Yoshio Tsujisaka

Purification and Characterization of Thermostable Maltooligosyl Trehalose Trehalohydrolase from the Thermoacidophilic Archaebacterium Sulfolobus acidocaldarius

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.

Purification and properties of a polyvinyl alcohol-degrading enzyme produced by a strain of Pseudomonas.

Abstract An enzyme which degraded polyvinyl alcohol, a water-soluble synthetic polymer, was isolated as a single protein from a culture of a strain of Pseudomonas . The pink-colored enzyme had absorption maxima at 280, 370, and 480 nm, a molecular weight of about 30,000, and an isoelectric point at about pH 10.3. The enzyme was most active at pH values from 7 to 9 and at 40 dgC and was stable at pH values from 3.5 to 9.5 and at temperatures below 45 dgC. The viscosity of the reaction mixture decreased and the pH dropped when the enzyme acted on polyvinyl alcohol as a substrate. Furthermore, the enzyme required O 2 for the reaction and produced 1 mol of H 2 O 2 , per 1 mol of O 2 consumed. The molecules of polyvinyl alcohol were cleaved into small fragments with a wide distribution of molecular weights. Inorganic Hg ions markedly inactivated the enzyme, and the activity was immediately recovered by glutathione. Enzyme inhibitors tested, which included p -chloromercuribenzoic acid, KCN, o -phenanthroline, and H 2 O 2 , showed no effect on the activity. Polyvinyl alcohol oxidized by periodic acid was similarly oxidized by the enzyme. The enzyme did not oxidize most of a variety of low molecular weight hydroxy compounds examined, such as primary alcohols, secondary alcohols, tertiary alcohols, diols, triols, and polyols, except for some secondary alcohols, such as 4-heptanol.

Purification and Characterization of a Thermostable Trehalose Synthase from Thermus aquaticus

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.

Purification and characterization of glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide in Bacillus globisporus C11.

Glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide (CTS; cyclo {→6}-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl- (1→)) from α-1,4-glucan were purified from Bacillus globisporus C11. The former was a 1,6-α-glucosyltransferase (6GT) catalyzing the α-1,6-transglucosylation of one glucosyl residue to the nonreducing end of maltooligosaccharides (MOS) to produce α-isomaltosyl-MOS from MOS. The latter was an isomaltosyl transferase (IMT) catalyzing α-1,3-, α-1,4-, and α,β-1,1-intermolecular transglycosylation of isomaltosyl residues. When IMT catalyzed α-1,3-transglycosylation, α-isomaltosyl-(1→3)-α-isomaltosyl-MOS was produced from α-isomaltosyl-MOS. In addition, IMT catalyzed cyclization, and produced CTS from α-isomaltosyl-(1→3)-α-isomaltosyl-MOS by intramolecular transglycosylation. Therefore, the mechanism of CTS synthesis from MOS by the two enzymes seemed to follow three steps: 1) MOS→α-isomaltosyl-MOS (by 6GT), 2) α-isomaltosyl-MOS→α-isomaltosyl-(1→3)-α- isomaltosyl-MOS (by IMT), and 3) α-isomaltosyl-(1→3)-α-isomaltosyl-MOS→CTS +MOS (by IMT). The molecular mass of 6GT was estimated to be 137 kDa by SDS-PAGE. The optimum pH and temperature for 6GT were pH 6.0 and 45°C, respectively. This enzyme was stable at from pH 5.5 to 10 and on being heated to 40°C for 60 min. 6GT was strongly activated and stabilized by various divalent cations. The molecular mass of IMT was estimated to be 102 kDa by SDS-PAGE. The optimum pH and temperature for IMT were pH 6.0 and 50°C, respectively. This enzyme was stable at from pH 4.5 to 9.0 and on being heated to 40°C for 60 min. Divalent cations had no effect on the stability or activity of this enzyme.

Purification and some enzymatic properties of the chitosanase from Bacillus R-4 which lyses Rhizopus cell walls

A strain of Bacillus sp (Bacillus R-4) produces a protease and a carbohydrolase both of which have the ability to lyse Rhizopus cell walls. Of the enzymes, the carbohydrolase has been purified to an ultracentrifugally and electrophoretically homogeneous state, and identified as a chitosanase. The enzyme was active on glycol chitosan as well as chitosan. Molecular weight of the purified enzyme was estimated as 31 000 and isoelectric point as pH 8.30. The enzyme was most active at pH 5.6 and at 40 degrees C with either Rhizopus cell wall or glycol chitosan as substrate, and was stable over a range of pH 4.5 to 7.5 at 40 degrees C for 3 h. The activity was lost by sulfhydryl reagents and restored by either reduced glutathione of L-cysteine. An abrupt decrease in viscosity of the reaction mixture suggested an endowise cleavage of chitosan by this enzyme.

Cloning and Sequencing of the Genes Encoding Cyclic Tetrasaccharide-synthesizing Enzymes from Bacillus globisporus C11

The genes for isomaltosyltransferase (CtsY) and 6-glucosyltransferase (CtsZ), involved in synthesis of a cyclic tetrasaccharide from α-glucan, have been cloned from the genome of Bacillus globisporus C11. The amino-acid sequence deduced from the ctsY gene is composed of 1093 residues having a signal sequence of 29 residues in its N-terminus. The ctsZ gene encodes a protein consisting of 1284 residues with a signal sequence of 35 residues. Both of the gene products show similarities to α-glucosidases belonging to glycoside hydrolase family 31 and conserve two aspartic acids corresponding to the putative catalytic residues of these enzymes. The two genes are linked together, forming ctsYZ. The DNA sequence of 16,515 bp analyzed in this study contains four open reading frames (ORFs) upstream of ctsYZ and one ORF downstream. The first six ORFs, including ctsYZ, form a gene cluster, ctsUVWXYZ. The amino-acid sequences deduced from ctsUV are similar in to a sequence permease and a sugar-binding protein for the sugar transport system from Thermococcus sp. B1001. The third ctsW encodes a protein similar to CtsY, suggested to be another isomaltosyltransferase preferring panose to high-molecular-mass substrates.

Enzymatic synthesis of kojioligosaccharides using kojibiose phosphorylase

We have attempted to synthesize kojioligosaccharides (oligosaccharides having the alpha-1,2 glycosidic linkage at the nonreducing end) using two methods. In the first, mixtures of various proportions of glucose and beta-D-glucose-1-phosphate (beta-G1P) were allowed to react in the presence of kojibiose phosphorylase (KPase). In the second, maltose was allowed to react with KPase and maltose phosphorylase (MPase) simultaneously. In the former method, kojioligosaccharides having only the alpha-1,2 glucosidic linkage were synthesized and the average degree of polymerization (D.P.) of oligosaccharides increased with decreasing proportions of glucose. In the second method, kojioligosaccharides were obtained at approximately 70% yields under optimum conditions. 4-alpha-D-Kojibiosyl-glucose, kojitriose and kojitetraose, the principal kojioligosaccharides synthesized, were not hydrolyzed by salivary amylase, artificial gastric juice, pancreatic amylase, or small intestinal enzymes.

Production of cyclic tetrasaccharide from starch using a novel enzyme system from Bacillus globisporus C11.

Production of cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS, cyclic tetrasaccharide) from starch was attempted using 1,6-alpha-glucosyltransferase (6GT) and 1,3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus C11. The optimal conditions for production from partially hydrolyzed starch were as follows: substrate concentration, 3%; pH 6-7; temperature, 30 degrees C; 6GT, 1 unit/g-dry solid (DS); IMT, 10 units/g-DS. The production of CTS was demonstrated and 544 g of CTS hydrate crystal powders were obtained from 3500 g of partially hydrolyzed starch. Two major by-products were also isolated from the reaction mixture and identified as the branched derivatives of CTSs, 4-O-alpha-D-glucopyranosyl-CTS and 3-O-alpha-isomaltosyl-CTS.

6-α-Glucosyltransferase and 3-α-isomaltosyltransferase from Bacillus globisporus N75

Abstract A bacterial strain, Bacillus globisporus N75, produced two glycosyltransferases, 6-α-glucosyltransferase (6GT) and 3-α-isomaltosyltransferase (IMT), jointly catalyzing formation of cyclo →6)-α- d -Glc p -(1→3)-α- d -Glc p -(1→6)-α- d -Glc p -(1→3)-α- d -Glc p -(1→ (CTS) from α-1,4-glucan. The N75 enzymes produced CTS from dextrin in a 43.8% yield at the reaction temperature of 50°C, which was 10°C higher than a critical temperature of CTS-forming by the enzymes from B. globisporus C11. The optimum temperatures for 6GT and IMT reactions were 55°C and 50°C, respectively. The thermal stability of both enzymes was 45°C under the condition at pH 6.0 for 60 min. The genes for 6GT and IMT were cloned from the genomic DNA of N75. The amino acid sequences deduced from the 6GT and IMT genes showed 82% and 85% identities, respectively, to the sequences of the enzymes from C11. CTS yield was decreased by high concentrations of the substrate. It was found that the reaction yield was improved by adding cyclomaltodextrin glucanotransferase (CGTase). We demonstrated mass-production of CTS from starch by using the N75 enzymes and CGTase.

Cloning and sequencing of kojibiose phosphorylase gene from Thermoanaerobacter brockii ATCC35047

A gene encoding kojibiose phosphorylase was cloned from Thermoanaerobacter brockii ATCC35047. The kojP gene encodes a polypeptide of 775 amino acid residues. The deduced amino acid sequence was homologous to those of trehalose phosphorylase from T. brockii and maltose phosphorylases from Bacillus sp. and Lactobacillus brevis with 35%, 29% and 28% identities, respectively. Kojibiose phosphorylase was efficiently overexpressed in Escherichia coli JM109. The DNA sequence of 3956 bp analyzed in this study contains three open reading frames (ORFs) downstream of kojP. The four ORFs, kojP, kojE, kojF, and kojG, form a gene cluster. The amino acid sequences deduced from kojE and kojF are similar to those of the N-terminal and C-terminal regions of a sugar-binding periplasmic protein from Thermoanaerobacter tengcongensis MB4. Furthermore, the amino acid sequence deduced from kojG is similar to that of a permease of the ABC-type sugar transport systems from T. tengcongensis MB4. Each of three amino acid substitutions, D362N, K614Q and E642Q, caused a complete loss of kojibiose phosphorylase activity. These results suggest that D362, K614 and E642 play an important role in catalysis. Another mutation, D459N, increased K(m) values for kojibiose (7-fold that for the wild type), beta-G1P (11-fold) and glucose (7-fold), whereas K(m) for inorganic phosphate was minimally affected by this mutation, suggesting that D459 may be involved in the binding to saccharides.