The specificity of the neopullulanase from Bacillus stearothermophilus was altered by protein engineering. The amino acid residues constituting the active center of the neopullulanase were tentatively identified according to a molecular model of Taka-amylase A and homology analysis of the amino acid sequences of neopullulanase, Taka-amylase A, and other amrlolytic enzymes. When one of the putative catalytic sites (Glu-357, Asp-424, and Asp-328) were replaced by the opposite charged (His) or non-charged (Gin or Asn) amino acid residue, neopullulanase activities toward α-(1→4)- and α-(1→6)-glucosidic linkages disappeared . When the putative substrate-binding sites were replaced, the specificities of the mutated neopuliulanases toward α-(1→4)- and α-(1→6)-glucosidic linkages were obviously different from that of the wild-type enzyme. This finding proves that one active center of the neopullulanase participated in the dual activity toward α-(1→4)- and α-(1→6)-glucosidic linkages. Some mutated neopullulanases exhibited higher specificities toward the α-(1→4) linkages relative to the wild-type enzyme. The mutated enzymes exhibiting higher specificities toward theα-(1→6) linkages were also obtained. These mutated neopullulanases were tested for the production of panose, a branched oligosaccharide which might be used as an anticariogenic sweetener . The production ratio of panose from puliulan was significantly increased by using the mutated neopullulanase which exhibited higher specificity toward the α-(1→4)-glucosidic linkage. In contrast, the production ratio of panose was obviously decreased by using the mutated neopullulanase which exhibited higher specificity toward the α-(1→6)-glucosidic linkage.

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The 210 th lysine residue in Saccharomycopsis α-amylase (Sfamy) molecule was replaced by arginine and asparagine residues. The resulting K210R and K210N enzymes cleave mainly the first glycosidic bond from the reducinng end of maltotetraose (G₄), while the native enzyme hydrolyzes mainly the second bond. We changed successfully the major cleavage point in the hydrolysis reaction of G₄. We estimated the 8th subsite affinities of the mutant enzymes and compared them with that of the native enzyme. These facts suggest that the K210 residue composes the 8th subsite, one of the major subsites, and that a positively charged s-amino residue is necessary for the 8th subsite affinity. The reduced catalytic activity specifically for the short substrates is also attributable to the remarkable decrease in the affinity of the 8th subsite. The 84th tryptophan residue was replaced by leucine residues. The resulting W84L enzyme showed an increase in transglycosylation activity. At a 40% digestion point of maltoheptaose (G₇), for example, maltooligosaccharide products larger than maltodecaose (G₁₀) amounted to approx. 60% of the total product from the mutant enzyme reaction, whereas no such large products were observed in the native enzyme reaction. These large products were formed by addition of the hydrolysis products on the nonreducing end side to the starting intact substrates. These results suggest that the W84 residue located at subsite 5 plays an important role in the addition of a water molecule to a carbonium ion intermediate and/or in the liberation of the hydrolysis product from the substrate binding pocket. The doubly mutated enzymes, W84LK210 N, are expected to form the transglycosylation products different in size from those produced by the single mutant, W84L enzyme.

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The gene coding for the maltopentaose (G₅)-forming enzyme of Pseudonaonas sp. KO-8940 was cloned into Escherichia coli and its nucleotides sequenced. It was found to have a long open reading frame composed of 1842 by that encoded 614 amino acid residues for a secretory precursor polypeptide including the typical signal sequence with an NH₂-terminal. In the deduced primary structure of this enzyme, a high degree of homology to four regions conserved by many α-amylases was found, and the COOH-terminal portion of this enzyme showed high homology with other raw starch digesting amylases. The G₅-forming enzyme was produced in large amount (52.7 IU/ml, 0.1 g/l) in E. coli under the tac promoter. This result showed that the G₅-forming enzyme can be produced in E. coli carrying this enzyme gene expression vector at levels up to 6 times greater than the native production system found in P, sp. KO-8940.

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A novel CGTase (Cyclomaltodextrin glucanotransferase) has been isolated from a strain of Thermoanaerobacter, a thermophilic anaerobe. The enzyme is extremely heat stable and has a temperature optimum of 90-95°C at pH 6.0. It is active over a broad pH range, and exhibits more than 80% activity from pH 5.0-6.7. In the presence of starch, the enzyme is stable at temperatures in excess of 100°C. In addition to producing cyclodextrins from starch, Thermoanaerobacter sp. CGTase has excellent starch liquefying properties. It is possible to liquefy a 35% DS starch slurry at pH 4. 5, in the absence of calcium, using the "jet cooker" process developed for B. licheniformis α-amylase (105°C for 5 min, 90-95°C for 90 min). Saccharification of the liquefied starch with A. niger glucoamylase can therefore be carried out without further pH adjustment. Some of the properties of Thermoanaerobacter sp. CGTase are described, and examples of the use of the enzyme for cyclodextrin production, intermolecular transglycosylation and starch liquefaction are given. The gene coding for Thermoanaerobacter sp. CGTase has been transfered to a Bacillus host, making large-scale production of the enzyme in commercially acceptable yields possible.

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An extracellular enzyme from Bacillus circulans OKUMZ 31B produced cycloinulo-oligosaccharides (CFs) from inulin. The enzyme, designated as cycloinulo-oligosaccharide fructanotransferase, was purified from the cultured broth to homogeneity. The enzyme is monomeric protein having molecular weight of about 132, 000 and optimum pH is 7-7.5. The enzyme catalyzes not only cyclization but also disproportionation, coupling and hydrolyzing reactions against β-2, 1 fructan. The molecular structure of cycloinulohexaose (CF₆), a main product of the enzyme reaction, was determined by X-ray crystallographic analysis. The molecule has C₃ symmetry with asymmetric units of inulobiosyl moieties in which two D-fructofuranosyl residues have 4T₃ conformations. The molecule has an 18-crown-6 moiety which shows the GTGTGT conformational arrangement of the six sequential -0-CH₂-C-O- units . Interactions of CFs and metal ions were examined by ligand exchange chromatography . Considerable interaction between CF₆ and Ba²⁺ was shown in H2O. In aq. 50% (v/v) methanol, CF₆ interacts with Bat, Pb²⁺, Ag⁺, K⁺, Rb⁺ and Cs⁺. A conductometric experiment suggested that CF6 and Ba²⁺ form a complex in the ratio of 1 : 1 in aq. 50% (v/v) methanol.

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Among several fungal β-xylosidases, Aspergillus niger β-xylosidase had the highest hydrolytic activity and stability in the presence of water miscible solvents such as acetone and alcohols. The enzymatic synthesis of alkyl β-xylosides from xylobiose and alcohols through the transxylosylation reaction of the enzyme was studied. Various alkyl β-xylosides were effectively synthesized from xylobiose and water miscible alcohol such as methanol, ethanol and 2-propanol. Water immiscible alcohol such as 1-butanol, 1-hexanol, benzyl alcohol and 2-butanol, also acted as effective acceptors for transxylosyl reaction, where a great part of synthesized β-xylosides were found in the insoluble alcohol layer. Therefore, the synthesized β-xyloside, such as l-hexyl β-xyloside, could be readily separated from the reaction mixture and crystallized. Xylooligomers and xylan hydrolyzates acted as an effective xylosyl donor . Accumulation factors of alkyl β-xyloside produced enzymatically in the transxylosylation and superiority of Asp, nigerβ-xylosidase for the enzymatic synthesis of alkyl β-xylosides were also described.

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We have established some novel synthetic transformations of simple sugars into useful oligosaccharides utilizing the transglycosylation of glycosidases in aqueous -organic solvent system. p-Nitrophenyl penta-N-acetyl-β-chitopentaoside, which is useful as a novel substrate for lysozyme assay, was efficiently synthesized through a lysozyme-catalyzed transglycosylation on penta-N-acetylchitopentaose and p-nitrophenyl β-N-acetylglucosaminide. In this case, the use of an aqueous-DMSO system in the reaction not only ensured the solubility of chromogenic substrate, but also resulted in the high yield of the desired compound. This concept was introduced for the preparation of p-nitrophenyl α-maltopentaoside from maltopentaose and p-nitrophenyl α-Dglucoside by the use of maltotetraose-forming amylase from Pseudomonas stutzeri. Furthermore, p-nitrophenyl N-acetyl-Iactosaminide and p-nitrophenyl N-acetyl -allolactosaminide were regioselectively synthesized from lactose and p-nitrophenyl N acetylglucosaminide by using the transglycosylation of β-D-galactosidase from Bacillus circulars by controlling the concentration of acetonitrile in the reaction system, respectively.

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Arthrobacter sp. K-1 β-fructofuranosidase catalyzed both transfructosylation and hydrolytic action, when it was incubated with sucrose alone. But in the presence of a suitable acceptor such as D-xylose and lactose, the enzyme catalyzed mostly transfructosylation and transferred the fructose residue preferentially to the acceptor. The enzyme had wide acceptor specificities. D-Xylose, D-galactose, L-sorbose, D- and L-fucose, D- and L-arabinose, maltose, isomaltose, cellobiose, lactose, melibiose, xylobiose, maltotriose, methyl β-glucoside, and galactoside were efficient acceptors in the transfructosylation. On the other hand, D-ribose, L-rhamnose, D-mannose, 2-deoxy-D-glucose, D-galactosamine, D-galacturonic acid, and 1-kestose were not efficient acceptors. Various primary alcohols, polyhydric alcohols including some sugar alcohols, and some glycosides acted as acceptors, but secondary alcohols with one hydroxyl group such as 2-propanol and 2-butanol were not effective as acceptors. The requirement for an acceptor of the transfructosylation by the enzyme is that the structure must have free hydroxyl groups of the equatorial bonds at C2 and C3 on ⁴C₁ or ¹C₄ conformation. The main transfer products to aldoses and ketoses by the enzyme were nonreducing oligosaccharides, which had a fructofuranosyl residue bounded to their hemiacetal hydroxyl groups. In the case of D-galactose and L-arabinose, the enzyme produced not only non-reducing oligosaccharides, but also reducing oligosaccharides, identified as 3-O-β-D-fructofuranosyl-D-galactose and 4-O-β-D-fructofuranosyl-L-arabinose, respectively. In the cases of glycosides such as methyl a-D-glucoside, the enzyme transferred the fructofuranosyl residue only to C6 hydroxyl group. This enzyme is useful to synthesize heterooligosaccharide containing fructose. The lactosucrose has been produced on industrial scale by using this enzyme, and supplied in large quantities for food applications. The lactosucrose is not digestible in the human small intestine, but it is fermented by human intestinal microorganisms, especially by Bif dobacterium. The administration of the saccharide will improve the intestinal bacterial flora.

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