Tsuchiyoshi Fujino

Inhibition by ajoene of skin-tumor promotion in mice.

Ajoene, a major compound containing sulfur in oil-macerated garlic products, inhibited in a two-stage carcinogenesis test on mouse skin. Treatment with ajoene suppressed skin tumor formation, depending on the amount. In particular, the group treated with 250 μg of ajoene had only 4.9% the number of tumors per mouse compared with the control group at 18 weeks.

α-Galactosidase Aga27A, an Enzymatic Component of the Clostridium josui Cellulosome

The Clostridium josui aga27A gene encodes the cellulosomal alpha-galactosidase Aga27A, which comprises a catalytic domain of family 27 of glycoside hydrolases and a dockerin domain responsible for cellulosome assembly. The catalytic domain is highly homologous to those of various alpha-galactosidases of family 27 of glycoside hydrolases from eukaryotic organisms, especially plants. The recombinant Aga27A alpha-galactosidase devoid of the dockerin domain preferred highly polymeric galactomannan as a substrate to small saccharides such as melibiose and raffinose.

Nucleotide sequences of the celB gene encoding endo-1,4-β-β-glucanase-2, ORF1 and ORF2 forming a putative cellulase gene cluster of Clostridium josui

Abstract The nucleotide sequence of the ce1B gene coding for endo-1,4-β-glucanase-2 (EG-2) of Clostridium josui was determined. The structural gene consists of an open reading frame of 1,380 by encoding a 460-amino acid polypeptide. The deduced amino acid sequence of EG-2 shows high similarities with the Clostridium cellulolyticum CelCCC (92.3%) and the Clostridium thermocellum endoglucanase CelA (61.6%) in the catalytic domain, but no homology with EG-1 of C. josui . In the COOH-terminal region of EG-2, there exists a reiterated segment similar to the segments present in many clostridial cellulases and hemicellulases. Upstream and downstream of the celB gene, two incomplete open reading frames (ORF1 and ORF2) were found to encode proteins homologous to the proteins which consist of the C. cellulolyticum cellulase gene cluster. ORF1 containing a reiterated segment showed high similarities with the proteins deduced from each ORF1 from C. cellulolyticum and Caldocellum saccharolyticum . Downstream of the ce1B gene, ORF2 was found to encode a protein homologous to CeICCG of C. cellulolyticum and CelF of C. thermocellum . These results indicate that the celB gene, ORF1 and ORF2 formed a cellulase gene cluster strictly homologous to that of C. cellulolyticum , which is proposed to be part of a single operon.

Analysis of a Clostridium josui Cellulase Gene Cluster Containing the man5A Gene and Characterization of Recombinant Man5A

A cellulase gene cluster of Clostridium josui was sequenced, and was found to encode 11 proteins responsible for cellulosome (cellulolytic complex) formation, viz., cipA, cel48A, cel8A, cel9A, cel9B, orfX, cel9C, cel9D, man5A, cel9E, and cel5B, in order from the upstream side. All the predicted enzymes had a dockerin module, suggesting that these proteins are members of the C. josui cellulosome. Among these genes, the man5A gene encoding β-mannanase was expressed in Escherichia coli and the recombinant enzyme (rMan5A) was characterized. rMan5A showed strong activity toward carob galactomannan and low activity toward guar gum, suggesting that it prefers non-galactosylated mannan to galactomannan. This enzyme hydrolyzed ivory nut mannan to produce mainly mannotriose and larger mannooligosaccharides, and was not active toward mannotriose. An antiserum raised against the recombinant enzyme detected Man5A in the culture supernatants of C. josui, which was grown on either ball-milled cellulose or glucose as a carbon source.

Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the translated product.

The nucleotide sequence of the Clostridium josui FERM P-9684 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,150 bp and encodes 1,050 amino acids with a molecular weight of 115,564. Xyn10A is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains. Immunological analysis indicated the presence of Xyn10A in the culture supernatant of C. josui FERM P-9684 and on the cell surface. The full-length Xyn10A expressed in a recombinant Escherichia coli strain bound to ball-milled cellulose (BMC) and the cell wall fragments of C. josui, indicating that both the CBM and the SLH domains are fully functional in the recombinant enzyme. An 85-kDa xylanase species derived from Xyn10A by partial proteolysis at the C-terminal side, most likely at the internal region of the CBM, retained the ability to bind to BMC. This observation suggests that the catalytic domain or the thermostabilizing domains are responsible for binding of the enzyme to BMC. Xyn10A-II, the 100-kDa derivative of Xyn10A, was purified from the recombinant E. coli strain and characterized. The enzyme was highly active toward xylan but not toward p-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-β-D-cellobioside, or carboxymethylcellulose.

Nucleotide sequence of an endo-1,4-β-glucanase gene (celA) from Clostridium josui

Abstract The nucleotide sequence of a DNA fragment containing an endo-1,4-β-glucanase (EG-1) gene of Clostridium josui was determined by the dideoxy-chain termination method. The EG-1 coding sequence was an open reading frame encoding 369 amino acids, and a putative promoter sequence was located in the upstream region of the open reading frame. The N-terminal amino acid sequence of the endoglucanase (EG-1C) purified from the Escherichia coli transformant (JM103/pUCJ1) was consistent with the deduced sequence from 30 Val to 44 Lys. The estimated molecular weights of the precursor and the mature enzymes were 41,774 and 38,352, respectively. The region of amino acids from 61st to 335th of the enzyme revealed high homology with those of Bacillus sp. and Clostridium acetobutylicum endoglucanases.

Cloning of the celB gene encoding endo-1,4-β-glucanase-2 from Clostridium josui in Escherichia coli and the properties of the translated product

Abstract The gene celB on a 3.9-kilobase-pair (kbp) EcoRI fragment encoding endo-1,4-β-glucanase of Clostridium josui was cloned into Escherichia coli. The structural gene located on the 1.6 kbp Sau3AI fragment excised from the EcoRI fragment was expressed by the lac promoter in the transformant E. coli JM103 (pUCJ2) in modified Luria-Bertani broth with an activity 1,000 times (1120 U/l) higher that on the EcoRI fragment. The translation product of celB in pUCJ2 was purified by CM Bio-Gel A column chromatography. The homogeneous enzyme was 42 kD of the molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optima for the temperature and pH of the enzyme were 60°C and 5.5, respectively. The enzyme hydrolyzed cellotetraose, cellopentaose and cellohexaose but not cellobiose and cellotriose. The mode of degradation of cellooligomers ( G4 a 2G2, G5 a G2+G3, G6 a 2G3 ) of the enzyme suggested that it acts as an endo-1,4-β-glucanase. This endoglucanase is distinguishable from those characterized by us previously with respect to its pH optimum and cellobiose-transferring activity.

Enhancement of Intestinal IgA Production by Ajoene in Mice

We investigated the effects of ajoene on intestinal IgA production. Ajoene (1.35, 4.5, and 13.5 µg/kg/d) was administered to mice for 4 weeks. The fecal IgA level in the 13.5 µg/kg/d group increased after 3 weeks. The intestinal IgA level also increased in a dose-dependent manner upon ajoene administration. An oil-macerated garlic extract, with 1500 µg/g of ajoene, enhanced the intestinal IgA production.

Purification and characterization of the Clostridium josui porphobilinogen deaminase encoded by the hemC gene from a recombinant Escherichia coli.

The porphobilinogen deaminase encoded by the Clostridium josui hemC gene was purified from a recombinant Escherichia coli strain and its properties were characterized. The optimal temperature and pH of the purified enzyme were 65 degrees C and 7.0, respectively. This enzyme was quite thermostable: it retained 86% of the original activity after incubation at 70 degrees C for 1 h. The Km and Vmax values of the enzyme were 65 microM and 3.3 micromol/h/mg for porphobilinogen, respectively.