Reduction of non-digestible oligosaccharides in soymilk: application of engineered lactic acid bacteria that produce alpha-galactosidase

Human consumption of soy-derived products has been limited by the presence of non-digestible oligosaccharides (NDO), such as the alpha-galactooligosaccharides raffinose and stachyose. Most mammals, including man, lack pancreatic alpha-galactosidase (alpha-Gal), which is necessary for the hydrolysis of these sugars. However, such NDO can be fermented by gas-producing microorganisms present in the cecum and large intestine, which in turn can induce flatulence and other gastrointestinal disorders in sensitive individuals. The use of microorganisms expressing alpha-Gal is a promising solution to the elimination of NDO before they reach the large intestine. In the present study, lactic acid bacteria engineered to degrade NDO have been constructed and are being used as a tool to evaluate this solution. The alpha-Gal structural genes from Lactobacillus plantarum ATCC8014 (previously characterized in our laboratory) and from guar have been cloned and expressed in Lactococcus lactis. The gene products were directed to different bacterial compartments to optimize their possible applications. The alpha-Gal-producing strains are being evaluated for their efficiency in degrading raffinose and stachyose: i) in soymilk fermentation when used as starters and ii) in situ in the upper gastrointestinal tract when administered to animals orally, as probiotic preparations. The expected outcomes and possible complications of this project are discussed.     

Human alpha-fucosidase. Purification and properties.

Human placental alpha-fucosidase (EC 3.2.1.51) has been extensively purified and partially characterized with respect to kinetic and structured properties. Although the enzyme seems to be separated by DEAE-cellulose chromatography in two forms which differ in their molecular weight and thermostability, an interconversion between the two forms takes place during storage and/or electrofocusing so that the same peaks of activity, revealed by the latter technique, are found before and after DEAE-cellulose chrome. The heterogeneous peaks of activity revealed by isoelectrofocusing show a reproducible pattern in the different tissues examined, except in serum where their pI values are consistently more acidic.     

Microorganisms that break down alkyl sulfates

The following bacteria assimilating alkyl sulphates as a sole source of carbon and energy were isolated from various substrates: Pseudomonas, Achromobacter, Flavobacterium, Citrobacter, Enterobacter. The bacteria decomposed sodium dodecyl sulphate at a high rate, and some of them, industrial preparations of alkyl sulphates. The ability to assimilate alkyl sulphates was tested in 257 collection cultures belonging to different taxonomic groups. Alkyl sulphates were found to be decomposed by heterotrophous gram-negative rod-like bacteria belonging to different families and genera. The frequency of bacteria destroying alkyl sulphates at a high rate was found most often in the Pseudomonas genus.     

Microorganisms from composting leaves: Ability to produce extracellular degradative enzymes

Mixed populations of bacteria, fungi, and actinomycetes in a leaf compost pile were examined over a 100-day test period for their ability to produce extracellular proteolytic, lipolytic, amylolytic, cellulolytic, pectolytic, and ureolytic enzymes and ability to utilize alkanes. Urea was added to the leaves to adjust the carbon to nitrogen ratio but was of little value in maintaining the proper ratio since it was degraded within the first few days. The degradative enzymes excreted by microorganisms was dependent on the temperature of the pile. In many cases organisms able to produce specific extracellular enzymes at medium temperatures were able to grow at high temperatures, but either did not excrete the specific enzymes or the enzymes were inactivated by the high temperature.     

Thermophilic bacteria, Thermus ruber, that produce a bright orange pigment

A non-sporeforming gram-negative bacterium isolated from soils of Africa was shown to belong to the species Thermus ruber in its morphologo-cytological and physiologobiochemical properties. It differed from the Thermus ruber species described elsewhere (Loginova et al., 1975; Loginova and Egorova, 1975) only on colour: the former contained a bright-red pigment while the latter produced bright-orange pigments.     

Resistance to thiostrepton, siomycin, and sporangiomycin in actinomycetes that produce them.

The antibiotics thiostrepton, siomycin, and sporangiomycin are closely related both in structure and in mode of action. Actinomycetes which produce this group of compounds possess ribonucleic acid-pentose methylases, which act upon 23S ribosomal ribonucleic acid and render ribosomes resistant to the action of these antibiotics. This is achieved via the formation of a single residue of 2'-O-methyladenosine per ribosome.     

Fungi that produce mycotoxins: Conditions and occurrence

The occurrence of mycotoxins, in agricultural commodities is a worldwide problem with almost all commodities being potentially susceptible to contamination under the proper conditions. The genera of fungi most implicated are Aspergillus, Penicillium and Fusarium, although the potential for toxin production varies considerably within any given species.Conditions that affect toxin production include fungal strain variation, genetic susceptibility of the host plant or commodity, moisture content, commodity composition, temperature, aeration, microbial population and stress factors. There is undoubtedly interaction between these factors so that laboratory studies involving limited variables can only, at best, approximate field conditions.Natural contamination with mycotoxins has been reported for most of the major agricultural commodities in the world including corn, wheat, rice, millet, barley, oats, sorghum, peanuts, beans, copra, some fruits and nuts and various forages; strangely, soybeans do not appear to be involved to any major extent. The major mycotoxins on commodities reported to date include aflatoxin, the trichothecenes, ochratoxin, citrinin, zearalenone, sporidesmins and some tremorgens. However, laboratory studies have shown that the fungi are capable of producing hundreds of toxic chemicals, most of which are not included in routine analyses. In addition, since toxin effects are often insidious and may go undetected, the true dimensions of the mycotoxin problem are unknown.     

Phosphoinositide kinases as enzymes that produce versatile signaling lipids, phosphoinositides

Phosphoinositide kinases comprise a unique family of enzymes that catalyze the phosphorylation of phosphatidylinositol and its phosphorylated metabolites to produce seven phosphoinositides. Recent advances have revealed that these phosphoinositides have specific physiological functions, such as in actin cytoskeletal reorganization, membrane transport, cell proliferation and survival, in eukaryotic cells and that each phosphoinositide kinase is differently and precisely regulated. Here we describe the diverse regulation and physiological functions of phosphoinositide kinases involving their products.     

Screening for microorganisms that produce only endo-inulinase

Sixteen fungal strains reported in the literature as endo-inulinase producers and three bacterial strains, isolated from the dahlia rhizosphere, were analysed for endo-inulinase production. From four isolated strains (one fungus and three bacteria) the results were evaluated in terms of substrate consumption, cell growth and production of endo-inulinases. All three bacterial strains were sole endo-inulinase producers and, among these, strain Paenibacillus sp. CDB 003 was the most suitable for endo-inulinase production, as this enzyme produced inulobiose as the principal substrate as well as inulo-oligosaccharides with polymerisation degrees of 3-5.     

Detection of microorganisms that produce extracellular maltases

Maltose is a disaccharide that is widely distributed in nature. Maltases (EC 3.2.1.20) are potentially useful, therefore, in industry, clinical laboratories, and basic research. Nevertheless, few microbial maltases have been described (1), and only one true maltase, prepared from yeast, is commercially available. Procedures are needed for screening microorganisms that produce extracellular maltases. This communication describes one method.     

Synthesis of an alpha-fucosidase inhibitor, 5a-carba-beta-L-fucopyranosylamine, and fucose-type alpha- and beta-DL-valienamine unsaturated derivatives

Discovery of a very potent alpha-fucosidase inhibitor 5a-carba-alpha-L-fucopyranosylamine led to preparation of its beta-anomer and the respective unsaturated derivatives, fucose-type alpha- and beta-valienamines, in order to elucidate the structure-activity relationship of carba-aminosugar inhibitors of this kind. Compound was demonstrated to be a potent inhibitor (K(i)=2.0 x 10(-7) M, bovine kidney), possessing ca. one-tenth of the activity of the parent. Interestingly, and were found to be rather weak inhibitors, contrary to the expectations based on the activity relationships between the alpha-glucosidase inhibitors, alpha-glucose-type validamine and valienamine.     

Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species

NADPH oxidases of the Nox family exist in various supergroups of eukaryotes but not in prokaryotes, and play crucial roles in a variety of biological processes, such as host defense, signal transduction, and hormone synthesis. In conjunction with NADPH oxidation, Nox enzymes reduce molecular oxygen to superoxide as a primary product, and this is further converted to various reactive oxygen species. The electron-transferring system in Nox is composed of the C-terminal cytoplasmic region homologous to the prokaryotic (and organelle) enzyme ferredoxin reductase and the N-terminal six transmembrane segments containing two hemes, a structure similar to that of cytochrome b of the mitochondrial bc(1) complex. During the course of eukaryote evolution, Nox enzymes have developed regulatory mechanisms, depending on their functions, by inserting a regulatory domain (or motif) into their own sequences or by obtaining a tightly associated protein as a regulatory subunit. For example, one to four Ca(2+)-binding EF-hand motifs are present at the N-termini in several subfamilies, such as the respiratory burst oxidase homolog (Rboh) subfamily in land plants (the supergroup Plantae), the NoxC subfamily in social amoebae (the Amoebozoa), and the Nox5 and dual oxidase (Duox) subfamilies in animals (the Opisthokonta), whereas an SH3 domain is inserted into the ferredoxin-NADP(+) reductase region of two Nox enzymes in Naegleria gruberi, a unicellular organism that belongs to the supergroup Excavata. Members of the Nox1-4 subfamily in animals form a stable heterodimer with the membrane protein p22(phox), which functions as a docking site for the SH3 domain-containing regulatory proteins p47(phox), p67(phox), and p40(phox); the small GTPase Rac binds to p67(phox) (or its homologous protein), which serves as a switch for Nox activation. Similarly, Rac activates the fungal NoxA via binding to the p67(phox)-like protein Nox regulator (NoxR). In plants, on the other hand, this GTPase directly interacts with the N-terminus of Rboh, leading to superoxide production. Here I describe the regulation of Nox-family oxidases on the basis of three-dimensional structures and evolutionary conservation.     

Search of Microorganisms that Degrade PAHs under Alkaline Conditions

Bacterial strains were enriched from building rubble contaminated with polycyclic aromatic hydrocarbons (PAHs). These strains were studied as an inoculum in bioremediation processes with contaminated building rubble. The selection criteria for the bacteria were broad profiles in PAH degradation, stable expression of the traits and tolerance to alkaline conditions. Various strains of Micrococcus sp., Dietzia sp., Rhodococcus sp. and Pseudomonas sp. met the selection criteria. In general, degradative activity was limited at higher pH values. Strains of Micrococcus were suitable for practical use as complete degradation of various PAHs was observed at pH values exceeding 10. Strains of Dietzia sp. showed broad PAH degradation profile, but in some cases degradation came to a halt leaving some of the PAHs unutilized. With Dietzia sp. this could be due to inhibitory effects from the accumulation of toxic PAH metabolic products and/or growth‐limiting media conditions.     

Two different genomes that produce the same result

Despite considerable differences in genomic sequence, the developmental program of gene expression between two similar Dictyostelium species is remarkably similar.     

Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans

Golgi alpha-mannosidase II (GlcNAc transferase I-dependent alpha 1,3[alpha 1,6] mannosidase, EC 3.2.1.114) catalyzes the final hydrolytic step in the N-glycan maturation pathway acting as the committed step in the conversion of high mannose to complex type structures. We have isolated overlapping clones from a murine cDNA library encoding the full length alpha-mannosidase II open reading frame and most of the 5' and 3' untranslated region. The coding sequence predicts a type II transmembrane protein with a short cytoplasmic tail (five amino acids), a single transmembrane domain (21 amino acids), and a large COOH-terminal catalytic domain (1,124 amino acids). This domain organization which is shared with the Golgi glycosyl-transferases suggests that the common structural motifs may have a functional role in Golgi enzyme function or localization. Three sets of polyadenylated clones were isolated extending 3' beyond the open reading frame by as much as 2,543 bp. Northern blots suggest that these polyadenylated clones totaling 6.1 kb in length correspond to minor message species smaller than the full length message. The largest and predominant message on Northern blots (7.5 kb) presumably extends another approximately 1.4-kb downstream beyond the longest of the isolated clones. Transient expression of the alpha-mannosidase II cDNA in COS cells resulted in 8-12-fold overexpression of enzyme activity, and the appearance of cross-reactive material in a perinuclear membrane array consistent with a Golgi localization. A region within the catalytic domain of the alpha-mannosidase II open reading frame bears a strong similarity to a corresponding sequence in the rat liver endoplasmic reticulum alpha-mannosidase and the vacuolar alpha-mannosidase of Saccharomyces cerevisiae. Partial human alpha-mannosidase II cDNA clones were also isolated and the gene was localized to human chromosome 5.     

Purification and characterization of neutral alpha-mannosidase that is activated by Co2+ from Japanese quail oviduct

An alpha-mannosidase was purified from the magnum section of Japanese quail oviduct by ammonium sulfate precipitation, DEAE-Sephacel chromatography, Sephacryl S-300 chromatography, mannan-Sepharose 4B chromatography, and hydroxyapatite chromatography. The purified alpha-mannosidase (referred to as neutral alpha-mannosidase) showed a single band on polyacrylamide gel with or without sodium dodecyl sulfate. Its molecular weight was found to be 330,000 by gel chromatography. Neutral alpha-mannosidase hydrolyzed p-nitrophenyl alpha-D-mannopyranoside and the pyridylamino derivative of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc (Km value was 3 mM). Mannosyl alpha 1-2 linkages in the pyridylamino derivative of Man alpha 1-2 Man alpha 1-6(Man alpha 1-2Man alpha 1-3)Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc were hardly hydrolyzed. Its optimum pH was found to be 7.0. The activity of the enzyme was activated by CO2+, and was potently inhibited by Cu2+, Hg2+, swainsonine, and 1-deoxymannojirimycin.