biochemical characterisation
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Agronomy ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 18
Author(s):  
Pasquale Tripodi ◽  
Rosa Pepe ◽  
Gianluca Francese ◽  
Macellaro Rosaria ◽  
Vincenzo Onofaro Sanajà ◽  
...  

Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops highly appreciated for the nutritional properties and content of beneficial compounds for human health. After its domestication, this crop has been spread throughout the world and found its secondary centre of diversification in the Mediterranean basin, where numerous landraces have been developed. Among these, ‘San Marzano’ is a famous and remunerative PDO variety for processing and fresh market consumption. ‘Re Fiascone’ is an ancient cultivar for which ongoing efforts aim to promote its diffusion and commercialisation. Both find their origin in the Campania region in Southern Italy where in the past decades, several accessions have been selected and handed down by farmers. This study reports a comprehensive assessment for morphological and biochemical traits of twenty-one accessions of the two landraces. Fruit morphology and content of sugars and flavonoids were the most discriminating parameters among cultivars. Among sugars and organic acids, fructose and citric acid were the most represented compounds, respectively. We found level of trans-lycopene and β-carotene up to 112.82 and 16.29 µg g−1 of fresh product, respectively, while ascorbic acid levels reached values up to 22.48 mg 100 g−1 of fresh weight. Molecular analysis has been performed using a double approach for microsatellite genotyping based on capillary electrophoresis and high-resolution melting. Results highlighted a separation of the accessions according to ‘Re Fiascone’ and ‘San Marzano’ identifying both unique and admixed accessions between the two groups. The study approach aims at the recovery and valorisation of local genetic resources, but also at the identification of traits of interest to transfer in breeding programs.


2021 ◽  
Author(s):  
Zumaira Nazeer ◽  
Eustace Fernando

A microbiological isolation and growth medium that can effectively discriminate electrochemically active exoelectrogenic bacteria from other non-exoelectrogenic bacteria, is currently unavailable. In this study, we developed a novel chromogenic growth and isolation solid medium based on MnO2 that can selectively allow the growth of exoelectrogenic bacteria and change the medium colour in the process. Known exoelectrogenic bacteria such as Shewanella oneidensis MR1 and other such bacteria from functional microbial fuel cell (MFC) anodes were capable of growing and changing colour in the novel growth medium. On the contrary, non-exoelectrogenic bacteria such as Escherichia coli ATCC 25922 were incapable of growing and inducing a colour changes in the novel medium. Further biochemical characterisation of these isolated exoelectrogenic bacteria by Raman micro-spectroscopy demonstrated that these bacteria over express cytochrome proteins that are vital in extracellular electron transfer events. This medium is a convenient method to isolate exoelectrogenic bacteria from complex environmental samples.


2021 ◽  
Vol 22 (22) ◽  
pp. 12448
Author(s):  
Zsuzsa Csobán-Szabó ◽  
Bálint Bécsi ◽  
Saïd El Alaoui ◽  
László Fésüs ◽  
Ilma Rita Korponay-Szabó ◽  
...  

Transglutaminases are protein-modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is involved in the development of autoimmune and tumour diseases. Although rodent TG4 is well characterised, biochemical characteristics of human TG4 that could help th e understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtitre plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell–cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.


2021 ◽  
Author(s):  
◽  
Jeremy George Owen

<p><b>Non-ribosomal peptide synthetases (NRPS) are large, modular enzymes that synthesisebiologically active secondary metabolites from amino acid precursors without the need for anucleic acid template. NRPS play an integral role in microbial physiology and also havepotential applications in the synthesis of novel peptide molecules. Both of these aspects areexamined in this thesis.</b></p> <p>Under conditions of iron starvation Pseudomonas syringae synthesises siderophores for activeuptake of iron. The primary siderophore of P. syringae is pyoverdine, a fluorescent moleculethat is assembled from amino acid (aa) precursors by NRPS. Five putative pyoverdine NRPSgenes in P. syringae pv. phaseolicola 1448a (Ps1448a) were identified and characterised insilico and their role in pyoverdine biosynthesis was confirmed by gene knockout. Creation ofpyoverdine null Ps1448a enabled identification of a previously uncharacterised temperatureregulatedsecondary siderophore, achromobactin, which is NRPS independent and has loweraffinity for iron. Pyoverdine and achromobactin null mutants were characterised in regard toiron uptake, virulence and growth in iron-limited conditions. Determination of the substratespecificity for the seven adenylation (A) domains of the Ps1448a pyoverdine sidechain NRPSwas also attempted. Although ultimately unsuccessful, these attempts provided a rigorousassessment of methods for the expression, purification and biochemical characterisation of Adomains.</p> <p>The Ps1448a NRPS were subsequently employed in domain swapping experiments to testcondensation (C) domain specificity for aa substrates during peptide formation in vivo.</p> <p>Experiments in which the terminal C- and/or A-domain of the Pseudomonas aeruginosa(PAO1) pyoverdine NRPS system were replaced with alternative domains from Ps1448a andPAO1 were consistent with previous in vitro observations that C-domains exhibit strongsidechain and stereo-selectivity at the downstream aa position, but only stereo-selectivity atthe upstream aa position.</p> <p>These results prompted investigation into the role of inter-domain communication in NRPSfunction, to test the hypothesis that the thiolation (T) domain enters into specific interactionswith other domains, which might provide an alternative explanation for the diminished activityof recombinant NRPS enzymes. A recently characterised single-module NRPS, bpsA, waschosen as a reporter gene for these experiments based on its ability to generate blue pigment inEscherichia coli. Substitution of the native bpsA T-domain consistently impaired function,consistent with the hypothesis. It was shown that directed evolution could be applied to restorefunction in substituted T-domains. Mutations that restored function were mapped in silico, anda structural model for interaction between the thioester (TE) and T-domain of BpsA wasderived.</p> <p>The utility of bpsA for discovery and characterisation of phosphopantetheinyl transferase(PPTase) enzymes was also investigated. In vivo and in vitro assays for determination ofPPTase activity were developed and a high-throughput screen for discovery of new PPTases inenvironmental DNA libraries was successfully implemented.</p>


2021 ◽  
Author(s):  
◽  
Jeremy George Owen

<p><b>Non-ribosomal peptide synthetases (NRPS) are large, modular enzymes that synthesisebiologically active secondary metabolites from amino acid precursors without the need for anucleic acid template. NRPS play an integral role in microbial physiology and also havepotential applications in the synthesis of novel peptide molecules. Both of these aspects areexamined in this thesis.</b></p> <p>Under conditions of iron starvation Pseudomonas syringae synthesises siderophores for activeuptake of iron. The primary siderophore of P. syringae is pyoverdine, a fluorescent moleculethat is assembled from amino acid (aa) precursors by NRPS. Five putative pyoverdine NRPSgenes in P. syringae pv. phaseolicola 1448a (Ps1448a) were identified and characterised insilico and their role in pyoverdine biosynthesis was confirmed by gene knockout. Creation ofpyoverdine null Ps1448a enabled identification of a previously uncharacterised temperatureregulatedsecondary siderophore, achromobactin, which is NRPS independent and has loweraffinity for iron. Pyoverdine and achromobactin null mutants were characterised in regard toiron uptake, virulence and growth in iron-limited conditions. Determination of the substratespecificity for the seven adenylation (A) domains of the Ps1448a pyoverdine sidechain NRPSwas also attempted. Although ultimately unsuccessful, these attempts provided a rigorousassessment of methods for the expression, purification and biochemical characterisation of Adomains.</p> <p>The Ps1448a NRPS were subsequently employed in domain swapping experiments to testcondensation (C) domain specificity for aa substrates during peptide formation in vivo.</p> <p>Experiments in which the terminal C- and/or A-domain of the Pseudomonas aeruginosa(PAO1) pyoverdine NRPS system were replaced with alternative domains from Ps1448a andPAO1 were consistent with previous in vitro observations that C-domains exhibit strongsidechain and stereo-selectivity at the downstream aa position, but only stereo-selectivity atthe upstream aa position.</p> <p>These results prompted investigation into the role of inter-domain communication in NRPSfunction, to test the hypothesis that the thiolation (T) domain enters into specific interactionswith other domains, which might provide an alternative explanation for the diminished activityof recombinant NRPS enzymes. A recently characterised single-module NRPS, bpsA, waschosen as a reporter gene for these experiments based on its ability to generate blue pigment inEscherichia coli. Substitution of the native bpsA T-domain consistently impaired function,consistent with the hypothesis. It was shown that directed evolution could be applied to restorefunction in substituted T-domains. Mutations that restored function were mapped in silico, anda structural model for interaction between the thioester (TE) and T-domain of BpsA wasderived.</p> <p>The utility of bpsA for discovery and characterisation of phosphopantetheinyl transferase(PPTase) enzymes was also investigated. In vivo and in vitro assays for determination ofPPTase activity were developed and a high-throughput screen for discovery of new PPTases inenvironmental DNA libraries was successfully implemented.</p>


BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Giovanna Pesante ◽  
Federico Sabbadin ◽  
Luisa Elias ◽  
Clare Steele-King ◽  
J. Reuben Shipway ◽  
...  

Abstract Background Shipworms are marine xylophagus bivalve molluscs, which can live on a diet solely of wood due to their ability to produce plant cell wall-degrading enzymes. Bacterial carbohydrate-active enzymes (CAZymes), synthesised by endosymbionts living in specialised shipworm cells called bacteriocytes and located in the animal’s gills, play an important role in wood digestion in shipworms. However, the main site of lignocellulose digestion within these wood-boring molluscs, which contains both endogenous lignocellulolytic enzymes and prokaryotic enzymes, is the caecum, and the mechanism by which bacterial enzymes reach the distant caecum lumen has remained so far mysterious. Here, we provide a characterisation of the path through which bacterial CAZymes produced in the gills of the shipworm Lyrodus pedicellatus reach the distant caecum to contribute to the digestion of wood. Results Through a combination of transcriptomics, proteomics, X-ray microtomography, electron microscopy studies and in vitro biochemical characterisation, we show that wood-digesting enzymes produced by symbiotic bacteria are localised not only in the gills, but also in the lumen of the food groove, a stream of mucus secreted by gill cells that carries food particles trapped by filter feeding to the mouth. Bacterial CAZymes are also present in the crystalline style and in the caecum of their shipworm host, suggesting a unique pathway by which enzymes involved in a symbiotic interaction are transported to their site of action. Finally, we characterise in vitro four new bacterial glycosyl hydrolases and a lytic polysaccharide monooxygenase identified in our transcriptomic and proteomic analyses as some of the major bacterial enzymes involved in this unusual biological system. Conclusion Based on our data, we propose that bacteria and their enzymes are transported from the gills along the food groove to the shipworm’s mouth and digestive tract, where they aid in wood digestion.


2021 ◽  
Vol 15 (10) ◽  
pp. e0009912
Author(s):  
Adriana Temporão ◽  
Margarida Sanches-Vaz ◽  
Rafael Luís ◽  
Helena Nunes-Cabaço ◽  
Terry K. Smith ◽  
...  

Malaria, a disease caused by Plasmodium parasites, remains a major threat to public health globally. It is the most common disease in patients with sleeping sickness, another parasitic illness, caused by Trypanosoma brucei. We have previously shown that a T. brucei infection impairs a secondary P. berghei liver infection and decreases malaria severity in mice. However, whether this effect requires an active trypanosome infection remained unknown. Here, we show that Plasmodium liver infection can also be inhibited by the serum of a mouse previously infected by T. brucei and by total protein lysates of this kinetoplastid. Biochemical characterisation showed that the anti-Plasmodium activity of the total T. brucei lysates depends on its protein fraction, but is independent of the abundant variant surface glycoprotein. Finally, we found that the protein(s) responsible for the inhibition of Plasmodium infection is/are present within a fraction of ~350 proteins that are excreted to the bloodstream of the host. We conclude that the defence mechanism developed by trypanosomes against Plasmodium relies on protein excretion. This study opens the door to the identification of novel antiplasmodial intervention strategies.


Author(s):  
Shoumo Bhattacharya ◽  
Patricia Anne Nuttall

Chemokines are structurally related proteins that activate leucocyte migration in response to injury or infection. Tick saliva contains chemokine-binding proteins or evasins which likely neutralize host chemokine function and inflammation. Biochemical characterisation of 50 evasins from Ixodes, Amblyomma and Rhipicephalus shows that they fall into two functional classes, A and B, with exclusive binding to either CC- or CXC- chemokines, respectively. Class A evasins, EVA1 and EVA4 have a four-disulfide-bonded core, whereas the class B evasin EVA3 has a three-disulfide-bonded “knottin” structure. All 29 class B evasins have six cysteine residues conserved with EVA3, arrangement of which defines a Cys6-motif. Nineteen of 21 class A evasins have eight cysteine residues conserved with EVA1/EVA4, the arrangement of which defines a Cys8-motif. Two class A evasins from Ixodes (IRI01, IHO01) have less than eight cysteines. Many evasin-like proteins have been identified in tick salivary transcriptomes, but their phylogenetic relationship with respect to biochemically characterized evasins is not clear. Here, using BLAST searches of tick transcriptomes with biochemically characterized evasins, we identify 292 class A and 157 class B evasins and evasin-like proteins from Prostriate (Ixodes), and Metastriate (Amblyomma, Dermacentor, Hyalomma, Rhipicephalus) ticks. Phylogenetic analysis shows that class A evasins/evasin-like proteins segregate into two classes, A1 and A2. Class A1 members are exclusive to Metastriate ticks and typically have a Cys8-motif and include EVA1 and EVA4. Class A2 members are exclusive to Prostriate ticks, lack the Cys8-motif, and include IHO01 and IRI01. Class B evasins/evasin-like proteins are present in both Prostriate and Metastriate lineages, typically have a Cys6-motif, and include EVA3. Most evasins/evasin-like proteins in Metastriate ticks belong to class A1, whereas in Prostriate species they are predominantly class B. In keeping with this, the majority of biochemically characterized Metastriate evasins bind CC-chemokines, whereas the majority of Prostriate evasins bind CXC-chemokines. While the origin of the structurally dissimilar classes A1 and A2 is yet unresolved, these results suggest that class B evasin-like proteins arose before the divergence of Prostriate and Metastriate lineages and likely functioned to neutralize CXC-chemokines and support blood feeding.


2021 ◽  
Author(s):  
Zsuzsa Csobán-Szabó ◽  
Bálint Bécsi ◽  
Saïd El Alaoui ◽  
László Fésüs ◽  
Ilma Rita Korponay-Szabó ◽  
...  

AbstractTransglutaminases are protein modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is frequently associated with pathological symptoms and particularly with cancer invasiveness. Although rodent TG4 is well characterised, bio-chemical characteristics of human TG4 that could help the understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtiter plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell-cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.


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