estrazione e purificazione di proteine enzimatiche dal terreno: studi preliminari e approccio metodologico

L'ecosistema suolo è ricco di proteine enzimatiche che possono essere raggruppate in due classi: intracellulari ed extracellulari. L'attività degli enzimi intracellulari dipende dalla proliferazione microbica a dierenza dell'attività di quelli extracellulari che vengono secreti nella fase acquosa del terreno da cellule microbiche e vegetali metabolicamente attive o in fase di lisi. Tali enzimi possono trovarsi liberi o stabilizzati dalla formazione di complessi con le sostanze umiche e le argille del terreno, sono considerati una riserva di materia ed energia biochimica del suolo in quanto la loro attività si svolge anche in condizioni proibitive per i microrganismi e, per questo, sono stati proposti come le ultime difese biologiche del suolo esposto a processi di degradazione irreversibili come la deserticazione. Il loro isolamento richiede studi laboriosi a causa della complessa matrice rappresentata dal terreno; e a causa dell'eventuale formazione dei suddetti complessi. In questo lavoro di tesi si mira alla messa a punto di tecniche atte ad estrarre e puricare tali proteine dal terreno, con lo scopo ultimo di saggiare la presenza e l'attività dell'enzima idrolitico beta-glucosidasi. Questo enzima catalizza l'idrolisi del cellobiosio in glucosio (ultimo step della via catabolica della cellulosa) ed è quindi importante per il ciclo del carbonio e per la "qualità" del suolo in generale. La metodologia utilizzata ha previsto l'estrazione delle proteine extracellulari libere e di quelle legate alla componente umica attraverso l'impiego di due estraenti. La componente microbica è stata allontanata dall'estratto attraverso ltrazione su membrana batteriologica 0,22 um. Per favorire la rottura dei complessi formati tra proteine e sostanze umiche, l'estratto è stato sottoposto a trattamenti sici (shock termico). In seguito è stato desalicato attraverso membrane da dialisi, e concentrato tramite ultraltrazione. A questo punto per allontanare le sostanze umiche in eccesso dall'estratto è stato eettuato un passaggio di puricazione utilizzando il polimero polivinilpirrolidone. Il successivo step ha previsto la precipitazione delle proteine con il metodo che utilizza deossicolato e acido tricloroacetico (DOC- TCA). Inne sono state applicate elettroforesi su gel di poliacrilammide in condizioni denaturanti e non denaturanti (SDS-PAGE e NATIVE-PAGE) per visualizzare le proteine estratte nella forma denaturata e nativa con lo scopo ultimo di ricercare la presenza dell'attività beta-glucosidasica

Environmental proteomics: A long march in the pedosphere

Environmental proteomics, the study of the expression profile of proteins extracted directly from living organisms and some stabilized extracellular proteins present in environmental samples, is a developing branch of soil science since the seminal papers appeared twenty years ago. Soil microbial communities hold the key to understanding terrestrial biodiversity; they are extremely complex and their physiological responses to dynamic environmental parameters are under-characterized. Therefore, the slow development of environment-related proteomic databases, and the high chemical reactivity of environmental matrices hamper the extraction, quantification, and characterization of proteins; and soil proteomics remains still in its infancy. We underscore the main achievements of environmental proteomics focusing on soil ecosystems, and we identify technical gaps that need to be bridged in the context of relevant ecological concepts that have received little attention in the development of proteomics methods. This analysis offers a new framework of research of soil proteomics toward improved understanding of the causal linkages between the structure and function of the soil microbiome, and a broader grasp of the sensitivity of terrestrial ecosystems to environmental change

Environmental proteomics: A long march in the pedosphere

Environmental proteomics, the study of the expression profile of proteins extracted directly from living organisms and some stabilized extracellular proteins present in environmental samples, is a developing branch of soil science since the seminal papers appeared twenty years ago. Soil microbial communities hold the key to understanding terrestrial biodiversity; they are extremely complex and their physiological responses to dynamic environmental parameters are under-characterized. Therefore, the slow development of environment-related proteomic databases, and the high chemical reactivity of environmental matrices hamper the extraction, quantification, and characterization of proteins; and soil proteomics remains still in its infancy. We underscore the main achievements of environmental proteomics focusing on soil ecosystems, and we identify technical gaps that need to be bridged in the context of relevant ecological concepts that have received little attention in the development of proteomics methods. This analysis offers a new framework of research of soil proteomics toward improved understanding of the causal linkages between the structure and function of the soil microbiome, and a broader grasp of the sensitivity of terrestrial ecosystems to environmental change

Environmental proteomics: A long march in the pedosphere

Environmental proteomics, the study of the expression profile of proteins extracted directly from living organisms and some stabilized extracellular proteins present in environmental samples, is a developing branch of soil science since the seminal papers appeared twenty years ago. Soil microbial communities hold the key to understanding terrestrial biodiversity; they are extremely complex and their physiological responses to dynamic environmental parameters are under-characterized. Therefore, the slow development of environment-related proteomic databases, and the high chemical reactivity of environmental matrices hamper the extraction, quantification, and characterization of proteins; and soil proteomics remains still in its infancy. We underscore the main achievements of environmental proteomics focusing on soil ecosystems, and we identify technical gaps that need to be bridged in the context of relevant ecological concepts that have received little attention in the development of proteomics methods. This analysis offers a new framework of research of soil proteomics toward improved understanding of the causal linkages between the structure and function of the soil microbiome, and a broader grasp of the sensitivity of terrestrial ecosystems to environmental change

Soil Proteomics

Proteomics is a post-genomic approach with the potential to interrogate natural complex systems such as soils. However, the great potentials of soil proteomics are currently limited by either the complexity of the soil matrix which is reactive, structured, teeming with microbial communities which are at the same time extremely diverse, in heterogeneous physiological state and normally poorly characterized. Taken together, these soil features pose problems of protein sampling, extraction and purification. This chapter, though not exhaustive, aims to illustrate the main approaches and achievements in soil proteomics and indicate some future directions for further developments soil proteomics

Soil volatile analysis by proton transfer reaction-time of flight mass spectrometry (PTR-TOF-MS)

We analyzed the volatile organic compounds (VOCs) emitted from different soils by using the PTR-MS-TOF technique under laboratory conditions and compared them with soil chemical biochemical activities. The emitted VOCs were related to soil microbial biomass, soil respiration and some soil enzyme activities so as to evaluate if size and activity of soil microbial communities influenced the soil VOCs profiles. Our results showed that the emitted VOCs discriminated between soils with different properties and management, and differences in the VOCs emission profiles were likely related to the active metabolic pathways in the microbial communities of the three studied soil. Our results also showed that some soil enzyme activities such as ß-glucosidase and arylsulfatase were possibly involved in the release of compounds fueling microbial metabolic pathways leading to the production of specific VOCs. It was concluded that the PTR-MS-TOF technique is suitable for analyze VOCs emission from soil and that studies comparing soil enzyme activities and soil volatile profiles can reveal the origin of VOCs and give further insights on microbial activity and soil functionality

Enzyme activity and microbial community structure in the rhizosphere of two maize lines differing in N use efficiency

AimsStudy of the changes in soil microbial biomass, enzyme activity and the microbial community structure in the rhizosphere of two contrasting maize lines differing in the nitrogen use efficiency (NUE).MethodsThe Lo5 and T250 inbred maize characterized by high and low NUE, respectively, were grown in rhizoboxes allowing precise sampling of rhizosphere and bulk soil and solution. We also determined microbial biomass, enzyme activities involved in the C, N, P and S cycles, and the microbial community structure using a phylogenetic group specific PCR-DGGE approach in the rhizosphere and bulk soil of both Lo5 and T250 maize lines.ResultsHigh NUE Lo5 maize induced faster inorganic N depletion in the rhizosphere and larger changes in microbial biomass and enzyme activities than the low NUE T250 maize line. The two maize lines induced differences in the studied microbial groups in the rhizosphere, with the larger modifications induced by the high NUE Lo5 maize line.ConclusionsThe Lo5 maize line with higher NUE induced larger changes in soil chemical properties and in the enzyme activity, soil microbial biomass and community structure than the low NUE T250 maize line, probably due to differences in the root exudates of the two maize lines

Protease encoding microbial communities and protease activity of the rhizosphere and bulk soils of two maize lines with different N uptake efficiency

none8siThis study was carried out to understand the interplay of plant Nitrogen Utilizing Efficiency (NUE) with protease activity and microbial proteolytic community composition in the rhizosphere and bulk soils. Protease activity, diversity and abundance of protease genes (using DGGE and qPCR respectively of two key bacterial protease encoding genes: alkaline metallo-peptidase (apr) and neutral-metallopeptidases (npr)) were monitored in both rhizosphere and bulk soils from two maize in-bred lines L05 and T250 with higher and lower NUE respectively, using a rhizobox approach. Illumina sequencing was employed to assess the diversity of proteolytic communities encoding for the above-mentioned protease genes. Our results show higher enzyme activity, higher abundance and diversity of proteolytic genes in L05 maize rhizosphere, with higher NUE than in T250 maize rhizosphere.restrictedBaraniya D.; Puglisi E.; Ceccherini M.T.; Pietramellara G.; Giagnoni L.; Arenella M.; Nannipieri P.; Renella G.Baraniya, D.; Puglisi, E.; Ceccherini, M. T.; Pietramellara, G.; Giagnoni, L.; Arenella, M.; Nannipieri, P.; Renella, G