The microbial biosynthesis of methionine

1. The enzymes leading to the methylation of homocysteine have been examined in three micro-organisms: a cobalamin-producing bacterium, Bacillus megaterium; a yeast, Candida utilis; and a basidiomycete fungus, Coprinus lagopus. The yeast and the fungus contain negligible endogenous cobalamin. 2. Extracts of each organism catalyse C(1)-transfer from serine to homocysteine with a polyglutamate folate coenzyme. 3. The enzymes generating the methyl group of methionine from C-3 of serine have similar properties in each case, but different mechanisms of homocysteine transmethylation from 5-methyltetrahydrofolates were found. 4. B. megaterium contains an enzyme with properties suggestive of a vitamin B(12)-dependent homocysteine transmethylase, whereas Cand. utilis and Cop. lagopus transfer the methyl group by a reaction characteristic of the cobalamin-independent mechanism established for Escherichia coli. 5. The specificity of each transmethylase for a 5-methyltetrahydropteroylpolyglutamate is consistent with the results of analyses of endogenous folates in these organisms, which showed only conjugated forms. 6. None of the extracts catalysed methionine production from S-adenosylmethionine and homocysteine. 7. These results are compared with results now available for methionine synthesis in other organisms, which show a considerable diversity of mechanisms.     

Batch production and growth kinetics of hybridomas

Batch cultures of mouse-mouse hybridoma cell lines were carried out and their growth and production kinetics investigated. Three main cell specific production patterns (expressed as pg IgG/cell x hour) were found, which can be used as a classification system for hybridoma cell lines (groups I–III). Cells showing the highest IgG-production at the beginning of the batch culture (during the lag and the onset of the log-phase) were classified as either group I and II. The difference was that cell lines of group II showed a second high cell specific production at the onset of the stationary and death phases. Cell lines of group III had a quite constant production of antibodies during their growth; but IgG secretion completely stopped after the beginning of the stationary phase. The implications of these three production patterns on the design of a production process are discussed.     

Protein–polysaccharide biosynthesis. Membrane-bound saccharides

Membrane-bound saccharides obtained from ear cartilage from rabbits recovering from papain injection were examined for glycosyl acceptor properties, alkali lability, molecular size and degree of sulphation. Although considerable glucuronosyl transfer from UDP-glucuronic acid could be demonstrated, less than one-third could be identified as representing chondroitin linkage formation; there was little or no effect of molecular size on acceptor properties. Approximately one-half of the membrane-associated chondroitin-like saccharide chains are solubilized by alkali, whereas one-half require proteolysis for solubilization. These fractions are analytically indistinguishable and both contain xylose and galactose as well as uronic acid and sulphated N-acetyl-galactosamine. All fragments that were examined contained stoicheiometric amounts of axial sulphate ester, strongly suggesting a close relationship between chain extension and sulphation.     

酵母微生物油脂的生物合成研究

摘要：目的 微生物油脂可作为制备绿色能源生物柴油的原料。对酵母微生物油脂的生物合成方法进行研究。方法 以斯达油脂酵母Lipomyces starkeyi AS 2.1560为菌种进行微生物油脂生物合成。首先获得大量细胞,将细胞收集后,转移至葡萄糖溶液中进行油脂合成。结果 斯达油脂酵母可在不含有其他营养成分的葡萄糖溶液中快速合成油脂,细胞油脂含量可达到细胞干重的60%以上。菌龄对油脂生成影响不明显,糖浓度过高抑制油脂生成,40 g/L葡萄糖溶液中60 h合成油脂最多,达到65.2%,并有进一步积累的可能,在（0.5～6）×108个/mL,接种细胞的密度越大,油脂合成能力越低。合成油脂成分主要为棕榈酸和油酸。结论 斯达油脂酵母细胞增殖与油脂生物合成可分开进行,其油脂成分与普通动植物油脂成分相似。     

Evidence for microbial polysaccharide preparations containing polyester substituents

CPMAS 13C-n.m.r. spectroscopy was employed to characterize the composition and solid phase morphology of gellan, welan, rhamsan and NW11. Spectra indicated that commercial preparations of these polysaccharides, which share a similar molecular backbone, contain a non-carbohydrate component exhibiting four inequivalent carbon atoms. Isolation of this component, followed by 13C-n.m.r. in CHCl3 and MS analysis, revealed its structure to be poly(beta-hydroxybutyrate). Evidence is presented which suggests that this polyester may be a covalent adduct to the above polysaccharides, although this cannot be unambiguously determined at this time. Further experimentation is in progress.     

Batch fermentation kinetics of sugar beet molasses by Zymomonas mobilis

A new osmotolerant mutant strain of Zymomonas mobilis was successfully used for ethanol production from beet molasses. Addition of magnesium sulfate to hydrolyzed molasses allowed repeated growth without the need of yeast extract addition. The kinetics and yields parameters of fermentation on media with different molasses concentrations were calculated. The anabolic parameters (specific growth rate, mu, and biomass yield, Y(X/S)) were inhibited at elevated molasses concentrations while the catabolic parameters (specific ethanol productivity, q(p), and ethanol yield, Y(p/s)) were not significantly affected. In addition to ethanol and substrate inhibition, osmotic pressure effects can explain the observed results.     

Batch production and secretion kinetics of hybridomas: Pulse-chase experiments

Pulse chase experiments of two mouse hybridoma lines were conducted in order to elucidate the kinetics of monoclonal antibody (mAb) production and secretion during different stages of batch cultures. The results indicate that a stock of cytoplasmic IgG exists in hybridoma cells and that the concentration of this stored IgG depends on the cell line used and the stage of the culture. This stored IgG can be released by dying cells, and a certain quantity of the secreted IgG is derived from this source. However, only between 0.3 and 9.3% of the released IgG of U0208 (average: 2.08%) and between 2.08 and 25.8% of the IgG, released from I.13.17 (average: 6.95%), were of storage origin, calculated on culture viability and intracellular IgG-stock. Comparing the accumulation of radio-labelled IgG (IgG^*) in the supernatant with the reduction of cytoplasmic IgG^* during the chase experiments, the percentages range between 14 and 50%, somewhat higher values probably caused by changes in the culture conditions. These changes led to a release of IgG during the chase experiments, which accounts for about 20–25% of the totally secreted IgG.It could be established that during the logarithmic growth phase of batch cultures a certain percentage of synthesized IgG was not released but stored within the cells: for U0208: 0.3–4.5%, for I.13.17: 1–7.6%. During the stationary and death phase, this percentage ranged between 1.5 and 20% for U0208 and between 0.5 and 8.1% for I.13.17. Finally, the chase experiments also revealed that the time of synthesis, assembly, and secretion of mAbs does not vary much during the different phases of batch cultures, and is within the range of 1.5 and 3 hrs.     

微生物多糖WL-26深层发酵工艺的优化

产碱杆菌Alcaligenes sp.JL-1能分泌一种高分子多糖WL-26.以生物量、发酵产量和黏度为主要指标,对其深层发酵培养基进行了筛选,并在此基础上对发酵条件进行优化.培养条件确定为:40g/L蔗糖,4g/L复合氮源(20%硝酸钠和80%的牛肉膏),2g/L KH2PO4,0.1g/L MgSO4,0.5mL/L FeSO4.初始pH为7.2～7.4,5%的接种量、装液量为50mL/250mL三角瓶、转速为200r/min、30℃恒温培养58h,WL-26产量从9.326g/L提高到21.767g/L.     

Production and rheological characteristics of the microbial polysaccharide gellan

B. MANNA, A. GAMBHIR AND P. GHOSH. 1996. Sphingomonas paucimobilis was used for the synthesis of the microbial polysaccharide gellan. In a 60 h fermentation, polysaccharide yield and productivity obtained were 0.45 g gellan per g glucose consumed and 0.21 g I⁻¹ h⁻¹ respectively. The broth showed pseudoplastic behaviour with yield stress. The requirement of the solvent propanol to precipitate gellan from the broth depended on the volume of the broth rather than on gellan concentration. The addition of salt to the broth reduced the propanol requirement. Attempts to separate cells from the highly viscous broth by microfiltration were not successful. Ultrafiltration reduced the propanol requirement but appreciable membrane fouling and loss of gellan was observed.     

Patterns of polysaccharide biosynthesis in differentiating cells of maize root-tips

1. The patterns of incorporation of radioactivity from d-[6-(3)H]-, d-[1-(14)C]-, d-[U-(14)C]- and d-[6-(14)C]-glucose and [U-(14)C]myoinositol into the neutral sugars and uronic acids of the polysaccharides synthesized in different regions of the root-tip of maize were determined. 2. The root-cap tissue synthesized a slime in which a polysaccharide that contained a high proportion of fucose (32%) and galactose (21%) was found. This polysaccharide is synthesized only by the root-cap cells, and very little polysaccharide containing fucose is synthesized in adjacent tissues. Part of the meristematic tissue of the root is surrounded by the cap cells. A section of the root that contains both these tissues can be analysed, and the polysaccharide synthesized by the meristematic region can be obtained since the contribution of the root-cap cells can be found by the amount of fucose formed. 3. It was shown that there is very little difference in the polysaccharide synthesis of the meristematic region from that of the cells immediately behind it. In the more mature cells, however, the amount of xylose synthesized relative to that of arabinose is increased, and the proportion of xylose and arabinose formed in the matrix polysaccharides is increased whereas that of galactose is decreased. 4. The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on polysaccharide synthesis was to bring about a decrease in the relative amount of galactose synthesized in the matrix polysaccharides of cells immediately adjacent to the meristematic region and also in the more mature tissue. The growth factor also increased the amount of xylose synthesized relative to that of arabinose in the more mature tissue. These metabolic effects were related to a very obvious change in the morphological appearance of the root-tips. 5. Radioactivity from [U-(14)C]myoinositol was incorporated mainly into xylose, arabinose and galacturonic acid rather than into the hexoses, although small amounts of these sugars were formed.     

Glycosaminoglycan polysaccharide biosynthesis and production: today and tomorrow

Glycosaminoglycans [GAGs] are essential heteropolysaccharides in vertebrate tissues that are also, in certain cases, employed as virulence factors by microbes. Hyaluronan [HA], heparin, and chondroitin sulfate [CS] are GAGs currently used in various medical applications and together are multi-billion dollar products thus targets for production by animal-free manufacture. By using bacteria as the source of GAGs, the pathogen’s sword may be converted into a plowshare to help avoid potential liabilities springing from the use of animal-derived GAGs including adventitious agents (e.g., prions, pathogens), antigenicity, degradation of the environment, and depletion of endangered species. HA from microbes, which have a chemical structure identical to human HA, has already been commercialized and sold at the ton-scale. Substantial progress towards microbial heparin and CS has been made, but these vertebrate polymers are more complicated structurally than the unsulfated bacterial polysaccharide precursors thus require additional processing steps. This review provides an overview of GAG structure, medical applications, microbial biosynthesis, and the state of bacterial GAG production systems. Representatives of all glycosyltransferase enzymes that polymerize the sugar chains of the three main GAGs have been identified and serve as the core technology to harness, but the proteins involved in sugar precursor formation and chain export steps of biosynthesis are also essential to the GAG production process. In addition, this review discusses future directions and potential important issues. Overall, this area is poised to make great headway to produce safer (both increased purity and more secure supply chains) non-animal GAG-based therapeutics.     

The microbial flora of sugary kefir grain (the gingerbeer plant): biosynthesis of the grain fromLactobacillus hilgardii producing a polysaccharide gel

Summary The microflora of sugary kefir grains was principally mesophilic and consisted chiefly of lactic acid bacteria [Lactobacillus casei, Lactobacillus hilgardii (=brevis),Leuconostoc mesenteroides ssp.dextranicum, Streptococcus lactis] and a small proportion of yeasts (Zygosaccharomyces florentinus, Torulospora pretoriensis, Kloeckera apiculata, Candida lambica andC. valida). Few coliforms and faecal streptococci were observed. Observation by scanning electron microscopy revealed that the filamentous yeasts adhered to the bacteria on the periphery of the grain.Lactobacillus hilgardii, the single microorganism isolated which was able to produce a gelling polysaccharide, was important in the biosynthesis of the grain. Pieces of gel produced by this strain, and transferred in a yeast extract-sucrose solution, grew and resembled the household kefir grains. This represents a new, cheap way of producing immobilized cells by self-embedding in a neutral polysaccharide.

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Flore microbienne du grain de kefir sucré (plant de la bière de gingembre): biosynthèse du grain par Lactobacillus hilgardii produisant un gel de polysaccharide

Résumé La microflore des grains de kefir sucré est essentiellement mésophile et comprend principalement des bactéries lactiquesLactobacillus casei, Lactobacillus hilgardii (= brevis),Leuconostoc mesenteroides ssp.dextranicum, Streptococcus lactis ainsi qu'une petite proportion de levures (Zygosaccharomyces florentinus, Torulospora pretoriensis, Kloeckera apiculata, Candida lambica etC. valida). On a observé peu de coliformes et de streptocoques fécaux. L'observation au microscope électronique à balayage a révélé que les levures filamenteuses adhèrent aux bactéries sur la périphérie du grain.Lactobacillus hilgardii, le seul microorganisme isolé susceptible de produire un polysaccharide gélifiant, est important dans la biosynthèse du grain. Des morceaux de gel produit par cette souche et transférés dans une solution d'extrait de levure et de saccharose, croissent et ressemblent aux grains de kefir domestique. Ceci représente une manière nouvelle, peu coûteuse de produire des cellules immobilisées par auto-piégeage dans un polysaccharide neutre.

     

Rapid estimation of polysaccharide content in complex microbial culture media

A rapid gravimetric assay for estimation of polysaccharide content in complex culture media was developed and validated. The quantitative assay showed great selectivity for the determination of xylan and pectin, eliminating all interferences from other medium components. In addition, our data showed this protocol could also be applied to the estimation of other polysaccharides in liquid media such as cellulose Avicel^®, chitin, starch and crude hemicellulose. Therefore, this method represents a useful tool for monitoring the in vitro microbial degradation of polysaccharides.     

Pseudomonas aeruginosa polysaccharide Psl supports airway microbial community development

Pseudomonas aeruginosa dominates the complex polymicrobial cystic fibrosis (CF) airway and is a leading cause of death in persons with CF. Oral streptococcal colonization has been associated with stable CF lung function. However, no studies have demonstrated how Streptococcus salivarius, the most abundant streptococcal species found in individuals with stable CF lung disease, potentially improves lung function or becomes incorporated into the CF airway biofilm. By utilizing a two-species biofilm model to probe interactions between S. salivarius and P. aeruginosa, we discovered that the P. aeruginosa exopolysaccharide Psl promoted S. salivarius biofilm formation. Further, we identified a S. salivarius maltose-binding protein (MalE) that is required for promotion of biofilm formation both in vitro and in a Drosophila melanogaster co-infection model. Finally, we demonstrate that promotion of dual biofilm formation with S. salivarius is common among environmental and clinical P. aeruginosa isolates. Overall, our data supports a model in which S. salivarius uses a sugar-binding protein to interact with P. aeruginosa exopolysaccharide, which may be a strategy by which S. salivarius establishes itself within the CF airway microbial community.Subject terms: Bacteriology, Biofilms, Microbiome, Clinical microbiology