RESUMO
PURPOSE: For growth of methylotrophic yeast, glycerol is usually used as a carbon source. Glucose is used in some cases, but not widely consumed due to strong repressive effect on AOX1 promoter. However, glucose is still considered as a carbon source of choice since it has low production cost and guarantees growth rate comparable to glycerol. RESULTS: In flask cultivation of the recombinant yeast, Pichia pastoris GS115(pPIC9K-appA38M), while methanol induction point(OD600) and methanol concentration significantly affected the phytase expression, glucose addition in induction phase could enhance phytase expression. The optimal flask cultivation conditions illustrated by Response Surface Methodology were 10.37 OD600 induction point, 2.02 h before methanol feeding, 1.16% methanol concentration and 40.36µL glucose feeding amount(for 20 mL culture volume) in which the expressed phytase activity was 613.4 ± 10.2U/mL, the highest activity in flask cultivation. In bioreactor fermentation, the intermittent glucose feeding showed several advantageous results such as 68 h longer activity increment, 149.2% higher cell density and 200.1% higher activity compared to the sole methanol feeding method. These results implied that remaining glucose at induction point might exhibit a positive effect on the phytase expression. CONCLUSION: Glucose intermittent feeding could be exploited for economic phytase production and the other recombinant protein expression by P. pastoris GS115.
Assuntos
6-Fitase , Reatores Biológicos , Fermentação , Glucose , Metanol , Proteínas Recombinantes , 6-Fitase/genética , 6-Fitase/metabolismo , Glucose/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Metanol/metabolismo , Reatores Biológicos/microbiologia , Meios de Cultura/química , Meios de Cultura/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Saccharomycetales/crescimento & desenvolvimento , Pichia/genética , Pichia/metabolismo , Pichia/crescimento & desenvolvimento , Expressão GênicaRESUMO
Male broiler chickens (384), Cobb 500, were housed in metabolic cages to assess the efficacy of phytase in diets with low and high phytate-phosphorus on the performance, bone physical characteristics, tissue and serum mineral deposits. Birds were distributed in four treatments with a 2 x 2 factorial arrangement in a completely randomized block design. Experimental diets based on maize-soybean meal were T1 - diet low phytate-phosphorus; T2 - diet low phytate-phosphorus and phytase (500 FTU/kg); T3 - diet high phytate-phosphorus; T4 - diet high phytate-phosphorus and phytase (500 FTU/kg). Feed intake, body weight, weight gain and feed conversion ratio were assessed. Two left tibias per experimental unit were analyzed for physical characteristics and mineral concentration; a section of skinless breast muscle and blood were collected to measure the concentration of calcium, phosphorus and sodium. Results showed interaction between bone stiffness and serum calcium. The inclusion of phytase in diets with low and high phytate-phosphorus did not alter performance, bone resistance and flexibility, mineral deposits in the tibia and breast muscle, but increased bone stiffness after 22 days of age. It also provided a higher serum calcium rate in broilers fed diets with low phytate-phosphorus up to 32 days of age.
Assuntos
6-Fitase , Fósforo , Animais , Masculino , 6-Fitase/metabolismo , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Cálcio/metabolismo , Galinhas/fisiologia , Dieta/veterinária , Suplementos Nutricionais , Minerais , Fósforo/metabolismo , Ácido FíticoRESUMO
Phosphatases are hydrolytic enzymes that cleave the phosphoester bond of numerous substrates containing phosphorylated residues. The typical classification divides them into acid or alkaline depending on the pH at which they have optimal activity. The histidine phosphatase (HP) superfamily is a large group of functionally diverse enzymes characterized by having an active-site His residue that becomes phosphorylated during catalysis. HP enzymes are relevant biomolecules due to their current and potential application in medicine and biotechnology. Entamoeba histolytica, the causative agent of human amoebiasis, contains a gene (EHI_146950) that encodes a putative secretory acid phosphatase (EhHAPp49), exhibiting sequence similarity to histidine acid phosphatase (HAP)/phytase enzymes, i.e., branch-2 of HP superfamily. To assess whether it has the potential as a biocatalyst in removing phosphate groups from natural substrates, we studied the EhHAPp49 structural and functional features using a computational-experimental approach. Although the combined outcome of computational analyses confirmed its structural similarity with HP branch-2 proteins, the experimental results showed that the recombinant enzyme (rEhHAPp49) has negligible HAP/phytase activity. Nonetheless, results from supplementary activity evaluations revealed that rEhHAPp49 exhibits Mg2+-dependent alkaline pyrophosphatase activity. To our knowledge, this study represents the first computational-experimental characterization of EhHAPp49, which offers further insights into the structure-function relationship and the basis for future research.
Assuntos
Entamoeba histolytica/enzimologia , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Relação Estrutura-Atividade , 6-Fitase/metabolismo , Sítios de Ligação , Domínio Catalítico , Difosfatos/metabolismo , Entamoeba histolytica/genética , Humanos , Simulação de Acoplamento Molecular , Monoéster Fosfórico Hidrolases/genética , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
6-Fitase/genética , 6-Fitase/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , 6-Fitase/química , Ração Animal , Animais , Aspergillus niger/enzimologia , Aspergillus niger/genética , Proteínas Fúngicas/química , Fungos/classificação , Fungos/enzimologia , Fungos/genética , Indústrias , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Bacillus subtilis is a remarkably diverse bacterial species that displays many ecological functions. Given its genomic diversity, the strain Bacillus subtilis EA-CB0575, isolated from the rhizosphere of a banana plant, was sequenced and assembled to determine the genomic potential associated with its plant growth promotion potential. The genome was sequenced by Illumina technology and assembled using Velvet 1.2.10, resulting in a whole genome of 4.09 Mb with 4332 genes. Genes involved in the production of indoles, siderophores, lipopeptides, volatile compounds, phytase, bacilibactin, and nitrogenase were predicted by gene annotation or by metabolic pathway prediction by RAST. These potential traits were determined using in vitro biochemical tests, finding that B. subtilis EA-CB0575 produces two families of lipopeptides (surfactin and fengycin), solubilizes phosphate, fixes nitrogen, and produces indole and siderophores compounds. Finally, strain EA-CB0575 increased 34.60% the total dry weight (TDW) of tomato plants with respect to non-inoculated plants at greenhouse level. These results suggest that the identification of strain-specific genes and predicted metabolic pathways might explain the strain potential to promote plant growth by several mechanisms of action, accelerating the development of plant biostimulants for sustainable agricultural.
Assuntos
Bacillus subtilis/genética , Genoma Bacteriano , Rizosfera , 6-Fitase/genética , 6-Fitase/metabolismo , Bacillus subtilis/metabolismo , Bacillus subtilis/patogenicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Produção Agrícola/métodos , Indóis/metabolismo , Lipopeptídeos/genética , Lipopeptídeos/metabolismo , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/microbiologia , Musa/crescimento & desenvolvimento , Musa/microbiologia , Nitrogenase , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/metabolismo , Sideróforos/genética , Sideróforos/metabolismoRESUMO
The enzyme phytase has important applications in animal feed, because it favors the bioavailability of phosphorus present in phytate, an antinutritional compound widely found associated with plant proteins. However, for feed applications, the phytase must withstand high temperatures during the feed pelleting process, as well as the gastrointestinal conditions of the animal. This work evaluates the feasibility of immobilizing phytase on hydroxyapatite (HA) nanoparticles, in order to improve its properties. HA is a material with excellent physicochemical characteristics for enzyme immobilization, and it can also act as an inorganic source of phosphorus and calcium in animal feed. The strong affinity of the phytase for the support resulted in rapid adsorption, with total immobilization yield and recovered activity greater than 100%. After immobilization, the phytase showed a broader activity profile in terms of pH and temperature, together with considerably higher thermoresistance at 80 and 90 °C. As a proof of concept, it was shown that the phytase immobilized on HA presented good resistance to acidic conditions and resistance to proteolysis when passing through simulated gastrointestinal conditions of fish. The findings showed that phytase immobilized onto HA presents suitable properties and has great potential for use in animal feed.
Assuntos
6-Fitase/metabolismo , Ração Animal , Durapatita/química , Enzimas Imobilizadas/metabolismo , Nanopartículas/química , Animais , Peixes , Temperatura Alta , Concentração de Íons de Hidrogênio , Concentração Osmolar , ProteóliseRESUMO
While many bacteria have been used as probiotics by industries, only two yeasts, Saccharomyces cerevisiae var. boulardii and Kluyveromyces fragilis (B0399), have been used for this purpose. In the present work, a total of 116 yeasts isolated from Brazilian indigenous fermented food, cocoa fermentation, and kefir were in vitro characterized for probiotic attributes. From 116 isolates, 36 were tolerant to gastrointestinal conditions evaluated by tolerance to pH 2.0, bile salts (0.3% w/v), and 37 °C temperature. From those, 15 isolates showed a similar or higher percentage (P < 0.05) of hydrophobicity, autoaggregation, and coaggregation with E. coli than the reference strain S. boulardii. All these strains showed a high percentage of adhesion to Caco-2 cells (> 63%) and antioxidant activity (ranging from 18 to 62%). Phytate hydrolysis was evaluated for these yeasts and 13 strains showed positive results, which is important for nutrient availability in plant-based foods. These results are important insights for characterization of novel probiotic yeast strains as well as to aggregate functional value to these food products.
Assuntos
6-Fitase/metabolismo , Alimentos Fermentados/microbiologia , Microbiologia de Alimentos , Kluyveromyces , Probióticos , Saccharomyces boulardii , Antioxidantes/isolamento & purificação , Brasil , Células CACO-2 , Escherichia coli , Humanos , Kluyveromyces/isolamento & purificação , Kluyveromyces/fisiologia , Probióticos/isolamento & purificação , Saccharomyces boulardii/isolamento & purificação , Saccharomyces boulardii/fisiologiaRESUMO
Cereals and pseudocereals are a rich source of nutrients and trace elements, but their dietary bioavailability is low due to the presence of phytate (IP6), an antinutritional compound with the ability to chelate cations and proteins. Phytase is an enzyme that catalyzes the hydrolysis of IP6 and it is used as an additive improving the nutritional quality of grain-based foods. The aim of this study was to select lactic acid bacteria (LAB) isolated from pseudocereals with phytase activity, characterize their production and activity, and purify the enzyme. LAB strains isolated from grains and spontaneous sourdough of quinoa and amaranth were grown in the Man Rogosa and Sharpe medium where the inorganic phosphate (Pi) was replaced by 1% of IP6. Phytase activity was determined by measuring the Pi released from IP6. Phytase of Lactobacillus (L.) plantarum CRL1964 (PhyLP) showed the highest specific activity from 73 LAB evaluated. IP6 induces PhyLP production, which is at its maximum at the end of the exponential phase. PhyLP was thermostable and maintained its activity under acidic conditions. The enzymatic activity is stimulated by ethylenediaminetetraacetic acid, Co2+, and ascorbic acid. PhyLP was partially purified and showed a molecular mass of 55 kDa. L. plantarum CRL1964 and/or PhyLP have the potential to be included in the processing of cereal/pseudocereals based products for animal feed and/or the food industry improving its nutritional value.
Assuntos
6-Fitase/metabolismo , Grão Comestível/microbiologia , Lactobacillus plantarum/enzimologia , 6-Fitase/química , 6-Fitase/isolamento & purificação , Meios de Cultura/química , Concentração de Íons de Hidrogênio , Hidrólise , Lactobacillales/enzimologia , Lactobacillales/crescimento & desenvolvimento , Lactobacillales/isolamento & purificação , Lactobacillus plantarum/crescimento & desenvolvimento , Lactobacillus plantarum/isolamento & purificação , Peso Molecular , Fosfatos/metabolismo , Ácido Fítico/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
The application of phytases for animal feed in developing countries is limited due to the high cost of these enzymes, determined by the importation fees and the expensive substrates used for their production. In this work, we have used agroindustrial byproducts for the production of extracts containing phytases, which were accessed for their stability focusing on the conditions found in the gastrointestinal tract of pigs. The fungus Acremonim zeae presented higher phytase production in medium containing cornmeal, while the yeast Kluyveromyces marxianus produced 10-fold more phytase when cultivated on rice bran. Process optimization increased the difference in productivity to more than 300 fold. The phytase from A. zeae was thermostable, with higher activity at neutral pH and 50 °C, but was inhibited at pH 2.5 and by various ions. The phytase activity in the K. marxianus extract was stable at a wide range of conditions, which indicates the presence of at least two enzymes. As far as we know, this manuscript describes for the first time the phytase production and the characteristics of the extracts produced by both these microbial species. These enzymes could be produced at low cost and have potential to replace enzymes currently imported for this purpose.
Assuntos
6-Fitase/metabolismo , Ração Animal/análise , Dieta/veterinária , Fungos/enzimologia , Trato Gastrointestinal/metabolismo , 6-Fitase/genética , Animais , SuínosRESUMO
The objective of this study was to evaluate the effect of increasing doses of bacterial phytase (RONOZYME HiPhos) on performance and carcass characteristics of growing and finishing pigs. The study included 120 castrated males with initial weight of 23.21 ± 1.91 kg and 68 days of age, distributed in a randomized block design with five treatments and eight replicates with three animals each. The pigs were fed five corn-soybean meal-based diets: positive control (PC), supplemented with inorganic phosphorus and calcium; negative control (NC), with 0.13% reduction in available phosphorus and 0.11% in calcium; and three NC diets supplemented with 1,000, 2,000, and 3,000 phytase units (FYT)/kg in the feed. Compared with the NC diets without phytase, diets with 1,000, 2,000, and 3,000 FYT/kg inclusion increased the daily weight gain by +12% (quadratic, p<0.05) during the growing I period; +2.9, +2.9, and +10.5% (linear, p<0.01), respectively, during the growing II period; and +4.1, +5.1, and +8.2% (linear, p<0.001), respectively, over the entire experimental period. The daily feed intake increased by 0, +2.8, and +4.3% (linear, p<0.05), respectively, considering the entire experimental period; and the final live weight increased by +3.2, +4.2, and +6.1% (linear, p<0.001), respectively. The phytase treatments did not influence feed conversion ratio, carcass weight and yield, backfat thickness, loin depth and carcass lean meat. According to the European Carcass Classification (SEUROP), however, the animals fed the PC diet and the three phytase levels had more carcasses classified as E (between 55-60% lean meat) when compared to carcasses of pigs fed the NC. Supplementing increasing levels of phytase to a corn- and soybean meal-based diet with inorganic P and Ca reduction improved daily weight gain and feed intake of growing pigs, and such effects were maintained until slaughter age.