Wheat phytase potentially protects HT-29 cells from inflammatory nucleotides-induced cytotoxicity.

OBJECTIVE: The aim of this study was to investigate the protective effect of wheat phytase as a structural decomposer of inflammatory nucleotides, extracellular adenosine triphosphate (ATP), and uridine diphosphate (UDP) on HT-29 cells. METHODS: Phosphatase activities of wheat phytase against ATP and UDP was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine using a Pi Color Lock gold phosphate detection kit. Viability of HT-29 cells exposed to intact- or dephosphorylated-nucleotides was analyzed with an EZ-CYTOX kit. Secretion levels of pro-inflammatory cytokines (IL-6 and IL-8) in HT-29 cells exposed to substrate treated with or without wheat phytase were measured with enzyme-linked immunosorbent assay kits. Activation of caspase-3 in HT-29 cells treated with intact ATP or dephosphorylated-ATP was investigated using a colorimetric assay kit. RESULTS: Wheat phytase dephosphorylated both nucleotides, ATP and UDP, in a dosedependent manner. Regardless of the presence or absence of enzyme inhibitors (L-phenylalanine and L-homoarginine), wheat phytase dephosphorylated UDP. Only L-phenylalanine inhibited the dephosphorylation of ATP by wheat phytase. However, the level of inhibition was less than 10%. Wheat phytase significantly enhanced the viability of HT-29 cells against ATP- and UDP-induced cytotoxicity. Interleukin (IL)-8 released from HT-29 cells with nucleotides dephosphorylated by wheat phytase was higher than that released from HT-29 cells with intact nucleotides. Moreover, the release of IL-6 was strongly induced from HT-29 cells with UDP dephosphorylated by wheat phytase. HT-29 cells with ATP degraded by wheat phytase showed significantly (13%) lower activity of caspase-3 than HT-29 cells with intact ATP. CONCLUSION: Wheat phytase can be a candidate for veterinary medicine to prevent cell death in animals. In this context, wheat phytase beyond its nutritional aspects might be a novel and promising tool for promoting growth and function of intestinal epithelial cells under luminal ATP and UDP surge in the gut.

Effect of sweet potato purple acid phosphatase on Pseudomonas aeruginosa flagellin-mediated inflammatory response in A549 cells.

OBJECTIVE: The study was conducted to investigate the dephosphorylation of Pseudomonas aeruginosa flagellin (PA FLA) by sweet potato purple acid phosphatase (PAP) and the effect of the enzyme on the flagellin-mediated inflammatory response in the A549 lung epithelial cell line. METHODS: The activity of sweet potato PAP on PA FLA was assayed at different pH (4, 5.5, 7, and 7.5) and temperature (25°C, 37°C, and 55°C) conditions. The release of interleukin-8 (IL-8) and the activation of nuclear factor kappa- light-chain-enhancer of activated B cells (NF-κB) in A549 cells exposed to PA FLA treated with or without sweet potato PAP was measured using IL-8 and NF-κB ELISA kits, respectively. The activation of toll-like receptor 5 (TLR5) in TLR5-overexpressing HEK-293 cells exposed to PA FLA treated with or without sweet potato PAP was determined by the secreted alkaline phosphatase-based assay. RESULTS: The dephosphorylation of PA FLA by sweet potato PAP was favorable at pH 4 and 5.5 and highest at 55°C. PA-FLA treated with the enzyme decreased IL-8 release from A549 cells to about 3.5-fold compared to intact PA FLA at 1,000 ng/mL of substrate. Moreover, PA-FLA dephosphorylated by the enzyme repressed the activation of NF-κB in the cells compared to intact PA FLA. The activation of TLR5 by PA-FLA was highest in TLR-overexpressing HEK293 cells at a substrate concentration of 5,000 ng/mL, whereas PA FLA treated with the enzyme strongly repressed the activation of TLR5. CONCLUSION: Sweet potato PAP has the potential to be a new alternative agent against the increased antibiotic resistance of P. aeruginosa and may be a new conceptual feed additive to control unwanted inflammatory responses caused by bacterial infections in animal husbandry.

Effect of long-chain inorganic polyphosphate treated with wheat phytase on interleukin 8 signaling in HT-29 cells.

OBJECTIVE: This study was performed to investigate the potential effect of wheat phytase on long-chain inorganic polyphosphate (polyP)-mediated interleukin 8 (IL-8) signaling in an intestinal epithelial cell line, HT-29 cells. METHODS: Cell viability and the release of the pro-inflammatory cytokine IL-8 in HT-29 cells exposed to polyP1150 (average of 1,150 phosphate residues) treated with or without wheat phytase were measured by the EZ-CYTOX kit and the IL-8 ELISA kit, respectively. Also, the activation of cellular inflammatory factors NF-κB and MAPK (p38 and ERK 1/2) in HT-29 cells was investigated using ELISA kits. RESULTS: PolyP1150 negatively affected the viability of HT-29 cells in a dose-dependent manner. However, 100 mM polyP1150 dephosphorylated by wheat phytase increased cell viability by 1.4-fold over that of the intact substrate. Moreover, the 24 h exposure of cells to enzyme-treated 50 mM polyP1150 reduced the secretion of IL-8 and the activation of NF-κB by 9% and 19%, respectively, compared to the intact substrate. PolyP1150 (25 and 50 mM) dephosphorylated by the enzyme induced the activation of p38 MAPK via phosphorylation to 2.3 and 1.4-fold, respectively, compared to intact substrate, even though it had little effect on the expression of ERK 1/2 via phosphorylation. CONCLUSION: Wheat phytase could attenuate polyP1150-induced IL-8 release in HT-29 cells through NF-κB, independent of MAP kinases p38 and ERK. Thus, wheat phytase may alleviate inflammatory responses including hypercytokinemia caused by bacterial polyP infection in animals. Therefore, wheat phytase has the potential as an anti-inflammatory therapeutic supplement in animal husbandry.

Wheat phytase can alleviate the cellular toxic and inflammatory effects of lipopolysaccharide.

The objective of this study was to characterize the enzymatic hydrolysis of lipopolysaccharide (LPS) by wheat phytase and to investigate the effects of wheat phytase-treated LPS on in vitro toxicity, cell viability and release of a pro-inflammatory cytokine, interleukin (IL)-8 by target cells compared with the intact LPS. The phosphatase activity of wheat phytase towards LPS was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine. In vitro toxicity of LPS hydrolyzed with wheat phytase in comparison to intact LPS was assessed. Cell viability in human aortic endothelial (HAE) cells exposed to LPS treated with wheat phytase in comparison to intact LPS was measured. The release of IL-8 in human intestinal epithelial cell line, HT-29 cells applied to LPS treated with wheat phytase in comparison to intact LPS was assayed. Wheat phytase hydrolyzed LPS, resulting in a significant release of inorganic phosphate for 1 h (p < 0.05). Furthermore, the degradation of LPS by wheat phytase was nearly unaffected by the addition of L-phenylalanine, the inhibitor of tissue-specific alkaline phosphatase or L-homoarginine, the inhibitor of tissue-non-specific alkaline phosphatase. Wheat phytase effectively reduced the in vitro toxicity of LPS, resulting in a retention of 63% and 54% of its initial toxicity after 1-3 h of the enzyme reaction, respectively (p < 0.05). Intact LPS decreased the cell viability of HAE cells. However, LPS dephosphorylated by wheat phytase counteracted the inhibitory effect on cell viability. LPS treated with wheat phytase decreased IL-8 secretion from intestinal epithelial cell line, HT-29 cell to 14% (p < 0.05) when compared with intact LPS. In conclusion, wheat phytase is a potential therapeutic candidate and prophylactic agent for control of infections induced by pathogenic Gram-negative bacteria and associated LPS-mediated inflammatory diseases in animal husbandry.

Potential immune-modulatory effects of wheat phytase on the performance of a mouse macrophage cell line, Raw 264.7, exposed to long-chain inorganic polyphosphate.

OBJECTIVE: This experiment was conducted to find out the immunological effects of wheat phytase when long-chain inorganic polyphosphate (polyP) treated with wheat phytase was added to a macrophage cell line, Raw 264.7, when compared to intact long-chain polyP. METHODS: Nitric oxide (NO) production of Raw 264.7 cells exposed to P700, a long-chain polyP with an average of 1,150 phosphate residues, treated with or without wheat phytase, was measured by Griess method. Phagocytosis assay of P700 treated with or without phytase in Raw 264.7 cells was investigated using neutral red uptake. The secretion of tumor necrosis factor α (TNF-α) by Raw 264.7 cells with wheat phytase-treated P700 compared to intact P700 was observed by using Mouse TNF-α enzyme-linked immunosorbent assay kit. RESULTS: P700 treated with wheat phytase effectively increased NO production of Raw 264.7 cells by 172% when compared with intact P700 at 12 h exposure. At 5 mM of P700 concentration, wheat phytase promoted NO production of macrophages most strongly. P700, treated with wheat phytase, stimulated phagocytosis in macrophages at 12 h exposure by about 1.7-fold compared to intact P700. In addition, P700 treated with wheat phytase effectively increased in vitro phagocytic activity of Raw 264.7 cells at a concentration above 5 mM when compared to intact P700. P700 dephosphorylated by wheat phytase increased the release of TNF-α from Raw 264.7 cells by 143% over that from intact P700 after 6 h exposure. At the concentration of 50 µM P700, wheat phytase increased the secretion of cytokine, TNF-α, by 124% over that from intact P700. CONCLUSION: In animal husbandry, wheat phytase can mitigate the long-chain polyP causing damage by improving the immune capabilities of macrophages in the host. Thus, wheat phytase has potential as an immunological modulator and future feed additive for regulating immune responses caused by inflammation induced by long-chain polyP from bacterial infection.

Catalytic properties of wheat phytase that favorably degrades long-chain inorganic polyphosphate.

OBJECTIVE: This study was conducted to determine catalytic properties of wheat phytase with exopolyphosphatase activity toward medium-chain and long-chain inorganic polyphosphate (polyP) substrates for comparative purpose. METHODS: Exopolyphosphatase assay of wheat phytase toward polyP75 (medium-chain polyP with average 75 phosphate residues) and polyP1150 (long-chain polyP with average 1150 phosphate residues) was performed at pH 5.2 and pH 7.5. Its activity toward these substrates was investigated in the presence of Mg2+, Ni+2, Co2+, Mn2+, or EDTA. Michaelis constant (Km) and maximum reaction velocity (Vmax) were determined from Lineweaver-Burk plot with polyP75 or polyP1150. Monophosphate esterase activity toward pNPP (p-nitrophenyl phosphate) was assayed in the presence of polyP75 or polyP1150. RESULTS: Wheat phytase dephosphorylated polyP75 and polyP1150 at pH 7.5 more effectively than that at pH 5.2. Its exopolyphosphatase activity toward polyP75 at pH 5.2 was 1.4-fold higher than that toward polyP1150 whereas its activity toward polyP75 at pH 7.5 was 1.4-fold lower than that toward polyP1150. Regarding enzyme kinetics, Km for polyP75 was 1.4-fold lower than that for polyP1150 while Vmax for polyP1150 was 2-fold higher than that for polyP75. The presence of Mg2+, Ni+2, Co2+, Mn2+, or EDTA (1 or 5 mM) exhibited no inhibitory effect on its activity toward polyP75. Its activity toward polyP1150 was inhibited by 1 mM of Ni+2 or Co2+ and 5 mM of Ni+2, Co2+, or Mg2+. Ni+2 inhibited its activity toward polyP1150 the most strongly among tested additives. Both polyP75 and polyP1150 inhibited the monophosphate esterase activity of wheat phytase toward pNPP in a dose-dependent manner. CONCLUSION: Wheat phytase with an unexpected exopolyphosphatase activity has potential as a therapeutic tool and a next-generational feed additive for controlling long-chain polyP-induced inappropriate inflammation from Campylobacter jejuni and Salmonella typhimurium infection in public health and animal husbandry.

Characterization, gene cloning, and sequencing of a fungal phytase, PhyA, from Penicillium oxalicum PJ3.

A phytase from Penicillium oxalicum PJ3, PhyA, was purified near to homogeneity with 427-fold increase in specific phytase activity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatographies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis of the purified enzyme indicated an estimated molecular mass of 65 kD. The optimal pH and temperature of the purified enzyme were pH 4.5 and 55°C, respectively. The enzyme activity was strongly inhibited by Ca(2+), Cu(2+), Zn(2+), and phenylmethylsulfonyl fluoride (PMSF). The Km value for sodium phytate was 0.545 mM with a Vmax of 600 U/mg of protein. The phyA gene was cloned, and it contains an open reading frame of 1,383 with a single intron (118 bp), and encodes a protein of 461 amino acids.

Production of extracellular α-galactosidase by Bacillus sp. LX-1 in solid state fermentation for application as a potential feed additive / Producción de α-galactosidasa por Bacillus sp. LX-1 en fermentación en estado sólido para su aplicación como potencial aditivo alimenticio / Produção de α-galactosidase por Bacillus sp. LX-1 em fermentação em estado sólido para o uso como um potencial aditivo na alimentação

Background: α-galacto-oligosaccharides, including raffinose and stachyose, are present in soybean meal and used widely as a protein source in poultry diets. These compounds have anti-nutritive effects that ultimately reduce performance and value of birds. Thus, the addition of exogenous α-galactosidase to poultry diets -which can initiate the digestion of these non-digestible sugars- can be an effective strategy to solve the nutritional disorders associated with consumption of these oligosacharides. Solid state fermentation (SSF) has drawn attention for the production of microbial enzymes, due to the possibility of using cheap and abundant agro-industrial residues as substrates. Objective: to present information on α-galactosidase production under SSF conditions by an Antarctic bacterial isolate, Bacillus LX-1. Methods: initially, wheat bran, soybean meal, corn flour and the combinations of these individual substrates with nutritive supplements containing 1% galactose, 0.5% yeast extract, 1% tryptone, and 0.001% MnSO(4)4H2O were evaluated to select an optimal medium in SSF to produce extracellular α-galactosidase. Certain fermentation parameters involving incubation time, moisture content, and initial pH were investigated separately. Additional studies were conducted to evaluate the influence on enzyme production of different carbon sources (glucose, sucrose, galactose, lactose, and maltose) and nitrogen sources (peptone, tryptone, sodium nitrate and ammonium sulfate). Results: a medium containing soybean meal resulted in best α-galactosidase synthesis and was used for further SSF explorations with Bacillus sp. LX-1. Maximum enzyme production was observed at a growth period of 72 h, 75% moisture content and pH 8.0. Enzyme activity was enhanced in the presence of galactose or lactose as the carbon source, and tryptone or peptone as the nitrogen source. Conclusion: this SSF technique could be potentially used to produce α-galactosidase for poultry feed.

Antecedentes: los α-galacto-oligosacáridos, incluyendo rafinosa y estaquiosa, están presentes en la harina de soja, la cual es utilizada ampliamente como fuente de proteína en dietas de aves. Estos compuestos tienen efectos anti-nutricionales que reducen el rendimiento de las aves. La adición de α-galactosidasa exógena a dietas de aves puede iniciar la digestión de esos azúcares, resultando en una estrategia eficaz para resolver los desórdenes nutricionales asociados con el consumo de dichos oligosacáridos. La fermentación en estado sólido (SSF) se puede usar para producir enzimas microbianas, debido a la posibilidad de utilizar residuos agroindustriales abundantes y baratos como sustrato. Objetivo: informar sobre la producción de α-galactosidasas por una bacteria antártica (Bacillus LX-1) bajo condiciones de SSF. Métodos: se evaluaron salvado de trigo, harina de soja, harina de maíz y las combinaciones individuales de estos sustratos con suplementos nutritivos conteniendo 1% de galactosa, 0,5% de extracto de levadura, 1% de triptona y 0,001% de MnSO(4)4H2O para seleccionar un medio óptimo de SSF para producir α-galactosidasa extracelular. Ciertos parámetros de fermentación incluyendo tiempo de incubación, contenido de humedad, y pH inicial se evaluaron por separado. Se realizaron estudios adicionales para evaluar la influencia de diferentes fuentes de carbono (glucosa, sucrosa, galactosa, lactosa y maltosa) y de nitrógeno (peptona, triptona, nitrato de sodio y sulfato de amonio) sobre la producción enzimatica. Resultados: un medio con harina de soja resultó ser el mejor para la síntesis de α-galactosidasa y se utilizó para nuevas exploraciones de SSF con Bacillus sp. LX-1. La producción máxima de la enzima se observó en un periodo de crecimiento de 72 h, a 75% de humedad y pH 8,0. La actividad enzimática mejoró en presencia de galactosa o lactosa como fuente de carbono, y triptona o peptona como fuente de nitrógeno. Conclusión: la técnica de SSF podría ser utilizada para producir α-galactosidasa destinada a la alimentación de aves de corral.

Antecedentes: os α-galacto-oligossacarídeos, incluindo rafinose e estaquiose, estão presentes na farinha de soja, que é amplamente utilizado como uma fonte de proteína em dietas de aves domésticas. Estes compostos têm efeitos anti-nutricionais que reduzem o desempenho das aves. A adição de α-galactosidase exógena em dietas de aves pode começar a digerir estes açúcares, resultando em uma estratégia eficaz para resolver os problemas nutricionais associados com o consumo desses oligossacarídeos. A fermentação em estado sólido (SSF) pode ser usada para produzir enzimas microbianas, devido à possibilidade de utilização de resíduos agroindustriais como substrato abundante e barato. Objetivo: relatar a produção de α-galactosidase por uma bactéria da Antártida (Bacillus LX- 1) em SSF. Métodos: foram avaliados o farelo de trigo, farelo de soja, farelo de milho e combinações específicas destes substratos com suplementos nutricionais contendo 1% de galactose, 0,5% de extrato de levedura, 1% de triptona e 0,001% de MnSO(4)4H2O para selecionar um meio ideal de SSF para produzir extracelular α-galactosidase. Certos parâmetros de fermentação, incluindo tempo de incubação, teor de umidade e pH inicial foram avaliados separadamente. Estudos adicionais foram conduzidos para avaliar a influência de diferentes fontes de carbono (glucose, sacarose, galactose, lactose e maltose) e de azoto (peptona, triptona, nitrato de sódio e sulfato de amónio) na produção da enzima. Resultados: um meio com farelo de soja acabou por ser o melhor para a síntese de α-galactosidase e foi usado para uma maior exploração de SSF com Bacillus sp. LX - 1. A produção máxima da enzima foi observada em um período de crescimento de 72 h, 75% de humidade e pH 8,0. Melhoria da atividade enzimática na presença de galactose ou lactose como fonte de carbono e de triptona e peptona como fonte de azoto. Conclusão: A técnica da SSF poderia ser usada para produzir α-galactosidase para a alimentação de aves domésticas.

Immobilization of the Antarctic Bacillus sp. LX-1 α-Galactosidase on Eudragit L-100 for the Production of a Functional Feed Additive.

Partially purified α-galactosidase from Bacillus sp. LX-1 was non-covalently immobilized on a reversibly soluble-insoluble polymer, Eudragit L-100, and an immobilization efficiency of 0.93 was obtained. The optimum pH of the free and immobilized enzyme was 6.5 to 7.0 and 7.0, respectively, while there was no change in optimum temperature between the free and immobilized α-galactosidase. The immobilized α-galactosidase was reutilized six times without significant loss in activity. The immobilized enzyme showed good storage stability at 37°C, retaining about 50% of its initial activity even after 18 d at this temperature, while the free enzyme was completely inactivated. The immobilization of α-galactosidase from Bacillus sp. LX-1 on Eudragit L-100 may be a promising strategy for removal of α-galacto-oligosaccharides such as raffinose and stachyose from soybean meal and other legume in feed industry.

Bacillus strains as feed additives: In vitro evaluation of its potential probiotic properties / Bacilos utilizados como aditivos para piensos: Evaluación in vitro de sus potenciales propiedades probióticas / Bacilos utilizados como aditivos para concentrados: Avaliação in vitro de suas propriedades probióticas

Background: much recent attention has been devoted to the genuine value of Bacillus species as multifunctional probiotic products, which produce various extracellular enzymes that enhance feed digestibility as well as many antimicrobial compounds for the purpose of improving animal performance. Objective: to describe novel, in vitro potential probiotic properties such as acid tolerance, bile tolerance, safety, and antimicrobial activity of mesophilic and psychrophilic Bacillus strains in conjunction with their extracellular enzymatic activities. Methods: four Bacillus strains (B. sp. T3, B. sp. T4, B. sp. SM2, and B. sp. JSP1) isolated from different sources were used. Strains were identified according to 16S rDNA sequences. Escherichia coli K88, E. coli O157:H7, Salmonella enteritidis KCCM 12021, Enterococcus faecalis, Listeria monocytogenes, and Staphylococcus aureus were used as indicator bacteria for the antimicrobial activity trial. Strains were activated and cultured in tryptic soy broth (pH 7.0) or broth solidified with 1.5% agar. Results: B. sp. JSP1 was fully resistant to both pH 2 and 3, whereas B. sp. SM2 showed relatively good viability at pH 3. All strains tolerated oxgall (0.3%) bile salt and were not cytotoxic to the HEK 293 human embryonic kidney cells. Three strains, except B. sp. T3, displayed differential production of extracellular enzymes including amylase, xylanase, cellulase, protease, phytase, and α-galactosidase. In particular, B. sp. SM2 inhibited six indicator pathogens: Escherichia coli K88, E. coli O157:H7, Salmonella enteritidis, Enterococcus faecalis, Listeria monocytogenes, and Staphylococcus aureus. Conclusion: the single use of B. sp. SM2 or the mixed use of the strain combined with acid or bile tolerant Bacillus strains secreting extracellular enzymes may be an alternative to antibiotics as a feed additive in farm animal production.

Antecedentes: recientemente el valor de las especies de Bacillus como productos probióticos multifuncionales ha recibido bastante atención, debido a que estos producen varias enzimas extracelulares que potencian la digestibilidad de los alimentos, así como también compuestos antimicrobianos que mejoran el desempeño del animal. Objetivo: describir y evaluar potenciales propiedades probióticas ''in vitro'' -tales como acidez, tolerancia a la bilis, seguridad y actividad antimicrobiana- de cuatro cepas de Bacilos (B. sp. T3, B. sp. T4, B. sp. SM2 y B. sp. JSP1) aisladas de diferentes fuentes, en conjunción con sus actividades enzimáticas extracelulares. Métodos: se usaron cuatro cepas de Bacillus (B. sp. T3, B. sp. T4, B. sp. SM2, and B. sp. JSP1) aisladas de diferentes fuentes. Las cepas se identificaron de acuerdo a secuencias 16S rDNA. Escherichia coli K88, E. coli O157:H7, Salmonella enteritidis KCCM 12021, Enterococcus faecalis, Listeria monocytogenes, y Staphylococcus aureus fueron empleadas como bacterias indicadoras para el ensayo de actividad antimicrobiana. Las cepas fueron activadas y cultivadas en caldo soya tripticasa (PH 7.0) o caldo solidificado con 1.5% de agar. Resultados: el B. sp. JSP1 resultó totalmente resistente tanto a pH 2 como a pH 3, mientras que el B. sp. SM2 mostró viabilidad relativamente alta a pH 3. Todas las cepas toleraron oxgall (0.3%) de sales biliares y no resultaron citotóxicas para las células humanas HEK 293 de riñón embrionario. Tres cepas, con excepción de B. sp. T3, presentaron producción diferencial de enzimas extracelulares -incluyendo amilasa, xilanasa, celulasa, proteasa, fitasa y α-galactosidasa. En particular, el B. sp. SM2 inhibió seis indicadores patógenos (Escherichia coli K88, E. coli O157: H7, Salmonella enteritidis, Enterococcus faecalis, Listeria monocytogenes y Staphylococcus aureus). Conclusiones: el uso específico de B. sp. SM2, o el uso combinado de esta cepa junto con cepas secretoras de enzimas extracelulares y tolerantes a ácidos o bilis puede ser una alternativa para reemplazar los antibióticos frecuentemente usados como aditivos en alimentación animal.

Antecedentes: o valor das espécies de Bacillus como produtos probióticos multifuncionais tem recebido bastante atenção recentemente, devido a que produzem várias enzimas extracelulares que potenciam a digestibilidade dos alimentos, como também compostos antimicrobianos que melhoram o desempenho do animal. Objetivo: descrever e avaliar as propriedades potenciais ''in vitro'' - como acidez, tolerância à bile, segurança e atividade antimicrobial- de quatro cepas de Bacilos (B. sp. T3, B. sp. T4, B. sp. SM2, and B. sp. JSP1) isoladas de diferentes fontes, em conjunto com as suas atividades enzimáticas extracelulares. Métodos: foram usadas quatro cepas de Bacillus (B. sp. T3, B. sp. T4, B. sp. SM2, and B. sp. JSP1) isoladas de diferentes fontes. As cepas foram identificadas de acordo às sequências 16S rDNA. As seguintes bactérias foram empregadas como indicadoras no teste de atividade antimicrobiana: Escherichia coli K88, E. coli O157:H7, Salmonella enteritidis KCCM 12021, Enterococcus faecalis, Listeria monocytogenes, y Staphylococcus aureus. As cepas foram ativadas e cultivadas em caldo soja tripticase (pH 7.0) ou caldo solidificado com 1.5 de Agar. Resultados: o B. sp. JSP1 foi totalmente resistente tanto no pH 2.0 como no pH 3.0, enquanto que o B. sp. SM2 mostrou viabilidade relativamente alta no pH 3.0. Todas as cepas toleraram oxgall (0.3%) de sais biliares e não foram citotóxicas para as células humanas HEK 293 de rim embrionário. Tres cepas, com exceção de B. sp. T3, apresentaram produção diferenciada de enzimas extracelulares -incluindo amilase, xilanase, celulase, protease, fitase e α-galactosidase. Particularmente, o B. sp, SM2 inibiu seis indicadores patógenos (Escherichia coli K88, E. coli O157: H7, Salmonella enteritidis, Enterococcus faecalis, Listeria monocytogenes y Staphylococcus aureus). Conclusões: O uso específico de B. sp. SM2 ou o uso combinado desta cepa junto com as cepas secretoras de enzimas extracelulares e tolerantes a ácidos ou bile, pode ser uma alternativa para substituir os antibióticos frequentemente usados como aditivos na alimentação animal.

Degradation of Phytate Pentamagnesium Salt by Bacillus sp. T4 Phytase as a Potential Eco-friendly Feed Additive.

A bacterial isolate derived from soil samples near a cattle farm was found to display extracellular phytase activity. Based on 16S rRNA sequence analysis, the strain was named Bacillus sp. T4. The optimum temperature for the phytase activity toward magnesium phytate (Mg-InsP6) was 40°C without 5 mM Ca(2+) and 50°C with 5 mM Ca(2+). T4 phytase had a characteristic bi-hump two pH optima of 6.0 to 6.5 and 7.4 for Mg-InsP6. The enzyme showed higher specificity for Mg-InsP6 than sodium phytate (Na-InsP6). Its activity was fairly inhibited by EDTA, Cu(2+), Mn(2+), Co(2+), Ba(2+) and Zn(2+). T4 phytase may have great potential for use as an eco-friendly feed additive to enhance the nutritive quality of phytate and reduce phosphorus pollution.

Partial Characterization of α-Galactosidic Activity from the Antarctic Bacterial Isolate, Paenibacillus sp. LX-20 as a Potential Feed Enzyme Source.

An Antarctic bacterial isolate displaying extracellular α-galactosidic activity was named Paenibacillus sp. LX-20 based on 16S rRNA gene sequence analysis. Optimal activity for the LX-20 α-galactosidase occurred at pH 6.0-6.5 and 45°C. The enzyme immobilized on the smart polymer Eudragit L-100 retained 70% of its original activity after incubation for 30 min at 50°C, while the free enzyme retained 58% of activity. The enzyme had relatively high specificity for α-D-galactosides such as p-nitrophenyl-α-galactopyranoside, melibiose, raffinose and stachyose, and was resistant to some proteases such as trypsin, pancreatin and pronase. Enzyme activity was almost completely inhibited by Ag(+), Hg(2+), Cu(2+), and sodium dodecyl sulfate, but activity was not affected by ß-mercaptoethanol or EDTA. LX-20 α-galactosidase may be potentially useful as an additive for soybean processing in the feed industry.

Production of an extracellular protease by an Antarctic bacterial isolate (Bacillus sp. JSP1)as a potential feed additive / Proteasa extracelular de un aislado bacteriano Antartico (Bacillus sp. JSP1) con uso potencial como aditivo alimenticio para animales / Produção de proteasa extracelular por bactérias antárcticas isoladas, Bacillus sp. JSP1 como um aditivo potencial em concentrado

Extracellular proteolytic activity was found in JSP1, an Antarctic bacterial isolate. The strain was related to Bacillus sp, based on 16S rRNA gene sequence analysis. The JSP1 protease was partially purified by ammonium sulfate precipitation. Optimal enzyme activity occurred at 40 C and pH 7.4. Enzyme activity was significantly enhanced in the presence of Mg2+ and Ca2+ and was completely inactivated in presence of Cu2+, Zn2+, Hg2+, EDTA and SDS. The enzyme hydrolyzed casein the most effectively among the protein substrates tested. The enzyme also exhibited relatively high activity on keratin and gluten, and was active against peptidyl conjugates such asL-Leu-p-Nitroanilide and N-Succinyl-L-Phe-p-Nitroanilide. This study suggests that JSP1 protease could be utilized as a potential environmentally-friendly feed additive in animal production.

Se reporta haber encontrado actividad proteolítica extracelular en una bacteria antártica denominada JSP1. Con base en el análisis de secuencia genética 16S ARNr, la cepa fué relacionada con Bacillus sp. La proteasa JSP1 fué parcialmente purificada por precipitación con sulfato de amonio. La actividad óptima de la enzima se produjo a 40 ºC y pH 7,4. La actividad enzimática fue significativamente mayor en presencia de Mg2+ y Ca2+ y se inactivó completamente en presencia de Cu2+, Zn2+ , Hg2+ , EDTA y SDS. Entre todos los sustratos ensayados, el más eficientemente hidrolizado por la enzima fue la caseína. La enzima tuvo actividad relativamente alta sobre la queratina y el gluten, y participó activamente contra conjugados peptídicos como la L-Leu-p-nitroanilida y la N-succinil-L-fenilalanina-p-nitroanilida. La enzima podría ofrecer potencial para su uso como aditivo alimenticio ecológico en producción animal.

A actividade proteolítica extracelular foi encontrada a partir de uma bactéria antárctica denominada JSP1, mediante uma análises de sequencia do gene 16S rRNA, a cepa foi relacionada para Bacillus sp. A proteasa JSP1 foi parcialmente purificada por precipitação com sulfato de amônia. Uma óptima actividade enzimática ocorreu em 40 ºC e pH 7,4. A actividade da enzima foi significativamente maior na presença de Mg2 + e Ca2 +, e foi completamente inactivada em presença de Cu2 + Zn2 +, Hg2 +, EDTA e SDS. A enzima hidrolisada de caseína foi mais eficaz entre os substratos de proteína testados, apresentando maior actividade na queratina e no glúten e foi activo contra os peptidil conjugados como L-Leu-p Nitroanilida e N-succinil-L-Phe-p-Nitroanilida. A enzima pode ser utilizado como aditivo ambiental na alimentação animal.

Dephosphorylation of 2,3-bisphosphoglycerate by MIPP expands the regulatory capacity of the Rapoport-Luebering glycolytic shunt.

The Rapoport-Luebering glycolytic bypass comprises evolutionarily conserved reactions that generate and dephosphorylate 2,3-bisphosphoglycerate (2,3-BPG). For >30 years, these reactions have been considered the responsibility of a single enzyme, the 2,3-BPG synthase/2-phosphatase (BPGM). Here, we show that Dictyostelium, birds, and mammals contain an additional 2,3-BPG phosphatase that, unlike BPGM, removes the 3-phosphate. This discovery reveals that the glycolytic pathway can bypass the formation of 3-phosphoglycerate, which is a precursor for serine biosynthesis and an activator of AMP-activated protein kinase. Our 2,3-BPG phosphatase activity is encoded by the previously identified gene for multiple inositol polyphosphate phosphatase (MIPP1), which we now show to have dual substrate specificity. By genetically manipulating Mipp1 expression in Dictyostelium, we demonstrated that this enzyme provides physiologically relevant regulation of cellular 2,3-BPG content. Mammalian erythrocytes possess the highest content of 2,3-BPG, which controls oxygen binding to hemoglobin. We determined that total MIPP1 activity in erythrocytes at 37 degrees C is 0.6 mmol 2,3-BPG hydrolyzed per liter of cells per h, matching previously published estimates of the phosphatase activity of BPGM. MIPP1 is active at 4 degrees C, revealing a clinically significant contribution to 2,3-BPG loss during the storage of erythrocytes for transfusion. Hydrolysis of 2,3-BPG by human MIPP1 is sensitive to physiologic alkalosis; activity decreases 50% when pH rises from 7.0 to 7.4. This phenomenon provides a homeostatic mechanism for elevating 2,3-BPG levels, thereby enhancing oxygen release to tissues. Our data indicate greater biological significance of the Rapoport-Luebering shunt than previously considered.

The nucleolus exhibits an osmotically regulated gatekeeping activity that controls the spatial dynamics and functions of nucleolin.

We demonstrate that physiologically relevant perturbations in the osmotic environment rheostatically regulate a gatekeeping function for the nucleolus that controls the spatial dynamics and functions of nucleolin. HeLa cells and U2-OS osteosarcoma cells were osmotically challenged with 100-200 mm sorbitol, and the intranuclear distribution of nucleolin was monitored by confocal microscopy. Nucleolin that normally resides in the innermost fibrillar core of the nucleolus, where it assists rDNA transcription and replication, was expelled within 30 min of sorbitol addition. The nucleolin was transferred into the nucleoplasm, but it distributed there non-uniformly; locally high levels accumulated in 4',6-diamidino-2-phenylindole-negative zones containing euchromatic (transcriptionally active) DNA. Inositol pyrophosphates also responded within 30 min of hyperosmotic stress: levels of bisdiphosphoinositol tetrakisphosphate increased 6-fold, and this was matched by decreased levels of its precursor, diphosphoinositol pentakisphosphate. Such fluctuations in inositol pyrophosphate levels are of considerable interest, because, according to previously published in vitro data, they regulate the degree of phosphorylation of nucleolin through a novel kinase-independent phosphotransferase reaction ( Saiardi, A., Bhandari, A., Resnick, R., Cain, A., Snowman, A. M., and Snyder, S. H. (2004) Science 306, 2101-2105 ). However, by pharmacologically intervening in inositol pyrophosphate metabolism, we found that it did not supervise the osmotically driven switch in the biological activities of nucleolin in vivo.

Purification, sequencing, and molecular identification of a mammalian PP-InsP5 kinase that is activated when cells are exposed to hyperosmotic stress.

Mammalian cells utilize multiple signaling mechanisms to protect against the osmotic stress that accompanies plasma membrane ion transport, solute uptake, and turnover of protein and carbohydrates (Schliess, F., and Haussinger, D. (2002) Biol. Chem. 383, 577-583). Recently, osmotic stress was found to increase synthesis of bisdiphosphoinositol tetrakisphosphate ((PP)2-InsP4), a high energy inositol pyrophosphate (Pesesse, X., Choi, K., Zhang, T., and Shears, S. B. (2004) J. Biol. Chem. 279, 43378-43381). Here, we describe the purification from rat brain of a diphosphoinositol pentakisphosphate kinase (PPIP5K) that synthesizes (PP)2-InsP4. Partial amino acid sequence, obtained by mass spectrometry, matched the sequence of a 160-kDa rat protein containing a putative ATP-grasp kinase domain. BLAST searches uncovered two human isoforms (PPIP5K1 (160 kDa) and PPIP5K2 (138 kDa)). Recombinant human PPIP5K1, expressed in Escherichia coli, was found to phosphorylate diphosphoinositol pentakisphosphate (PP-InsP5) to (PP)2-InsP4 (Vmax = 8.3 nmol/mg of protein/min; Km = 0.34 microM). Overexpression in human embryonic kidney cells of either PPIP5K1 or PPIP5K2 substantially increased levels of (PP)2-InsP4, whereas overexpression of a catalytically dead PPIP5K1(D332A) mutant had no effect. PPIP5K1 and PPIP5K2 were more active against PP-InsP5 than InsP6, both in vitro and in vivo. Analysis by confocal immunofluorescence showed PPIP5K1 to be distributed throughout the cytoplasm but excluded from the nucleus. Immunopurification of overexpressed PPIP5K1 from osmotically stressed HEK cells (0.2 M sorbitol; 30 min) revealed a persistent, 3.9 +/- 0.4-fold activation when compared with control cells. PPIP5Ks are likely to be important signaling enzymes.

Integration of inositol phosphate signaling pathways via human ITPK1.

Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a reversible, poly-specific inositol phosphate kinase that has been implicated as a modifier gene in cystic fibrosis. Upon activation of phospholipase C at the plasma membrane, inositol 1,4,5-trisphosphate enters the cytosol and is inter-converted by an array of kinases and phosphatases into other inositol phosphates with diverse and critical cellular activities. In mammals it has been established that inositol 1,3,4-trisphosphate, produced from inositol 1,4,5-trisphosphate, lies in a branch of the metabolic pathway that is separate from inositol 3,4,5,6-tetrakisphosphate, which inhibits plasma membrane chloride channels. We have determined the molecular mechanism for communication between these two pathways, showing that phosphate is transferred between inositol phosphates via ITPK1-bound nucleotide. Intersubstrate phosphate transfer explains how competing substrates are able to stimulate each others' catalysis by ITPK1. We further show that these features occur in the human protein, but not in plant or protozoan homologues. The high resolution structure of human ITPK1 identifies novel secondary structural features able to impart substrate selectivity and enhance nucleotide binding, thereby promoting intersubstrate phosphate transfer. Our work describes a novel mode of substrate regulation and provides insight into the enzyme evolution of a signaling mechanism from a metabolic role.

Avian multiple inositol polyphosphate phosphatase is an active phytase that can be engineered to help ameliorate the planet's "phosphate crisis".

Contemporary phytase research is primarily concerned with ameliorating the problem of inadequate digestion of inositol hexakisphosphate (phytate; InsP6) in monogastric farm animal feed, so as to reduce the pollution that results from the high phosphate content of the manure. In the current study we pursue a new, safe and cost-effective solution. We demonstrate that the rate of hydrolysis of InsP6 by recombinant avian MINPP (0.7 micromol/mg protein/min) defines it as by far the most active phytase found to date in any animal cell (the corresponding activity of recombinant mammalian MINPP is only 0.006 micromol/mg protein/min). Although avian MINPP has less than 20% sequence identity with microbial phytases, we create a homology model of MINPP in which it is predicted that the structure of the phytase active site is well-conserved. This model is validated by site-directed mutagenesis and by use of a substrate analogue, scyllo-InsP6, which we demonstrate is only a weak MINPP substrate. In a model chicken cell line, we overexpressed a mutant form of MINPP that is secretion-competent. This version of the enzyme was actively secreted without affecting either cell viability or the cellular levels of any inositol phosphates. Our studies offer a genetic strategy for greatly improving dietary InsP6 digestion in poultry.

scyllo-inositol pentakisphosphate as an analogue of myo-inositol 1,3,4,5,6-pentakisphosphate: chemical synthesis, physicochemistry and biological applications.

myo-Inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P(5)), an inositol polyphosphate of emerging significance in cellular signalling, and its C-2 epimer scyllo-inositol pentakisphosphate (scyllo-InsP(5)) were synthesised from the same myo-inositol-based precursor. Potentiometric and NMR titrations show that both pentakisphosphates undergo a conformational ring-flip at higher pH, beginning at pH 8 for scyllo-InsP(5) and pH 9 for Ins(1,3,4,5,6)P(5). Over the physiological pH range, however, the conformation of the inositol rings and the microprotonation patterns of the phosphate groups in Ins(1,3,4,5,6)P(5) and scyllo-InsP(5) are similar. Thus, scyllo-InsP(5) should be a useful tool for identifying biologically relevant actions of Ins(1,3,4,5,6)P(5), mediated by specific binding sites, and distinguishing them from nonspecific electrostatic effects. We also demonstrate that, although scyllo-InsP(5) and Ins(1,3,4,5,6)P(5) are both hydrolysed by multiple inositol polyphosphate phosphatase (MINPP), scyllo-InsP(5) is not dephosphorylated by PTEN or phosphorylated by Ins(1,3,4,5,6)P(5) 2-kinases. This finding both reinforces the value of scyllo-InsP(5) as a biological control and shows that the axial 2-OH group of Ins(1,3,4,5,6)P(5) plays a part in substrate recognition by PTEN and the Ins(1,3,4,5,6)P(5) 2-kinases.

Physiological levels of PTEN control the size of the cellular Ins(1,3,4,5,6)P(5) pool.

To understand how a signaling molecule's activities are regulated, we need insight into the processes controlling the dynamic balance between its synthesis and degradation. For the Ins(1,3,4,5,6)P5 signal, this information is woefully inadequate. For example, the only known cytosolic enzyme with the capacity to degrade Ins(1,3,4,5,6)P5 is the tumour-suppressor PTEN [J.J. Caffrey, T. Darden, M.R. Wenk, S.B. Shears, FEBS Lett. 499 (2001) 6 ], but the biological relevance has been questioned by others [E.A. Orchiston, D. Bennett, N.R. Leslie, R.G. Clarke, L. Winward, C.P. Downes, S.T. Safrany, J. Biol. Chem. 279 (2004) 1116 ]. The current study emphasizes the role of physiological levels of PTEN in Ins(1,3,4,5,6)P5 homeostasis. We employed two cell models. First, we used a human U87MG glioblastoma PTEN-null cell line that hosts an ecdysone-inducible PTEN expression system. Second, the human H1299 bronchial cell line, in which PTEN is hypomorphic due to promoter methylation, has been stably transfected with physiologically relevant levels of PTEN. In both models, a novel consequence of PTEN expression was to increase Ins(1,3,4,5,6)P5 pool size by 30-40% (p<0.01); this response was wortmannin-insensitive and, therefore, independent of the PtdIns 3-kinase pathway. In U87MG cells, induction of the G129R catalytically inactive PTEN mutant did not affect Ins(1,3,4,5,6)P(5) levels. PTEN induction did not alter the expression of enzymes participating in Ins(1,3,4,5,6)P5 synthesis. Another effect of PTEN expression in U87MG cells was to decrease InsP6 levels by 13% (p<0.02). The InsP6-phosphatase, MIPP, may be responsible for the latter effect; we show that recombinant human MIPP dephosphorylates InsP6 to D/L-Ins(1,2,4,5,6)P5, levels of which increased 60% (p<0.05) following PTEN expression in U87MG cells. Overall, our data add higher inositol phosphates to the list of important cellular regulators [Y. Huang, R.P. Wernyj, D.D. Norton, P. Precht, M.C. Seminario, R.L. Wange, Oncogene, 24 (2005) 3819 ] the levels of which are modulated by expression of the highly pleiotropic PTEN protein.