RESUMEN
Phosphate release from inorganic and organic phosphorus compounds can be enzymatically mediated. Phosphate-releasing enzymes, comprising acid and alkaline phosphatases, are recognized as useful biocatalysts in applications such as plant and animal nutrition, bioremediation, and diagnostic analysis. Here, we describe a functional metagenomics approach enabling rapid identification of genes encoding these enzymes. The target genes are detected based on small- and large-insert metagenomic libraries derived from diverse environments. This approach has the potential to unveil entirely new phosphatase families or subfamilies and members of known enzyme classes that hydrolyze phosphomonoester bonds such as phytases. Additionally, we provide a strategy for efficient heterologous expression of phosphatase genes.
Asunto(s)
6-Fitasa , Metagenómica , Metagenoma , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , 6-Fitasa/genética , FosfatosRESUMEN
O desmame é caracterizado como o período mais crítico da produção de suínos devido à imaturidade digestiva e à baixa concentração de enzimas digestivas, como carboidrases, proteases e fitases, nas primeiras semanas do pós-desmame, o que ocasiona o baixo desempenho dos leitões. Neste caso, há a necessidade de suplementação exógena de enzimas que atuam na degradação de frações do alimento, aumentando o aproveitamento da dieta. A nutrição enzimática exógena tem sido considerada uma alternativa eficaz frente à baixa concentração de enzimas endógenas nas primeiras semanas após o desmame por contribuir para o aumento na digestibilidade das dietas sólidas e, deste modo, para um maior aproveitamento dos ingredientes. Na nutrição de suínos, as enzimas exógenas mais utilizadas são as carboidrases, as proteases e as fitases. Ademais, a utilização de blends enzimáticos também é considerada uma ótima alternativa, pois contribui para a inibição de fatores antinutricionais, os quais são responsáveis por indisponibilizar nutrientes necessários para o desenvolvimento e desempenho dos animais. Diante do exposto, o objetivo deste estudo foi apresentar as principais enzimas exógenas utilizadas na nutrição de leitões desmamados, bem como seus efeitos no desempenho animal.
Weaning is characterized as the most critical period in swine production due to digestive immaturity and low concentration of digestive enzymes, such as carbohydrases, proteases, and phytases, in the first weeks after weaning, which causes poor performance of piglets. In this case, there is the need for exogenous supplementation of enzymes that act on the degradation of food fractions, increasing diet utilization. Exogenous enzyme nutrition has been considered an effective alternative to the low concentration of endogenous enzymes in the first weeks after weaning, as it contributes to an increase in the digestibility of solid diets and, consequently, greater use of the ingredients. In swine nutrition, the most used exogenous enzymes are carbohydrases, proteases, and phytases. Furthermore, the use of enzymatic blends is also considered a great alternative, as they contribute to the inhibition of anti-nutritional factors, which are responsible for making nutrients necessary for the development and performance of animals unavailable. Therefore, this study aimed to present the main exogenous enzymes used in the nutrition of weaned piglets, as well as their effects on animal performance.
Asunto(s)
Animales , Péptido Hidrolasas/administración & dosificación , Porcinos , Enzimas/análisis , Hidrolasas/administración & dosificación , Destete , Dieta/veterinaria , Digestión/fisiologíaRESUMEN
This work intended to prospect new phytase-producing organisms. In silico genomic analyses allowed the selection of twelve potential phytase-producing fungi. Based on gene sequence, it was possible to identify four well-defined groups of phytate-degrading enzymes: esterase-like, ß-propeller phytases (ßPP), phosphoglycerate mutase-like, and phytases of the histidine acid phosphatases (HAP) family. Analysis of the predicted genes encoding phytases belonging to the HAP family and ßPP phytases and in silico characterization of these enzymes indicated divergence among the catalytic activities. Predicted fungal ßPP phytases exhibited higher molecular mass (around 77 kDa) probably due to the epidermal growth factor-like domain. Twelve sequences of phytases contained signal peptides, of which seven were classified as HAP and five as ßPP phytases, while ten sequences were predicted as phytases secreted by non-classical pathways. These fungi were grown in liquid or semi-solid medium, and the fungal enzymatic extracts were evaluated for their ability to hydrolyze sodium phytate at 50 °C and pH ranging from 2.0 to 9.0. Seven fungi were identified as phytase producers based on phosphate release under enzyme assay conditions. Results obtained from in silico analyses combining experimental enzymatic activities suggest that some selected fungi could secrete ßPP phytases and HAP phytases.
Asunto(s)
6-Fitasa/química , Fosfatasa Ácida/química , Proteínas Fúngicas/química , Histidina/química , Alimentación Animal , Biotecnología , Catálisis , Dominio Catalítico , Simulación por Computador , Factor de Crecimiento Epidérmico/química , Fermentación , Genómica , Concentración de Iones de Hidrógeno , Ácido Fítico/metabolismo , Dominios Proteicos , Señales de Clasificación de Proteína , TemperaturaRESUMEN
Phytic acid stores 60-90% of the inorganic phosphorus in legumes, oil seeds, and cereals, making it inaccessible for metabolic processes in living systems. In addition, given its negative charge, phytic acid complexes with divalent cations, starch, and proteins. Inorganic phosphorous can be released from phytic acid upon the action of phytases. Phytases are phosphatases produced by animals, plants, and microorganisms, notably Aspergillus niger, and are employed as animal feed additive, in chemical industry and for ethanol production. Given the industrial relevance of phytases produced by filamentous fungi, this work discusses the functional characterization of fungal phytase-coding genes/proteins, highlighting the physicochemical parameters that govern the enzymatic activity, the development of phytase super-producing strains, and key features for industrial applications.
Asunto(s)
6-Fitasa/genética , 6-Fitasa/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , 6-Fitasa/química , Alimentación Animal , Animales , Aspergillus niger/enzimología , Aspergillus niger/genética , Proteínas Fúngicas/química , Hongos/clasificación , Hongos/enzimología , Hongos/genética , Industrias , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMEN
BACKGROUND: Phytases are enzymes capable of degrading phytic acid and are used in animal feed supplementation in order to improve digestibility through the release of minerals such as phosphorus. Recent inventions show interest in production and optimization of recombinant phytases with biochemical and physicochemical characteristics promising for animal feed industry. METHODS: This review article is focused on relevant patents of promising phytases, together with the commonly used expression systems for their production and tools currently employed to generate new phytases. We revised all patents related to recombinant phytases and their application in the animal feed industry. The following patents databases were consulted: European Patent Office (Espacenet), the United States Patent and Trademark Office (USPTO), the United States Latin America Patents (LATIPAT), Patent scope -Search International and National Patent Collections (WIPO) and Google Patents. RESULTS: In this review, information was collected from recent publications, including 38 patents related production systems for different recombinant phytases and their application in the animal feed industry. CONCLUSION: We showed that important recombinant phytases were successfully produced in different expression systems. In addition, this work highlights certain biotechnological tools such as mutagenesis for generation of novel enzymes with biochemical properties of use in the animal feed industry.
Asunto(s)
6-Fitasa/metabolismo , Alimentación Animal/análisis , 6-Fitasa/genética , Animales , Bacterias/genética , Bacterias/metabolismo , Reactores Biológicos , Hongos/genética , Hongos/metabolismo , Patentes como Asunto , Ácido Fítico/metabolismo , Ingeniería de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
UNLABELLED: The ecology of microbial communities associated with organic phosphorus (P) mineralization in soils is still understudied. Here, we assessed the abundance and diversity of bacteria harbouring genes encoding ß-propeller phytases (BPP) in the rhizosphere of traditional and transgenic maize cultivated in two Brazilian soils. We found a soil-dependent effect towards a higher abundance of phytase genes in the rhizosphere, and an absence of any impact of plant genotype. Phylogenetic analyses indicated members of the genera Pseudomonas, Caulobacter, Idiomarina and Maricaulis, close to 'uncultured bacteria', to constitute the dominant bacteria hosting this gene. The results obtained validate a methodology to target bacteria that are involved in the organic P cycle, and depict the responsiveness of such bacteria to the rhizosphere, albeit in dependency of the soil in which maize is cultivated. The data also identified the major bacterial groups that are associated with the organic P mineralization function. SIGNIFICANCE AND IMPACT OF THE STUDY: Micro-organisms play a key role in nutrient balance in soil ecosystems that are essential to life on the planet. However, some processes such as organic phosphorus mineralization, an important source of phosphorus supply in soil, is poorly studied mainly due the absence of an efficient methodology to assess the phytase-producing micro-organisms. In this study, a method to assess beta-propeller phytase (BPP)-carrying bacteria in soil was validated. This method may contribute to the knowledge of how these micro-organisms behave in the environment and contribute for plant growth promotion.