gel electrophoresis of this amylase showed that the activities on starch and isopanose migrated exactly the same position as the enzyme protein band. The optimum pH for its action on maltotriose and isopanose was 4.5, while was almost the same as the values for starch and pullulan. Action patterns on isopanose were dependent on the substrate concentration. At low substrate concentration (0.5%) equimolar maltose and glucose were produced from isopanose. At high substrate concentration (4.0%) small amount of isomaltose was found besides maltose and glucose, equimolar glucose and sum of maltose and isomaltose were pro-duced at the early reaction stages. Action patterns on reducing end-(¹⁴C)-labeled maltotriose was also dependent on substrate concentration. Increasing the substrate concentration from 0.5 to 4.0%, the molar ratio of labeled glucose to labeled maltose in the products was decreased from 6 to 1.5. Apparent formation of labeled glucose was depressed by the addition of isopanose to the labeled maltotriose-hydrolyzing mixture. The results above supported the view that this enzyme can hydrolyze α-1, 6-glucosidic linkages as well as α-1, 4-glucosidic linkages in isopanose or maltotriose at the same catalytic site. Moreover the action of this enzyme on IP6 (a-D-Glcp-(1→4)-α-D-Glcp-(1→6)-α-D-Glcp- (1→4) -α-D-Glcp- (1→4) -α-D-Glcp- (1→6) -D-Glcp), TV6 (α-D-Glcp- (1→6) -α-D-Glcp- (1→4) -α-D-Glcp- (1→4) -α-D-Glcp- (1→6) -α-D-Glcp- (1→4) -D-Glcp) and P6 (α-D-Glcp- (1→4) -α-D-Glcp- (1→4) -α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-D-Glcp)was examined by using paper chromatography. Other four kinds of α-amylases tested cleaved isopanose to produce glucose, maltose and isomaltose; especially Bacillus subtilis saccharifying a-amylase and Taka amylase A produced larger amounts of isomaltose than others.

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The majority of starches used in the manu-facture of glucose syrups contain 75-85% amylopectin.1) Amylopectin is a highly branched polysaccharide consisting of linear chains of 1, 4-α-linked D-glucose residues, joined together by 1, 6-α-glucosidic linkages. The branch points occur on average every 20-25 D-glucose units, so that amylopectin contains 4-5% of 1, 6-α-glucosidic linkages.^2-4) The 1, 6-α-glucosidic linkages act as a kind of barrier to the action of exo-acting, saccharif ying amylases such as glucoamylases or maltogenic /3-amylases. Endo-acting α-amylases are able to by-pass the branch points, ⁵⁾ but in general are not capable cf hydrolyzing the 1, 6-α-glucosidic linkage. Recent work by Kobayashi and co-workers has shown that at least one α-amylase (from Thermoactinomyces vulgaris) can hydro-lyze 1, 6-α-glucosidic linkages, in addition to 1, 4-α-glucosidic linkages.⁶⁾ Glucoamylases can slowly hydrolyze 1, 6-α-glucosidic linkages in amylopectin and partially hydrolyzed amylopectin, ⁷⁾ but the action of maltogenic exo-amylases ceases as a branch point is approached.⁸⁾ It is therefore obvious that the efficiency of the saccharification reaction could be improved by incorporating a specific amylopectin debranchina enzyme in the system. Debranching enzyme such as isoamylase [EC 3.2.1.68, glycogen 6-glucanohydrolase] and pullulanase [EC 3.2.1.41, pullulan 6-glucanohy-drolase] have been known for many years, ⁹⁾ but their use in the glucose syrups industry is far from widespread. Pullulanases from Klebsiella pneumoniae, ^{10, 11)} Streptomyces sp., ¹²⁾ and Bacillus cereus var, mycoides¹³⁾ and isoamylases from Pseudomonas amyloderamosa^{14, 15)} and Cy-tophaga sp.¹⁶⁾ are not sufficiently thermostable to be used at 60°C. Moreover the Pseudomonas isoamylase was the only debranching enzyme sufficiently acidophilic to be used at a pH of around 4. 5.¹⁷, ¹⁸⁾ After an extensive screening programme, our research laboratories succeeded in isolating a species of Bacillus which produced a thermostable, acidophilic pullulanase which was free from glucoamylase, β-amylase and a-amylase side activities. Some of the properties of this enzyme will now be described.

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A branching enzyme was extracted from the mycelia of Neurospora crassa, and was purified to electrophortic homogeneity by a procedure including DEAE-Sephacel column chromatography, 6-aminohexyl-Sepharose column chromatography and gel filtration on Toyopearl HW-55S. The subunit molecular weight of this enzyme was estimated to be 80, 000 by electrophoresis in sodium dodecyl sulfate (SDS)-polyacrylamide gel, and 84, 800 by analysis of its amino acid composition and carbohydrate content. The optimum pH of this enzyme was around 8, and the optimum temperature was 27°C. The branching activity of the enzyme was confirmed by its, action on amylopectin and other substrates as well as by the combined action of this enzyme and N. crassa glycogen synthase. Action of this enzyme on various substrates induced decrease in the wave length maximum of absorption spectrum of the glucan-iodine complex, β-amylolysis limit and in the unit chain length of the debranched product. In the combined action, the branching activity stimulated incorporation of glucose into α-glucan. The products formed by the combined action had glycogenlike form, but the unit chain profiles of synthetic products were different from that of the native glycogen on Toyopearl HW-40F.

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Branching enzyme [α-1, 4-glucan : α-1, 4-glucan 6-glycosyltransferase EC 2.4.1.18] from Bacillus megaterium strain No.10-5 was purified to an enzyme preparation completely free of enzyme contaminants that were capable of modifying the substrates and products of the branching reaction. The molecular weight of the enzyme was determined to be 85, 000 by sucrose density gradient ultracentrifugation. The isoelectric point of the enzyme was pH 4.5. The enzyme was most active at pH 7.6 and 25°C, and stable up to 40t at pH 7.0 and in the range of pH 6.5-8.5 at 25°C in the 2 hr incubation. This enzyme converts amylose, soluble starch and amylopectin into a glycogenlike molecule. The minimum length of maltodextrin chain on which the branching enzyme can act as a substrate was found to be maltononaose. Furthermore, it was shown that the branched dextrin, the substrate of the branching enzyme, had 12 glucose units for its minimum chain length.

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Cyclodextrins (CDs) are used in various fields of application because its ability forming inclusion compounds or complexes with various kinds of substances. Accordingly, recent advances in the research on CD production and application are remarkable. CDs are now commercially produced and the main products are β-CD, CD syrup and powdered CD syrup. And powdered CD syrup (commercial name: Cyclo-TC, Toyoderin) is especially useful for making powdered oily materials with it. Though there are several kinds of special CDs, branched-CDs have not been studied so farr because the production of the special CDs was extremely difficult. Recently, we established a method of producing branched-CDs, in which branched-CDs were prepared by the action of Bacillus m.acerans cyclomaltodextrin glucanotransferase (naacerans enzyme) on branched dextrin of waxy corn starch prepared by removing the CDs with bromobenzene and tetrachloroethane from the macerans enzyme-starch digest. Branched-CDs were effectively produced by the action of macerans enzyme on branched dextrin in the presence of SDS and were further degraded to glucosyl-CDs by the combined action of Aspergillus oryzae a-amylase and glucoamylase. These enzymes also degrade β and γ-CDs, which interfere with the separation of glucosyl-CDs by paper and column chromatography, to glucose. Glucosyl-CDs were separated by paper and column chromatography respectively. Glucosyl-α-CD was crystallized from water and made into a crystal complex with iodine. Glucosyl-CDs have extremely high solubility compared with the original CDs, and solubilize various kinds of oily substances and are extraordinary resistant to the action of Aspergillus oryzae α-amylase. Possible uses of branched-CDs are also discussed in the text.

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α-Cyclodextrin (CD) (1500mg) was orally fed to fasted rats and time courses of sugar compositions in gastrointestinal tract, the amount of serum glucose and liver glycogen and total lipid were followed. The results indicated that α-CD might be difficult to be digested and utilized by rats. However, liver total lipid levels were higher in α-CD fed rats than in starch fed control rats. In addition, after digestion of 62.5 mg of carbohydrate, as dessert, liver total lipid levels elevated similarly in α-CD, sucrose, fructose and glucose fed mice during the 2-hr sleeping period. There was no difference in weight gain or in the weights of organs and tissues of rats, when 20% soluble starch or Cyclo-TC (α, β, γ-CD and dextrin: 30, 15, 5 and 50%) was added to a commercial basal diet.

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This paper is mainly concerned with some applications of cyclodextrin homologues (α-, β-, γ-CyD) in pharmaceutical preparations. Various examples such as improvements of solubility, physico chemical stabilities and bioavailability, and alleviation of local irritation of drug molecules are presented. Furthermore, the possible utilities of methylated CyD derivatives and CyD copolymers in the pharmaceutical fields are discussed.

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