Localization and release of β-glucosidase in the thermophilic and cellulolytic fungus, Sporotrichum thermophile

n a medium containing cellulose as the carbon source, the rapid growth of Sporotrichum thermophile, the secretion of cellulases and the utilization of cellulose were well-correlated events. The production of beta-glucosidase in culture medium lagged behind cellulases, coinciding with the time of extensive autolysis of mycelia. By contrast, neither apparent autolysis nor secretion of beta-glucosidase occurred when S. thermophile was grown in medium containing cellobiose; the enzyme activity remained associated with mycelia. The release of beta-glucosidase in cellulose-grown cultures was correlated with the activity of the lytic enzyme in the cell wall. Immunocytochemical localization and biochemical characterization showed that a beta-glucosidase released in the cellulose medium was the same as that which remained associated with mycelia grown on cellobiose. The results indicated that the release of beta-glucosidase in the cellulose culture is incidental to the activity of the lytic enzymes which are strongly induced by cellulose. The observations minimize a functional role of the culture fluid beta-glucosidase in cellulolysis by the fungus. Rather, the available information suggests that the cellulases and beta-glucosidases associated with the hyphal cell wall may play a role in cellulolysis by the fungus. (C) 1994 Academic Press, Inc.

Purification and partial characterization of acyl carrier proteins from developing oil seeds of pisa (Actinodaphne hookeri) and ground nut (Arachis hypogaea)

Acyl carrier proteins (ACP) were purified to homogeneity in the active form from developing seeds of pisa (Actinodaphne hookeri) which synthesizes exclusively trilaurin and from ground nut (Arachis hypogaea) which synthesizes triacylglycerols containing long chain fatty acids. Two major isoforms of ACPs were purified from developing pisa seeds using DEAE-cellulose, Superose-6 FPLC and C-4 reversed phase HPLC chromatographic methods. In contrast, only a single form of ACP was present in ground nut seeds which was purified by anion-exchange and activated thiol-Sepharose 4B affinity chromatography. The two isoforms of ACPs from pisa showed nearly the same specific activity of 6,706 and 7,175 pmol per min per mg protein while ground nut ACP showed a specific activity of 3,893 pmol per min per mg protein when assayed using E. coli acyl-ACP synthetase and [1-C-14]palmitic acid. When compared with E. coli ACP, the purified ACPs from both the seeds showed considerable difference in their mobility in native PAGE, but showed similar mobility in SDS-PAGE under reducing conditions. In the absence of reducing agents formation of dimers was quite prominent. The ACPs from both the seed sources were acid- and heat-stable. The major isoform of pisa seed ACP and the ground nut ACP contain 91 amino acids with M(r) 11,616 and 1,228 respectively. However, there is significant variation in their amino acid composition. A comparision of the amino acid sequence in the N-terminal region of pisa and ground nut seed ACPs showed considerable homology between themselves and with other plant ACPs but not with E. coli ACP.

Role of length scales on microwave thawing dynamics in 2D cylinders

Microwave (MW) thawing of 2D frozen cylinders exposed to uniform plane waves from one face, is modeled using the effective heat capacity formulation with the MW power obtained from the electric field equations. Computations are illustrated for tylose (23% methyl cellulose gel) which melts over a range of temperatures giving rise to a mushy zone. Within the mushy region the dielectric properties are functions of the liquid volume fraction. The resulting coupled, time dependent non-linear equations are solved using the Galerkin finite element method with a fixed mesh. Our method efficiently captures the multiple connected thawed domains that arise due to the penetration of MWs in the sample. For a cylinder of diameter D, the two length scales that control the thawing dynamics are D/D-p and D/lambda(m), where D-p and lambda(m) are the penetration depth and wavelength of radiation in the sample respectively. For D/D-p, D/lambda(m) much less than 1 power absorption is uniform and thawing occurs almost simultaneously across the sample (Regime I). For D/D-p much greater than 1 thawing is seen to occur from the incident face, since the power decays exponentially into the sample (Regime III). At intermediate values, 0.2 < D/D-p, D/lambda(m) < 2.0 (Regime II) thawing occurs from the unexposed face at smaller diameters, from both faces at intermediate diameters and from the exposed and central regions at larger diameters. Average power absorption during thawing indicates a monotonic rise in Regime I and a monotonic decrease in Regime III. Local maxima in the average power observed for samples in Regime II are due to internal resonances within the sample. Thawing time increases monotonically with sample diameter and temperature gradients in the sample generally increase from Regime I to Regime III. (C) 2002 Elsevier Science Ltd. All rights reserved.

Grafting of m-isopropenyl-alpha, alpha-dimethylbenzyl-isocyanate (m-TMI) onto isotactic polypropylene: Synthesis and characterization

A novel vinyl monomer with an isocyanate functional group, m-isopropenyl-alpha,alpha-dimethylbenzyl-isocyanate (m-TMI), was grafted onto isotactic polypropylene (i-PP) using dicumyl peroxide (DCP) as the initiator. This would open up the possibility of using the grafted polymer with the reactive isocyanate group as compatibilizer for blending carbohydrates such as cellulose with. polypropylene. The grafting was carried out in a Brabender Plasticoder at 180degreesC. The effects of monomer and initiator concentrations on the yield of grafting were investigated by performing statistical analysis. While the grafting yield increased with the concentration of DCP at any given concentration of m-TMI, the variation of the grafting yield with m-TMI concentration, for a given concentration of DCP, went through a maximum, the optimum yield of 7.8% (w/w) being obtained at 10 wt.% concentration of both DCP and m-TMI. The grafting reaction is. accompanied by considerable chain scission of I-PP, resulting in a decrease in the molecular weight of the grafted polymer. While the molecular weight drops sharply even at a low concentration of DCP, there occurs no further significant change in the molecular weight even at much higher concentrations of the initiator.

The selection of a sugar for transport and storage of carbon in plants

Sugars perform two vital functions in plants: as compatible solutes protecting the cell against osmotic stress and as mobile source of immediate and long-term energy requirement for growth and development. The two sugars that occur commonly in nature are sucrose and trehalose. Sucrose comprises one glucose and one fructose molecule; trehalose comprises two glucose molecules. Trehalose occurs in significant amounts in insects and fungi which greatly outnumber the plants. Surprisingly, in plants trehalose has been found in barely detectable amounts, if at all, raising the question `why did nature select sucrose instead of trehalose as the mobile energy source and as storage sugar for the plants'? Modelling revealed that when attached to the ribbon-shaped beta-1,4 glucan a trehalose molecule is shaped like a hook. This suggests that the beta-1,4 glucan chains with attached trehalose will fail to align to form inter-chain hydrogen bonds and coalesce into a cellulose microfibril, as a result of which in trehalose-accumulating plant cells, the cell wall will tend to become leaky. Thus in plants an evolutionary selection was made in favour of sucrose as the mobile energy source. Genetic engineering of plant cells for combating abiotic stresses through microbial trehalose-producing genes is fraught with risk of damage to plant cell walls.

Enhanced Survival of Probiotic Lactobacillus acidophilus by Encapsulation with Nanostructured Polyelectrolyte Layers through Layer-by-Layer Approach

The encapsulation of probiotic Lactobacillus acidophilus through layer-by-layer self-assembly of polyelectrolytes (PE) chitosan (CHI) and carboxymethyl cellulose (CMC) has been investigated,to enhance its survival m adverse conditions encountered in the GI tract The survival of encapsulated cells in simulated gastric (SGF) and intestinal fluids (SIF) is significant when compared to nonencapsulated cells On sequential exposure to SGF and SIF fox 120 nun, almost complete death of free cells is observed However, for cells coated with three nanolayers of PEs (CHI/CMC/CHI) about 33 log % of the cells (6 log cfu/500 mg) survived under the same conditions The enhanced survival rate of encapsulated L acidophilus can be attributed to the impermeability of polyelectrolyte nanolayers to large enzyme molecules like pepsin, and pancreatin that cause proteolysis and to the stability of the polyelectrolyte nanolayers in gastric and intestinal pH The PE coating also serves to reduce viability losses during freezing and freeze- drying About 73 and 92 log % of uncoated and coated cells survived after freeze:drying, and the losses occurring between freezing and freeze-drying were found to be lower for coated cells

Impact of heavy metals on bacterial communities from mangrove soils of the Mahanadi Delta (India)

This study aimed to assess soil nutrient status and heavy metal content and their impact on the predominant soil bacterial communities of mangroves of the Mahanadi Delta. Mangrove soil of the Mahanadi Delta is slightly acidic and the levels of soil nutrients such as carbon, nitrogen, phosphorous and potash vary with season and site. The seasonal average concentrations (g/g) of various heavy metals were in the range: 14810-63370 (Fe), 2.8-32.6 (Cu), 13.4-55.7 (Ni), 1.8-7.9 (Cd), 16.6-54.7 (Pb), 24.4-132.5 (Zn) and 13.3-48.2 (Co). Among the different heavy metals analysed, Co, Cu and Cd were above their permissible limits, as prescribed by Indian Standards (Co=17g/g, Cu=30 g/g, Cd=3-6 g/g), indicating pollution in the mangrove soil. A viable plate count revealed the presence of different groups of bacteria in the mangrove soil, i.e. heterotrophs, free-living N-2 fixers, nitrifyers, denitrifyers, phosphate solubilisers, cellulose degraders and sulfur oxidisers. Principal component analysis performed using multivariate statistical methods showed a positive relationship between soil nutrients and microbial load. Whereas metal content such as Cu, Co and Ni showed a negative impact on some of the studied soil bacteria.

Alumina and Silica based Epoxy Nano-composites for Electrical Insulation

Plain epoxy resins or resin impregnated cellulose have found application as electrical insulation in power equipment. In the past, their performance was improved by the use of inorganic oxide fillers of microscopic dimensions. In the recent past nano-particle doped epoxy insulation came into use with a view to further enhance the dielectric properties. This paper reports dielectric investigations into epoxy nano-composites based on a class of metal oxides, Al2O3 and SiO2. In particular, consideration has been given to the partial discharge performance and electrical breakdown under different voltage profiles as a function of the volumetric composition of the nano-particles in epoxy resin.

DECT evaluation of noncalc fied coronary artery plaque

Purpose: Composition of the coronary artery plaque is known to have critical role in heart attack. While calcified plaque can easily be diagnosed by conventional CT, it fails to distinguish between fibrous and lipid rich plaques. In the present paper, the authors discuss the experimental techniques and obtain a numerical algorithm by which the electron density (rho(e)) and the effective atomic number (Z(eff)) can be obtained from the dual energy computed tomography (DECT) data. The idea is to use this inversion method to characterize and distinguish between the lipid and fibrous coronary artery plaques. Methods: For the purpose of calibration of the CT machine, the authors prepare aqueous samples whose calculated values of (rho(e), Z(eff)) lie in the range of (2.65 x 10(23) <= rho(e) <= 3.64 x 10(23)/cm(3)) and (6.80 <= Z(eff) <= 8.90). The authors fill the phantom with these known samples and experimentally determine HU(V-1) and HU(V-2), with V-1,V-2 = 100 and 140 kVp, for the same pixels and thus determine the coefficients of inversion that allow us to determine (rho(e), Z(eff)) from the DECT data. The HU(100) and HU(140) for the coronary artery plaque are obtained by filling the channel of the coronary artery with a viscous solution of methyl cellulose in water, containing 2% contrast. These (rho(e), Z(eff)) values of the coronary artery plaque are used for their characterization on the basis of theoretical models of atomic compositions of the plaque materials. These results are compared with histopathological report. Results: The authors find that the calibration gives Pc with an accuracy of 3.5% while Z(eff) is found within 1% of the actual value, the confidence being 95%. The HU(100) and HU(140) are found to be considerably different for the same plaque at the same position and there is a linear trend between these two HU values. It is noted that pure lipid type plaques are practically nonexistent, and microcalcification, as observed in histopathology, has to be taken into account to explain the nature of the observed (rho(e), Z(eff)) data. This also enables us to judge the composition of the plaque in terms of basic model which considers the plaque to be composed of fibres, lipids, and microcalcification. Conclusions: This simple and reliable method has the potential as an effective modality to investigate the composition of noncalcified coronary artery plaques and thus help in their characterization. In this inversion method, (rho(e), Z(eff)) of the scanned sample can be found by eliminating the effects of the CT machine and also by ensuring that the determination of the two unknowns (rho(e), Z(eff)) does not interfere with each other and the nature of the plaque can be identified in terms of a three component model. (C) 2015 American Association of Physicists in Medicine.

Infiltration of Matrix-Non-producers Weakens the Salmonella Biofilm and Impairs Its Antimicrobial Tolerance and Pathogenicity

Bacterial biofilms display a collective lifestyle, wherein the cells secrete extracellular polymeric substances (EPS) that helps in adhesion, aggregation, stability, and to protect the bacteria from antimicrobials. We asked whether the BPS could act as a public good for the biofilm and observed that infiltration of cells that do not produce matrix components weakened the biofilm of Salmonella enterica serovar Typhimurium. PS production was costly for the producing cells, as indicated by a significant reduction in the fitness of wild type (WT) cells during competitive planktonic growth relative to the non-producers. Infiltration frequency of non-producers in the biofilm showed a concomitant decrease in overall productivity. It was apparent in the confocal images that the non producing cells benefit from the BPS produced by the Wild Type (WT) to stay in the biofilm. The biofilm containing non-producing cells were more significantly susceptible to sodium hypochlorite and ciprofloxacin treatment than the WT biofilm. Biofilm infiltrated with non-producers delayed the pathogenesis, as tested in a murine model. The cell types were spatially assorted, with non producers being edged out in the biofilm. However, cellulose was found to act as a barrier to keep the non-producers away from the WT microcolony. Our results show that the infiltration of non-cooperating cell types can substantially weaken the biofilm making it vulnerable to antibacterials and delay their pathogenesis. Cellulose, a component of BPS, was shown to play a pivotal role of acting as the main public good, and to edge-out the non-producers away from the cooperating microcolony.

Covalent interlocking of glucose oxidase and peroxidase in the voids of paper: enzyme-polymer ``spider webs''

A modular, general method for trapping enzymes within the voids of paper, without chemical activation of cellulose, is reported. Glucose oxidase and peroxidase were crosslinked with poly(acrylic acid) via carbodiimide chemistry, producing 3-dimensional networks interlocked in cellulose fibers. Interlocking prevented enzyme activity loss and enhanced the washability and stability.

Aplicaciones enzimáticas en procesos de conservación y restauración de obras de arte. Consolidación de celulosa

690 p.

Engineering enzyme systems by recombination

Homologous recombination is a source of diversity in both natural and directed evolution. Standing genetic variation that has passed the test of natural selection is combined in new ways, generating functional and sometimes unexpected changes. In this work we evaluate the utility of homologous recombination as a protein engineering tool, both in comparison with and combined with other protein engineering techniques, and apply it to an industrially important enzyme: Hypocrea jecorina Cel5a.

Chapter 1 reviews work over the last five years on protein engineering by recombination. Chapter 2 describes the recombination of Hypocrea jecorina Cel5a endoglucanase with homologous enzymes in order to improve its activity at high temperatures. A chimeric Cel5a that is 10.1 °C more stable than wild-type and hydrolyzes 25% more cellulose at elevated temperatures is reported. Chapter 3 describes an investigation into the synergy of thermostable cellulases that have been engineered by recombination and other methods. An engineered endoglucanase and two engineered cellobiohydrolases synergistically hydrolyzed cellulose at high temperatures, releasing over 200% more reducing sugars over 60 h at their optimal mixture relative to the best mixture of wild-type enzymes. These results provide a framework for engineering cellulolytic enzyme mixtures for the industrial conditions of high temperatures and long incubation times.

In addition to this work on recombination, we explored three other problems in protein engineering. Chapter 4 describes an investigation into replacing enzymes with complex cofactors with simple cofactors, using an E. coli enolase as a model system. Chapter 5 describes engineering broad-spectrum aldehyde resistance in Saccharomyces cerevisiae by evolving an alcohol dehydrogenase simultaneously for activity and promiscuity. Chapter 6 describes an attempt to engineer gene-targeted hypermutagenesis into E. coli to facilitate continuous in vivo selection systems.

Gene enrichment using antibodies to DNA/RNA hybrids : mapping the ribosomal DNA of slime mold and rat

A novel method for gene enrichment has been developed and applied to mapping the rRNA genes of two eucaryotic organisms. The method makes use of antibodies to DNA/RNA hybrids prepared by injecting rabbits with the synthetic hybrid poly(rA)•poly(dT). Antibodies which cross-react with non-hybrid nucleic acids were removed from the purified IgG fraction by adsorption on columns of DNA-Sepharose, oligo(dT)-cellulose, and poly(rA)-Sepharose. Subsequent purification of the specific DNA/RNA hybrid antibody was carried out on a column of oligo(dT)-cellulose to which poly(rA) was hybridized. Attachment of these antibodies to CNBr-activated Sepharose produced an affinity resin which specifically binds DNA/RNA hybrids.

In order to map the rDNA of the slime mold Dictyostelium discoideum, R-loops were formed using unsheared nuclear DNA and the 178 and 268 rRNAs of this organism. This mixture was passed through a column containing the affinity resin, and bound molecules containing R- loops were eluted by high salt. This purified rDN A was observed directly in the electron microscope. Evidence was obtained that there is a physical end to Dictyostelium rDN A molecules approximately 10 kilobase pairs (kbp) from the region which codes for the 268 rRNA. This finding is consistent with reports of other investigators that the rRNA genes exist as inverse repeats on extra-chromosomal molecules of DNA unattached to the remainder of the nuclear DNA in this organism.

The same general procedure was used to map the rRNA genes of the rat. Molecules of DNA which contained R-loops formed with the 188 and 288 rRNAs were enriched approximately 150- fold from total genomal rat DNA by two cycles of purification on the affinity column. Electron microscopic measurements of these molecules enabled the construction of an R-loop map of rat rDNA. Eleven of the observed molecules contained three or four R-loops or else two R-loops separated by a long spacer. These observations indicated that the rat rRNA genes are arranged as tandem repeats. The mean length of the repeating units was 37.2 kbp with a standard deviation of 1.3 kbp. These eleven molecules may represent repeating units of exactly the same length within the errors of the measurements, although a certain degree of length heterogeneity cannot be ruled out. If significantly shorter or longer repeating units exist, they are probably much less common than the 37.2 kbp unit.

The last section of the thesis describes the production of antibodies to non-histone chromosomal proteins which have been exposed to the ionic detergent sodium dodecyl sulfate (SDS). The presence of low concentrations of SDS did not seem to affect either production of antibodies or their general specificity. Also, a technique is described for the in situ immunofluorescent detection of protein antigens in polyacrylamide gels.

Estrategias de arranque de biofiltros orgánicos para el tratamiento de corrientes gaseosas contaminadas con compuestos de azufre

[ES]El disulfuro de carbono (CS2) y el sulfuro de hidrógeno (H2S) son gases especialmente tóxicos presentes en una gran cantidad de emisiones gaseosas industriales, principalmente en la producción de fibras de rayón a partir de celulosa en el caso del CS2, y en la industria petroquímica en el caso del H2S. La legislación vigente en cuanto a las emisiones de estos compuestos se refiere hace que su tratamiento sea, en muchos casos, necesario. La biofiltración como tecnología para el tratamiento de corrientes gaseosas contaminadas se ha convertido en los últimos años en una alternativa a los tratamientos físico-químicos empleados hasta la fecha. Sin embargo, uno de los principales obstáculos a la hora de la implantación de este tratamiento biológico a escala industrial es la duración del periodo de aclimatación de la biomasa encargada de degradar los contaminantes, que en función del compuesto a tratar puede resultar demasiado largo para la aplicación comercial de esta tecnología. El presente trabajo se centra en el estudio de estos periodos de arranque y aclimatación, especialmente lentos en el caso del CS2, y propone una estrategia de arranque basada en la reutilización de biomasa capaz de degradar los compuestos de interés, almacenada tras usos anteriores, que permite reducir el tiempo necesario para alcanzar eficacias de eliminación elevadas. Paralelamente, se ha concluido que paradas de corta duración no afectan de manera importante a la operación del sistema.