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Thermostable trehalose synthase, which catalyzes the conversion of maltose into trehalose by intramolecular transglucosylation, was purified from a cell-free extract of the thermophilic bacterium Thermus aquaticus ATCC 33923 to an electrophoretically homogeneity by successive column chromatographies. The purified enzyme had a molecular weight of 105,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.6 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The optimum pH and temperature were pH 6.5 and 65°C, respectively. The enzyme was stable from pH 5.5 to 9.5 and up to 80°C for 60min. The trehalose synthase from Thermus aquaticus is more thermoactive and thermostable than that from Pimelobacter sp. R48. The yield of trehalose from maltose by the enzyme was independent of the substrate concentration, and tended to increase at lower temperatures. The maximum yield of trehalose from maltose by the enzyme reached 80-82% at 30-40°C. The activity was inhibited by Cu(2+) , Hg(2+), Zn(2+), and Tris.

Highly branched α-glucan molecules exhibit low digestibility for α-amylase and glucoamylase, and abundant in α-(1→3)-, α-(1→6)-glucosidic linkages and α-(1→6)-linked branch points where another glucosyl chain is initiated through an α-(1→3)-linkage. From a culture supernatant of Paenibacillus sp. PP710, we purified α-glucosidase (AGL) and α-amylase (AMY), which were involved in the production of highly branched α-glucan from maltodextrin. AGL catalyzed the transglucosylation reaction of a glucosyl residue to a nonreducing-end glucosyl residue by α-1,6-, α-1,4-, and α-1,3-linkages. AMY catalyzed the hydrolysis of the α-1,4-linkage and the intermolecular or intramolecular transfer of maltooligosaccharide like cyclodextrin glucanotransferase (CGTase). It also catalyzed the transfer of an α-1,4-glucosyl chain to a C3- or C4-hydroxyl group in the α-1,4- or α-1,6-linked nonreducing-end residue or the α-1,6-linked residue located in the other chains. Hence AMY was regarded as a novel enzyme. We think that the mechanism of formation of highly branched α-glucan from maltodextrin is as follows: α-1,6- and α-1,3-linked residues are generated by the transglucosylation of AGL at the nonreducing ends of glucosyl chains. Then AMY catalyzes the transfer of α-1,4-chains to C3- or C4-hydroxyl groups in the α-1,4- or α-1,6-linked residues generated by AGL. Thus the concerted reactions of both AGL and AMY are necessary to produce the highly branched α-glucan from maltodextrin.

Lactosucrose (LS, 4(G)-beta-D-galactosylsucrose) is a non-digestible oligosaccharide, and the consumption of LS selectively increases the proportion of intestinal bifidobacteria. We examined in this study the hypolipidemic potential of LS. An oral triolein tolerance test on rats indicated that LS reduced the elevation of serum triglyceride (TG) and free fatty acids (FFA). Furthermore, LS inhibited the enzymatic digestion of triolein by pancreatic lipase in vitro. NMR spectroscopy showed that LS formed an intermolecular complex with triolein. The long-term consumption of a diet containing 5% LS for 8 weeks significantly decreased the weight of abdominal adipose tissue when compared with that of the control group. Thus, LS may reduce adipose tissue accumulation by inhibiting intestinal lipid absorption via a direct interaction with TG.

Lactosucrose (4G-β-D-galactosylsucrose, LS) is suggested to be an oligosaccharide required for the proliferation of Bifidobacteria in the intestine. We have examined the dietary effects of LS on the intestinal immune function of mice. BALB/c mice were fed with 2 and 5% LS for 4 weeks, and the intestinal mucosal immune responses were determined. In the 2 and 5% LS fed groups, the amounts of IgA in feces and in cecum contents were significantly increased. In addition, IgA, transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6) secretion by Peyer’s patch (PP) cells were enhanced in LS fed mice. In LS fed mice, pH in the cecum was decreased. LS, in addition, suppressed serum IgG1. These results suggest that LS supplementation changes the intestinal environment of microflora, and indirectly enhances the immune function in the gut, and suppresses the systemic immune response to the dominant type 2 helper T (Th2).

Production of a novel cyclomaltopentaose cyclized by an alpha-1,6-linkage, [ICG5; cyclo-{--6)-alpha-D-Glcp-(1--4)-alpha-D-Glcp-(1--4)-alpha-D-Glcp-(1--4)-alpha-D-Glcp-(1--4)-alpha-D-Glcp-(1--}], from starch was performed using isocyclomaltooligosaccharide glucanotransferase (IGTase) derived from Bacillus circulans AM7. The optimal conditions for ICG5-production from partially hydrolyzed starch were as follows: substrate concentration, 1.0% (w/v); pH, 5.5; temperature, 45 degrees C; reaction time, 24 h, IGTase, 1.0 unit/g-dry solid (DS); isoamylase, 2,500 units/g-DS. The yield of ICG5 reached 25.9% under optimal conditions. ICG5-production was achieved from partially hydrolyzed starch using a crude enzyme preparation containing IGTase. Finally, ICG5 was obtained in a yield of 17.9% (99.3% purity, 2,681 g-DS). A digestive test with a human salivary amylase, an artificial gastric juice, a pancreatic amylase, and small intestinal enzymes showed that ICG5 was an indigestible oligosaccharide.

We examined the dietary effects of cyclic nigerosylnigerose (CNN), a dietary indigestible oligosaccharide with four D-glucopyranosyl residues linked by alternating alpha-(1-->3)- and alpha-(1-->6) glucosidic linkages, on the intestinal immune function of mice, and the effects were compared with those of alpha-(1-->3)-linked oligosaccharide (nigerooligosaccharides, NOS) or alpha-(1-->6)-linked oligosaccharide (isomaltooligosaccharides, IMO). BALB/c mice were fed with 1-5% CNN, 5% IMO, or 12.5% NOS for 4 weeks, and the intestinal mucosal immune responses were determined. In the 1-5% CNN fed groups, the amounts of IgA in feces increased significantly. In addition, IgA, transforming growth factor-beta1 (TGF-beta1), and interleukin-6 (IL-6) secretion by Peyer's patch (PP) cells were enhanced in CNN fed mice. In the 5% CNN group, pH in the cecum decreased, and the amounts of lactic acid and butyric acid increased. These findings were not observed in the NOS- or IMO-fed group of mice. They suggest that CNN supplementation changes the intestinal environment of microflora and indirectly enhances the immune function in the gut.

A bacterial strain AM7, isolated from soil and identified as Bacillus circulans , produced two kinds of novel cyclic oligosaccharides. The cyclic oligosaccharides were produced from amylose using a culture supernatant of the strain as the enzyme preparation. The major product was a cyclomaltopentaose cyclized by an α-(1→6)-linkage, cyclo -{→6)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→}. The other minor product was cyclomaltohexaose cyclized by an α-(1→6)-linkage, cyclo -{→6)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→}. We propose the names isocyclomaltopentaose (ICG5) and isocyclomaltohexaose (ICG6) for these novel cyclic maltooligosaccharides having one α-(1→6)-linkage. ICG5 was digested by α-amylase derived from Aspergillus oryzae , cyclomaltodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus , and maltogenic α-amylase. On the other hand, ICG6 was digested by CGTase from B. stearothermophilus and B. circulans , and maltogenic α-amylase. This is the first report of enzymatically produced cyclomaltopentaose and cyclomaltohexaose, which have an α-(1→6)-linkage in their molecules.

We investigated the acceptor specificity of a thermostable trehalose phosphorylase from Thermoanaerobacter brockii ATCC 35047 (TbTP) was examined using beta-D-glucose-1-phosphate (beta-G1P) as a glucosyl donor and oligosaccharides as the acceptor. Oligosaccharides with a reducing-end glucose residue as the C-6 substituent (e.g., isomaltose, gentiobiose, melibiose, isomaltotriose, and isopanose) were found to be successful acceptors. The transfer products of isomaltose, gentiobiose, and melibiose were isolated and characterized as 6-O-alpha-D-glucopyranosyl trehalose (alpha-GlcTre), 6-O-beta-D-glucopyranosyl trehalose (beta-GlcTre), and 6-O-alpha-D-galactopyranosyl trehalose (alpha-GalTre), respectively. To produce alpha-GalTre, a novel nonreducing trisaccharide, the reaction conditions of alpha-GalTre were examined using trehalose as a glucosyl donor. As a result, the yield of alpha-GalTre reached 40.5%.

Malto-oligosyltrehalose synthase (EC 5.4.99.15, MTSase) catalyzes the conversion of α-1,4-glucan to glycosyltrehalose by forming an α,α-1,1-glucosidic linkage on the reducing side of the α-1,4-glucan. This enzyme also slightly hydrolyses the glucan and releases glucose from the reducing end of the glucan. We mutated the gene of MTSase from Sulfolobus acidocaldarius ATCC 33909 and expressed the mutated gene in E. coli. The mutants of Asp228, Glu255 or Asp443 corresponding to the catalytic residues of the α-amylase family enzymes showed no enzymatic activity. The transglycosylation activity of the mutants of Lys390 or Lys445 decreased, but the hydrolytic activity of the mutants increased in comparison to the wild-type enzyme. The substitution of Lys390 or Lys445 for a bulky residue, tryptophan, caused the loss of the transglycosylation activity of the enzyme, and provided a novel hydrolase reacting on the reducing side of the α-1,4-glucan.

We examined the digestibility of cyclic tetrasaccharide (CTS; cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}). CTS was resistant to two types of α-amylase and to artificial gastric juice. Enzymes in the rat small intestinal mucosa slightly hydrolyzed CTS. CTS was not utilized by 22 strains of human intestinal bacteria. CTS was not fermented by rat cecal content. In the single administration to rats, more than 90% of the administered CTS was recovered in the feces. From the above results, CTS was confirmed as an indigestible and unfermentable saccharide. As a result of 4 weeks’ feeding to rats, CTS was effective in the suppression of body fat accumulation and lowering of serum triglyceride of the rats. These effects of CTS depended on the amount of CTS in the diet.

A nonreducing trisaccharide was formed in a reaction mixture containing trehalose and the cell-free extract of a thermophilic anaerobe, Thermoanaerobium brockii ATCC 35047. This saccharide was isolated from the mixture by trehalase digestion, alkaline treatment and preparative HPLC. From the results of partial acid hydrolysis, methylation analysis, and 13C-NMR analysis, the chemical structure of the saccharide was determined to be 2-O-α-D-glucopyranosyl α-D-glucopyranosyl α-D-glucopyrano- side (selaginose). Two enzymes related to Selaginose synthesis were partially purified by DEAEToyopearl column chromatography from the cell-free extract of T brockii. One was a trehalose phosphorylase (EC 2.4.1.64), which catalyzes the reversible phosphorolysis of trehalose into β-glucose-1-phosphate and glucose. The other synthesized kojibiose from β-glucose 1-phosphate and glucose. The latter enzyme was considered to be a novel phosphorylase. The hypothetical mechanism of selaginose synthesis was proposed as follows: trehalose+Pi→glucose+β-glucose 1-phosphate, β-glucose 1-phosphate +trehalose→selaginose+Pi.

The thermophilic anaerobe Thermoanaerobium brockii ATCC 35047 produces a novel phosphorylase, kojibiose phosphorylase, which catalyzes the reversible phosphorolysis of kojibiose to form β-glucose 1-phosphate and D-glucose. The enzyme was purified from a cell-free extract to an electrophoretically homogeneous state by successive column chromatography on DEAE-Toyopearl 650S, CM-Toyopearl 6505, Hydroxyapatite, Ultrogel AcA44, Mono Q, and Butyl-Toyopearl 650 M . The enzyme had a molecular weight of 83, 000 by SDS-polyacrylamide gel electrophoresis and a pl of 4 .3 to 4.4 by gel isoelectrofocusing. The enzyme showed the highest activity at pH 5 .5 and 65°C, and was stable from pH 5.5 to 9.7 and up to 65°C. The enzyme activity was inhibited by Hg2+ and Pb2+0. The Km values for kojibiose, Pi, glucose, and β-glucose 1-phosphate were 0 .77, 0.85, 3.52, and 0.77 mM, respectively.

A thermostable trehalose phosphorylase (EC 2.4.1.64) from a thermophilic anaerobe, Thermoanaero bium brockii ATCC 35047, was purified to an electrophoretically homogeneous state by successive column chromatographies on DEAE-Toyopearl 6505, Butyl-Toyopearl 650 M and Ultrogel AcA 44 . The molecular weight of the enzyme was estimated to be 190, 000 Da by gel filtration, and 88, 000 Da by SDS-polyacrylamide gel electrophoresis. The enzyme showed the highest activity in the range of pH 7.0 to 7.5 for phosphorolysis, and pH 6.0 to 7.0 for synthesis . The optimum temperature of the enzyme was 70°C in both directions. The enzyme was stable from pH 6.0 to 9.0, and up to 60°C . This result showed that our trehalose phosphorylase was the most thermostable in those reported to date . Activity was inhibited by Cue, Hg2+, Mg2+, Mn2+, Pb2+, and Zn2+ The Kms for trehalose, Pi, D-glucose and R-D-glucose 1-phosphate were 0.97, 0.57, 2.4 and 0.75 mM, respectively.

Cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus produced a series of glycosyl-trehaloses through the transglycosylation reaction with cyclomaltohexaose as the glycosyl donor and trehalose as its acceptor. After beta-amylase treatment, five species of glycosyl-trehaloses were isolated by column chromatography. After chemical and enzymatic analyses, it was concluded that these oligosaccharides were alpha-maltosyl alpha-D-glucopyranoside, alpha-maltotriosyl alpha-D-glucopyranoside, alpha-maltosyl alpha-maltoside, alpha-maltotriosyl alpha-maltoside, and alpha-maltotriosyl alpha-maltotrioside. These were not hydrolyzed by salivary amylase, artificial gastric juice, or pancreatic amylase, however they were hydrolyzed by enzymes of the small intestine.

Glycoside hydrolase (GH) family 65 contains phosphorylases acting on maltose (Glc-α1,4-Glc), kojibiose (Glc-α1,2-Glc), trehalose (Glc-α1,α1,-Glc), and nigerose (Glc-α1,3-Glc). These phosphorylases can efficiently catalyze the reverse reactions with high specificities, and thus can be applied to the practical synthesis of α-glucosyl oligosaccharides. Here, we determined the crystal structures of kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus in complex with glucose and phosphate and in complex with kojibiose and sulfate, providing the first structural insights into the substrate recognition of a glycoside hydrolase family 65 enzyme. The loop 3 region comprising the active site of kojibiose phosphorylase is significantly longer than the active sites of other enzymes, and three residues around this loop, Trp391, Glu392, and Thr417, recognize kojibiose. Various mutants mimicking the residue conservation patterns of other phosphorylases were constructed by mutation at these three residues. Activity measurements of the mutants against four substrates indicated that Trp391 and Glu392, especially the latter, are required for the kojibiose activity. Database Structural data are available in the Protein Data Bank database under accession numbers 3WIQ and 3WIR. Structured digital abstract CsKP and CsKP bind by x-ray crystallography (View interaction) .

A novel enzyme, trehalose synthase, was purified from a cell-free extract of Pimelobacter sp. R48 to an electrophoretically homogeneous state by successive chromatographies on DEAE-Toyopearl 650, Butyl-Toyopearl 650, and Mono Q HR5/5 columns. The molecular weight of the enzyme was estimated to be 62,000 by SDS-polyacrylamide gel electrophoresis, and the enzyme had a pI of 4.6 by gel isoelectrofocusing. The enzyme catalyzed the conversion of maltose into trehalose by intramolecular transglucosylation. The enzyme also converted into maltose but was inactive on other saccharides. The N-terminal amino acid of the enzyme was serine. The optimum pH and temperature were pH7.5 and 20 degrees C, respectively. The enzyme was stable in the range of pH 6.0-9.0 and up to 30 degrees C for 60 min. The rate of conversion of maltose into trehalose was independent of the maltose concentration. The maximum yield of trehalose from maltose were 81.8%, 80.9%, and 76.7% at 5, 15, and 25 degrees C, respectively. The activity was inhibited by Cu2+, Hg2+, Ni2+, Zn2+, and Tris.

A novel enzyme, maltooligosyl trehalose trehalohydrolase from Arthrobacter sp. Q36 was purified from a cell-free extract to an electrophoretically pure state by successive column chromatography on Sepabeads FP-DA13, Butyl-Toyopearl 650M, DEAE-Toyopearl 650S, and Toyopearl HW-55S. The enzyme specifically catalyzed the hydrolysis of the alpha-1,4-glucosidic linkage that bound the maltooligosyl and trehalose moieties of maltooligosyl trehalose. The Km of the enzyme for maltosyl trehalose, maltotriosyl trehalose, maltotetraosyl trehalose, and maltopentaosyl trehalose was 5.5 mM, 4.6 mM, 7.0 mM, and 4.2 mM, respectively. The enzyme had a molecular mass of 62,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.1 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was threonine. The enzyme showed the highest activity at pH 6.5 and 45 degrees C, and was stable from pH 5.0 to 10.0 and up to 45 degrees C. The activity was inhibited by Hg2+, Cu2+, Fe2+, and Zn2+.

One kojibiose phoshorylase (KP) homolog gene was cloned from Caldicellulosiruptor saccharolyticus ATCC43494. Recombinant KP from C. saccharolyticus (Cs-KP) expressed in Escherichia coli showed highest activity at pH 6.0 at 85 °C, and was stable from pH 3.5 to 10.0 and up to 85 °C for phosphorolysis. Cs-KP showed higher productivity of kojioligosaccharides of DP ≧ 4 than KP from Thermoanaerobacter brockii ATCC35047.

B. stearothermophzlus由来のCGTaseを高濃度のα-CDに作用させることによりアミロース粒子が生成することを見出し,その生成条件ならびにその諸性質を調べ,以下の結果を得た.1) アミロース粒子の生成は,主に反応温度,基質濃度に影響され,α-CD濃度35%,反応温度30℃で最高の生成率96%を示した.2) 他の細菌起源のCGTaseにおいてもアミロース粒子の生成が認められた.3) 得られるアミロース粒子は3～20gumの球状で,そのλmaxは560～580nmを示し,数平均分子量は5500～7000で,グルコース・ユニットとして34～43個からなるアミロースであった.4) 本物質はX線回折により澱粉のB形X線回折図を示し,偏光十字が観察されることから規則性のある結晶構造を有するアミロースであることがわかった.

The bacterial strain PP710, isolated from soil and identified as Paenibacillus species, produced a low-digestibility alpha-glucan containing a large amylase-resistant portion. This alpha-glucan was obtained in high yields from maltodextrin (dextrose equivalent 3) by using the condensed culture supernatant of the strain as the enzyme preparation. The water-soluble dietary fiber content of the low-digestibility alpha-glucan was 80.2%, and showed resistance to a rat intestinal enzyme preparation. The alpha-glucan was found to be a novel highly branched alpha-glucan by acid hydrolysis, NMR analysis, gel permeation chromatography, methylation analysis, and enzymatic digestion.

A bacterial strain M6, isolated from soil and identified as Arthrobacter globiformis, produced a novel nonreducing oligosaccharide. The nonreducing oligosaccharide was produced from starch using a culture supernatant of the strain as enzyme preparation. The oligosaccharide was purified as a crystal preparation after alkaline treatment and deionization of the reaction mixture. The structure of the oligosaccharide was determined by methylation analysis, mass spectrometry, and (1)H and (13)C NMR spectroscopy, and it was demonstrated that the oligosaccharide had a cyclic structure consisting of four glucose residues joined by alternate alpha-(1--4)- and alpha-(1--6)-linkages. The cyclic tetrasaccharide, cyclo-{--6)-alpha-D-Glcp(1--4)-alpha-D-Glcp(1--6)-alpha-D-Glcp(1--4)-alpha-D-Glcp(1--}, was found to be a novel oligosaccharide, and was tentatively called cyclic maltosyl-maltose (CMM). CMM was not hydrolyzed by various amylases, such as alpha-amylase, beta-amylase, glucoamylase, isoamylase, pullulanase, maltogenic alpha-amylase, and alpha-glucosidase, but hydrolyzed by isomalto-dextranase to give rise to isomaltose. This is the first report of the cyclic tetrasaccharide, which has alternate alpha-(1--4)- and alpha-(1--6)-glucosidic linkages.

A gene encoding a trehalose phosphorylase was cloned from Thermoanaerobacter brockii ATCC 35047. The gene encodes a polypeptide of 774 amino acid residues. The deduced amino acid sequence was homologous to bacterial maltose phosphorylases and a trehalose 6-phosphate phosphorylase catalyzing anomer-inverting reactions. On the other hand, no homology was found between the T. brockii enzyme and an anomer-retaining trehalose phosphorylase from Grifola frondosa.

Glucosyl-L-sorbose, -maltose, and -sucrose were synthesized using kojibiose phosphorylase (KPase) from Thermoanaerobacter brockii ATCC35047 with beta-D-glucose-1-phosphate (beta-G1P) as a glucosyl donor. One disaccharide and two trisaccharides thus synthesized were isolated by Toyopearl HW-40S column chromatography. The results of KPase digestion, methylation analysis, and 13C-NMR studies indicated that these oligosaccharides were alpha-D-glucopyranosyl-(1-->5)-alpha-L-sorbopyranose, alpha-D-glucopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->4)-D-glucopyranose (4-alpha-D-kojibiosyl-glucose), and alpha-D-glucopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->2)-beta-D-fructofuranoside, which are all novel oligosaccharides. Glucosyl-L-sorbose was partially hydrolyzed to glucose and L-sorbose by alpha-glucosidases, while glucosyl-sucrose and glucosyl-maltose were not hydrolyzed by glucoamylase, alpha-glucosidases, or CGTase.

Glycosyl-trehaloses with an isomaltosyl residue were synthesized by alpha-glucosidase from Aspergillus niger by using maltotetraose as a glucosyl donor and trehalose as the acceptor. The one trisaccharide and two tetrasaccharides formed were isolated by successive column chromatography. The results of an enzymatic digestion, methylation analysis, and 13C-NMR studies indicated that these oligosaccharides were alpha-isomaltosyl alpha-glucoside, alpha-isomaltotriosyl alpha-glucoside and alpha-isomaltoside. These oligosaccharides were not fermented to an acid by Streptococcus mutans, and they effectively inhibited water-insoluble glucan synthesis from sucrose by glucosyltransferase. In an in vitro utilization test with human intestinal bacteria, these oligosaccharides were predominantly utilized by Bifidobacteria.

A thermostable maltooligosyl trehalose trehalohydrolase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius ATCC 33909 was purified from a cell-free extract to an electrophoretically pure state by successive column chromatographies on Sepabeads FP-DA13, Butyl-Toyopearl 650M, DEAE-Toyopearl 650S, Toyopearl HW-55S and Ultrogel AcA44. The enzyme had a molecular mass of 59,000 by SDS-polyacrylamide gel electrophoresis and a pI of 6.1 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The enzyme showed the highest activity from pH 5.5 to 6.0 and at 75 degrees C, and was stable from pH 5.5 to 9.5 and up to 85 degrees C. The activity was inhibited by Hg2+, Cu2+, Fe2+, Pb2+, and Zn2+. The Km values of the enzyme for maltosyl trehalose, maltotriosyl trehalose, maltotetraosyl trehalose, and maltopentaosyl trehalose were 16.7 mM, 2.7 mM, 3.7 mM, and 4.9 mM, respectively.

Arthrobacter sp. Q36 produces a novel enzyme, maltooligosyl trehalose synthase, which catalyzes the conversion of maltooligosaccharide into the non-reducing saccharide, maltooligosyl trehalose (alpha-maltooligosyl alpha-D-glucoside) by intramolecular transglycosylation. The enzyme was purified from a cell-free extract to an electrophoretically homogeneous state by successive column chromatography on Sepabeads FP-DA13, DEAE-Sephadex A-50, Ultrogel AcA44, and Butyl-Toyopearl 650M. The enzyme was specific for maltooligosaccharides except maltose, and catalyzed the conversion to form maltooligosyl trehalose. The Km of the enzyme for maltotetraose, maltopentaose, maltohexaose, and maltoheptaose were 22.9 mM, 8.7 mM, 1.4 mM, and 0.9 mM, respectively. The enzyme had a molecular mass of 81,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.1 by gel isoelectrofocusing. The N-terminal and C-terminal amino acids of the enzyme were methionine and serine, respectively. The enzyme showed the highest activity at pH 7.0 and 40 degrees C, and was stable from pH 6.0 to 9.5 and up to 40 degrees C. The enzyme activity was inhibited by Hg2+ and Cu2+.

A trehalose-producing bacterium, Arthrobacter sp. strain Q36, was isolated from soil. From a supernatant of the culture broth, two novel enzymes related to trehalose synthesis were partially purified by Sepabeads FP-DA column chromatography. One enzyme catalyzed the conversion of maltopentaose into maltotriosyl trehalose by intramolecular transglycosylation, showing it to be maltooligosyl trehalose synthase. The other hydrolyzed the product transferred by the former into maltotriose and trehalose specifically, showing it to be maltooligosyl trehalose trehalohydrolase. In addition to the bacterial strain isolated, several bacteria kept in our laboratory were found to produce these enzymes. The enzymatic system was proposed to be a novel biosynthesis of trehalose in bacteria involving the following reactions : maltodextrin→maltooligosyl trehalose, maltooligosyl trehalose→maltodextrin + trehalose. When these enzymes acted on amylose simultaneously, the trehalose in the reaction mixture reached more than 80% in content.

A thermostable trehalose synthase from Thermus aquaticus ATCC 33923, which catalyzes the interconversion between maltose and trehalose by intramolecular transglucosylation, converted sucrose into trehalulose (1-O-α-d-glucopyranosyl-d-fructose). The trehalulose-forming activity of the enzyme was very low compared with that of maltose and trehalose. Kinetic studies showed that sucrose competitively inhibited the interconversion activity between maltose and trehalose. Consequently, these three substrates, maltose, trehalose, and sucrose, are thought to bind the same active site of trehalose synthase.

A bacterium, Pimelobacter sp. R48, isolated from soil, showed the ability to produce trehalose from maltose. The partially purified enzyme from a cell-free extract catalyzed the conversion of maltose into trehalose without requiring phosphate. The enzyme was considered to be a new intramolecular glucosyltransferase. The enzyme was also tentatively found to exist in Pseudomonas putida H262 isolated from soil and in some Thermus strains.

At least, four kinds of amylase inhibitors are found in culture of Streptomyces sp. No.280.1) A large amount of amylase inhibitors were produced by Streptomyces sp. No. 280 when cultivated on 3% oatmeal medium and it was found that the molecular weight of the inhibitors were transformed to smaller molecules during the cultivation time. The transformation of the amylase inhibitor was found to result from degradation of its carbohydrate moiety by α-amylase in the culture broth. The amylase inhibitor was hydrolyzed partially by the action of taka-amylase A or hog pancreatic α-amylase. With hydrolyzation of amylase inhibitor by α-amylase, neutral sugars (mainly maltose) were liberated from the amylase inhibitor and a modified inhibitor was newly formed, but amylase inhibitory activity against glucoamylase was not changed. The inhibitory activity against muscle Phosphorylase a, however, was almost completely lost.

We found that the new amylase inhibitor produced by Streptomyces sp . No. 280 strongly inhibited animal a-amylase, bacterial saccharogenic a-amylase, and fungal glucoamylase, but not pullulanase, nor plant jS-amylase. This amylase inhibitor can be separated by chromatography into four fractions (inhibitor A, B, B' and C) that were homogeneous by paper chromatography and paper electrophoresis . These inhibitors acted not only on glucoamylase and a-amylase, but also on a-glucosidase, cycloamylose glucanotransferase, and phosphorylase. The amylase inhibitors A and B slightly activated yeast a-glucosidase. These inhibitors contained both carbohydrates (mainly D-glucose) and amino acids; their molecular weights ranged from 1, 000 to 3, 000.

Pullulanase was immobilized successfully by simple, inexpensive methods that may be useful for industrial application of this enzyme. A tannin--pullulanase(TP) complex was obtained by addition of tannic acid to the culture filtrate of thermophilic Streptomyces flavochromogenes. TP could be bound to TEAE--cellulose (TTCP). Immobilization in this manner took place with quantitative retention of activity. The immobilized enzymes were stable for more than six months. The optimum temperatures of the native enzyme and TP were both 50 degrees C; that of TTCP was 45 degrees C. In the presence of 5mM Ca2+, the activity of TTCP was increased approximately twofold and the optimum temperature was raised to 50--60 degrees C. Pullulanase was not significantly eluted from TP or TTCP by NaCl solution (0.1--0.5M).