The amino acid sequence of soybean β-amylase (isozyme 2) revealed 60 and 30% homology with those of β-amylase from barley and Bacillus polymyxa, respectively. Two SH groups (SH2 and SH4) of soybean β-amylase are located in the highly conserved regions between the sequences of the higher plants and the bacterial enzymes . The chemical modification of the two SH groups resulted in the loss of the enzymatic activity . SH2 and SH4 were identified to be Cys 95 and Cys 343 by the analyses of chymotryptic digest of the enzyme after selective fluorescein-labeling of the two SH groups. As the modification of SH2 decreased the Vmax for amylopectin and the binding ability for glucose depending on the size of the substituents at SH2, it was concluded that SH2 is located near the subsite 1 of the enzyme. The X-ray crystallography of trigonal crystals of soybean β-amylase indicated that the molecule is composed of a large and of a small domain . The high resolution study of soybean β-amylase is now in progress for the hexagonal crystals using the oscillation camera method.

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Three sulfhydryl groups occur in Bacillus cereus β-amylase molecule : two of them, which are located closely on the polypeptide chain, form intramolecular -S-S- linkage ; one of them is free -SH form, which is involved in enzyme activity. The amino acid sequences around these sulfhydryl groups are HQCGGNVGDDCNVPIPSW or TCLEM, respectively. The free sulfhydryl group in B.cereus β-amylase corresponds to cys324 in B. polymyxa β-amylase, cys343 in soybean β-amylase, and cys34l in barley β-amylase. The amino acid sequences around the sulfhydryl groups are highly homologous in each β-amylase. These data support the idea that the sulf hydrylgroups in each β-amylase molecule are of importance to maintain an environment of enzyme activity.

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The affinity labeling of a functional carboxyl group in the active site of B-amylase is described. 2, 3-Epoxypropyl α-D-glucopyranoside (α-EPG) is a specific irreversible inactivator for β-amylases (soybean, sweet potato, barley, and Bacillus cereus), and the binding of α-EPG is stoichiometric, i, e., one α-EPG molecule per an active site of enzyme, α-EPG acts as an affinity labeling reagent and a mechanism-based inactivator for soybean p-amylase . It is demonstrated that the carboxylate of Glu 186 affinity-labeled by α-EPG is a functional group (pKa3.5) at the catalytic site of soybean β-amylase. The amino acid sequence around the Glu 186 is well conserved among all of the five β-amylases with known sequences.

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β- Amylase [EC 3.2.1.2 a-1, 4-glucan maltohydrolase] is an exo-acting amylase, cleaving α-1, 4 linkages sequentially from the non-reducing end of starch, producing β-maltose and β-limit dextrins as the end products, ?A-Amylases are widely distributed in higher plants and some bacteria. Sweet potato β-amylase was first crystallized by Balls et al. in 1948. Some physicochemical properties, substrate specificity, and action mechanism have been reported. Sweet potato β-amylase is a homotetrameric enzyme with MW of 215; 000, judging from the experimental results. The complete amino acid sequence was established by the strategy of protein chemistry. The subunit of the enzyme consisted of 496 amino acid residues giving Mr of 55, 707. The native enzyme was affinity labeled with 2, 3-epoxypropyl-α-D-glucopyranoside (α-EPG). The α-EPG was incorporated stoichiometrically (one mol α-EPG/mol subunit) into the enzyme, resulting in the complete loss of the enzymatic activity. The Glu-187 affinity labeled with α-EPG was identified by isolating and sequencing the ¹⁴C-α-EPG-peptide. Sequence homology of sweet potato enzyme with those from soybean and barley was 64 and 53%, respectively. Ba polymyxa and B. circulans enzymes were 80% homologous with each other. It is noteworthy that there are at least six highly conserved sequences throughout plants and bacterial β-amylases and that the active site Glu-187 in sweet potato enzyme is the invariant residue in one of these conserved regions. These results suggest that glutamate residue corresponding to the Glu-187 in sweet potato β-amylase is the active site of other β-amylases from different origins.

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A crude "inulinase" system from A. ficuum has been partly characterized and shown to contain at least 3 major enzyme components: Exo-inulinase [EC 3.2.1.80], Endo-inulinase [EC3.2.1.7] and fructosyltransferase [EC 2.4.1.99]. The fructosyltransferase has a pH-optimum of about 5.0 and a temperature optimum of 50°C. It can be used to produce "Neosugar" (a mixture of 1-kestose, nystose etc.) from sucrose. The endo-inulinase has a pH-optimum of about 5.0 and a temperature optimum of 55-60°C. It can convert inulin to a mixture of fructooligosaccharides, mainly inulotriose and inulotetraose. When used in combination with exo-inulinase, it provides an efficient system for producing fructose from inulin.

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We have studied a new procedure for the production of di- or trisaccharides using various glycosidases. In this "column system reaction, " the solution of substrates is circulated through columns of immobilized enzyme and activated carbon connected in series . The procedure is based on the preferred adsorption, by activated carbon, of di- or trisaccharides over monosac charides. By eliminating the products from the system, the equilibrium is expected to shift continually toward di- or trisaccharide formation . After an appropriate time of circulation, dior trisaccharides accumulated in the activated carbon column could be eluted with an aqueous ethanol solution. In the present study, we have applied this procedure to the synthesis of the following oligosaccharides. 1. D-Glucobioses from D-glucose using α- or β-glucosidases . 2. D-Galactosyl-D-fructoses from D-galactose and D-fructose using β-galactosidases. 3. D-Galactosyl sucroses from D-galactose and sucrose using α- or β-galactosidases. The column system reaction was shown to be exceedingly regioselective . In the batch system reaction, starting materials and products are in equilibrium after a prolonged incubation. In the column system reaction, on the other hand, the products are not in equilibrium because they are immediately adsorbed onto the activated carbon column as soon as they are produced . Consequently, the composition of the products by the column system reaction would reflect the substrate specificity of enzymes.

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β-Galactosidases (lactases) from various origins give different kinds of linkage and size of oligosaccharides. In the first chapter, enzyme sources, mechanism of transgalactosylation reactions, and structures of oligosaccharides were briefly introduced. Galactooligosaccharides (TOS) which consist of tri, tetra-, penta-, and hexasaccharides were formed from lactose by transgalactosylation reactions of Aspergillus oryzae β-galactosidase.TOS were a non-absorbable sugar and selectively utilized by all of the Bifidobacterium species in vitro. Administration of TOS caused remarkable increase in the number of Bifidobacterium and decrease in the number of Bacteroides in fecal flora. The improvement of intestinal microflora by TOS led to the suppression of putrefaction in the gut. It was concluded that TOS were a superior bifidus growth-promoting factor. For the large-scale production of TOS, the optimum conditions of transgalactosylation reactions and the manufacturing process were investigated. The amount of TOS were increased together with increase in initial lactose by A. oryzae S-galactosidase, and the maximum TOS formed were 30% by weight of total sugar. After transgalactosylation reactions with A .oryzae enzyme, second reactions with Streptococcus thermophilus β-galactosidase gave rise to TOS content and to sweetness, which were of a desirable sugar composition for food applications. A, oryzae β-galactosidase was immobilized by glutaraldehyde crosslinking on the ion-exchange resin for the continuous production of TOS. Half-life of this immobilized enzyme was 192 days at 55°C. High-grade TOS could be obtained from the reaction mixture by chromatographic separation with sodium-form cation-exchange resin.

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1. Two galactanases (I and II) from Penicillium citrinum were purified to a homogeneous state. These enzymes were electrophoretically distinctive, but were very similar in proteochemical and enzymatic properties. The enzymes hydrolyzed soybean arabinogalactan (SAG) in an endo manner. They also acted on o-nitrophenyl-β-D-galactoside (ONPG), p-nitrophenyl-β-Dgalactoside and, β-1, 4-galactobiose (Gal₂) after lag phases. 2. The action of galactanase I on ONPG was studied. In the course of the reaction, the accumulation of various transfer products was observed by thin-layer chromatography. The products were isolated from the reaction mixture and their structures were examined. They were a series of β-1, 4-linked galactooligosaccharides (Gal₂-Gal₄) and o-nitrophenol (ONP)-substituted oligosaccharides (G₂-ONP-G₅-ONP). The enzyme liberated ONP without significant lag phases from G₂-ONP-G₅-ONP at a much faster rate than that from ONPG. Furthermore, the lag phase of ONP liberation from ONPG could be eliminated by the addition of Gala or Gal4 to the mixture. Thus a reaction mechanism that involved transfer reaction in addition to hydrolysis was proposed for the cause of the observed lag . 3. The transfer reaction catalyzed by galactanase I was studied using SAG as a donor. The enzyme had a broad acceptor specificity and gave transfer products from several alcohols, monosaccharides, sugar alcohols, glycerol and its derivatives, and catechol. Formation of transfer products from SAG and glycerol was examined in detail. In the course of the reaction, transfer products with various degrees of polymerization were accumulated, suggesting the enzyme activity as transferring galactosyl and galactooligosyl units to the acceptor. The amounts of transfer products depended on the glycerol concentration, and about 50% of the galactose residues which could be liberated from SAG by the enzyme were transferred to glycerol at an acceptor concentration of 2.5%. Two transfer products were isolated and their structures were examined. They were 2-O-β-galactosyl glycerol and 2-O-β-galactobiosyl glycerol, whereas the transfer product from ONPG and glycerol by Escherichia coli β-galactosidase was 1-O-β-galactosyl glycerol.

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