-
[show abstract]
[hide abstract]
ABSTRACT: 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%.
Journal of Bioscience and Bioengineering 06/2006; 101(5):385-90. · 1.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The kojibiose phosphorylase (KP) gene and trehalose phosphorylase (TP) gene from Thermoanaerobacter brockii ATCC35047 were intracellularly hyper-expressed under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be 2.1 g of KP and 4.9 g of TP per liter of medium. Selaginose, non-reducing trisaccharide, was synthesized from trehalose utilizing the recombinant KP and TP from B. subtilis. Selaginose was not hydrolyzed by salivary amylase, artificial gastric juice, pancreatic amylase, or small intestinal enzymes.
Journal of Bioscience and Bioengineering 10/2005; 100(3):343-6. · 1.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A bacterial strain Arthrobacter globiformis A19 producing cyclic tetrasaccharide (CTS) was isolated from soil. The enzymes, 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT), involved in the synthesis of CTS were purified to homogeneity. The molecular and enzymatic properties of IMT from A. globiformis were similar to those of enzymes from Bacillus globisporus C11 and N75. Arthrobacter 6GT had a smaller molecular mass of 108 kDa and a higher optimum pH of 8.4 than the enzymes from strains of B. globisporus. The genes for IMT (ctsY) and 6GT (ctsZ) were cloned from the genome of A. globiformis A19. The two genes linked together in tandem and formed a gene cluster, ctsYZ. Both of the gene products showed similarities to alpha-glucosidases belonging to glycoside hydrolase family 31, and conserved two aspartic acids corresponding to the putative catalytic residues of the family enzymes. The enzymatic system for the production of CTS consisting of 6GT and IMT might be widespread among bacteria.
Bioscience Biotechnology and Biochemistry 01/2005; 68(12):2529-40. · 1.28 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Bioscience and Bioengineering 02/2004; 98(2):99-106. · 1.79 Impact Factor
-
Hajime Aga,
Tomoyuki Nishimoto,
Mieko Kuniyoshi, Kazuhiko Maruta,
Hiroshi Yamashita,
Takanobu Higashiyama,
Tetsuya Nakada,
Michio Kubota,
Shigeharu Fukuda,
Masashi Kurimoto,
Yoshio Tsujisaka
[show abstract]
[hide abstract]
ABSTRACT: A bacterial strain, Bacillus globisporus N75, produced two glycosyltransferases, 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT), jointly catalyzing formation of cyclo-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1--> (CTS) from alpha-1,4-glucan. The N75 enzymes produced CTS from dextrin in a 43.8% yield at the reaction temperature of 50 degrees C, which was 10 degrees 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 degrees C and 50 degrees C, respectively. The thermal stability of both enzymes was 45 degrees 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.
Journal of Bioscience and Bioengineering 02/2003; 95(3):215-24. · 1.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Bioscience Biotechnology and Biochemistry 10/2002; 66(9):1976-80. · 1.28 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The genes for isomaltosyltransferase (CtsY) and 6-glucosyltransferase (CtsZ), involved in synthesis of a cyclic tetrasaccharide from alpha-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 alpha-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.
Bioscience Biotechnology and Biochemistry 06/2002; 66(5):1057-68. · 1.28 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A 5.1-kbp genomic DNA fragment was cloned from trehalose-producing bacterium Arthrobacter sp. strain Q36. Sequence analysis of the DNA fragment revealed two open reading frames of 2325 and 1794 bp, encoding maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ), respectively. The 3′ end of treY overlaps with the 5′ end of treZ by one nucleotide, and it is suggested that treYZ constitutes an operon. The deduced amino acid sequences of both enzymes have several regions common to amylolytic enzymes belonging to an ‘α-amylase family’.
Biochimica et Biophysica Acta (BBA) - General Subjects 1289(1):10-13. · 5.00 Impact Factor
