Multidimensional engineering of Chymosin for efficient cheese production by machine learning guided directed evolution

The global cheese market today exceeds $100B/year. Chymosin (a.k.a. rennin) is an aspartic endopeptidase produced by the stomach lining of new-born mammals. During cheese production chymosin is added to the milk where it cleaves the glycomacropeptide (GMP) from the surface of casein micelles to initiate milk coagulation. Current commercial recombinant chymosin enzymes derived from Bos taurus (cow) or Camelus dromedarius (camel) are limited in their proteolytic specificity leading to incomplete milk-to-cheese conversion. Increasing the chymosin specificity for GMP cleavage would significantly decrease the amount of milk needed for cheese production thereby reducing cost and decreasing environmental footprint of the dairy industry. Separate from milk coagulation, chymosin dependent release of N-terminal peptides from alphaS1 casein during cheese ripening leads to unwanted softening, accompanied with cheese loss during industrial processing such as slicing and shredding. Furthermore, chymosin dependent cleavage of the C-terminal end of beta casein contributes to unwanted bitterness of the cheese. Improvement of chymosin proteolytic specificity in both milk coagulation and cheese ripening is consequently of high commercial relevance. Please click Additional Files below to see the full abstract

An engineered gastrointestinally stable microbial leucine decarboxylase for potential treatment of maple syrup urine disease

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Controlling self-assembling and tumor cell-targeting of protein-only nanoparticles through modular protein engineering

Altres ajuts: EU COST Action CA 17140. Villaverde A received an ICREA ACADEMIA award. Unzueta was supported by PERIS program from the Health Department of la Generalitat de Catalunya.Modular protein engineering is suited to recruit complex and multiple functionalities in single-chain polypeptides. Although still unexplored in a systematic way, it is anticipated that the positioning of functional domains would impact and refine these activities, including the ability to organize as supramolecular entities and to generate multifunctional protein materials. To explore this concept, we have repositioned functional segments in the modular protein T22-GFP-H6 and characterized the resulting alternative fusions. In T22-GFP-H6, the combination of T22 and H6 promotes self-assembling as regular nanoparticles and selective binding and internalization of this material in CXCR4-overexpressing tumor cells, making them appealing as vehicles for selective drug delivery. The results show that the pleiotropic activities are dramatically affected in module-swapped constructs, proving the need of a carboxy terminal positioning of H6 for protein self-assembling, and the accommodation of T22 at the amino terminus as a requisite for CXCR4 cell binding and internalization. Furthermore, the failure of self-assembling as regular oligomers reduces cellular penetrability of the fusions while keeping the specificity of the T22-CXCR4 interaction. All these data instruct how multifunctional nanoscale protein carriers can be designed for smart, protein-driven drug delivery, not only for the treatment of CXCR4 human neoplasias, but also for the development of anti-HIV drugs and other pathologies in which CXCR4 is a relevant homing marker

Nanostructured antimicrobial peptides : The last push towards clinics

Altres ajuts: to EU COST Action CA 17140 and ICREA ACADEMIA awardPeptide drugs hold great potential for the treatment of infectious diseases due to their unconventional mechanisms of action, biocompatibility, biodegradability and ease of synthesis and modification. The increasing rising of bacterial strains resistant to classical antibiotics have pushed the development of new peptide-based antimicrobial therapies. In this context, over the past few years, different approaches have reached a clinical approval. Furthermore, the application of nanotechnological principles to the design of antimicrobial peptide-based composites increases even more the already known benefits of antimicrobial peptides as competent protein drugs. Then, we provide here an overview of the current strategies for antimicrobial peptide discovery and modification and the status of such peptides already under clinical development. In addition, we summarize the innovative formulation strategies for their application, focusing on the controlled self-assembly for the fabrication of antimicrobial nanostructures without the assistance of external nanocarriers, and with emphasis on bioengineering, design of ultra-short peptides and rising insights in bacterial selectivity

Validazione di prodotti a base di carne senza aggiunta di nitriti, nitrati e fosfati

The challenge for the meat industry is developing meat products that contain no additives, have similar sensory properties as traditional nitrite-cured products and will persist throughout their shelf life with the same level of quality and safety. In this study, new recipes with no additives or with food additive substitutes of natural origin were compared with commercially available products. It was concluded that the new meat products – cooked ham (as representative of thermally processed meat products) and fermented sausage had concentrations of nitrite and nitrate below the method’s limit of quantification during the entire shelf life period. Acerola has successfully replaced sodium ascorbate as a natural source of ascorbic acid in meat products. Furthermore, it was proved that no phosphates, comparable to those from raw meat, were added to meat products. Using sensory evaluation, cooked ham and fermented sausage were classified into the highest quality category, in terms of color, flavor, odor and texture during the entire shelf life, and were evaluated as microbiologically safe, according to the Guide for Microbiological Criteria for Foodstuffs (Ministry of Agriculture, March 2011).Izazov je mesne industrije razvoj novih mesnih proizvoda bez dodanih aditiva sa senzorskim i mikrobiološkim svojstvima sličnim onima tradicionalnih proizvoda s nitritnom soli kroz cijeli rok trajanja. U ovom radu, novi recepti bez aditiva ili sa zamjenom aditiva iz prirodnog izvora uspoređeni su s komercijalno dostupnim proizvodima. Zaključeno je da novi mesni proizvodi – kuhana šunka (kao predstavnik termički obrađenih proizvoda) i trajna kobasica imaju koncentraciju nitrita i nitrata ispod granice kvantifikacije za cijelog roka trajanja. Acerola je uspješno zamijenila natrijev askorbat kao prirodni izvor askorbinske kiseline u mesnim proizvodima. Nadalje, dokazano je da fosfati, uspoređujući rezultate s onima iz neobrađenog mesa, nisu dodani u mesne proizvode. Kuhana šunka i trajna kobasica senzorskom su evaluacijom svrstane u najvišu kategoriju kvalitete u pogledu boje, okusa, mirisa i teksture za cijelog roka trajanja te su ocijenjene mikrobiološki ispravnima prema Vodiču za mikrobiološke kriterije za hranu (Ministarstvo poljoprivrede, ožujak 2011.).Die Herausforderung für die Fleischindustrie besteht in der Entwicklung von Fleischerzeugnissen, die keine Zusatzstoffe enthalten und während ihrer gesamten Haltbarkeitsdauer ähnliche sensorische Eigenschaften wie herkömmliche nitritgepökelte Produkte aufweisen. In dieser Studie wurden neue Rezepturen ohne Zusatzstoffe oder mit Ersatzstoffen natürlichen Ursprungs mit handelsüblichen Produkten verglichen. Es wurde festgestellt, dass die neuen Fleischerzeugnisse - gekochter Schinken (stellvertretend für thermisch verarbeitete Fleischerzeugnisse) und fermentierte Wurst - während der gesamten Haltbarkeitsdauer Nitrit- und Nitratkonzentrationen unterhalb der Bestimmungsgrenze der Methode aufwiesen. Acerola hat erfolgreich Natriumascorbat als natürliche Quelle für Ascorbinsäure in Fleischerzeugnissen ersetzt. Außerdem wurde nachgewiesen, dass den Fleischerzeugnissen keine Phosphate zugesetzt wurden, die mit denen aus rohem Fleisch vergleichbar sind. Bei der sensorischen Bewertung wurden Kochschinken und fermentierte Wurst in die höchste Qualitätskategorie eingestuft, was Farbe, Geschmack, Geruch und Textur während der gesamten Haltbarkeitsdauer betrifft, und wurden als mikrobiologisch sicher bewertet, gemäß dem Leitfaden für mikrobiologische Kriterien für Lebensmittel (Landwirtschaftsministerium, März 2011).El desafío de la industria cárnica es el desarrollo de nuevos productos cárnicos sin aditivos con propiedades sensoriales y microbiológicas similares a los productos tradicionales con sal de nitrito a lo largo de su vida útil. En este trabajo se compararon nuevas recetas sin aditivos o con la sustitución de aditivos de origen natural con productos disponibles comercialmente. Se concluyó que los nuevos productos cárnicos, como el jamón cocido (como representante de productos procesados térmicamente) y la salchicha curada, tienen concentraciones de nitritos y nitratos por debajo del límite de cuantificación durante toda su vida útil. Acerola ha reemplazado con éxito al ácido ascórbico sódico como fuente natural de ácido ascórbico en productos cárnicos. Además, se demostró que los fosfatos no se añaden a los productos cárnicos, comparando los resultados con los de la carne no procesada. El jamón cocido y la salchicha curada se clasificaron en la categoría más alta de calidad en términos de color, sabor, aroma y textura durante toda su vida útil según los criterios sensoriales y se evaluaron como microbiológicamente seguros según la Guía de criterios microbiológicos para alimentos (Ministerio de Agricultura, marzo de 2011).Lo sviluppo di nuovi prodotti a base di carne senza additivi aggiunti, che abbiano per tutta la loro durata di conservazione proprietà sensoriali e microbiologiche simili a quelle dei prodotti tradizionali con nitrito di sodio, è una vera e propria sfida per l’industria della carne. In questo lavoro, nuove ricette senza additivi o con sostituzione di additivi di origine naturale vengono confrontate con prodotti disponibili in commercio. Si è concluso che i nuovi prodotti a base di carne - prosciutto cotto (come rappresentante dei prodotti trasformati termicamente) e la salsiccia secca stagionata - presentano una concentrazione di nitriti e nitrati inferiore al limite di quantificazione per l\u27intera durata di conservazione. L’acerola ha sostituito con successo l’ascorbato di sodio come fonte naturale di acido ascorbico nei prodotti a base di carne. Inoltre, è stato dimostrato che i fosfati, confrontando i risultati con quelli della carne non lavorata, non sono stati aggiunti ai prodotti a base di carne. Il prosciutto cotto e la salsiccia secca stagionata sono stati classificati mediante valutazione sensoriale nella categoria di massima qualità in termini di colore, sapore, odore e consistenza per tutta la loro durata di conservazione e sono stati valutati microbiologicamente idonei secondo la Guida ai criteri microbiologici per gli alimenti (Ministero dell\u27Agricoltura, marzo 2011)

Explorando el nivel de especialización de un corpus de textos científicos y las percepciones y necesidades de los alumnos en un contexto de inglés para fines específicos

The incorporation of some original texts combining the academic component with a particular scientific domain can be seen as an opportunity, in higher education and, in particular, in an English for Specific and Academic Purposes (ESP and EAP, respectively) context, to enable students to access authentic examples of the use of the language. This article investigates the degree of specialisation of some scientific texts that could be read and analysed in class in order to determine the pedagogical effect these might have when teaching the reading skill. In so doing, a corpus of twelve recent texts belonging to the domain of Biotechnology was analysed. Moreover, two different empirical studies were conducted in order to know more about students’ needs and perceptions throughout two consecutive academic years; 2017-2018 (qualitative analysis of their initial perceptions) and 2018-2019 (qualitative and quantitative analysis of the data provided by a questionnaire). Preliminary findings suggested that the selection of texts showed different degrees of specialisation.La incorporación de textos originales capaces de combinar el componente académico con el de un campo científico concreto puede verse, en el ámbito de la educación superior y, en concreto, en el del inglés para fines específicos y académicos, como una oportunidad para que los estudiantes accedan a ejemplos auténticos del uso de la lengua. En el presente artículo se estudia el grado de especialización de varios textos científicos con el fin de utilizarlos en el aula y determinar su efecto a nivel pedagógico al enseñar la destreza de la comprensión escrita. Para ello, se analizó un corpus de doce textos relacionados con la Biotecnología publicados recientemente y se llevaron a cabo dos estudios empíricos para indagar en las necesidades y percepciones de los estudiantes durante dos cursos académicos; 2017-2018 (análisis cualitativo de las primeras percepciones) y 2018-2019 (análisis cualitativo y cuantitativo de los datos aportados a través de un cuestionario). Los primeros resultados indicaron que los textos seleccionados mostraban diferentes grados de especialización

Assessing Computational Amino Acid β-Turn Propensities with a Phage-Displayed Combinatorial Library and Directed Evolution

SummaryStructure propensities of amino acids are important determinants in guiding proteins' local and global structure formation. We constructed a phage display library—a hexa-HIS tag upstream of a CXXC (X stands for any of the 20 natural amino acids) motif appending N-terminal to the minor capsid protein pIII of M13KE filamentous phage—and developed a novel directed-evolution procedure to select for amino acid sequences forming increasingly stable β-turns in the disulfide-bridged CXXC motif. The sequences that emerged from the directed-evolution cycles were in good agreement with type II β-turn propensities derived from surveys of known protein structures, in particular, Pro-Gly forming a type II β-turn. The agreement strongly supported the notion that β-turn formation plays an active role in initiating local structure folding in proteins

Structural Basis for the Propagation of Homing Endonuclease-Associated Inteins

Inteins catalyze their removal from a host protein through protein splicing. Inteins that contain an additional site-specific endonuclease domain display genetic mobility via a process termed "homing" and thereby act as selfish DNA elements. We elucidated the crystal structures of two archaeal inteins associated with an active or inactive homing endonuclease domain. This analysis illustrated structural diversity in the accessory domains (ACDs) associated with the homing endonuclease domain. To augment homing endonucleases with highly specific DNA cleaving activity using the intein scaffold, we engineered the ACDs and characterized their homing site recognition. Protein engineering of the ACDs in the inteins illuminated a possible strategy for how inteins could avoid their extinction but spread via the acquisition of a diverse accessory domain.Peer reviewe