l-Asparaginase from E. coli

l-Asparaginase [EC 3.5.1.1], antitumor enzyme, was purified to a crystalline form from the cell free extract of Escherichia coli A-l-3 KY3598, by ethanol fractionation and chromatographies on DEAE cellulose and CM Sephadex. The crystalline enzyme was homogeneous by the criteria of ultracentrifugation: s_{20, w} was 7.87S.

The molecular weight was estimated to be 141,000 by the short column method. The pI of the enzyme protein was 4.75 according to isoelectric electrofocusing.

Amino acid analysis revealed the absence of cysteine or cystine residues in the molecule.

The enzyme exhibited optimal activity between pH 6 and 8. It was stable in the pH range 5.5 ~ 9.0.

The enzyme activity was cleared very slowly in the plasma of dog. Intravenous administration of the enzyme caused a complete regression of the Gardner lymphoma implanted in the C3H mice.     

Collagenase in Stomach of Crown-of-thorns Starfish Acanthaster planci

Acetobacter pasteurianus strains IFO3283, SKU1108, and MSU10 were grown under acetic acid fermentation conditions, and their growth behavior was examined together with their capacity for acetic acid resistance and pellicle formation. In the fermentation process, the cells became aggregated and covered by amorphous materials in the late-log and stationary phases, but dispersed again in the second growth phase (due to overoxidation). The morphological change in the cells was accompanied by changes in sugar contents, which might be related to pellicle polysaccharide formation. To determine the relationship between pellicle formation and acetic acid resistance, a pellicle-forming R strain and a non-forming S strain were isolated, and their fermentation ability and acetic acid diffusion activity were compared. The results suggest that pellicle formation is directly related to acetic acid resistance ability, and thus is important to acetic acid fermentation in these A. pasteurianus strains.     

An Arabinogalactan from Extracellular Polysaccharides of Suspension-cultured Tobacco Cells

The structure of an arabinogalactan, separated from extracellular polysaccharides of cultured tobacco cells, has been investigated by methylation analysis of the original polysaccharide and of the products obtained after mild acid hydrolysis and after controlled Smith degradation.

The arabinogalactan consists of l-arabinose, d-galactose and l-rhamnose in the molar ratio of 47: 45: 8. The arabinogalactan has a main chain of (1→3)-linked d-galactopyranosyl residues, half of which are substituted at the 6-position. Most of the side chains consist of three (1→6)-linked D-galactopyranosyl residues, to which l-arabinose residues are attached at C-3. The l-arabinofuranosyl and pyranosyl residues are present as end groups, and l-arabinopyranosyl residues are attached to C-5 of l-arabinofuranosyl residues. Non-reducing terminal l-rhamnopyranosyl residues are also present.     

乙醇脱氢酶（ADH）产酶菌株的选育

从食醋生产企业的醋醅中采集样品,以乙醇为唯一碳源,用碳酸钙透明圈平板法分离出185株菌株,然后以产酸量和耐乙醇能力为标准,瓶发酵选育出20株ADH产酶菌株;A5-2产酸量为49.85 g/L,耐乙醇能力强,A5-2的菌种形态学和16S rDNA序列分析初步鉴定为巴斯德醋酸杆菌（ Acetobacter pasteurianus）;A5-2乙醇脱氢酶酶学性质研究表明：最适作用温度和pH分别为45℃和pH 4.0,具有一定的耐热性和良好的耐酸碱性;A5-2乙醇脱氢酶粗酶制备条件为硫酸铵饱和度70%~80%,回收率84%。     

Proteome analysis of Acetobacter pasteurianus during acetic acid fermentation

Acetic acid bacteria (AAB) are Gram-negative, strictly aerobic microorganisms that show a unique resistance to ethanol (EtOH) and acetic acid (AcH). Members of the Acetobacter and Gluconacetobacter genera are capable of transforming EtOH into AcH via the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes and are used for the industrial production of vinegar.Several mechanisms have been proposed to explain how AAB resist high concentrations of AcH, such as the assimilation of acetate through the tricarboxylic acid (TCA) cycle, the export of acetate by various transporters and modifications of the outer membrane. However, except for a few acetate-specific proteins, little is known about the global proteome responses to AcH.In this study, we used 2D-DIGE to compare the proteome of Acetobacter pasteurianus LMG 1262^T when growing in glucose or ethanol and in the presence of acetic acid. Interesting protein spots were selected using the ANOVA p-value of 0.05 as threshold and 1.5-fold as the minimal level of differential expression, and a total of 53 proteins were successfully identified.Additionally, the size of AAB was reduced by approximately 30% in length as a consequence of the acidity. A modification in the membrane polysaccharides was also revealed by PATAg specific staining.     

Biological denitrification by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> immobilized on microbial cellulose

This study demonstrated the feasibility of a biological denitrification process using immobilized Pseudomonas stutzeri. The microbial cellulose (MC) from Acetobacter xylinum was used as the support material for immobilization of the bacterium. Nitrate removal took place mainly in the anoxic system. The effects of various operating conditions such as the initial nitrate concentration, pH, and carbon source on biological denitrification were demonstrated experimentally. The system demonstrated a high capacity for reducing nitrate concentrations under optimum conditions. The denitrification rate increased up to a maximal value of 1.6 kg NO₃-N m⁻³ day⁻¹ with increasing nitrate loading rate. Because of its porosity and purity, MC may be considered as appropriate supports for adsorbed immobilized cells. The simplicity of immobilization and high efficiency in operation are the main advantages of such systems. To date, the immobilization of microorganisms onto MC has not been carried out. The results of this research shows that a pilot bioreactor containing P. stutzeri immobilized on MC exhibited efficient denitrification with a relatively low retention time.     

A shaking bioreactor equipped with twin ceramic membranes for acetic acid production using Acetobacter pasteurianus

A shaking bioreactor system with twin internal ceramic membranes was developed for effective perfusion culture and applied to the continuous production of acetic acid using Acetobacter pasteurianus. The system makes it possible to carry out the back-washing of the membrane without stopping the continuous operation because one membrane can be washed by medium feed flow while another membrane provides filtration of the broth by the simple switching of the medium and the broth flow direction. The medium flow through the membrane could successfully wash the surface of the membrane thereby effectively maintaining the filtration ability. By using the system, continuous operation of more than 800 h was achieved and the maximum acetic acid productivity reached 13.4 g l^–1 h^–1 using air enriched with 40% O₂.     

Infection and Colonization of Sugar Cane and Other Graminaceous Plants by Endophytic Diazotrophs

Agriculturally important grasses such as sugar cane (Saccharum sp.), rice (Oryza sativa), wheat (Triticum aestivum) sorghum (Sorghum bicolor), maize (Zea mays), Panicum maximum, Brachiaria spp., and Pennisetum purpureum contain numerous diazotrophic bacteria, such as, Acetobacter diazotrophicus, Herbaspirillum spp., Azospirillum spp. These bacteria do not usually cause disease symptoms in the plants with which they are associated and the more numerous of them, for example, Herbaspirillum spp. and A. diazotrophicus, are obligate or facultative endo-phytes that do not survive well (or at all) in native soil; these are thought to be spread from plant generation to plant generation via seeds, vegetative propagation, dead plant material, and possibly by insect sap feeders. By contrast, Azospirillum spp. are not wholly endophytic but are root-associated, soil-dwelling bacteria that are also often found within plants, probably entering host plants via seeds or via wounds/cracks at lateral root junctions. Endophytic diazotrophs have been isolated from a number of grasses in which significant biological N₂ fixation (BNF) has been demonstrated, particularly Brazilian sugar cane varieties, but also in rice, maize, and sorghum. However, although the endophytic diazotrophs are held to be the causative agents of the observed BNF, direct evidence for this is lacking. Therefore, in this review we examine probable sites of bacterial multiplication and/or BNF within endophyte-containing grasses and discuss these in terms of potential benefits (or not) to both host plants and bacteria. In particular, we examine how potentially large numbers of bacteria, especially Herbaspirillum spp., A. diazotrophicus, and Azospirillum spp., can exist extracellularly within non-specialized (for symbiotic purposes) regions such as xylem vessels and intercellular spaces. The processes of infection and colonization of various grasses (particularly sugar cane) by diazotrophic endophytes are also described, and these are compared with those of important (nondiazotrophic) endophytic sugar cane pathogens such as Clavibacter xyli subsp. xyli and Xanthomonas albilineans.     

The linear oxidation of formaldehyde to CO2 as the proper energy generating sequence for the assimilation of methanol in Acetobacter methanolicus MB58

Acetobacter methanolicus MB58 can grow on methanol. Since this substrate exhibits to be energy deficient there must be a chance to oxidize methanol to CO₂ merely for purpose of energy generation. For the assimilation of methanol the FBP variant of the RuMP pathway is used. Hence methanol can be oxidized cyclically via 6-phosphogluconate. Since Acetobacter methanolicus MB58 possesses all enzymes for a linear oxidation via formate the question arises which of both sequences is responsible for generation of the energy required. In order to clarify this the linear sequence was blocked by inhibiting the formate dehydrogenase with hypophosphite and by mutagenesis inducing mutants defective in formaldehyde or formate dehydrogenase. It has been shown that the linear dissimilatory sequence is indispensable for methylotrophic growth. Although the cyclic oxidation of formaldehyde to CO₂ has not been influenced by hypophosphite and with mutants both the wild type and the formaldehyde dehydrogenase defect mutants cannot grown on methanol. The cyclic oxidation of formaldehyde does not seem to be coupled to a sufficient energy generation, probably it operates only detoxifying and provides reducing equivalents for syntheses. The regulation between assimilation and dissimilation of formaldehyde in Acetobacter methanolicus MB58 is discussed.Abbreviations ATP Adenosine-5-triphosphate - DCPIP 2,6-dichlorphenolindophenol - DW dry weight - ETP electron transport phosphorylation - FBP fructose-1,6-bisphosphate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PMS phenazine methosulfate - RuMP ribulose monophosphate - Ru5P ribulose-5-phosphate - SDS sodiumdodecylsulphate - TCA tricarboxylic acid - TYB toluylene blue Dedicated to Prof. Dr. Dr. S. M. Rapoport on occasion of his 75th birthday     

Lipopolysaccharides from six strains of Acetobacter diazotrophicus

Abstract Lipopolysaccharides from six nitrogen-fixing strains of Acetobacter diazotrophicus (PR2, PAL3, PAL5, PR4, PR14, PR20), isolated from sugarcane, were purified by phenol-water extraction and ultracentrifugation. The relatively large molecular mass observed by SDS-PAGE indicated that the lipopolysaccharides of each strain possessed an O-side chain. Analysis of each lipopolysaccharide by colorimetric assays and by gas liquid chromatography/mass spectrometry combination showed that the core and lipid A composition was similar for all strains, containing 3-deoxy-d-manno-2-octulosonic acid, glucosamine and fatty acid (16-0, 3-OH-14, 2-OH-16:0, 3-OH-16:0). The neutral sugar composition showed the predominance of 6-deoxy-hexose (rhamnose and fucose) and ribose, in comparison with hexose (glucose, galactose, mannose). The presence of 6-deoxy-hexose and ribose containing O-side chains is discussed as a way of discriminating A. diazotrophicus from other Acetobacter species.