Characterization of PhlG, a hydrolase that specifically degrades the antifungal compound 2,4-diacetylphloroglucinol in the biocontrol agent Pseudomonas fluorescens CHA0.

The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial Pseudomonas fluorescens CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the phlG gene, the function of which has not been elucidated thus far. The phlG gene is located upstream of the phlACBD biosynthetic operon, between the phlF and phlH genes which encode pathway-specific regulators. In this study, we assigned a function to PhlG as a hydrolase specifically degrades DAPG to equimolar amounts of mildly toxic monoacetylphloroglucinol (MAPG) and acetate. DAPG added to cultures of a DAPG-negative DeltaphlA mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a DeltaphlA DeltaphlG double mutant. To confirm the enzymatic nature of PhlG in vitro, the protein was histidine tagged, overexpressed in Escherichia coli, and purified by affinity chromatography. Purified PhlG had a molecular mass of about 40 kDa and catalyzed the degradation of DAPG to MAPG. The enzyme had a kcat of 33 s(-1) and a Km of 140 microM at 30 degrees C and pH 7. The PhlG enzyme did not degrade other compounds with structures similar to DAPG, such as MAPG and triacetylphloroglucinol, suggesting strict substrate specificity. Interestingly, PhlG activity was strongly reduced by pyoluteorin, a further antifungal compound produced by the bacterium. Expression of phlG was not influenced by the substrate DAPG or the degradation product MAPG but was subject to positive control by the GacS/GacA two-component system and to negative control by the pathway-specific regulators PhlF and PhlH.

Identification and functional characterization of auxiliary enzymes of the β-oxidation in " Arabidopsis thaliana "

Abstract: The ß-oxidation is the universal pathway that allows living organisms to degrade fatty acids. leading to lipid homeostasis and carbon and energy recovery from the fatty acid molecules. This pathway is centred on four core enzymatic activities sufficient to degrade saturated fatty acids. Additional auxiliary enzymes of the ß-oxidation are necessary for the complete degradation of a larger array of molecules encompassing the unsaturated fatty acids. The main pathways of the ßoxidation of fatty acids have been investigated extensively and auxiliary enzymes are well-known in mammals and yeast. The comparison of the established ß-oxidation systems suggests that the activities that are required to proceed to the full degradation of unsaturated fatty acids are present regardless of the organism and rely on common active site templates. The precise identity of the plant enzymes was unknown. By homology searches in the genome of Arabidopsis thaliana, I identified genes. encoding for proteins that could be orthologous to the yeast or animal auxiliary enzymes Δ 3, Δ 2-enoyl-CoA isomerase, Δ 3,5, Δ 2,4 -dienoyl-CoA isomerase, and type 2 enoyl-CoA hydratase. I established that these genes are expressed in Arabidopsis and that their expression can be correlated to the expression of core ß-oxidation genes. Through the observation of chimeric fluorescent protein fusions, I demonstrated that the identified proteins are localized in the peroxisóme, the only organelle where the ß-oxidation occurs in plants. Enzymatic assays were performed with the partially purified enzymes to demonstrate that the identified enzymes can catalyze the same in vitro reactions as their non-plant orthologs. The activities in vivo of the plant enzymes were demonstrated by heterologous complementation of the corresponding yeast Saccharomyces cerevisiae mutants. The complementation was visualized using the artificial polyhydroxyalkanoate (PHA) production in yeast peroxisomes. The recombinant strains, expressing a Pseudomonas aeruginosa PHA synthase modified for a peroxisomal localization, produce this polymer that serves as a trap for the 3-hydroxyacyl-CoA intermediaries of the ßoxidation and that reflects qualitatively and quantitatively the array of molecules that are processed through the ß-oxidation. This complementation demonstrated the implication of the plant Δ 3, Δ 2-enoyl-CoA isomerases and Δ3,5, Δ2,4-dienoyl-CoA isomerase in the degradation of odd chain position unsaturated fatty acids. The presence of a monofunctional type 2 enoyl-CoA hydratase is a novel in eukaryotes. Downregulation of the corresponding gene expression in an Arabidopsis line, modified to produce PHA in the peroxisome, demonstrated thàt this enzyme participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2Eenoyl-CoA for further degradation through the core ß-oxidation cycle. Résumé: La ß-oxydation est une voie universelle de dégradation des acides gras qui permet aux organismes vivants d'assurer une homéostasie lipidique et de récupérer l'énergie et le carbone contenus dans les acides gras. Le coeur de cette voie est composé de quatre réactions enzymatiques suffisantes à la dégradation des acides gras saturés. La présence des enzymes auxiliaires de la ß-oxydation est nécessaire à la dégradation d'une gamme plus étendue de molécules comprenant les acides gras insaturés. Les voies principales de la ß-oxydation des acides gras ont été étudiées en détail et les enzymes auxiliaires sont déterminées chez les mammifères et la levure. La comparaison entre les systèmes de ß-oxydation connus suggère que les activités requises pour la dégradation complète des acides gras insaturés reposent sur la présence de site actifs similaires. L'identité précise des enzymes auxiliaires chez les plantes était inconnue. En cherchant par homologie dans le génome de la plante modèle Arabidopsis thaliana, j'ai identifié des gènes codant pour des protéines pouvant être orthologues aux enzymes auxiliaires Δ3 Δ2-enoyl-CoA isomérase, Δ 3,5 Δ 2,4-dienoyl-CoA isomérase et enoyl-CoA hydratase de type 2 d'origine fongique ou mammalienne. J'ai établi la corrélation de l'expression de ces gènes dans Arabidopsis avec celle de gènes des enzymes du coeur de la ß-oxydation. En observant des chimères de fusion avec des protéines fluorescentes, j'ai démontré que les protéines identifiées sont localisées dans le péroxysomes, le seul organelle où la ß-oxydation se déroule chez les plantes. Des essais enzymatiques ont été conduits avec ces enzymes partiellement purifiées pour démontrer que les enzymes identifiées sont capables de catalyser in vitro les mêmes réactions que leurs orthologues non végétaux. Les activités des enzymes végétales in vivo ont été .démontrées par complémentation hétérologue des mutants de délétion correspondants de levure Saccharomyces cerevisiae. La visualisation de la complémentation est rendue possible par la synthèse de polyhydroxyalcanoate (PHA) dans les péroxysomes de levure. Les souches recombinantes expriment la PHA synthase de Pseudomonas aeruginosa modifiée pour être localisée dans le péroxysome produisent ce polymère qui sert de piège pour les 3-hydroxyacylCoAs intermédiaires de la ß-oxydation et qui reflète qualitativement et quantitativement la gamme de molécules qui subit la ß-oxydation. Cette complémentation a permis de démontrer que les Δ3, Δ2-enoyl-CoA isomérases, et la Δ3.5, Δ2,4-dienoyl-CoA isomérase végétales sont impliquées dans la dégradation des acides gras insaturés en position impaire. L'enoyl-CoA hydratase de type 2 monofonctionelle est une enzyme nouvelle chez les eucaryotes. La sous-expression du gène correspondant dans une lignée d'Arabidopsis modifiée pour produite du PHA dans le péroxysome a permis de démontrer que cette enzyme participe in vivo à la dégradation des acides gras ayant une double liaison en conformation cis (Z) en position paire.

Identification and characterization of novel classes of macrophage migration inhibitory factor (MIF) inhibitors with distinct mechanisms of action.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC(50) values in the range of 0.2-15.5 microm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro(1); 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.

Characterization of membrane vesicles circulating in the serum of patients with common acute lymphoblastic leukemia.

Common acute lymphoblastic leukemia antigen detected by radioimmunoassay in the serum of patients with common acute lymphoblastic leukemia was found to be exclusively associated with the pellet of the serum samples obtained by ultracentrifugation at 100,000 X g. The pellets were shown to contain membrane vesicles or fragments which were characterized by electron microscopy and determination of enzymatic activity. The pelleted fragments had an apparent diameter ranging between 60 and 260 nm and showed a trilaminar membrane structure. On freeze-fracture preparations, the fragments with concave profile, corresponding to the external fracture face of plasma membrane, displayed an intramembrane particle density (ranging from 0 to 750 particles per micron2) which is similar to that recorded on the corresponding fracture face of intact cells from the common lymphoblastic leukemia antigen positive leukemic cell line (Nalm-1) or of vesicles shed in the culture medium by Nalm-1 cells. Furthermore, analysis of the membrane enzyme marker 5'-nucleotidase in the pellet of patient's sera, showed that the presence of this enzyme correlated with that of common lymphoblastic leukemia antigen, but the quantitative relationship between the two surface constituents was not linear. The results suggest that the two markers are located on the same membrane fragments, but that their individual distribution on the shed fragments is heterogeneous.

Biosynthesis of pyranonaphthoquinone polyketides:characterization of the alnumycin pathway

Alnumycin A is an aromatic pyranonaphthoquinone (PNQ) polyketide closely related to the model compound actinorhodin. While some PNQ polyketides are glycosylated, alnumycin A contains a unique sugar-like dioxane moiety. This unusual structural feature made alnumycin A an interesting research target, since no information was available about its biosynthesis. Thus, the main objective of the thesis work became to identify the steps and the enzymes responsible for the biosynthesis of the dioxane moiety. Cloning, sequencing and heterologous expression of the complete alnumycin gene cluster from Streptomyces sp. CM020 enabled the inactivation of several alnumycin biosynthetic genes and preliminary identification of the gene products responsible for pyran ring formation, quinone formation and dioxane biosynthesis. The individual deletions of the genes resulted in the production of several novel metabolites, which in many cases turned out to be pathway intermediates and could be used for stepwise enzymatic reconstruction of the complete dioxane biosynthetic pathway in vitro. Furthermore, the in vitro reactions with purified alnumycin biosynthetic enzymes resulted in the production of other novel compounds, both pathway intermediates and side products. Identification and molecular level studies of the enzymes AlnA and AlnB catalyzing the first step of dioxane biosynthesis – an unusual C-ribosylation step – led to a mechanistic proposal for the C-ribosylation of the polyketide aglycone. The next step on the dioxane biosynthetic pathway was found to be the oxidative conversion of the attached ribose into a highly unusual dioxolane unit by Aln6 belonging to an uncharacterized protein family, which unexpectedly occurred without any apparent cofactors. Finally, the last step of the pathway was found to be catalyzed by the NADPH-dependent reductase Aln4, which is able to catalyze the conversion of the formed dioxolane into a dioxane moiety. The work presented here and the knowledge gained of the enzymes involved in dioxane biosynthesis enables their use in the rational design of novel compounds containing C–C bound ribose, dioxolane and dioxane moieties.

Characterization of nitrate reductase activity in vitro in Gracilaria caudata J. Agardh (Rhodophyta, Gracilariales)

The marine red alga Gracilaria caudata J. Agardh has been used in Brazil for agar extraction, mainly in the northeast region of the country. Nitrogen availability is the most important abiotic factor in seawater that limits the growth of seaweeds. The enzyme nitrate reductase (NR) is the key regulatory point in the nitrogen assimilation in photosynthetic organisms. This study describes an in vitro assay, characterizing the enzymatic activity of NR in terms of kinetic constants and stability, its oscillation during the day and glucose effect on NR modulation. Maximal peaks of NR activity were recorded at 20 ºC and pH 8.0. The enzymatic stability in crude extracts stored at 3 ± 1 ºC decreased significantly after 48 hours. Apparent Michaelis-Menten constants (K M) for NADH and nitrate were 22 µM and 3.95 mM, respectively. Gracilaria caudata NR activity showed an oscillation under light:dark photoperiod (14:10 hours LD) with 3-fold higher activity during the light phase, peaking after 10 hours of light. Under optimal assay conditions, the maximal activity was 92.9 10-3 U g-1. The addition of glucose induced the enzymatic activity during the light and dark phase, evidencing a possible modulation of this enzyme by the photosynthesis. This relationship can be explained by the need of carbon skeletons, produced by the photosynthetic process, to incorporate the intermediary metabolites of nitrate assimilatory pathway, avoiding the toxic intracellular accumulation of nitrite and ammonium. The optimization of enzymatic assay protocols for NR is essential to establish appropriate conditions to study nutritional behaviour, compare different taxonomic groups and to understand its regulatory mechanism.

Proteolytic release and partial characterization of human sperm-surface glycopeptides

Sperm-surface glycopeptides were obtained from intact sperm membranes after proteolytic release by different enzymatic treatments such as autoproteolysis, trypsin, papain and pronase. Glycopeptides were isolated, their properties and composition were examined, and their monosaccharide and amino acid constituents were characterized. The monosaccharides identified were fucose, mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine, which form part of more than one type of oligosaccharide units. Autoproteolytic treatment mainly provided O-glycosidic type oligosaccharides, while a mixture of O- and N-glycosidic oligosaccharides was obtained in variable proportions when treated with trypsin, papain or pronase. The highest degree of peptide cleavage was obtained with pronase. Despite the higher yields reached with trypsin, these glycopeptides contain the lowest percentage of oligosaccharide chains. Proteolytic treatment provides a simple, rapid procedure for the isolation of glycopeptides from the sperm surface

Production, isolation and characterization of bioactive peptides with antihypertensive properties from rapeseed and potato protein

Consumers’ increasing awareness of healthiness and sustainability of food presents a great challenge to food industry to develop healthier, biologically active and sustainable food products. Bioactive peptides derived from food proteins are known to possess various biological activities. Among the activities, the most widely studied are antioxidant activities and angiotensin I converting enzyme (ACE) inhibitory activity related to blood pressure regulation and antihypertensive effects. Meanwhile, vast amounts of byproducts with high protein content are produced in food industry, for example potato and rapeseed industries. The utilization of these by-products could be enhanced by using them as a raw material for bioactive peptides. The objective of the present study was to investigate the production of bioactive peptides with ACE inhibitory and antioxidant properties from rapeseed and potato proteins. Enzymatic hydrolysis and fermentation were utilized for peptide production, ultrafiltration and solid-phase extraction were used to concentrate the active peptides, the peptides were fractionated with liquid chromatographic processes, and the peptides with the highest ACE inhibitory capacities were putified and analyzed with Maldi-Tof/Tof to identify the active peptide sequences. The bioavailability of the ACE inhibitory peptides was elucidated with an in vitro digestion model and the antihypertensive effects in vivo of rapeseed peptide concentrates were investigated with a preventive premise in 2K1C rats. The results showed that rapeseed and potato proteins are rich sources of ACE inhibitory and antioxidant peptides. Enzymatic hydrolysis released the peptides effectively whereas fermentation produced lower activities.The native enzymes of potato were also able to release ACE inhibitory peptides from potato proteins without the addition of exogenous enzymes. The rapeseed peptide concentrate was capable of preventing the development of hypertension in vivo in 2K1C rats, but the quality of rapeseed meal used as raw material was found to affect considerably the antihypertensive effects and the composition of the peptide fraction.

High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes

Isolation and Characterization of Ovomucin - a Bioactive Agent of Egg White

The hen’s egg is a source of new life. Therefore, it contains many biologically active compounds. In addition to being a very nutritious food and also commonly used in the food industry due to its many techno-functional properties, the egg can serve as a source of compounds used as nutra-, pharmaand cosmeceuticals. One such interesting compound is ovomucin, an egg white protein responsible for the gel-like properties of thick egg white. Previous studies have indicated that ovomucin and ovomucin-derived peptides have several different bioactive properties. The objectives of the present study were to develop isolation methods for ovomucin, to characterize the structure of ovomucin, to compare various egg fractions as sources of ovomucin, to study the effects of various dissolving methods for ovomucin, and to investigate the bioactive properties of ovomucin and ovomucin-derived peptides. A simple and rapid method for crude ovomucin separation was developed. By using this method crude ovomucin was isolated within hours, compared to the 1-2 days (including a dialysis step) needed when using several other methods. Structural characterization revealed that ovomucin is composed of two subunits, α- and β-ovomucin, as egg white protein formerly called α1-ovomucin seemed to be ovostatin. However, it might be possible that ovostatin is associated within β- and α-ovomucin. This interaction could even have some effect on the physical nature of various egg white layers. Although filtration by-product fraction was a very prominent source of both crude and β-ovomucin, process development has reduced its amount so significantly that it has no practical meaning anymore. Thus, the commercial liquid egg white is probably the best option, especially if it generally contains amounts of β-ovomucin as high as were found in these studies. Crude ovomucin was dissolved both by using physical and enzymic methods. Although sonication was the most effective physical method for ovomucin solubilisation, colloid milling seemed to be a very promising alternative. A milk-like, smooth and opaque crude ovomucin suspension was attained by using a colloid mill. The dissolved ovomucin fractions were further tested for bioactive properties, and it was found that three dissolving methods tested produced moderate antiviral activity against Newcastle disease virus, namely colloid milling, enzymatic hydrolysis and a combination of sonicaton and enzymatic hydrolysis. Moreover, trypsin-digested crude ovomucin was found to have moderate antiviral activity against avian influenza virus: both subtype H5 and H7.

Characterization of the interdependency between residues that bind the substrate in a β-glycosidase

The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the β-glycosidase from Spodoptera frugiperda (Sfβgly), both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfβgly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl β-galactoside and p-nitrophenyl β-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfβgly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ES‡ (enzyme-transition state complex) of the double mutations (∆∆G‡xy) is not the sum of the effects resulting from the single mutations (∆∆G‡x and ∆∆G‡y). This difference in ∆∆G‡ indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in ∆∆G‡xy. Crystallographic data on β-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.

Production and partial characterization of lipase from Penicillium verrucosum obtained by submerged fermentation of conventional and industrial media

The growing interest in lipase production is related to the potential biotechnological applications that these enzymes present. Current studies on lipase production by submerged fermentation involve the use of agro-industrial residues aiming at increasing economic attractiveness. Based on these aspects, the objective of this work was to investigate lipase production by Penicillium verrucosum in submerged fermentation using a conventional medium based on peptone, yeast extract, NaCl and olive oil, and an industrial medium based on corn steep liquor, Prodex Lac (yeast hydrolysate), NaCl and olive oil, as well as to characterize the crude enzymatic extracts obtained. Kinetics of lipase production was evaluated and the highest enzymatic activities, of 3.15 and 2.22 U.mL-1, were observed when conventional and industrial media were used, respectively. The enzymatic extract showed optimal activity in the range from 30 to 40 °C and at pH 7.0. Although the industrial medium presents economical advantages over the conventional medium, the presence of agro-industrial residues rich in nitrogen and other important nutrients seemed to contribute to a reduction in lipase activity.

Characterization of the chitinolytic system during the mycoparasitic interaction between Trichoderma aggressivum f. aggressivum and different host strains of Agaricus bisporus /

Green mould is a serious disease of commercially grown mushrooms, the causal agent being attributed to the filamentous soil fungus Triclzodenna aggressivum f. aggressivu11l and T. aggressivum f. ellropaellm. Found worldwide, and capable of devastating crops, this disease has caused millions of dollars in lost revenue within the mushroom industry. One mechanism used by TricllOdenlla spp. in the antagonism of other fungi, is the secretion of lytic enzymes such as chitinases, which actively degrade a host's cell wall. Therefore, the intent of this study was to examine the production of chitinase enzymes during the host-parasite interaction of Agaricus bisporus (commercial mushroom) and Triclzodemza aggressivum, focusing specifically on chitinase involvement in the differential resistance of white, off-white, and brown commercial mushroom strains. Chitinases isolated from cultures of A. bisporus and T. aggressivu11l grown together and separately, were identified following native PAGE, and analysis of fluorescence based on specific enzymatic cleavage of 4-methylumbelliferyl glucoside substrates. Results indicate that the interaction between T. aggressivulll and A. bisporus involves a complex enzyme battle. It was determined that T. aggressivum produces a number of chitinases that appear to correlate to those isolated in previous studies using biocontrol strains of T. Izarziallilm. A 122 kDa N-acetylglucosaminidase of T. aggressivu11l revealed the highest and most variable activity, and is therefore believed to be an important predictor of antifungal activity. Furthermore, results indicate that brown strain resistance of mushrooms may be related to high levels of a 96 kDa N-acetylglucosaminidase, which showed elevated activity in both solitary and dual cultures with T. aggressivum. Overall, each host-parasite combination produced unique enzyme profiles, with the majority of the differences seen between day 0 and day 6 for the extracellular chitinases. Therefore, it was concluded that the antagonistic behaviour of T. aggressivli1ll does not involve a typical response, always producing the same types and levels of enzymes, but that mycoparasitism, specifically in the form of chitinase production, may be induced and regulated based on the host presented.

Further characterization and determination of the single amino acid change in the tsI138 reovirus thermosensitive mutant

Many temperature-sensitive mutants have been isolated in early studies of mammalian reovirus. However, the bio- logical properties and nature of the genetic alterations remain incompletely explored for most of these mutants. The mutation harbored by the tsI138 mutant was already assigned to the L3 gene encoding the l1 protein. In the present study, this mu- tant was further studied as a possible tool to establish the role of the putative l1 enzymatic activities in viral multiplication. It was observed that synthesis of viral proteins is only marginally reduced, while it was difficult to recover viral particles at the nonpermissive temperature. A single nucleotide substitution resulting in an amino acid change was found; the position of this amino acid is consistent with a probable defect in assembly of the inner capsid at the nonpermissive temperature.

Zeolite Encapsulated Complexes Of Fe,Co,Ni,Cu And Pd:Synthesis , Characterization And Catalysis-2003

This thesis deals with the synthesis, characterization and catalysis activity studies of some zeolite encapsulated complexes. Encapsulation inside the zeolite cages makes the catalysts more stable. Further, the framework prevents the complexes from dimerising. Catalysis by metal complexes encapsulated in the cavities of zeolites and other molecular sieves has many features of homogeneous, heterogenous and enzymatic catalysis. Serious attempts has been made to gain product selectivity in catalysis .The catalytic activity shown by the encapsulated complexes can be correlated to the structure of the active site inside the zeolite pore. It deals with the studies on the partial oxidation of benzyl alcohol to benzaldehyde. The oxidatio was carried out using hydrogen peroxide as oxidant in presence of PdYDMG and CuYSPP as catalysts. The product (benzaldehyde) was detected using TLC and confirmed using GC.The catalytic activity of the complexes was tested for oxidation under various conditions. The operating conditions like the amount of the catalyst, reaction time, oxidant to substrate ratio, reaction temprature, and solvents have been optimized. No further oxidation products were obtained on continuing the reaction for four hours beyond the optimum time. Maximum conversion was obtained at room temperature and the percentage conversion decreased with increase in temperature. Activity was found to be dependent on the solvent used. With increasing awareness about the dangers of environmental degradation, research in chemistry is getting increasing geared to the development of “green chemistry,” by designing environmentally friendly products and processes that bring down the generation and use of hazardous substances.