Unraveling the structure of sugarcane bagasse after soaking in concentrated aqueous ammonia (SCAA) and ethanol production by Scheffersomyces (Pichia) stipitis

Abstract Background Fuel ethanol production from sustainable and largely abundant agro-residues such as sugarcane bagasse (SB) provides long term, geopolitical and strategic benefits. Pretreatment of SB is an inevitable process for improved saccharification of cell wall carbohydrates. Recently, ammonium hydroxide-based pretreatment technologies have gained significance as an effective and economical pretreatment strategy. We hypothesized that soaking in concentrated aqueous ammonia-mediated thermochemical pretreatment (SCAA) would overcome the native recalcitrance of SB by enhancing cellulase accessibility of the embedded holocellulosic microfibrils. Results In this study, we designed an experiment considering response surface methodology (Taguchi method, L8 orthogonal array) to optimize sugar recovery from ammonia pretreated sugarcane bagasse (SB) by using the method of soaking in concentrated aqueous ammonia (SCAA-SB). Three independent variables: ammonia concentration, temperature and time, were selected at two levels with center point. The ammonia pretreated bagasse (SCAA-SB) was enzymatically hydrolysed by commercial enzymes (Celluclast 1.5 L and Novozym 188) using 15 FPU/g dry biomass and 17.5 Units of β-glucosidase/g dry biomass at 50°C, 150 rpm for 96 h. A maximum of 28.43 g/l reducing sugars corresponding to 0.57 g sugars/g pretreated bagasse was obtained from the SCAA-SB derived using a 20% v/v ammonia solution, at 70°C for 24 h after enzymatic hydrolysis. Among the tested parameters, pretreatment time showed the maximum influence (p value, 0.053282) while ammonia concentration showed the least influence (p value, 0.612552) on sugar recovery. The changes in the ultra-structure and crystallinity of native SCAA-SB and enzymatically hydrolysed SB were observed by scanning electron microscopy (SEM), x-ray diffraction (XRD) and solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. The enzymatic hydrolysates and solid SCAA-SB were subjected to ethanol fermentation under separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) by Scheffersomyces (Pichia) stipitis NRRL Y-7124 respectively. Higher ethanol production (10.31 g/l and yield, 0.387 g/g) was obtained through SSF than SHF (3.83 g/l and yield, 0.289 g/g). Conclusions SCAA treatment showed marked lignin removal from SB thus improving the accessibility of cellulases towards holocellulose substrate as evidenced by efficient sugar release. The ultrastructure of SB after SCAA and enzymatic hydrolysis of holocellulose provided insights of the degradation process at the molecular level.

Bioprocess and biotecnology: effect of xylanase from Aspergillus niger and Aspergillus flavus on pulp biobleaching and enzyme production using agroindustrial residues as substract

This study compares two xylanases produced by filamentous fungi such as A. niger and A. flavus using agroindustrial residues as substract and evaluated the effect of these enzymes on cellulose pulp biobleaching process. Wheat bran was the best carbon source for xylanase production by A. niger and A. flavus. The production of xylanase was 18 and 21% higher on wheat bran when we compare the xylanase production with xylan. At 50°C, the xylanase of A. niger retained over 85% activity with 2 h of incubation, and A. flavus had a half-life of more than 75 minutes. At 55°C, the xylanase produced by A. niger showed more stable than from A. flavus showing a half-life of more than 45 minutes. The xylanase activity of A. niger and A. flavus were somehow protected in the presence of glycerol 5% when compared to the control (without additives). On the biobleaching assay it was observed that the xylanase from A. flavus was more effective in comparison to A. niger. The kappa efficiency corresponded to 36.32 and 25.93, respectively. That is important to emphasize that the cellulase activity was either analyzed and significant levels were not detected, which explain why the viscosity was not significantly modified.

New insights regarding HCV-NS5A structure/function and indication of genotypic differences

Abstract Background HCV is prevalent throughout the world. It is a major cause of chronic liver disease. There is no effective vaccine and the most common therapy, based on Peginterferon, has a success rate of ~50%. The mechanisms underlying viral resistance have not been elucidated but it has been suggested that both host and virus contribute to therapy outcome. Non-structural 5A (NS5A) protein, a critical virus component, is involved in cellular and viral processes. Methods The present study analyzed structural and functional features of 345 sequences of HCV-NS5A genotypes 1 or 3, using in silico tools. Results There was residue type composition and secondary structure differences between the genotypes. In addition, second structural variance were statistical different for each response group in genotype 3. A motif search indicated conserved glycosylation, phosphorylation and myristoylation sites that could be important in structural stabilization and function. Furthermore, a highly conserved integrin ligation site was identified, and could be linked to nuclear forms of NS5A. ProtFun indicated NS5A to have diverse enzymatic and nonenzymatic activities, participating in a great range of cell functions, with statistical difference between genotypes. Conclusion This study presents new insights into the HCV-NS5A. It is the first study that using bioinformatics tools, suggests differences between genotypes and response to therapy that can be related to NS5A protein features. Therefore, it emphasizes the importance of using bioinformatics tools in viral studies. Data acquired herein will aid in clarifying the structure/function of this protein and in the development of antiviral agents.

Ultra-structural mapping of sugarcane bagasse after oxalic acid fiber expansion (OAFEX) and ethanol production by Candida shehatae and Saccharomyces cerevisiae

Background Diminishing supplies of fossil fuels and oil spills are rousing to explore the alternative sources of energy that can be produced from non-food/feed-based substrates. Due to its abundance, sugarcane bagasse (SB) could be a model substrate for the second-generation biofuel cellulosic ethanol. However, the efficient bioconversion of SB remains a challenge for the commercial production of cellulosic ethanol. We hypothesized that oxalic-acid-mediated thermochemical pretreatment (OAFEX) would overcome the native recalcitrance of SB by enhancing the cellulase amenability toward the embedded cellulosic microfibrils. Results OAFEX treatment revealed the solubilization of hemicellulose releasing sugars (12.56 g/l xylose and 1.85 g/l glucose), leaving cellulignin in an accessible form for enzymatic hydrolysis. The highest hydrolytic efficiency (66.51%) of cellulignin was achieved by enzymatic hydrolysis (Celluclast 1.5 L and Novozym 188). The ultrastructure characterization of SB using scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy, Fourier transform–near infrared spectroscopy (FT-NIR), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) revealed structural differences before and after OAFEX treatment with enzymatic hydrolysis. Furthermore, fermentation mediated by C. shehatae UFMG HM52.2 and S. cerevisiae 174 showed fuel ethanol production from detoxified acid (3.2 g/l, yield 0.353 g/g; 0.52 g/l, yield, 0.246 g/g) and enzymatic hydrolysates (4.83 g/l, yield, 0.28 g/g; 6.6 g/l, yield 0.46 g/g). Conclusions OAFEX treatment revealed marked hemicellulose degradation, improving the cellulases’ ability to access the cellulignin and release fermentable sugars from the pretreated substrate. The ultrastructure of SB after OAFEX and enzymatic hydrolysis of cellulignin established thorough insights at the molecular level.

Functional characterisation of the non-essential protein kinases and phosphatases regulating Aspergillus nidulans hydrolytic enzyme production

Abstract Background Despite recent advances in the understanding of lignocellulolytic enzyme regulation, less is known about how different carbon sources are sensed and the signaling cascades that result in the adaptation of cellular metabolism and hydrolase secretion. Therefore, the role played by non-essential protein kinases (NPK) and phosphatases (NPP) in the sensing of carbon and/or energetic status was investigated in the model filamentous fungus Aspergillus nidulans. Results Eleven NPKs and seven NPPs were identified as being involved in cellulase, and in some cases also hemicellulase, production in A. nidulans. The regulation of CreA-mediated carbon catabolite repression (CCR) in the parental strain was determined by fluorescence microscopy, utilising a CreA: GFP fusion protein. The sensing of phosphorylated glucose, via the RAS signalling pathway induced CreA repression, while carbon starvation resulted in derepression. Growth on cellulose represented carbon starvation and derepressing conditions. The involvement of the identified NPKs in the regulation of cellulose-induced responses and CreA derepression was assessed by genome-wide transcriptomics (GEO accession 47810). CreA:GFP localisation and the restoration of endocellulase activity via the introduction of the ∆creA mutation, was assessed in the NPK-deficient backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses, including the expression of hydrolytic enzymes and transporters. The mechanism by which these two NPKs controlled gene transcription was identified, as the NPK-deficient mutants were not able to unlock CreA-mediated carbon catabolite repression under derepressing conditions, such as carbon starvation or growth on cellulose. Conclusions Collectively, this study identified multiple kinases and phosphatases involved in the sensing of carbon and/or energetic status, while demonstrating the overlapping, synergistic roles of schA and snfA in the regulation of CreA derepression and hydrolytic enzyme production in A. nidulans. The importance of a carbon starvation-induced signal for CreA derepression, permitting transcriptional activator binding, appeared paramount for hydrolase secretion.

Analysis of 16S rRNA and mxaF genes reveling insights into Methylobacterium niche-specific plant association

The genus Methylobacterium comprises pink-pigmented facultative methylotrophic (PPFM) bacteria, known to be an important plant-associated bacterial group. Species of this group, described as plant-nodulating, have the dual capacity of producing cytokinin and enzymes, such as pectinase and cellulase, involved in systemic resistance induction and nitrogen fixation under specific plant environmental conditions. The aim hereby was to evaluate the phylogenetic distribution of Methylobacterium spp. isolates from different host plants. Thus, a comparative analysis between sequences from structural (16S rRNA) and functional mxaF (which codifies for a subunit of the enzyme methanol dehydrogenase) ubiquitous genes, was undertaken. Notably, some Methylobacterium spp. isolates are generalists through colonizing more than one host plant, whereas others are exclusively found in certain specific plant-species. Congruency between phylogeny and specific host inhabitance was higher in the mxaF gene than in the 16S rRNA, a possible indication of function-based selection in this niche. Therefore, in a first stage, plant colonization by Methylobacterium spp. could represent generalist behavior, possibly related to microbial competition and adaptation to a plant environment. Otherwise, niche-specific colonization is apparently impelled by the host plant.

Frequency of human platelet antigens in oncohematological patients with thrombocytopenia and the probability of incompatibility to platelet transfusions

OBJECTIVE: The objective of this study was to evaluate the frequencies of human platelet antigens in oncohematological patients with thrombocytopenia and to analyze the probability of their incompatibility with platelet transfusions. METHODS: Platelet antigen genotyping was performed by sequence-specific primer polymerase chain reaction (SSP-PCR) for the HPA-1a, HPA-1b, HPA-2a, HPA-2b, HPA-3a, HPA-3b, HPA-4a, HPA-4b, HPA-5a, HPA-5b; HPA-15a, HPA-15b alleles in 150 patients of the Hematology Service of the Hospital das Clínicas (FMUSP). RESULTS: The allele frequencies found were: HPA-1a: 0.837; HPA-1b: 0.163; HPA-2a: 0.830; HPA-2b: 0.170; HPA-3a: 0.700; HPA-3b: 0.300; HPA-4a: 1; HPA-4b: 0; HPA-5a: 0.887; HPA-5b: 0.113; HPA-15a: 0.457 and HPA-15b: 0.543. CONCLUSIONS: Data from the present study showed that the A allele is more common in the population than the B allele, except for HPA-15. This suggests that patients homozygous for the B allele are more predisposed to present alloimmunization and refractoriness to platelet transfusions by immune causes. Platelet genotyping could be of great value in the diagnosis of alloimmune thrombocytopenia and to provide compatible platelet concentrates for these patients.

Environmental assessment of residues generated after consecutive acid-base pretreatment of sugarcane bagasse by advanced oxidative process

Abstract Background Biofuels produced from sugarcane bagasse (SB) have shown promising results as a suitable alternative of gasoline. Biofuels provide unique, strategic, environmental and socio-economic benefits. However, production of biofuels from SB has negative impact on environment due to the use of harsh chemicals during pretreatment. Consecutive sulfuric acid-sodium hydroxide pretreatment of SB is an effective process which eventually ameliorates the accessibility of cellulase towards cellulose for the sugars production. Alkaline hydrolysate of SB is black liquor containing high amount of dissolved lignin. Results This work evaluates the environmental impact of residues generated during the consecutive acid-base pretreatment of SB. Advanced oxidative process (AOP) was used based on photo-Fenton reaction mechanism (Fenton Reagent/UV). Experiments were performed in batch mode following factorial design L9 (Taguchi orthogonal array design of experiments), considering the three operation variables: temperature (°C), pH, Fenton Reagent (Fe2+/H2O2) + ultraviolet. Reduction of total phenolics (TP) and total organic carbon (TOC) were responsive variables. Among the tested conditions, experiment 7 (temperature, 35°C; pH, 2.5; Fenton reagent, 144 ml H2O2+153 ml Fe2+; UV, 16W) revealed the maximum reduction in TP (98.65%) and TOC (95.73%). Parameters such as chemical oxygen demand (COD), biochemical oxygen demand (BOD), BOD/COD ratio, color intensity and turbidity also showed a significant change in AOP mediated lignin solution than the native alkaline hydrolysate. Conclusion AOP based on Fenton Reagent/UV reaction mechanism showed efficient removal of TP and TOC from sugarcane bagasse alkaline hydrolysate (lignin solution). To the best of our knowledge, this is the first report on statistical optimization of the removal of TP and TOC from sugarcane bagasse alkaline hydrolysate employing Fenton reagent mediated AOP process.

Estudio ultrasonográfico e histológico de la evolución de un modelo canino de hiperplasia benigna de próstata inducida por hormonas

[ES] Introducción: A pesar de exhibir diferencias histológicas con el proceso en humanos, la hiperplasia benigna de prostáta (HBP) inducida por hormonas en perros representa el modelo más utilizado actualmente para la evaluación de tratamientos. Objetivos: El objetivo del presente estudio es determinar el momento óptimo para la realización de un ensayo terapéutico en este modelo. Material y métodos: En el estudio se utilizaron 6 perros beagle, machos mayores de un año, a los que se administró una combinación de hormonas, 17b-estradiol y 5a-androsteno 3a 17b-diol, en días alternos durante 3 (grupo 1, n = 3) o 5 meses (grupo 2, n = 3). Se realizaron seguimientos ultrasonográficos mensuales, con determinación del tamaño prostático. A los 5 meses se procedió al sacrificio de todos los animales y el estudio histológico de las glándulas prostáticas. Resultados: Todos los animales desarrollaron una HBP, con un volumen prostático que aumentaba con el tiempo de administración hormonal (r = 0,910), evidenciándose en todos los estudios ecográficos hasta el tercer mes un aumento significativo de tamaño frente al estudio anterior. El tamaño prostático disminuyó considerablemente en el grupo 1 al retirar la hormonoterapia, al tiempo que en el grupo 2 continuó aumentando. Los animales del grupo 1 apenas evidenciaron signos histológicos de HBP, frente a los del grupo 2, que presentaron una clara hiperplasia del epitelio glandular. Conclusiones: La administración de una combinación de hormonas esteroides es efectiva para inducir hiperplasia de próstata en perro, si bien ésta desaparece en el momento que se retira la hormonoterapia. Para utilizar este modelo en estudios experimentales, se deben administrar hormonas al menos durante 3 meses antes de la actuación terapéutica, y continuar durante todo el ensayo

Enzimi, acidi organici ed altri metaboliti coinvolti nella patogenesi di Penicillium spp

Blue mould caused by Penicillium expansum Link is one of the most destructive rot of pome fruit in all growing areas (Snowdon, 1990; Jones and Aldwinckle, 1991; Tonini,1996) In the past, Penicillium rot has been controlled by fungicide postharvest treatment mainly by thiabendazole (TBZ) and benomyl (Hardenburg and Spalding, 1972), but their intense use produced the appearance of resistant strains with a great reduction of their activity The aims of the present study were to characterize the isolates of Pencillium sp causing blue mold on pear in Italy by physiological and biochemical parameters. In particular differencing also the behavior of isolates to relationship with sensitivity or resistance to TBZ treatments. We have examined the early stage of infection in relation to enzyme activity, local modulation of pH, production of organic acids, and to secondary metabolism of pathogen. The results described here confirm that the majority of P. expansum isolates from pears packing houses are resistant to TBZ, Among the TBZ-resistant isolates scored in this work, different isolates (RR) showed higher percentage of conidial germination on TBZ-amended medium compared to non amended medium. This may indicate a stimulatory effect of TBZ on conidial germination. Therefore TBZ treatments are not only ineffective for controlling P. expansum, but they may also increase the severity of blue mould on fruits. In the absence of fungicide, isolates showed a significant difference for infection severity, R and RR isolates are characterized by higher pathogenic fitness on fruits, producing larger lesions than S isolates. These data are supported by the study with laboratory-induced resistant isolates, which shows the lack of correlation between TBZ resistance and osmotic sensitivity, and highlights the association between TBZ resistance and infection severity (Baraldi et al 2003). Enzymatic screening gave a positive reaction to esterase, urease, pectinase activity, in addition, the pathogen is able to synthesize a complex enzyme act to degrade the main components of the cell wall especially pectin and cellulose. Isolated sensitive and resistant are characterized by a good activity of pectinase, especially from poligactoronase, which, as already reported by several studies (D'hallewin et al, 2004; Prusky et al, 2004), are the basis of degradative process of cell wall. Also, although the measure was minor also highlighted some activities of cellulase, but even note in the production of this kind of cellulase and hemicellulase P. Expansum were not targeted, studies have found no other source of information in this regard. Twenty isolates of Penicillium expansum, were tested in vitro ad in vivo for acid production ability and pH drop. We have found that modulation of pH and the organic acids extrusion were influence to various parameter:  Initial pH: in general, the greatest reduction of pH was observed in isolates grown at pH 7, except for four isolates that maintained the pH of the medium close to 7, the others significantly decreased the pH, ranging from 5.5 to 4.1.. In extreme acid condition (pH 3,0) growth and modulation of pH is most lower respect optimal condition (pH 5,0). Also isolates R and RR have showed a greater adaptation to environmental condition more than isolates S.  Time: although the acidification continues for some days, PH modulation is strongest in early hours (48-72 hours)of inoculation process. Time also affects the quality of organic acids, for example in vitro results showed an initial abundant production of succinc acid, followed to important production of galacturoinc acid.  Substrates: there are many differences for the type of acids produced in vitro and in vivo. Results showed in vivo an abundant production of galacturonic, malic, and citric acids and some unknown organic acids in smaller concentrations. Secondary metabolite analysis revealed intra-specific differences, and patulin was found in all isolates, but most significant reduction was observed between in vitro and in vivo samples. There was no correlation between the concentration of patulin, and the percentage of infected fruits, but sample with a lower infection severity of rotten area than the others, showed a significantly lower mycotoxin concentration than samples with a higher lesion diameter of rotten area. Beyond of patulin was detected the presence of another secondary metabolite, penitrem A.

Charakterisierung von Tumorantigenen des kutanen T-Zell Lymphoms

Immuntherapien stellen basierend auf gut charakterisierten, tumorspezifischen Antigenen ein vielversprechendes Konzept in der Tumor-Therapie dar. Für das kutane T-Zell Lymphom (CTCL) sind bislang nur wenige tumorassoziierte Antigene bekannt. In dieser Arbeit konnten ein mögliches Onkogen (PAR-3ta), ein weiteres, putativ Virus-induziertes Antigen (se57-1), neue Antigene und ihre Splicing-Varianten (se70-2, cTAGE-1/2, cTAGE-5), sowie lymphozytenspezifische Differenzierungsantigene (se20-10-Familie) für das CTCL gefunden werden. Auf Grund des Cancer-Testis spezifischen Expressionsprofils kommen die Antigene cTAGE-1 (46% CTCL-Expression), cTAGE-1B (31%) und cTAGE-5A (44%) für eine CTCL-Immuntherapie in Frage. Einige bekannte, spezifische Tumorantigene konnten auch im CTCL nachgewiesen werden: MAGE-A9 (21%), GAGE 3-7b (46%) und MUC-1 (44%). Die Immunogenität konnte mittels der SEREX-Technologie durch reaktive Antikörper gegen rekombinantes MAGE-A9 in 22% der getesteten CTCL-Seren, gegen GAGE in 16% und gegen cTAGE-5A in 15% bewiesen werden. Die cTAGE-mRNAs bilden eine neue Cancer-Testis Antigenfamilie. Die Multigenfamilie ist durch Retrotransposition eines aktiven Gens (cTAGE-5) entstanden, wobei 7 aktive, transkribierte und 7 nicht transkribierte Pseudogene unterschieden werden müssen. Die aktiven Gene cTAGE-1, cTAGE-1B und cTAGE-5A werden tumorspezifisch aus den Chromosomen 18p11.2 und 14 gespliced. Die Testis-CTCL spezifischen mRNAs können häufig auch in anderen Tumoren gefunden werden. Die Antigene cTAGE-1, cTAGE-5, MAGE-A9 und GAGE 3-7b gehören zu den ersten spezifischen Zielstrukturen des CTCLs, die somit für mögliche Immuntherapien in Betracht kommen.

Production, purification, properties and application of the cellulases from a wild type strain of a yeast isolate

Kurzzusammenfassung Produktion, Reinigung, Eigenschaften und Anwendung von Cellulasen eines Wildtyp-Hefeisolates. Die effiziente Verwendung von Cellulose wird in naher Zukonft ein wichtiges Instrument zur Vermeidung einer Nahrungsmittel- und Energieknappheit werden. Deshalb haben wir uns intensiv mit Cellulasen befaßt, die aus Hefestämmen isoliert wurden. Die Fähigkeit der Cellulase-produktion eines Hefe-Stammes der Feuerwanze Pyrrhocoris apterus wurde genauer untersucht. Die systematische Stellung des Hefe-Isolates PAG1 wurde durch Sequenzierung der 18S rDNA bestimmt. Es zeigte eine nahe Verwandtschaft zu einem bereits beschriebenen Stämme der Gattung Trichosporon. Außerdem wurden die Wachstums-bedingungen für eine optimale Cellulase–Produktion bestimmt. Anschließend konnte eine der produzierten Cellulasen mit FPLC aufgereinigt und deren biochemische Eigenschaften (z.B. Substratspezifität, Temperatur optimum, optimaler pH-Wert, Einfluß von Chemikalien) untersucht werden. Eine Analyse der Abbau-Produkte zeigte, daß kristalline Cellulose und CMC zu Cellobiose, Cellulotriose, Cellulotetraose und Cellulopentaose in einem molaren Verhältnis von 32:16:8:1 umgesetezt wurden. Bei Zusatz von ?-Glykosidase aus demselben Hefestamm entstand nur Glucose und Cellobiose in einem molaren Verhältnis von 1:10. Da bisher nur eine Publikation über Cellulase-produzierende Hefe-Stämme erschienen ist, zeigen auch unsere Untersuchungen, daß Wildtyp-Hefestämme Cellulasen mit interessanten Eigenschaften produzieren können.

Ricerca sulle Caratteristiche di Filtrabilità della Birra e Ottimizzazione del Processo di Filtrazione

Il presente lavoro ha avuto lo scopo di identificare le caratteristiche di filtrabilità della birra attraverso lo svolgimento di prove tecnologiche di filtrazione in scala di laboratorio e su scala pilota, utilizzando diverse tipologie di sistemi filtranti e sperimentando diversi materiali di membrana. La fase preliminare della caratterizzazione della filtrabilità della birra è stata condotta presso i laboratori del Campden-BRI – The Brewing Research International, Inghilterra, mentre le prove tecnologiche su scala pilota si sono svolte presso il CERB – Centro di Eccellenza per la Ricerca sulla Birra. Le prove di filtrazione e le analisi sui campioni hanno permesso di verificare le performance delle diverse membrane utilizzate in risposta alla variazione dei principali parametri del processo di filtrazione tangenziale. Sono stati analizzati diversi parametri di qualità della birra filtrata attraverso il monitoraggio del processo e lo svolgimento di prove analitiche sul prodotto volte ad evidenziare gli effetti delle differenti tecnologie adottate. Per quanto riguarda le prove di laboratorio per la caratterizzazione della filtrabilità della birra, l’analisi della PCS (Photon Correlation Spectroscopy) è stata utilizzata per verificare l’influenza di diversi trattamenti enzimatici sulla efficienza del processo di filtrazione e la loro influenza sulla stabilità colloidale della birra filtrata. Dai risultati ottenuti è emerso che il PCS è un valido strumento per determinare la distribuzione in classi di diametro e il diametro medio effettivo delle particelle solide in sospensione nella birra e può essere utilizzato sia per predire la stabilità della birra filtrata, sia per monitorare il processo di filtrazione in tempo reale.

N-Heterocyclic carbene complexes of rhodium: structures, dynamics and catalysis

A series of imidazolium salts of the type [BocNHCH2CH2ImR]X (Boc = t-Bu carbamates; Im = imidazole) (R = Me, X = I, 1a; R = Bn, X = Br, 1b; R = Trityl, X = Cl, 1c) and [BnImR’]X (R’ = Me, X = Br, 1d; R’ = Bn, X = Br, 1e; R’ = Trityl, X = Cl, 1g; R’ = tBu, X = Br, 1h) bearing increasingly bulky substituents were synthetized and characterized. Subsequently, these precursors were employed in the synthesis of silver(I)-N-heterocyclic (NHC) complexes as transmetallating reagents for the preparation of rhodium(I) complexes [RhX(NBD)(NHC)] (NHC = 1-(2-NHBoc-ethyl)-3-R-imidazolin-2-ylidene; X = Cl; R = Me, 4a; R = Bn, 4b; R = Trityl, 4c; X = I, R = Me, 5a; NHC = 1-Bn-3-R’-imidazolin-2-ylidene; X = Cl; R’ = Me, 4d, R’ = Bn, 4e, R’ = Trityl, 4g; R’ = tBu, 4h). VT NMR studies of these complexes revealed a restricted rotation barriers about the metal-carbene bond. While the rotation barriers calculated for the complexes in which R = Me, Bn (4a,b,d,e and 5a) matched the experimental values, this was not true for the complexes 4c,g, bearing a trityl group for which the values are much smaller than the calculated ones. Energy barriers for 4c,g, derived from a line shape simulation, showed a strong dependence on the temperature while for 4h the rotational energy barrier is stopped at room temperature. The catalytic activity of the new rhodium compounds was investigated in the hydrosilylation of terminal alkynes and in the addition of phenylboronic acid to benzaldehyde. The imidazolium salts 1d,e were also employed in the synthesis of new iron(II)-NHC complexes. Finally, during a six-months stay at the University of York a new ligand derived from Norharman was prepared and employed in palladium-mediated cross-coupling.

Sistemi per la produzione di enzimi industriali finalizzati alla valorizzazione di scarti agroalimentari

La demolizione idrolitica delle pareti cellulari delle piante tramite enzimi lignocellulosici è quindi uno degli approcci più studiati della valorizzazione di scarti agricoli per il recupero di fitochimici di valore come secondary chemical building block per la chimica industriale. White rot fungi come il Pleurotus ostreatus producono una vasta gamma di enzimi extracellulari che degradano substrati lignocellulosici complessi in sostanze solubili per essere utilizzati come nutrienti. In questo lavoro abbiamo studiato la produzione di diversi tipi di enzimi lignocellulosici quali cellulase, xilanase, pectinase, laccase, perossidase e arylesterase (caffeoilesterase e feruloilesterase), indotte dalla crescita di Pleurotus ostreatus in fermentazione allo stato solido (SSF) di sottoprodotti agroalimentari (graspi d’uva, vinaccioli, lolla di riso, paglia di grano e crusca di grano) come substrati. Negli ultimi anni, SSF ha ricevuto sempre più interesse da parte dei ricercatori, dal momento che diversi studi per produzioni di enzimi, aromi, coloranti e altre sostanze di interesse per l' industria alimentare hanno dimostrato che SSF può dare rendimenti più elevati o migliorare le caratteristiche del prodotto rispetto alla fermentazione sommersa. L’utilizzo dei sottoprodotti agroalimentari come substrati nei processi SSF, fornisce una via alternativa e di valore, alternativa a questi residui altrimenti sotto/o non utilizzati. L'efficienza del processo di fermentazione è stato ulteriormente studiato attraverso trattamenti meccanici di estrusione del substrato , in grado di promuovere il recupero dell’enzima e di aumentare l'attività prodotta. Le attività enzimatiche prodotte dalla fermentazione sono strettamente dipendente della rimozione periodica degli enzimi prodotti. Le diverse matrici vegetali utilizzate hanno presentato diversi fenomeni induttivi delle specifiche attività enzimatiche. I processi SSF hanno dimostrato una buona capacità di produrre enzimi extracellulari in grado di essere utilizzati successivamente nei processi idrolitici di bioraffinazione per la valorizzazione dei prodotti agroalimentari.