A new technique for the determination of phosphorus by the molybdenum blue method

Holman, W. I. M.

doi:10.1042/bj0370256

Cited by 90 publications

(54 citation statements)

References 4 publications

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“…Standard phytase activity was assayed with the ferrous sulfate-molybdenum blue method (40). A 50-l volume of each enzyme was incubated with 950 l of substrate solution (1.5 mM sodium phytate, 0.25 M sodium acetate [pH 4.5]) at 37°C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Niu

Luo

Shi

et al. 2016

Appl Environ Microbiol

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N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed.A s much as 80% of the total phosphorus in cereal grains, oilseeds, and legumes exists in the form of phytate (myo-inositol hexakisphosphate), one of the most important functional ingredients in animal feed (1). However, phytate usually forms indigestible complexes with mineral cations and proteins, thereby causing reductions in nutrient utilization (2, 3). The dietary phytate undigested by monogastric animals is also released into the environment, where it causes phosphorus pollution (4, 5).The enzyme phytase catalyzes the sequential release of inorganic phosphate and lower myo-inositol phosphates from phytate (6); thus, inclusion of exogenous phytases in animal feeds as enzyme additives can enhance the nutrient uptake, lower the feedstuff production costs, and protect the environment in regions where intensive animal farming is conducted (7,8). Numerous phytases have been identified from microorganisms, animals, and plants (9-11), but only a few of them are commercially produced (12). The extensive application of phytases is limited by enzyme lability and protease sensitivity under high processing temperatures and low physiological pHs. Therefore, improving phytase stability at low pHs and high protease concentrations is desirable.Pepsin, a monomeric protein composed of two ␤-barrel-like domains, represents the main proteolytic enzyme in gastric juice (13). The acidic residues D32 and D215 are mainly responsible for proteoly...

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Section: Methodsmentioning

confidence: 99%

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Niu

Luo

Shi

et al. 2016

Appl Environ Microbiol

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“…A diferença consiste na utilização do método do azul de molibdênio (HOLMAN, 1943;SILVA, 2009) para as amostras de solo, que apresentam teores inferiores, já que este método é mais sensível quando comparado ao método do amarelo de metavanadato (HOLMAN, 1943;KISTON;MELLON, 1944), utilizado para amostras de tecido vegetal. O vanádio ( V ), presente no ânion metavanadato ( VO 3 -), é tóxico, pois tem efeito mutagênico em células germinativas e pode provocar problemas respiratórios (CETESB, 2012).…”

Section: Gerenciamento De Resíduos Sólidos No Laaunclassified

Gerenciamento de Resíduos de Ensaios Químicos de Solo e Planta no Centro Nacional de Pesquisa de Arroz e Feijão (CNPAF)

et al. 2016

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resumo: Este trabalho relata o gerenciamento de resíduos sólidos gerados nas análises de solo e planta do Laboratório de Análise Agroambiental (LAA) do Centro Nacional de Pesquisa de Arroz e Feijão (CNPAF) como experiência única da bibliografia nacional por englobar conjuntamente: identificação dos riscos toxicológicos, descrição da composição química, classificação, redução da geração e tratamento dos resíduos. Por isso, esse gerenciamento de resíduos sólidos está em consonância com a Política Nacional de Resíduos Sólidos -PNRS (Lei nº 12.305, de 2010). Detalham-se a origem, toxicidade e tratamento dos resíduos tóxicos do LAA e as medidas adotadas para diminuição do passivo ambiental gerado. Os resíduos desse laboratório advêm das análises químicas de solo e tecido vegetal, dentre os quais se destacam pela toxicidade: o cianeto que é tratado por oxidação a gás carbônico; e o cromo que é tratado por precipitação, reduzindo seu volume em 90%. O LAA avaliou ainda a possibilidade de reciclagem ou reutilização como pigmento cerâmico do resíduo contendo cromo. O LAA destaca-se por aplicar de forma eficaz a PNRS, mas os resultados apontam necessidade de automatização para viabilizar a reciclagem ou reutilização dos resíduos.

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“…A solution of refolded protein was obtained. The phytase activity of this solution was determined by measuring the amount of phosphate released from InsP6 using a modified ferrous sulfate molybdenum blue method (Holman, 1943;Makarewicz et al, 2006). The clear supernatant of the protein solution after centrifugation at 13 000 rev min À1 at 277 K for 30 min was applied directly onto a 3 ml Ni Sepharose Fast Flow column (GE Healthcare) equilibrated with buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM CaCl 2 ).…”

Section: Refolding and Purification Of Phyhtmentioning

confidence: 99%

Preparation, purification, crystallization and preliminary crystallographic analysis of dual-domain β-propeller phytase fromBacillussp. HJB17

Liu

Guo

et al. 2014

Acta Cryst Sect F

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-Propeller phytases (BPPs) are abundant in nature. Recently, dual-domain BPPs have been found in which the typical BPP domain is responsible for phytate hydrolysis. The dual-domain BPP (PhyH) from Bacillus sp. HJB17 was obtained with an incomplete N-terminal BPP domain (PhyH-DI; residues 41-318) and a typical BPP domain (PhyH-DII; residues 319-644) at the C-terminus. PhyH-DI was found to act synergistically (with a 1.2-2.5-fold increase in phosphate release) with PhyH-DII, other BPPs (PhyP and 168PhyA) and a histidine acid phosphatase. The structure of PhyH was therefore studied with the aim of explaining these functions. PhyH with the secreted signal peptide of the first 40 amino acids deleted (PhyHT) was cloned and expressed in Escherichia coli. Purified and active PhyHT protein was obtained by refolding from the precipitant. PhyHT was crystallized using the vapour-diffusion method. The crystal grew in a condition consisting of 0.2 M sodium acetate trihydrate, 0.1 M Tris-HCl pH 9.5, 25%(w/v) polyethylene glycol 4000 using 1 mg ml À1 protein solution at 289 K. A complete data set was collected from a crystal to 2.85 Å resolution using synchrotron radiation at 100 K. The crystal belonged to space group P12 1 1, with unit-cell parameters a = 46.82, b = 140.19, c = 81.94 Å , = 90.00, = 92.00, = 90.00 . The asymmetric unit was estimated to contain one molecule of PhyHT.

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A new technique for the determination of phosphorus by the molybdenum blue method

Cited by 90 publications

References 4 publications

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Gerenciamento de Resíduos de Ensaios Químicos de Solo e Planta no Centro Nacional de Pesquisa de Arroz e Feijão (CNPAF)

Preparation, purification, crystallization and preliminary crystallographic analysis of dual-domain β-propeller phytase fromBacillussp. HJB17

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