Cellulases from a mutant (cellulase higher producer) obtained by UV treatment, Aspergillus niger UC were fractionated into Avicel hydrolyzing cellulase (Avicelase), carboxymethyl-cellulase (CMCase) and β-glucosidase by DEAE-Sepharose CL-6B column chromatography. Avicelase and CMCase were further purified by a multistep procedure involving Amberlite CG-50 and Sephadex G-100. Purification of β-glucosidase was carried out in three steps: by Sephadex G-100 followed by Amberlite CG-50 and Concanavalin A-Sepharose. All purified enzymes were homogeneous as judged by SDS polyacrylamide slab gel electrophoresis. The activities of both Avicelase and CMCase were optimum at pH 4.5 and 40-50°C. Avicelase was stable in the pH range 5.0 to 6.5 at temperatures below 50°C, while CMCase was stable in the pH range 4.0 to 7.0 at temperatures below 60°C. The molecular weights of Avicelase, CMCase and β-glucosidase were estimated to be 80, 000, 32, 000 and 120, 000, respectively. A strong synergistic action was shown by the combination of Avicelase along with CMCase and β-glucosidase.
Purified Avicelase and CMCase from a mutant Aspergillus niger UC hydrolyzed cellotetraose, and cellohexaose releasing glucose, cellobiose and cellotriose. The enzymes showed a straight line slope (1/ηsp/reducing sugar) with a roughly 45° angle in the relationship between the decrease in viscosity and the increase in the reducing sugar from CM-cellulose. The enzymes also hydrolyzed 4-methylumbelliferyl (MU) trisaccharide and 4-MU disaccharide at a different rate. We obtained no evidence that this cellulase is an exo-type one so far named cellobiohydrolase (CBH) as the systematic nomenclature.
Three strains of sulfate-reducing bacteria utilizing H2 as the electron donor of sulfate reduction were isolated from anaerobic digester slurry of municipal sewage sludge. The isolates were Gram-negative, non-spore- forming, motile, curved rods with a single polar flagellum. Cytochrome c3 and desulfoviridin were present. The DNA base compositions (mol% Three strains of sulfate-reducing bacteria utilizing H2 as the electron donor of sulfate reduction were isolated from anaerobic digester slurry of municipal sewage sludge. The isolates were Gram-negative, non-spore-forming, motile, curved rods with a single polar flagellum. Cytochrome c3 and desulfoviridin were present. The DNA base compositions (mol%G+C) were 59.6±0.7). The isolates used H2, formate, lactate, pyruvate, fumarate, malate, alcohols and amino acids as the electron donor for sulfate reduction. Alcohols were oxidized to corresponding monocarboxylates, and other organic compounds were to acetate. Sulfate, Three strains of sulfate-reducing bacteria utilizing H2 as the electron donor of sulfate reduction were isolated from anaerobic digester slurry of municipal sewage sludge. The isolates were Gram-negative, non-spore-forming, motile, curved rods with a single polar flagellum. Cytochrome c3 and desulfoviridin were present. The DNA base compositions (mol% G+C) were 59.6±0.7. The isolates used H2, formate, lactate, pyruvate, fumarate, malate, alcohols and amino acids as the electron donor for sulfate reduction. Alcohols were oxidized to corresponding monocarboxylates, and other organic compounds were to acetate. Sulfate, sulfite and thiosulfate, but not nitrate, were used as the electron acceptor and reduced to sulfide. The isolates grew with pyruvate, fumarate, or malate as the energy source in the absence of oxidized sulfur compounds. yruvate was oxidized to acetate with the production of formate and H2. Fumarate and malate were oxidized to acetate, and the oxidation was coupled with the reduction of fumarate or malate to succinate. From the morphological and physiological properties, the isolates were considered to belong to a Desulfovibrio sp. In association with Methanobacteriumformicicum as a hydrogen-consuming partner, one of the isolates could also grow in the absence of oxidized sulfur compounds by utilizing organic substrates other than pyruvate, malate and fumarate. In the coculture of the isolate and the methanogen, all the organic compounds other than malate and fumarate, which could serve as the electron donor for sulfate reduction in the pure culture of isolates, were syntrophically oxidized with the production of methane. These results indicate that the isolates of sulfate-reducing bacteria can play a role as a syntrophic degrader of a wide range of organic compounds in the methanogenic sewage sludge devoid of sulfate.
Aerobic bacteriochlorophyll (BChl) a-containing bacteria (designated as strains NS89, NS102, and NS130) were isolated from soil samples in Japan and their taxonomic characteristics were investigated. These strains were obligately aerobic, Gram-negative, and non-motile cocci, and formed irregular colonies on agar media. The strains could not utilize methanol as a sole carbon and energy source and sulfate as a sole sulfur source. Strain NS130 required divalent cations (Ca2+ or Mg2+) for growth in liquid culture. The cations were not necessary for the growth of strains NS89 and NS102 in liquid culture, however, the cations promoted growth. Light stimulated the growth of strains NS89 and NS 102, but not the growth of strain NS130. The absorption spectra of membrane fractions of strains NS89 and NS102 had a large peak at 856 nm that was derived from BChl a, while the spectrum of strain NS130 had a large peak at 872nm. Membrane fractions of the three strains showed photochemical activity. The guanine plus cytosine contents of DNA of strains NS89, NS102, and NS130 were 70.3, 71.0, and 70.5mol%, respectively. The ubiquinone system of the strains was Q-10, and the major cellular fatty acid was octadecenoic acid (C18:1) (57-68%). The three strains contained 2-hydroxy and 3-hydroxy fatty acids, and 2-hydroxy-octadecenoic acid (2-OH C18:1) was 11-12%.
The nucleotide sequence of the whole gerK spore germination locus of Bacillus subtilis has been determined by sequencing the remaining first and third genes of the locus. The gerK locus contained three open reading frames capable of encoding polypeptides of 546, 407 and 373 amino acids. Each predicted gene product had homologues in the gerA and gerB operon, but the gene order was different from that of gerA and gerB. Sequences regarded as -10 and -35 promoter sequences for the sigma-G type RNA polymerase was found upstream of the first gene suggesting that the gerK also is an operon expressed specifically in the forespore during sporulation. All three genes were suggested to be essential for spore germination in response to glucose.
evgS and evgA located at 51min on the Escherichia coli chromosome were previously found as a new two-component system, which is highly homologous to bvgS and bvgA in Bordetella pertussis. In this study, the putative phosphorylation and phospho-transferred sites of EvgS and EvgA, respectively, were changed using modified primers during PCR amplification of their structural genes, and their functions were analyzed in vivo. The ompC expression was induced in a ΔenvZ E. coli in the presence of pSK-001, which contains evgS and evgA, but was not changed in the presence of pKM18 (evgS721Arg) or pKM12 (evgA52Ala). Moreover, phosphotransfer from phosphorylated EvgS to EvgA was observed in vitro using the membrane fraction containing EvgS and His-tagged EvgA. These results suggested that the signal transduction via EvgS and EvgA involves the previously reported phosphorelay in the two-component system via BvgS and BvgA.
A trypsin-like serine proteinase was found in a plasma membrane fraction from the plasmodia of Physarum polycephalum. It was solubilized from the plasma membrane fraction with 1% (w/v) Triton X-100 and purified by a series of column chromatographic steps followed by polyacrylamide gel electrophoresis (PAGE). The molecular mass of the enzyme was estimated to be 130kDa by SDS-PAGE under non-reducing conditions. This enzyme exhibited maximal activity at pH 9 and at 35°C with the substrate Boc-Phe-Ser-Arg-MCA, and was sensitive to various serine proteinase inhibitors and trypsin inhibitors. The enzyme was significantly labeled with [3H]diisopropylfluorophosphate (DFP); however, a clear decrease in the amount of the enzyme bound-[3H]DFP was observed in the presence of p-nitrophenyl-p′-guanidinobenzoate (NPGB), an active site titrant for trypsin. The activity was completely inhibited in the presence of EDTA or EGTA, but the metal-depleted enzyme was reactivated by the addition of Ca2+ Moreover, this enzyme was capable of cleaving several synthetic peptides at the carboxyl side of arginine, but did not work on synthetic substrates for chymotrypsin, elastase and aminopeptidase. The enzyme showed an affinity to Con A, indicating that it contains a carbohydrate moiety. Inhibition of the activity by a serine proteinase inhibitor resulted in cessation of the DNA synthesis in growing plasmodia without loss of the protein synthesis. Thus, this trypsin-like serine proteinase may have a regulatory role in the growth and development of the slime mould.