Aislamiento, purificación y caracterización de las proteasas presentes en el látex de frutos de Maclura pomifera (Raf.) Schneid. (Moraceae)

En la búsqueda de una especie vegetal que resultara buena productora de proteasas, utilizando como criterios selectivos la biomasa disponible, la accesibilidad de su obtención y la posibilidad de su aprovechamiento industrial, se realizaron ensayos preliminares en especies pertenecientes a taxones promisorios en este sentido. Finalmente se seleccionaron los frutos de Maclura pomífera (Raf.) Schneid. (Moraceae) para estudiar las proteasas presentes en el látex que contienen los mismos. Una vez elegido el material vegetal, se elaboró la estrategia experimental que permitiera conseguir los siguientes objetivos: a) Establecer la relación entre el grado de madurez de los frutos, la producción de látex y el contenido de proteasas de los mismos. b) Obtener preparaciones enzimáticas crudas y determinar las condiciones óptimas para su conservación. c) Caracterizar las preparaciones crudas en cuanto al efecto del pH, la temperatura, la fuerza iónica y el agregado de activadores c inhibidores, así como conocer su estabilidad durante lapsos variables en diferentes condiciones de pH y temperatura y ensayar la actividad protcolítica frente a diferentes sustratos naturales y sintéticos. d) Someter las preparaciones a un proceso de purificación que permita obtener una o más fracciones de elevada actividad específica con alto grado de rendimiento. e) Caracterizar las fracciones así purificadas.Tesis digitalizada en SEDICI gracias a la colaboración de la Biblioteca Central de la Facultad de Ciencias Exactas (UNLP).Facultad de Ciencias Exacta

Proteolytic extracts of three bromeliaceae species as eco-compatible tools for leather industry

In addition to the large quantities of solid waste rich in protein, most tanneries use high proportions of Na2S and CaO during the dehairing step, resulting in effluents of high alkalinity and large amounts of suspended solid, besides the risk of liberating the toxic hydrogen sulphide. The current worldwide legislation on environmental requires tanneries to reduce pollution and to replace conventional processes by greener technologies. Enzymes are a technological tool of interest for industry because are able to achieve a high reaction rate under soft pH, temperature, and pressure conditions, besides a high specificity of reaction, biodegradability, non-toxic nature and non-polluting effluent generation. In leather industry enzymes are principally used in pre-tanning operations (soaking, dehairing, bating, and degreasing) and waste treatment. Particularly, proteases have been chosen as a promising eco-friendly alternative to lime and sodium sulphide dehairing. Extracts rich in cysteine proteases with high proteolytic activity (CU) have been obtained from fruits of Bromeliaceae species: Bromelia balansae (Bb), B. hieronymi (Bh), and Pseudananas macrodontes (Pm). In this work, Bb, Bh, and Pm have been studied for application in leather industry compared with commercial enzyme, focusing in their dehairing properties. Enzymatic activities against representative substrates of skin proteins were spectrophotometrically measured at 25, 35, and 55ºC (Tris-HCl, 0.1 M, pH 8, Cys 20 mM). Keratin Azure (KA), Elastin-Congo Red (E), epidermis substrate (EP), and Hide Powder Azure (HPA) were used as representative substrates of keratin, elastin, epidermis, and collagen, respectively. Ability to dehairing was evaluated by incubating soaked cow skins with different concentrations of extracts at 25ºC and pH 8 during 24 h. Grain surface and cross section of skins were observed by scanning electron microscopy. Extracts were able to degrade representative substrates of skin proteins and when compared to the same CU showed similar activity on collagen and epidermis; however, Bh and Pm were the most actives against keratin, while Bh was the only active against elastin. Extracts showing different proteolytic activity (Bb required 1 CU/ml, Bh 1.5 CU/ml, and Pm 0.5 CU/ml) were able to depilate cow skin after a gentle scraping. Although depilated skins with Bb, Bh, and Pm showed different surface aspects, desirable characteristics of dehairing were observed for all extracts since hair pores did not show residual hair, grain surface were clean and intact, and collagen fiber bundles of dermis were not damaged. In conclusion, results here presented show that proteolytic extracts of Bromeliaceae species are promising eco-compatible tools for leather industry, principally in treatment of their waste and dehairing process.Centro de Investigación de Proteínas VegetalesComisión de Investigaciones Científicas de la provincia de Buenos Aire

Proteolytic extracts of three bromeliaceae species as eco-compatible tools for leather industry

In addition to the large quantities of solid waste rich in protein, most tanneries use high proportions of Na2S and CaO during the dehairing step, resulting in effluents of high alkalinity and large amounts of suspended solid, besides the risk of liberating the toxic hydrogen sulphide. The current worldwide legislation on environmental requires tanneries to reduce pollution and to replace conventional processes by greener technologies. Enzymes are a technological tool of interest for industry because are able to achieve a high reaction rate under soft pH, temperature, and pressure conditions, besides a high specificity of reaction, biodegradability, non-toxic nature and non-polluting effluent generation. In leather industry enzymes are principally used in pre-tanning operations (soaking, dehairing, bating, and degreasing) and waste treatment. Particularly, proteases have been chosen as a promising eco-friendly alternative to lime and sodium sulphide dehairing. Extracts rich in cysteine proteases with high proteolytic activity (CU) have been obtained from fruits of Bromeliaceae species: Bromelia balansae (Bb), B. hieronymi (Bh), and Pseudananas macrodontes (Pm). In this work, Bb, Bh, and Pm have been studied for application in leather industry compared with commercial enzyme, focusing in their dehairing properties. Enzymatic activities against representative substrates of skin proteins were spectrophotometrically measured at 25, 35, and 55ºC (Tris-HCl, 0.1 M, pH 8, Cys 20 mM). Keratin Azure (KA), Elastin-Congo Red (E), epidermis substrate (EP), and Hide Powder Azure (HPA) were used as representative substrates of keratin, elastin, epidermis, and collagen, respectively. Ability to dehairing was evaluated by incubating soaked cow skins with different concentrations of extracts at 25ºC and pH 8 during 24 h. Grain surface and cross section of skins were observed by scanning electron microscopy. Extracts were able to degrade representative substrates of skin proteins and when compared to the same CU showed similar activity on collagen and epidermis; however, Bh and Pm were the most actives against keratin, while Bh was the only active against elastin. Extracts showing different proteolytic activity (Bb required 1 CU/ml, Bh 1.5 CU/ml, and Pm 0.5 CU/ml) were able to depilate cow skin after a gentle scraping. Although depilated skins with Bb, Bh, and Pm showed different surface aspects, desirable characteristics of dehairing were observed for all extracts since hair pores did not show residual hair, grain surface were clean and intact, and collagen fiber bundles of dermis were not damaged. In conclusion, results here presented show that proteolytic extracts of Bromeliaceae species are promising eco-compatible tools for leather industry, principally in treatment of their waste and dehairing process.Centro de Investigación de Proteínas VegetalesComisión de Investigaciones Científicas de la provincia de Buenos Aire

Proteolytic extracts of three bromeliaceae species as eco-compatible tools for leather industry

In addition to the large quantities of solid waste rich in protein, most tanneries use high proportions of Na2S and CaO during the dehairing step, resulting in effluents of high alkalinity and large amounts of suspended solid, besides the risk of liberating the toxic hydrogen sulphide. The current worldwide legislation on environmental requires tanneries to reduce pollution and to replace conventional processes by greener technologies. Enzymes are a technological tool of interest for industry because are able to achieve a high reaction rate under soft pH, temperature, and pressure conditions, besides a high specificity of reaction, biodegradability, non-toxic nature and non-polluting effluent generation. In leather industry enzymes are principally used in pre-tanning operations (soaking, dehairing, bating, and degreasing) and waste treatment. Particularly, proteases have been chosen as a promising eco-friendly alternative to lime and sodium sulphide dehairing. Extracts rich in cysteine proteases with high proteolytic activity (CU) have been obtained from fruits of Bromeliaceae species: Bromelia balansae (Bb), B. hieronymi (Bh), and Pseudananas macrodontes (Pm). In this work, Bb, Bh, and Pm have been studied for application in leather industry compared with commercial enzyme, focusing in their dehairing properties. Enzymatic activities against representative substrates of skin proteins were spectrophotometrically measured at 25, 35, and 55ºC (Tris-HCl, 0.1 M, pH 8, Cys 20 mM). Keratin Azure (KA), Elastin-Congo Red (E), epidermis substrate (EP), and Hide Powder Azure (HPA) were used as representative substrates of keratin, elastin, epidermis, and collagen, respectively. Ability to dehairing was evaluated by incubating soaked cow skins with different concentrations of extracts at 25ºC and pH 8 during 24 h. Grain surface and cross section of skins were observed by scanning electron microscopy. Extracts were able to degrade representative substrates of skin proteins and when compared to the same CU showed similar activity on collagen and epidermis; however, Bh and Pm were the most actives against keratin, while Bh was the only active against elastin. Extracts showing different proteolytic activity (Bb required 1 CU/ml, Bh 1.5 CU/ml, and Pm 0.5 CU/ml) were able to depilate cow skin after a gentle scraping. Although depilated skins with Bb, Bh, and Pm showed different surface aspects, desirable characteristics of dehairing were observed for all extracts since hair pores did not show residual hair, grain surface were clean and intact, and collagen fiber bundles of dermis were not damaged. In conclusion, results here presented show that proteolytic extracts of Bromeliaceae species are promising eco-compatible tools for leather industry, principally in treatment of their waste and dehairing process.Centro de Investigación de Proteínas VegetalesComisión de Investigaciones Científicas de la provincia de Buenos Aire

Desarrollo de procesos destinados a minimizar el impacto ambiental de las curtiembres de la provincia de Buenos Aires

La industria curtidora es una de las más tradicionales de nuestro país y al mismo tiempo genera, a través del curtido, una variedad de desechos que resultan contaminantes para el medioambiente. La implementación de tecnologías limpias y el tratamiento integral de los residuos sólidos y líquidos resulta fundamental para minimizar el impacto ambiental y cumplir con la legislación vigente. Mediante la obtención, producción y caracterización de extractos enzimáticos vegetales y fúngicos se logró depilar la piel bovina a escala laboratorio, sin daño apreciable en la capa reticular colagénica. Además, el residuo sólido pelo bovino actuó como sustrato para el crecimiento de especies fúngicas y para la producción de un pool enzimático aplicable en la curtiembre. Por medio del tratamiento biológico de los efluentes líquidos se logró disminuir la demanda química de oxígeno (DQO) hasta valores aceptables para su descarga, con un tiempo de retención hidráulico de 60 h para una relación alimento/microorganismo (F/M= 0,47/día), DQO inicial 5000 mg O2/L y DQO final 250 mg O2/L.Trabajo presentado por el Centro de Investigación de tecnología del Cuero (CITEC)

Granulosain I, a cysteine protease isolated from ripe fruits of Solanum granuloso-leprosum (Solanaceae)

A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family.Facultad de Ciencias ExactasCentro de Investigación de Proteínas Vegetale

Granulosain I, a cysteine protease isolated from ripe fruits of Solanum granuloso-leprosum (Solanaceae)

A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family.Facultad de Ciencias ExactasCentro de Investigación de Proteínas Vegetale

High-Throughput System for the Early Quantification of Major Architectural Traits in Olive Breeding Trials Using UAV Images and OBIA Techniques

The need for the olive farm modernization have encouraged the research of more efficient crop management strategies through cross-breeding programs to release new olive cultivars more suitable for mechanization and use in intensive orchards, with high quality production and resistance to biotic and abiotic stresses. The advancement of breeding programs are hampered by the lack of efficient phenotyping methods to quickly and accurately acquire crop traits such as morphological attributes (tree vigor and vegetative growth habits), which are key to identify desirable genotypes as early as possible. In this context, an UAV-based high-throughput system for olive breeding program applications was developed to extract tree traits in large-scale phenotyping studies under field conditions. The system consisted of UAV-flight configurations, in terms of flight altitude and image overlaps, and a novel, automatic, and accurate object-based image analysis (OBIA) algorithm based on point clouds, which was evaluated in two experimental trials in the framework of a table olive breeding program, with the aim to determine the earliest date for suitable quantifying of tree architectural traits. Two training systems (intensive and hedgerow) were evaluated at two very early stages of tree growth: 15 and 27 months after planting. Digital Terrain Models (DTMs) were automatically and accurately generated by the algorithm as well as every olive tree identified, independently of the training system and tree age. The architectural traits, specially tree height and crown area, were estimated with high accuracy in the second flight campaign, i.e. 27 months after planting. Differences in the quality of 3D crown reconstruction were found for the growth patterns derived from each training system. These key phenotyping traits could be used in several olive breeding programs, as well as to address some agronomical goals. In addition, this system is cost and time optimized, so that requested architectural traits could be provided in the same day as UAV flights. This high-throughput system may solve the actual bottleneck of plant phenotyping of "linking genotype and phenotype," considered a major challenge for crop research in the 21st century, and bring forward the crucial time of decision making for breeders

Propiedades funcionales de proteínas de soja parcialmente hidrolizadas empleando la peptidasa de Asclepias fruticosa

La harina de soja es el subproducto más relevante en el procesamiento de esta leguminosa y contiene un alto contenido de proteínas. La hidrólisis controlada de dichas proteínas puede generar ingredientes de alto valor añadido, con mejores características nutricionales y propiedades funcionales. La preparación proteolítica denominada Af, obtenida por centrifugación del látex de Asclepias fruticosa (Asclepiadaceae) recolectado en buffer cítrico-citrato, posee una única peptidasa cisteínica de PM 23,7 kDa y pI>9,3 denominada asclepaína f, la cual ha sido clonada y expresada en Pichia pastoris. La preparación Af posee una actividad proteolítica de 1,1 Ucas/ml, alta estabilidad térmica a 37° y 45°C, un rango de pH óptimo de actividad entre 7,5 y 10,5 y ha demostrado capacidad para hidrolizar las proteínas de soja, degradando fuertemente la fracción 7S y en menor medida las globulinas 11S. El objetivo del trabajo fue evaluar las propiedades funcionales (solubilidad, capacidad de retención de agua y aceite, y espumado) de un hidrolizado de proteínas de soja obtenido con la preparación Af. Se utilizó una dispersión de concentrado de soja (conteniendo 62,1 % de proteínas determinadas por Kjeldhal) al 8% p/v y una cantidad de extracto enzimático equivalente a 8,9 Ucas por gramo de proteína, con el agregado de cisteína 20 mM. La hidrólisis se llevó a cabo a diferentes valores de pH (8, 9 y 10) durante 180 minutos a 45ºC. La actividad enzimática se detuvo por calentamiento en microondas, las suspensiones fueron centrifugadas y los pellets secados en estufa. Se realizó un blanco de reacción en las mismas condiciones, reemplazando la preparación enzimática por igual volumen de agua. Posteriormente, se determinó la concentración de proteínas solubles mediante el método de Bradford, la capacidad de retención de agua a diferentes temperaturas (5, 20 y 45°C), la retención de aceite de soja y girasol, y la formación de espuma. La capacidad de retención de agua se ensayó mezclando 1 gr de muestra con 10 ml de agua destilada a diferentes temperaturas, centrifugando las suspensiones y determinando el peso de las muestras húmedas; del mismo modo se determinó la capacidad de retención de aceite. Para ensayar la capacidad espumante se prepararon suspensiones al 1% p/v de los hidrolizados, se agitaron durante 1 minuto a temperatura ambiente y posteriormente se midió el volumen de espuma y de suspensión. El mayor grado de hidrólisis de las proteínas de soja con la preparación Af se obtuvo a pH 10 y fue del 7%. A través de dicho proceso de hidrólisis se logró incrementar la concentración de proteínas solubles un 60%, la capacidad de retención de agua a 20°C se incrementó un 71% y a 45°C un 134%, mientras que la capacidad de retención de aceite de soja a 20°C aumentó un 92% y la de girasol 73,5%. En tanto que la capacidad espumante casi no fue afectada, se detectó solo un ligero aumento (3%). Empleando la preparación Af se logró obtener un hidrolizado de proteínas de soja con propiedades funcionales particulares que podrá emplearse en el desarrollo de alimentosInstituto Multidisciplinario de Biología Celula

Radiotherapy-Related Fatigue Associated Impairments in Lung Cancer Survivors during COVID-19 Voluntary Isolation

The author Alejandro Heredia Ciuro and the author Laura Lopez Lopez have received financial support through a FPU ("Formacion Profesorado Universitario") grant of the Spanish Ministry of Education (Spain), (FPU: 17/00408, and FPU: 20/01670).The main objective of this study was to investigate the impairments presented after COVID-19 voluntary isolation by lung cancer survivors that experienced radiotherapy-related fatigue. In this observational study, data were collected after COVID-19 voluntary isolation. Patients were divided into two groups according to their fatigue severity reported with the Fatigue Severity Scale. Health status was assessed by the EuroQol-5D, anxiety and depression by the Hospital Anxiety and Depression Scale, and disability by theWorld Health Organization Disability Assessment Schedule 2.0. A total of 120 patients were included in the study. Patients with severe fatigue obtained higher impairment results compared to patients without severe fatigue, with significant differences in all the variables (p < 0.05). Lung cancer survivors who experienced severe radiotherapy-related fatigue presented higher impairments after COVID-19 voluntary isolation than lung cancer patients who did not experience severe radiotherapy-related fatigue, and showed high levels of anxiety, depression and disability, and a poor self-perceived health status.FPU ("Formacion Profesorado Universitario") grant of the Spanish Ministry of Education (Spain) FPU: 17/00408 FPU: 20/0167