Taka-amylase has a (αβ)₈ barrel structure and the active site is located at the C-terminal end of a β-strand as reported earlier. In this paper, we describe about the direction of substrate amylose binding with respect to the barrel structure. A possible mechanism of hydrolysis is also proposed, in which Glu 230, Asp 297 and Asp 206 located near the active site are essentially involved.

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The 3-dimensional X-ray crystallographic structure of cyclodextrin glucanotransferase (CGT-ase) from B. stearothermophilus showed that the CGTase molecule fold into four globular domains, A, B, C and D. The N-terminal domains, A and B, are similar to those of Taka-amylase. The C and D domains, which are unique to this enzyme, both consist of antiparallel β-barrel structure. With a substrate binding analysis in the crystal, two binding sites have been found on the enzyme molecule, one in a cleft of the A-domain and the other in the D-domain. The first one is considered to be related to the enzyme activity in a same manner with a-amylase and the second to the raw starch binding activity of the CGTase.

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The structure of soybean β-amylase has been determined by X-ray crystallography by using multiple isomorphous replacement technique. The low resolution analysis at 6 Å revealed that the enzyme is composed of a large and a small domain. The difference Fourier synthesis for the enzyme-a-cyclodextrin complex and the enzyme-maltose complex showed that the substrate analogs bind to a deep cleft between the two domains. One maltose molecule is supposed to occupy the binding site of nonreducing ends of the substrate (subsite 1). The higher resolution analysis at 3 Å of the enzyme-a-cyclodextrin complex clearly showsthat the large domain contains a (αβ)₈ supersecondary structure. The chain following shows that the smaller domain is inserted in the loop region after β4. The structure of β-amylase is quite different from that of β-amylases except for the (αβ)₈ barrel structure. The chain following also shows that the two SH groups, Cys95 and Cys343, are located in the edges of the active cleft. Cys95 is near the maltose specific binding site and Cys343 is near the α-cyclodextrin binding site. These two SH groups were demonstrated to be responsible for the inactivation of the enzyme by chemical modification.

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The active monomer of sweet potato tetrameric β-amylase prepared by modification with periodate-oxidized maltohexaose was a labile molecule. However, the stability of the active monomer was found to be very enhanced at pHs lower than 5 and higher than 9, and also at 60°C in the presence of α-cyclodextrin. The monomer, therefore, was prepared by the same modification in the presence of α-cyclodextrin. The specific activity of the resulting active monomer was 2700 units per mg protein while that of the original tetrameric enzyme was 3000, and the yield of the monomer was about 50% by weight of protein. Both the specific activity and yield of the active monomer were greatly improved comparing with those of the active monomer obtained in the absence of α-cyclodextrin. Also, the stabilization with α-cyclodextrin was observed for the original tetrameric enzyme.

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Soybean α-amylase mediates three kinds of reactions: (1) the hydrolysis of amylose, etc.; (2) an esterification of a functional carboxylate of G1u186 with 2, 3-epoxypropyl α-D-glucopyranoside (α-EPG); (3) a hydration of maltal. Thus, what the functional groups for each reaction are, and whether the catalytic sites are the same, are interesting questions. For hydration of maltal, we here studied the binding properties and the pH dependence of the rate parameter (pK_m, k₂). It was found that the binding of two maltal molecules to the enzyme is required for the hydration, and that only one protonated functional group with pK=6.75 is concerned with the catalysis, although two functional groups with pK=3.5 and pK=8.2 for both the hydrolysis of amylose and the esterification of G1u186 with a-EPG. Hehre et al. (J. Biol. Chem., 261, 2147(1986)) have proposed a possible mechanism for the hydration of maltal catalyzed by sweet potato β-amylase and assumed a functional carboxyl group (pK=3.7) for the hydrolysis of amylose as catalytic group. If this assumption is true in the case of soybean B-amylase, the pK of the functional carboxyl group (normally pK=3.5) must be raised to 6.75 as a result of the maltal binding to the enzyme. From inhibition study with 4-O-α-glucopyranosyl (1→4)-1-deoxynojirimycin as strong competitive inhibitor, it was presumed that three reactions are catalyzed at the same place in the active site.

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Disproportionating enzyme (D-enzyme, EC 2.4.1.25) is a transglycosylase and its reaction involves the participation of more than two molecules of a substrate; E+2×Gn→G_n-i+G_n+i. The HPLC analysis of digests of maltooligosaccharides (G₃-G₇) showed that maltose is not formed in any case. The products from all substrates except G₄ are those resulting from maltosyl transfer as the predominant reaction. Glucan phosphorylase(EC 2.4.1.1) has a rapid equilibrium-random Bi Bi mechanism involving the two kinds of substrate; E+G_n+G1P→E+G_n-1+Pi. Purified G3 is of poor primer ability, and the time course of the reaction shows an accelerating curve. By incorporating a sufficient quantity of β-amylase in the digests, the true rates of the G3-primed reaction could be determined from the linear time courses to give the K₄ value of 9.3 mM. Other kinetic parameters for a series of maltooligosaccharides (G₄-G₈) were also determined in both the synthetic and the phosphorolytic directions. The reaction mechanisms of both enzymes are more complicated than the hydrolytic reaction of amylases and do not obey the simple mechanism of Michaelis-Menten type. We attempted to apply the subsite theory to the two enzyme reactions to analyze the characteristics of their action patterns. The two enzymes were isolated from a β-amylase-deficient variety of sweet potato.

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Exo-α-glucanases (β-amylase, glucoamylase, glucodextranase) that catalyze the hydrolysis of specific a-glucosidic substrates with inversion of configuration have usually been assumed to act by a base assisted nucleophilic displacement mechanism. On the other hand, the possibility of exo-carbonium ion mediation of such reaction has been recognized, but no supporting experimental evidence for this type of mechanism appears to have been reported. In order to examine this possibility, we have studied the α-secondary hydrogen isotope effects on the hydrolysis of α-glucosyl fluoride catalyzed by glucoamylases of several origins, and by a glucodextranase. α-Secondary deuterium isotope effects were 1.11-1.26 on these reactions. α-Secondary tritium isotope effects ranging from 1.17 to 1.26 were also measured for the hydrolysis of α-glucosyl fluoride catalyzed by these exo-α-glucanases. These results indicate that the reactions of hydrolysis of the C-F glycosylic bond of glycosyl fluoride by glucoamylase and glucodextranase proceed via an intermediate with oxo-carbonium ion character.

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Pseudomonas amyloderamosa isoamylase and Klebsiella pneumoniae pullulanase were crystalline preparations obtained from Hayashibara Biochemical Laboratory, Inc., Okayama, Japan. Bacillus acidopullulyticus pullulanase was purified from Promozyme 200 L (Novo Nordisk Bioindustry, Ltd., Copenhagen, Denmark) by the method of Kusano et al. (Agric. Biol. Chem., 52, 2293). These three enzyme preparations showed a single band on PAGE. Optimum pH of Pseudomonas isoamylase for amylopectin was 3.5 with a shoulder near pH 5. The optimum pH for Br-CDs shifted from 3.5 for amylopectin to 4.5-4.7 (Br-α- and -β-CDs) and 4.0 (G3-, G4-γ-CDs). The pH curve for Br-γ-CDs had a shoulder near pH 5.0. Optimum pHs of Klebsiella and Bacillus pullulanases for pullulan and Br-γ-CDs were 5.5 and 5. 0, but those for Br-α-CDs were 6.0 and 4. 0, respectively. Kinetic parameters of these enzymes for Br-CDs indicated that (1) all of them cleaved more easily the a(1→6) linkages of G3-G5-CDs than those of G2-CDs, (2) they split more easily the a(1→6) linkages of Br-r-CDs than those of the other Br-CDs, (3) Pseudomonas isoamylase hydrolyzed readily the a(1→6) linkage of G2-7-CD and (4) Br-r-CDs were better substrates for kinetical analysis of debranching amylase than amylopectin and pullulan.

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yHXA is the gene product of a newly found human a-amylase gene (AMY2B) expressed in yeast. Its mode of action on a derivative of p-nitrophenyl α-maltopentaoside, FG5P (FG-G-G-GG-P), was compared with those of the α-amylases (yHSA, yHPA), which were produced by expression of human salivary and pancreatic α-amylase genes (AMY1, AMY2A) in yeast. The product analysis of the digests by HPLC showed that the enzymes hydrolyzed FG5P to FG3 (FG-G-G) and p-nitrophenyl α-maltoside (G-G-P) and to FG4 (FG-G-G-G) and p-nitrophenyl α-glucoside (G-P) and the ratio of the two reactions changed with pH. The three enzymes differed from each other in the mode of action at pH 5.5. The molar ratio of FG4 to FG3 inthe digest with yHXA was the largest and that with yHSA was the least.

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Cyclodextrins (CD_s, tt cyclomaltooligosaccharides) with maltosyl and panosyl branches were produced from maltose or panose and CDs by the reverse action of pullulanase. Purification on columns of octadesylated silica gave G₂-α-CD, G₂-β-CD, Pan-α-CD, and Pan-β-CD. The solubility of G₂-β-CD in aqueous 80% ethanol was higher than that of β-CD in water. The relative rates of degradation of maltose, G₂-α-CD, and G₂-β-CD with glucoamylase were 1 : 3.6 : 5.0 and those of panose, Pan-α-CD, and Pan-β-CD were 1 : 3.0 : 2.2. The rates of degradation of branched and unbranched maltooligosaccharides were markedly different from those of G₂-CD_s and Pan-CD_s.

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Transgalactosylated derivatives of branched cyclodextrins (CDs) were synthesized with Bacillus circulars β-galactosidase under the co-existence of lactose and branched CDs. The structure of the transfer products were determined by β-galactosidase digestion, FAB-MS, and 13C-NMR analysis. B. circulans β-galactosidase produced B-galactosyl-(1→4)-α-glucosyl-(1→6)-βCD and β-galactosyl-(1→4)-β-galactosyl-(1→4)-α-glucosyl-(1→6)-1SCD, or β-galactosyl-(1→4)-α-glucosyl-(1→4)-α-glucosyl-(1→6)-CD and β-galactosyl-(1→4)-β-galactosyl-(1→4)-α-glucosyl-(1→4)-α-glucosyl-(1→6)-αCD from the mixture of lactose and glucosyl-BCD or maltosyl-αCD, respectively. Aspergillus oryzae and Penicillium multicolor β-galactosidases also produced transgalactosylated products of branched CDs. These structures are now under investigation.

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3A, 3C, 3E-O-tris(β-naphthylsulfonyl)-β-cyclodextrin was selectively prepared by the reaction of β-cyclodextrin with β-naphthylsulfonyl chloride in an alkaline solution. The structure determination was carried out by use of a regiochemical relationship between a 3-O-sulfonylcyclodextrin with a 6-O-sulfonylcyclodextrin through a 3, 6-anhydrocyclodextrin.

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During the studies on application of amylases, some a-amylases showed a remarkable transglycosylation action in a high concentration of starch. Using the action, the production of novel and useful saccharides was undertaken, and CGTase was selected. The enzyme catalyzes “cyclization” forming cyclodextrins, “disproportionation” and “coupling reaction, ” in which glycosyl residues are transferred from α-1, 4-glucan or cyclodextrins to an acceptor such as glucose or sucrose. Through the coupling reaction of CGTase we succeeded in the conjugation with glycosyl residue and various substances, and established their usages in food industry and related fields.

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It was observed that a considerable amount of fructosyltransferase, levansucrase, was produced when microorganisms (Bacillus subtilis, Bacillus natto, Zymomonas mobilis, etc.) were grown on a medium containing sucrose and the activity was found in culture filtrate and cells. But most of the activity was retained by the cells together with viscous levan in the case of Bacillus natto. The cells harvested and washed with buffer after cultivation were able to be used as reactors repeatedly for the production of levan by soaking in sucrose solution. A complete liberation of the enzyme from the cells was achieved by treating with 2 M sodium chloride solution. Oligosaccharides synthesized from sucrose in the presence of some acceptor sugars were useful for screening of microorganisms which produce exo-type carbohydrases such as a-glucosidase. Low molecular weight levan synthesized under the condition of high concentration of sodium chloride (2 M) was also useful for screening the microorganisms which produced levanbiose-producing enzyme because of its few branchings. The low molecular levan may be useful as a carbon source for finding enzyme having special specificity such as producing cyclic f ructan. Sucrase (β-fructofuranosidase) obtained from microorganisms produced mainly 1-kestose except yeast invertase, which produced mainly 6-kestose; levansucrase produced three types of kestose (1-kestose, neokestose and 6-kestose) and levan, regardless of enzyme source.

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