Phytase production through response surface methodology and molecular characterization of Aspergillus fumigatus NF191.

Phytase play an important role in phytic acid catalysis that act as a food inhibitor in cereals. Here, we isolated high phytase producing isolates NF191 closely related to Aspergillus fumigatus sp. from piggery soil. DNA was isolated from the fungal culture and amplified the ITS region using ITS1 and ITS4 primer using PCR. The 400-900 bp amplicon was gel eluted and subjected to sequencing. The sequencing results were assembled and compared with NCBI data base which showed the 99% identity of Aspergilllus fumigatus. Different carbon sources viz., fructose, galactose, lactose, dextrose, sucrose, maltose and different nitrogen sources (organic & inorganic) NH4Cl, NH4NO3, (NH4)2SO4, KNO3, NaNO3, urea, yeast extract, peptone, beef extract were tested for optimal production. The 0.3% dextrose, 0.5% NH4NO3 and 96 h incubation time showed the best production and enzyme activity at 45 ºC incubation temperature. The selected parameters, dextrose, ammonium sulphate and incubation time, when employed with statistical optimization approach involving response surface optimization using Box Behnken Design, gave a 1.3 fold increase in phytase production compared to unoptimized condition.

Isolation of a thermostable acid phytase from Aspergillus niger UFV-1 with strong proteolysis resistance

An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The KM for sodium phytate hydrolysis was 30.9 mM, while the kcat and kcat/KM were 1.46 ×105 s−1 and 4.7 × 106 s−1.M−1, respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg2+, Cd2+, K+ and Ca2+, and it was drastically inhibited by F−. The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment.

Purification, characterization and properties of phytase from Shigella sp. CD2.

Phytases catalyze the release of phosphate from phytic acid. In this study, a phytase producing bacterial strain Shigella sp. CD2 was isolated from the wheat rhizosphere. Phytase production started from the exponential phase of bacterial growth, showing the highest activity during the stationary phase. The enzyme activity was detected in both periplasmic and intracellular fractions. The enzyme was purified by about 133-fold with specific activity 780 U mg-1 protein. The optimum pH and temperature of the enzyme was 5.5 and 60oC, respectively. The enzyme was thermostable and retained 100% and 75% of its activity on pre-incubation at 70o and 80oC for 30 min, respectively. The Km value for the substrate sodium phytate was 0.25 mM. The enzyme was highly specific to substrate phytate, and no activity was detected in presence of other phosphorylated substrates, such as ATP, ADP, glucose 6-phosphate, fructose 6-phosphate and p-nirophenyl phosphate. The activity declined dramatically in presence of Cu2+, Zn2+ and Fe2+ and SDS, whereas Mg2+ and Co2+ slightly enhanced the enzyme activity. The addition of other metal ions or chemicals had little or no effect on phytase activity. The enzyme was resistant to both pepsin and trypsin. Due to high specific activity, substrate specificity, good pH profile, protease insensitivity and thermostability, phytase encoding gene from Shigella sp. CD2 could be an interesting candidate for industrial applications. Further studies on cloning and expression of Shigella phytase gene are currently in progress.

Influence of calcium and non-phytate phosphorus deficient diets with phytase on the performance of broilers, serum concentrations of minerals and the activity of alanin transaminase, aspartate transaminase and lactate dehydrogenase

An experiment was conducted to study the effect of microbial phytase [phyzyme XP5000G] supplementation in broiler chicks' diets on the performance, serum total protein [TP], minerals [Ca, Mg and P] and the serum enzyme activities [AST, ALT, LDH]. A reference -1 -1 diet adequate in calcium and non-phytate phosphorus [10.0 gkg Ca and 5 gkg nPP] and two -1 -1 -1 -1 deficient diets in Ca and nPP [8.5 gkg Ca and 3.5 gkg nPP, and 7.5 gkg Ca and 2.5 gkg nPP] -1 with or without phytase [0 and 100mgkg] were offered to broiler chicks from 1 to 21 days of age. Although the low-nPP diets had no significant effect on body weight gain [BWG] of chicks [p > 0.05] They increased [p < 0.01] feed intake [FI] and feed conversion ratio [FCR] when compared to the low-nPP diet supplemented with enzyme. Phytase had a favorable effect, although non-significantly, on BWG of chicks fed very low level of nPP. Enzyme reduced the feed intake [p < 0.05] and improved the FCR of Ca-nPP deficient chicks [p < 0.01]. The decrease in Ca-nPP content in the diet caused a significant increase in serum concentration of Ca [p < 0.05] and decrease in P concentration [p < 0.05]. Low Ca-nPP diets had no influence on serum Mg concentration. Dietary phytase reduced the Ca level and increased the P level [p < 0.05] of serum in chicks fed with Ca-nPP deficient diets. The activity of LDH increased [p < 0.01] in response to low dietary Ca and nPP, deficient but there was no influence on serum ALT and AST activity and TP content [p > 0.05]. Phytase supplementation reduced serum ALT [p < 0.05] and had no effect on AST [p > 0.05]. Serum LDH activity further increased [p < 0.01] by phytase supplementation. These results demonstrated that the performance of the chicks received low

Functional properties of phytate / Propriedades funcionais do fitato

Phytate [myo-inositol (1,2,3,4,5,6) hexakisphosphate], a naturally compound formed during maturation of plant seeds and grains, is a common constituent of plant-derived foods. The major concern about the presence of phytate in thediet is its negative effect on mineral uptake. Minerals of concern in this regardin clude Zn2+, Fe2+/3+, Ca2+, Mg2+, Mn2+, and Cu2+. Especially zinc and iron deficiencies were reported as a consequence of high phytate intakes. In addition, a negative effect on the nutritional value of protein by dietary phytate is discussed. Consumption of phytate, however, seems not to have only negative effects on human health. Dietary phytate was reported to prevent kidney stone formation, protect against diabetes mellitus, caries, atherosclerosis and coronary heart disease as well as against a variety of cancers. Furthermore, individual myo-inositol phosphate esters have been proposed to be metabolicall y active. D-myo-inositol(1,2,6)trisphosphate, for example, has been studied in respect to prevention of diabetes complications and treatment of chronic inflammations as well as cardiovascular diseases and due to its antiangiogenic and antitumour effects myo-inositol(1,3,4,5,6) pentakisphosphate was suggested as a promising compound for anticancer therapeutic strategies

El fitato, (1,2,3,4,5,6) hexafosfato de mio-inositol, compuesto que se forma naturalmente durante la maduración de las semillas ygranos, es un constituyente común de los alimentos vegetales. La mayor preocupación con la presencia de fitato es su efecto negativo en la absorción de minerales, particularmente Zn2+, Fe2+/3+, Ca2+, Mg2+, Mn2+, e Cu2+.Las deficiencias de zinc y hierro, fueron relacionadas con altas ingestas de fitato. Es discutido también su efecto negativo en el valor biológico de las proteínas. Sin embargo, el consumo de fitato parece no tener solamente efectos negativos para la salud humana. Fue descripto, por ejemplo, un efecto protector del fitato contra la formación de cálculos renales, contra la diabetes mellitus, formación de caries, ateriosclerosis y enfermedades coronarias, como también contra una gran variedad de tipos de tumores malignos. Además, ha sido propuesto que, individualmente, algunos ésteres de fosfato de mio-inositol sean metabólicamente activos. Hay estudios relacionando el (1,2,6) trisfosfato de D-mio-inositol con la prevención de complicaciones de la diabetes y con el tratamiento de inflamaciones crónicas y de enfermedades cardiovasculares; el (1,3,4,5,6)pentafosfato de mio-inositol, debido a sus efectosanti-angiogénicos y anti-tumorales, fue sugerido como un compuesto promisor en las estratégias terapéuticas contra el cáncer

O fitato, (1,2,3,4,5,6) hexafosfato de mio-inositol], composto que ocorre naturalmente e é formado durante a maturação de sementes e grãos, é um constituinte comum de alimentos vegetais. A maior preocupação com a presença de fitato na dieta é seu efeito negativo na absorção de minerais, particularmente Zn2+, Fe2+/3+, Ca2+, Mg2+, Mn2+, e Cu2+. As deficiências de zinco e de ferro, em especial, foram relacionadas com altas ingestões de fitato. É discutido, também, o efeito negativo do fitato no valor biológico de proteínas. Por outro lado, o consumo de fitato parece não ter somente efeitos negativos para a saúde humana. Foi descrito, por exemplo, um efeito protetor do fitato contra a formação de cálculos renais, contra diabetes Mellitus, formação de cáries, aterosclerose e doença coronariana, bem como contra uma grande variedade de tipos de tumores malignos. Além disso, foi proposto que, individualmente, alguns ésteres de fosfato de mio-inositol sejam metabolicamente ativos. Assim, há estudos relacionando o (1,2,6) trisfosfato de D-mio-inositol com a prevenção das complicações do diabetes e com o tratamento de inflamações crônicas e de doenças cardiovasculares; o(1,3,4,5,6) pentafosfato de mio-inositol, devido a seus efeitos anti-angiogênicos e anti-tumorais, foi sugerido como um composto promissor nas estratégias terapêuticas contra o câncer

Adições crescentes de ácido fítico à dieta não interferiram na digestibilidade da caseína e no ganho de peso em ratos / Increasing quantities of phytic acid in the diet did not affect casein digestibility and weight gain in rats

O crescente consumo de alimentos de origem vegetal, sejam como fontes protéicas com baixo teor de gordura ou como fontes de fibras, tem acrescido à dieta humana o ácido fítico. Devido à sua carga altamente negativa, o ácido fítico tem sido visto como componente de ação antinutricional capaz de quelar minerais bivalentes, proteínas e amido, podendo comprometer a biodisponibilidade destes nutrientes. No presente estudo investigou-se a influência da adição de ácido fítico à dieta de caseína, em concentrações iguais ou até oito vezes superiores àquelas encontradas no feijão-comum Phaseolus vulgaris, cultivar IAC-Carioca (14,7mg de ácido fítico/g feijão cru), durante período experimental de dez dias, sobre os índices nutricionais Ganho de Peso, Quociente de Eficiência da Dieta, Quociente de Eficiência Protéica Líquida, Digestibilidade Aparente e Digestibilidade Verdadeira. Trinta e seis ratos machos SPF da linhagem Wistar, recém-desmamados, divididos em grupos experimentais com seis ratos cada, foram alimentados com dieta purificada AIN-93G isenta de ácido fítico (Controle) e dietas teste AIN-93G acrescidas de 218, 436, 872 e 1744mg de ácido fítico/kg de dieta (Tratamentos). Os ganhos de peso (g) e os índices de qualidade dietética e protéica não apresentaram diferença estatística (p>0,05), e os valores médios entre os grupos foram: Ganho de Peso: 59,5 ± 5,0g; Quociente de Eficiência da Dieta: 0,39 ± 0,01; Quociente de Eficiência Protéica Líquida: 3,64 ± 0,12; Digestibilidade Aparente: 92,7 ± 1,1 por cento e Digestibilidade Verdadeira: 94,4 ± 0,9 por cento. Os resultados demonstraram que nas condições experimentais utilizadas, o ácido fítico não foi capaz de alterar o valor nutritivo da caseína.

Acido fítico: aspectos nutricionales e implicaciones analíticas / Phytic acid: nutritional aspects and analytical implications

This review provides a current summary of the literature concerning various aspects of phytic acid. These include data relative to its chemical structure and physicochemical properties, its occurrence in numerous cereals and legumes, and its role in plants. In addition, the nutritional significance of phytate with regard to its protein and mineral binding abilities, its health benefits and the methods commonly used for the analysis of phytate are discussed.

Effect of myo-inositol(1,4,5)trisphosphate on the hydrolysis of phytic acid by phytase.

Phytase is a monomeric enzyme of molecular mass 160 kDa which catalyzes the hydrolysis of phytic acid (D-myo inositol hexakisphosphate, InsP6) in a stepwise manner to myo-inositol. The enzyme-InsPn (n = 1-6) interaction at the catalytic site has a dissociation constant in the micro molar range. There also exists in the enzyme, a non-catalytic site specific for InsP3 with dissociation constant in the nano molar range. We have probed the effect of the high affinity InsP3 binding on the dissociation constant (Kd) of the phytase-InsP6 interaction and the kinetics of hydrolysis. These studies demonstrate the effect exerted by the high affinity InsP3 binding on the catalytic site of the enzyme.

Response of sugar interconversion, starch and protein accumulation to supplied metabolites during pod filling in chickpea.

Detached chickpea inflorescences bearing pods at 20 days after flowering (DAF) were cultured for 5 days in complete liquid medium supplemented separately with asparate, myo-inositol, alpha-ketoglutarate and phytic acid. Effect of these metabolites on sugar interconvestion and starch and protein accumulation in developing pods was studied. Substituting asparate (62.5 mM) for glutamine in culture medium decreased relative proportion of sucrose in all pod tissues but increased the level of sugars, starch and protein in pod wall and cotyledons. In cotyledons, whereas myo-inositol (75 mM) reduced the accumulation of starch without affecting protein level, alpha-ketoglutarate (44 mM) increased both starch and protein accumulation. Both myo-inositol and alpha-ketoglutarate increased relative proportion of sucrose in cotyledons. Phytic acid (1 mM) decreased in cotyledons 14C incorporation from glucose into EtOH extract (principally constituted by sugars), amino acids and proteins but increased the same into starch. In cotyledons, phytic acid also increased 14C incorporation from glutamate into amino acids but this increase was negatively correlated with protein synthesis. Phytic acid decreased the relative distribution of 14C from glucose and glutamate into sucrose from pod wall but enhanced the same into EtOH extract from embryo. Based on the results, it is suggested that mode of metabolic response to exogenously supplied metabolites widely differs in pod tissues of chickpea.

Polyphosphate binding sites in Liophis miliaris hemoglobin: evidence with reduced nicotinamide adenine dinucleotide phosphate

The water-snake Liophis miliaris presentes hemoglobin which binds organic polyphosphate through a simple single-site per tetramer (Mol. Wt. 64500) as judged by titration curves of reduced nicotinamide adenine dinucleotide phosphate either in the presence or absence of inositol hexaphosphate. The site seems to have the same structural nature of that found on other hemoglobins and is able to strongly bind most of the known protein effectors such as inositol hexaphosphate, adenosine triphosphate or 2,3-disphosphoglicerate. The high association constant at pØ7 of reduced nicotinamide for the deoxy hemoglobin of about K (D) = 7 x 10***6 M***-1 comparaed to human hemoglobin (K(D) = 7 x 10***5 M***-1), and to that of adenosine triphosphate (its natural erythrocytic polyphosphate) still higher of about K(D) = 10***11 M ***-1, shows clearly the very high affinity of this snake hemoglobin for such allosteric effector. The results besides corroborating the dimer-tetramer transition mechanism proposed to describe the oxygen transport by the hemoglobin of Liophis miliaris - may explain the difficulties to obtain the oxy dimeric conformation of the protein by usual hemolysis and stripped off procedures