RESUMEN
BACKGROUND: Laccases and laccase-like multicopper oxidases (LMCOs) oxidize a vast array of phenolic compounds and amines, releasing water as a byproduct. Their low substrate specificity is responsible for their tremendous biotechnological interest, since they have been used for numerous applications. However, the laccases characterized so far correspond to only a small fraction of the laccase genes identified in fungal genomes. Therefore, the knowledge regarding the biochemistry and physiological role of minor laccase-like isoforms is still limited. RESULTS: In the present work, we describe the isolation, purification and characterization of two novel LMCOs, PcLac1 and PcLac2, from Pleurotus citrinopileatus. Both LMCOs were purified with ion-exchange chromatographic methods. PcLac2 was found to oxidize a broader substrate range than PcLac1, but both LMCOs showed similar formal potentials, lower than those reported previously for laccases from white-rot fungi. Proteomic analysis of both proteins revealed their similarity with other well-characterized laccases from Pleurotus strains. Both LMCOs were applied to the oxidation of ferulic and sinapic acid, yielding oligomers with possible antioxidant activity. CONCLUSIONS: Overall, the findings of the present work can offer new insights regarding the biochemistry and variability of low-redox potential laccases of fungal origin. Low-redox potential biocatalysts could offer higher substrate selectivity than their high-redox counterparts, and thus, they could be of applied value in the field of biocatalysis.
RESUMEN
Ingestion of gluten proteins (gliadins and glutenins) from wheat, barley and rye can cause coeliac disease (CD) in genetically predisposed individuals. The only remedy is a strict and lifelong gluten-free diet. There is a growing desire for coeliac-safe, whole-grain wheat-based products, as consumption of whole-grain foods reduces the risk of chronic diseases. However, due to the large number of gluten genes and the complexity of the wheat genome, wheat that is coeliac-safe but retains baking quality cannot be produced by conventional breeding alone. CD is triggered by immunogenic epitopes, notably those present in α-, γ-, and ω-gliadins. RNA interference (RNAi) silencing has been used to down-regulate gliadin families. Recently, targeted gene editing using CRISPR/Cas9 has been applied to gliadins. These methods produce offspring with silenced, deleted, and/or edited gliadins, that overall may reduce the exposure of patients to CD epitopes. Here we review methods to efficiently screen and select the lines from gliadin gene editing programs for CD epitopes at the DNA and protein level, for baking quality, and ultimately in clinical trials. The application of gene editing for the production of coeliac-safe wheat is further considered within the context of food production and in view of current national and international regulatory frameworks.
RESUMEN
Membrane proteins are an interesting class of proteins because of their functional importance. Unfortunately their analysis is hampered by low abundance and poor solubility in aqueous media. Since shotgun methods are high-throughput and partly overcome these problems, they are preferred for membrane proteomics. However, their application in non-model plants demands special precautions to prevent false positive identification of proteins. In the current paper, a workflow for membrane proteomics in banana, a poorly sequenced plant, is proposed. The main steps of this workflow are (i) optimization of the peptide separation, (ii) performing de novo sequencing to allow a sequence homology search and (iii) visualization of identified peptide-protein associations using Cytoscape to remove redundancy and wrongly assigned peptides, based on species-specific information. By applying this workflow, integral plasma membrane proteins from banana leaves were successfully identified.
