Effects of different solid state fermentation substrate on biochemical properties of cutinase from Fusarium sp.

The present results demonstrate that the catalytic characteristics of cutinase produced by the same strain differ depending on the culture medium used. This conclusion was possible after the study of biochemical characterization and enantioselective properties of cutinases produced by Fusarium oxysporum in four different culture mediums. The mediums were composed of wheat bran, soybean rind, rice bran and Jatropha curcas seed cake, different Brazilian agricultural by-products. The largest difference can be observed on cutinase produced by J. curcas seed cake. This enzyme has been activated in most metal ions tested and exhibited excellent stability in organic solvent, especially hexane. The cutinase produced in rice bran showed greatest activity in the presence of p-nitrophenyl butyrate as a substrate, whereas the other enzymes showed greatest activity in the presence of p-nitrophenyl caprilate. Regarding enantioselective properties the cutinase produced in soybean rind showed the best result compared to enzymes produced in wheat bran.     

Carbon Source Requirements for Exopolysaccharide Production by Lactobacillus casei CG11 and Partial Structure Analysis of the Polymer

Exopolysaccharide production by Lactobacillus casei CG11 was studied in basal minimum medium containing various carbon sources (galactose, glucose, lactose, sucrose, maltose, melibiose) at concentrations of 2, 5, 10, and 20 g/liter. L. casei CG11 produced exopolysaccharides in basal minimum medium containing each of the sugars tested; lactose and galactose were the poorest carbon sources, and glucose was by far the most efficient carbon source. Sugar concentrations had a marked effect on polymer yield. Plasmid-cured Muc^- derivatives grew better in the presence of glucose and attained slightly higher populations than the wild-type strain. The values obtained with lactose were considerably lower for both growth and exopolysaccharide yield. The level of specific polymer production per cell obtained with glucose was distinctively lower for Muc^- derivatives than for the Muc⁺ strain. The polymer produced by L. casei CG11 in the presence of glucose was different from that formed in the presence of lactose. The polysaccharide produced by L. casei CG11 in basal minimum medium containing 20 g of glucose per liter had an intrinsic viscosity of 1.13 dl/g. It was rich in glucose (76%), which was present mostly as 2- or 3-linked residues along with some 2,3 doubly substituted glucose units, and in rhamnose (21%), which was present as 2-linked or terminal rhamnose; traces of mannose and galactose were also present.     

Fermentation media and production of exotoxin by three varieties of Bacillus thuringiensis

The insecticidal activities of the exotoxin produced by three varieties of Bacillus thuringiensis grown in six fermentation media were determined by testing the supernatants against larvae of the house fly, Musca domestica, and the black cutworm, Agrotis ipsilon. The activities of the exotoxins from the isolates varied when they were grown in the same medium and also when they were grown in different media. When an isolate of B. thuringiensis var. thuringiensis, and of var. tolworthi were grown in proflo broth, the supernatants produced were more toxic to house fly than to black cutworm larvae, indicating the presence of more than one exotoxin. Autoclaving the supernatants for 15 and 30 min further demonstrated the presence of several exotoxins.     

Byssus production rate of the White Sea blue mussel <Emphasis Type="Italic">Mytilus edulis</Emphasis> (Linnaeus, 1758) in the presence of metabolites of some hydrobionts

The byssus production of the blue mussel Mytilus edulis L. was studied in the laboratory in the presence of the metabolites of the following animals: a predator (a starfish Asterias rubens L.) and several species competing with the mussel in White Sea fouling communities (a bivalve Hiatella arctica L. and a solitary ascidian Styela rustica L.). The byssus threads and attachment plaques produced by each mussel per day were counted. The number of byssus threads and plaques was smallest in pure sea water and in the presence of metabolites produced by conspecific individuals.     

The effect of tunicamycin on the molecular heterogeneity of guinea pig migration inhibitory factor

Migration inhibitory factor (MIF) was produced by guinea pig lymph node cells stimulated with concanavalin A in the absence and presence of the glycosylation inhibitor tunicamycin. The active supernatants were purified on Sephadex G-100 and fractionated into pH 3-MIF and pH 5-MIF using isoelectrofocusing. When produced in the absence of tunicamycin pH 3-MIF shows extensive charge heterogeneity with activity focusing from pH 3.0 to 4.5; it elutes from Sephadex G-75 with molecules of an apparent MW of 70,000. In contrast, pH 3-MIF produced in the presence of tunicamycin (TM-pH 3-MIF) focuses as a sharp homogeneous peak with a pI of 3.6 to 4.0 and elutes from Sephadex G-75 with molecules of an apparent MW of 25,000–35,000. TM-pH 3-MIF is trypsin sensitive and displays a buoyant density similar to that of proteins which contain little or no carbohydrate (?₂₅ 1.26?1.34). Tunicamycin caused no detectable change in the characteristics of pH 5-MIF. This study indicates that lymphocytes stimulated in the presence of tunicamycin produce a novel species of pH 3-MIF with characteristics distinct from classical pH 3-MIF.     

Single cell and in vivo analyses elucidate the effect of xylC lactonase during production of D-xylonate in Saccharomyces cerevisiae

D-xylonate is a potential platform chemical which can be produced by engineered Saccharomyces cerevisiae strains. In order to address production constraints in more detail, we analysed the role of lactone ring opening in single cells and populations. Both D-xylono-γ-lactone and D-xylonate were produced when the Caulobacter crescentus xylB (D-xylose dehydrogenase) was expressed in S. cerevisiae, with or without co-expression of xylC (D-xylonolactonase), as seen by ¹H NMR. XylC facilitated rapid opening of the lactone and more D-xylonate was initially produced than in its absence. Using in vivo ¹H NMR analysis of cell extracts, culture media and intact cells we observed that the lactone and linear forms of D-xylonic acid were produced, accumulated intracellularly, and partially exported within 15–60 min of D-xylose provision.During single-cell analysis of cells expressing the pH sensitive fluorescent probe pHluorin, pHluorin fluorescence was gradually lost from the cells during D-xylonate production, as expected for cells with decreasing intracellular pH. However, in the presence of D-xylose, only 9% of cells expressing xylB lost pHluorin fluorescence within 4.5 h, whereas 99% of cells co-expressing xylB and xylC lost fluorescence, a large proportion of which also lost vitality, during this interval. Loss of vitality in the presence of D-xylose was correlated to the extracellular pH, but fluorescence was lost from xylB and xylC expressing cells regardless of the extracellular condition.     

Phytochrome: A Re-examination of the Quaternary Structure

Highly purified phytochrome samples from rye (Secale Cereale cv. Cougar) were fractionated by ultracentrifugation in isokinetic sucrose density gradients. Three protein species were separated with estimated sedimentation coefficients of 6.5S, 8.0S, and 11.5S. The 6.5S and 8.0S forms contained photoreversible phytochrome and produced a single subunit of 120,000 molecular weight upon reduction and electrophoresis in the presence of sodium dodecyl sulfate. The 11.5S species contained no detectable phytochrome. Reduction and electrophoresis of the 11.5S species in the presence of sodium dodecyl sulfate produced a major polypeptide of 32,000 molecular weight and a minor polypeptide of 48,000 molecular weight. The square tetrameric structures, observed by electron microscopy and previously thought to be phytochrome molecules, were found to be due to the presence of this 11.5S species in phytochrome preparations.     

Target cell specificity of the Pasteurella haemolytica leukotoxin is unaffected by the nature of the fatty-acyl group used to activate the toxin in vitro

The leukotoxin (LktA) of Pasteurella haemolytica is active only against cells of ruminant origin. It is synthesised as an inactive protoxin encoded by the lktA gene and post-translationally modified to the active toxin by the product of the lktC gene. The LktA and LktC proteins were expressed separately in Escherichia coli and partially purified. Active LktA was produced in vitro in the presence of LktC and acyl-acyl carrier protein (ACP) charged separately in vitro with a fatty-acyl group. The toxic activity and target cell specificity of LktA and adenylate cyclase toxin (CyaA), a toxin active against a wide variety of mammalian cells, were investigated after activation with ACP charged with different fatty acids. Palmitoyl-ACP produced the most active toxin in both cases and, although other fatty acids were also effective, the fatty acid preference was the same for the in vitro activation of both toxins. Activated LktA remained ruminant cell-specific whichever acyl group was used to acylate the A protoxin.     

1,2 Propanediol utilization by Lactobacillus reuteri DSM 20016, role in bioconversion of glycerol to 1,3 propanediol, 3-hydroxypropionaldehyde and 3-hydroxypropionic acid

The objective of the presented work is to demonstrate the metabolism of 1,2 propandiol by Lactobacillus reuteri and to elucidate the metabolites produced during the process. This Metabolic pathway is crucial for biotechnological applications using L. reuteri in bioconversion of glycerol to industrially important plate-form chemicals. L. reuteri grown on minimal media containing 1,2 propanediol was able to utilize the compound as a sole carbon and energy source. The growth of the bacteria was linear with time; however the specific growth rate was significantly low compared to bacteria grown on the same media in the presence of glucose.The fermentation of 1,2 propanediol by L. reuteri in presence and absence of glucose was followed for 72 h and the metabolites produced during the process were detected using HPLC. 1,2 Propanediol was completely converted to propionaldhyde in a time dependent fashion, this process had a higher rate in presence of glucose. Consequently the produced propionaldhyde was converted to propionic acid and propanol in a skewed equimolar manner. In presence of glucose: acetic acid, lactic acid, succinic acid and ethanol were detected while in absence of glucose only minute amounts of acetic acid and lactic acid were detected which indicates presence of different metabolic pathways for glucose and 1,2 propanediol metabolism. Resting cells of L. reuteri induced in presence of 1,2 propanediol have shown significant capabilities to convert aqueous glycerol to 1,3 propanediol, 3-hydroxypropionaldhyde and a compound proposed to be 3-hydroxypropionic acid as detected by gas chromatographic technique.     

Production of Cellulose Microfibrils by Rhizobium

Electron microscope examination of Rhizobium spp. revealed microfibrils produced by flocculating strains but not by nonflocculating strains. The microfibrils from R. trifolii (NA30) were isolated and identified as cellulose by enzymatic, X-ray diffraction, and infrared spectral analyses. Both infective and noninfective strains of R. trifolii flocculated and produced microfibrils. More infection threads were observed in clover root hairs growing in the presence of flocs in comparison with root hairs where single bacterial cells predominated.     

Importance of the methyl-citrate cycle on glycerol metabolism in the yeast Yarrowia lipolytica

A novel approach to trigger lipid accumulation and/or citrate production in vivo through the inactivation of the 2-methyl-citrate dehydratase in Yarrowia lipolytica was developed. In nitrogen-limited cultures with biodiesel-derived glycerol utilized as substrate, the Δphd1 mutant (JMY1203) produced 57.7 g/L of total citrate, 1.6-fold more than the wild-type strain, with a concomitant glycerol to citrate yield of 0.91 g/g. Storage lipid in cells increased at the early growth stages, suggesting that inactivation of the 2-methyl-citrate dehydratase would mimic nitrogen limitation. Thus, a trial of JMY1203 strain was performed with glycerol under nitrogen-excess conditions. Compared with the equivalent nitrogen-limited culture, significant quantities of lipid (up to ∼31% w/w in dry weight, 1.6-fold higher than the nitrogen-limited experiment) were produced. Also, non-negligible quantities of citric acid (up to ∼26 g/L, though 0.57-fold lower than the nitrogen-limited experiment) were produced, despite remarkable nitrogen presence into the medium, indicating the construction of phenotype that constitutively accumulated lipid and secreted citrate in Y. lipolytica during growth on waste glycerol utilized as substrate.     

Production of volatile organic compounds by an antagonistic strain Paenibacillus polymyxa WR-2 in the presence of root exudates and organic fertilizer and their antifungal activity against Fusarium oxysporum f. sp. niveum

The volatile organic compounds (VOCs) produced by antagonistic microbes have great antifungal potential against soil-borne fungal pathogens. The VOCs produced by Paenibacillus polymyxa strain WR-2 in the presence of root exudates and organic fertilizer were identified and their effects on the growth and spore germination of Fusarium oxysporum f. sp. niveum were evaluated. The VOCs produced by WR-2 inhibited the growth of F. oxysporum by 38%, 36% and 40% in agar medium, sterilized soil and natural soil, respectively. This inhibitory effect was increased to 60%, 58% and 64% with the addition of organic fertilizer in agar medium, sterilized soil and natural soil, respectively. The addition of root exudates did not affect the production of antifungal VOCs by WR-2. The VOCs produced by WR-2 completely inhibited the germination of F. oxysporum spores. Out of 42 identified VOCs, seven VOCs; benzothiazole, benzaldehyde, undecanal, dodecanal, hexadecanal, 2-tridecanone and phenol were found to inhibit the growth of F. oxysporum. The results of these experiments suggest another significance of using organic fertilizer as a carrier material with the biocontrol agents to control soil-borne fungal pathogens.     

Predator-induced morphological shift in the pea aphid

Aphids exhibit a polymorphism whereby individual aphids are either winged or unwinged. The winged dispersal morph is mainly responsible for the colonization of new plants and, in many species, is produced in response to adverse environmental conditions. Aphids are attacked by a wide range of specialized predators and predation has been shown to strongly influence the growth and persistence of aphid colonies. In two experiments, we reared two clones of pea aphid (Acyrthosiphon pisum) in the presence and absence of predatory ladybirds (Coccinella septempunctata or Adalia bipunctata). In both experiments, the presence of a predator enhanced the proportion of winged morphs among the offspring produced by the aphids. The aphid clones differed in their reaction to the presence of a ladybird, suggesting the presence of genetic variation for this trait. A treatment that simulated disturbance caused by predators did not enhance winged offspring production. The experiments indicate that aphids respond to the presence of a predator by producing the dispersal morph which can escape by flight to colonize other plants. In contrast to previous examples of predator-induced defence this shift in prey morphology does not lead to better protection against predator attack, but enables aphids to leave plants when mortality risks are high.     

The degradation of l-threose at Maillard reaction conditions

l-Threose, a comparatively unstable aldose, is produced from l-ascorbic acid in the presence of oxygen and participates vigorously in Maillard reactions, even at comparatively mild conditions. In the present study, the degradation of l-threose at pH 7.0 alone, in the presence of N-α-acetyl-l-lysine, and at pH 2.0 alone at 37°C was investigated by identification of some of the products produced in the reactions by means of GLC and GLC-MS. Among the compounds identified were3-deoxy-tetros-2-ulose (1), the predicted alkaline rearrangement product derived from 1 (2,4-dihydroxybutyrate, the 4-carbon metasaccharinic acid), as well as glyceraldehyde. Isotopic tracer studies clearly show that the glyceraldehyde is produced by loss of C-1 from the starting l-threose molecule. The presence of N-acetyl lysine in incubation solutions appears to accelerate the production of 1, but the formation of glyceraldehyde appears to be independent of the lysine derivative.     

The effect of acid proteinases on the activity and stability of glucoamylase preparations

It was demonstrated that the presence of acid proteinases in the preparation isolated from Aspergillus awamori decreased the activity and stability of glucoamylase. Patterns of changes in the enzymatic activity and stability of glucoamylase at increased temperature and various pH values were studied over a long-term storage. A biospecific sorbent for removal of acid proteinases was synthesized and glucoamylase preparations free of proteolytic activity were produced.     

The NADPH oxidase AoNoxA in <Emphasis Type="Italic">Arthrobotrys oligospora</Emphasis> functions as an initial factor in the infection of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Reactive oxygen species (ROS) produced by NADPH oxidases can serve as signaling molecules to regulate a variety of physiological processes in multi-cellular organisms. In the nematophagous fungus Arthrobotrys oligospora, we found that ROS were produced during conidial germination, hyphal extension, and trap formation in the presence of nematodes. Generation of an AoNoxA knockout strain demonstrated the crucial role of NADPH oxidase in the production of ROS in A. oligospora, with trap formation impaired in the AoNoxA mutant, even in the presence of the nematode host. In addition, the expression of virulence factor serine protease P186 was up-regulated in the wild-type strain, but not in the mutant strain, in the presence of Caenorhabditis elegans. These results indicate that ROS derived from AoNoxA are essential for full virulence of A. oligospora in nematodes.     

Transgenerational plasticity in the eye size of Daphnia

It is well established that environmental signals can induce phenotypic responses that persist for multiple generations. The induction of such ‘transgenerational plasticity’ (TGP) depends upon the ability of organisms to accurately receive and process information from environmental signals. Thus, sensory systems are likely intertwined with TGP. Here we tested the link between an environmental stressor and transgenerational responses in a component of the sensory system (eye size) that is linked to enhanced vision and ecologically relevant behaviours. We reared 45 clones of Daphnia pulicaria in the presence and absence of a low-quality resource (cyanobacteria) and evaluated shifts in relative eye size in offspring. Our results revealed divergent shifts in relative eye size within- and across-generations. Parental Daphnia that were fed cyanobacteria produced a smaller eye than Daphnia fed high-quality algae. Such differences were then reversed in the offspring generation; Daphnia whose mothers were fed cyanobacteria produced larger eyes than Daphnia that were continually fed green algae. We discuss the extent to which this maternal effect on eye size is an adaptive response linked to improved foraging.     

Polygalacturonase Production by Agrobacterium tumefaciens Biovar 3

Agrobacterium tumefaciens biovar 3 causes both crown gall and root decay of grape. Twenty-two Agrobacterium strains representing biovars 1, 2, and 3 were analyzed for tumorigenicity, presence of a Ti plasmid, ability to cause grape seedling root decay, and pectolytic activity. All of the biovar 3 strains, regardless of their tumorigenicity or presence of a Ti plasmid, caused root decay and were pectolytic, whereas none of the biovar 1 and 2 strains had these capacities. Isoelectrically focused gels that were activity stained with differentially buffered polygalacturonate-agarose overlays revealed that all of the biovar 3 strains produced a single polygalacturonase with a pH optimum of 4.5 and pIs ranging from 4.8 to 5.2. The enzyme was largely extracellular and was produced constitutively in basal medium supplemented with a variety of carbon sources including polygalacturonic acid. Lesions on grape seedling roots inoculated with A. tumefaciens biovar 3 strain CG49 yielded polygalacturonase activity with a pI similar to that of the enzyme produced by the bacterium in culture. These observations support the hypothesis that the polygalacturonase produced by A. tumefaciens biovar 3 has a role in grape root decay.     

On the subdivision of the genusCeratocystis

The desirability is discussed of a subdivision of the genusCeratocystis into a group characterized by the presence of a phialidic conidial state, and a group in which the conidia are usually produced exogenously. Corroborative evidence for this arrangement is found in the carbohydrate constitution of the cells: in all examined species of the former group (Ceratocystis s.str.) rhamnose and cellulose are absent, whereas in the latter both components are present (Ophiostoma H. et P. Sydow).     

Properties of gallic acid-induced extracellular laccase of Botrytis cinerea

The properties of the extracellular laccase produced by Botrytis cinerea induced by garlic acid are compared with the laccase produced by Botrytis in the presence of grape juice. The extra- and intracellular laccases are compared and found to be very similar.