http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. 2013-05-24T05:35:47Z http://hdl.handle.net/2067/1495 Title: Vanillin production using metabolically engineered Escherichia coli under non-growing conditions Authors: Barghini, Paolo; Di Gioia, Diana; Fava, Fabio; Ruzzi, Maurizio Abstract: Vanillin is one of the most important aromatic flavour compounds used in the food and cosmetic industries. Natural vanillin is extracted from vanilla beans and is relatively expensive. Moreover, the consumer demand for natural vanillin highly exceeds the amount of vanillin extracted by plant sources. This has led to the investigation of other routes to obtain this flavour such as the biotechnological production from ferulic acid. Studies concerning the use of engineered recombinant Escherichia coli cells as biocatalysts for vanillin production are described in the literature, but yield optimization and biotransformation conditions have not been investigated in details. 2006-12-31T23:00:00Z http://hdl.handle.net/2067/1499 Title: Regulation of ferulic catabolic genes in Pseudomonas fluorescens BF13: involvement of a MarR family regulator Authors: Calisti, Cecilia; Ficca, Anna Grazia; Barghini, Paolo; Ruzzi, Maurizio Abstract: In Pseudomonas fluorescens BF13, the cluster of genes essential for degradation of ferulic to vanillic acid (ech, vdh and fcs) is expressed in ferulic but not in succinic grown cells. In the upstream region, we identified a gene, ferR, encoding a protein homologous to transcriptional regulators of the MarR family. A ferR knockout mutant (BF13–89) showed a 3.5-fold increase in expression of an ech-reporter gene fusion compared with the parent strain in succinic-grown cells, indicating that the ferR gene product negatively regulates expression of the ferulic catabolic operon in P. fluorescens BF13. Consistent with the increased expression of the catabolic genes in the ferR mutant, BF13-89 showed a shorter (relative to its FerR+ parent) lag phase during carbon source shift from succinic to ferulic acid. However, expression of ech-lacZ fusion did not increase in BF13–89 grown in the presence of ferulic acid, indicating that FerR has a second function as transcriptional activator. Expression of ech-lacZ in a feruloyl-CoA synthetase- eficient strain revealed unambiguously that FerR-mediated activation of the ferulic catabolic operon is dependent on the thioester product of the feruloyl-CoA synthetase reaction. Description: L'articolo è disponibile sul sito dell'editore http://www.springerlink.com 2007-12-31T23:00:00Z http://hdl.handle.net/2067/1505 Title: Optimization of capsaicin acylase production from Streptomyces mobaraensis in bench-top fermenter Authors: Crognale, Silvia; Barghini, Paolo; Di Matteo, Paola; Federici, Federico; Ruzzi, Maurizio Abstract: Capsaicin, the major pungent principle in hot pepper fruit, can be hydrolyzed enzymatically to vanillylamine (a natural precursor of vanillin) using a specific acylase from Streptomyces mobaraensis. Production of this enzyme using strain DSM40847 was studied under batch fermentation conditions in stirred tank (STR) and airlift (AR) bioreactors. The process performance in both fermentation devices was different with respect to biomass, enzyme concentration and specific yield (enzyme activity/biomass content); in particular the specific yield was lower in the AR (5.7 mU/g of biomass) than in the STR (6.25 mU/g of biomass). Experiments carried out in STR bioreactors at controlled (DO = 20% of saturation) and uncontrolled dissolved oxygen concentration, and constant stirred speeds (300, 450 and 600 rpm) demonstrated that the DO level has no remarkable effect on the production of the capsaicin-hydrolyzing enzyme, which is mainly produced in a cell-associated form. 2007-12-31T23:00:00Z http://hdl.handle.net/2067/1725 Title: Development of Innovative Molecular Methods for the Detection and the Identification of Pseudomonas spp. in Environmental and Clinical Samples Authors: Calisti, Cecilia; Ruzzi, Maurizio Abstract: We have developed a rapid, reliable and sensitive method to analyze, detect and trace dissemination of pathogenic and spoilage Pseudomonas species in foods and environments. The molecular identification of Pseudomonas species was achieved using a PCR-based assay with primer sets specific for 16S ribosomal DNA and gyrB genes. This PCR assay was found to provide highly genus-specific detection and could be successfully used to identify Pseudomonas in microbial consortia where these bacteria were not abundant. By coupling the PCR assay for 16S rDNA gene to a T-RFLP technique, identification of Pseudomonas strains at species level could be obtained without cultivation. PCR with a combination of two target sequences (multiplex PCR) appeared to be the optimum choice for discriminating between Pseudomonas and closely related genera. 2008-12-31T23:00:00Z http://hdl.handle.net/2067/2172 Title: In vitro system for studying the interaction between Erwinia amylovora and genotypes of pear Authors: Abdollahi, Hamid; Rugini, Eddo; Ruzzi, Maurizio; Muleo, Rosario Abstract: A new in vitro system is described for studying an interaction between Erwinia amylovoraand Pyrus communis (L.). The system uses single shoots placed onto the solid medium, and it enables to detect changes in pH of the medium and differential appearance of shoot necrosis. Shoots of susceptible cultivar (Williams) and tolerant cultivar (Harrow Sweet) were compared measuring the necrosis rate along the in vitroshoots and the pH variation following proton extrusion of both plant and pathogen. Shoots acidified differentially the culture medium depending on the presence of the pathogen, cultivar susceptibility and shoot inoculation methods. Differences in the tolerance level against pathogen among the cultivars were distinguishable only when the shoots were inoculated at the basal end. In susceptible cultivar, the necrosis appeared after 48 h of inoculation, while in tolerant cultivars after 72 h. This system is repeatable and more reliable than already known methods, such as in vitroleaf explants or in vivoplants; it can be used all around the year to test the gene expression and products essential to characterize the genes involved in the pathogenesis. This system showed the effects of E. amylovoraon the photosystem dependent system of host cells, confirmed by the effects of pathogen attack on the variation of chlorophyll a and chlorophyll b ratios and positive effects of light on the appearance of the first disease symptoms. Description: L'articolo è disponibile sul sito del nuovo editore http://www.springerlink.com 2003-12-31T23:00:00Z