Isophenoxazine synthase

An enzyme which catalyses the oxidation of o-aminophenol to o-quinoneimine and the subsequent condensation of o-aminophenol and o-quinoneime to give isophenoxazine has been isolated from the leaves of Tecoma stans. The reaction had an optimum pH of 6.2 and an optimum temperature of 45°. Heavy-metal ions like Hg2+, Co2+, Mg2+, Fe3+, were inhibitory. Mn2+ activated the reaction to about 40%. The reaction requires intact sulfhydryl groups. A study of the coenzyme requirements showed that isophenoxazine synthase (o-aminophenol: O2 oxidoreductase) is a flavoprotein requiring FAD for maximum activity. Stoichiometric studies showed that 2 moles of o-aminophenol gave 1 mole of isophhenoxazine.

Enzymatic conversion of anthranilic acid to catechol by chloroplast from the leaves of Tecoma stans

An enzyme system which converts anthranilic acid to catechol was detected in the leaves of Tecoma stans, and its properties studied. The system is present exclusively in the chloroplast fraction of the leaves. The optimum pH of the reaction is 5·2 and maximum activity was obtained with citrate-phosphate buffer. There was good stoichiometry between the amounts of anthranilic acid disappeared and the amounts of catechol and ammonia formed. The enzyme system showed an absolute requirement for oxygen and evidence was obtained for the probable participation of NADPH and FAD in the hydroxylation step. The optimum concentration of anthranilic acid was 10−4 M; at higher concentrations the reaction was inhibited to a considerable extent. Cyanide, pyrophosphate, and EDTA also caused inhibition indicating a requirement for metal ions.

Studies on nucleotide pyrophosphatase I. Partial purification and properties of a sheep liver enzyme that catalyzes the hydrolysis of dinucleotides

A partially purified sheep liver enzyme that hydrolyzed dinucleotides at the pyrophosphate bond was obtained by solubilizing the 18,000g sediment with n-butanol and fractionating the solubilized enzyme with acetone. The enzyme activity when measured using FAD as substrate, (FAD → FMN + AMP), was optimal at pH 9.7 and temperatures between 30 °–36 ° and at 60 °. The rate of release of FMN with time occurred with an initial lag of 30 sec, a linear increase for 1 min, and a subsequent irregular rate. In the presence of orthophosphate (Pi; 10 μImage ), FMN was released at an uniformly continuous and enhanced rate. 32Pi was not incorporated into the substrate or products. Sodium arsenate counteracted the effects of Pi. The apparent Km and Vmax were 0.133 mImage and 100 units; and 0.133 mImage and 200 units, in the absence and presence of Pi, respectively. The temperature optimum was 42 ° in the presence of Pi.Negative cooperative interactions observed at low concentrations of FAD were abolished by the addition of Pi. The inhibition by AMP was sigmoid and Pi abolished this sigmoidal response. The enzyme hydrolyzed in addition to FAD, NAD+ and NADP+. Nucleoside triphosphates were potent inhibitors of the enzyme activity. The partial inhibition of the enzyme by o-phenanthroline and by p-hydroxymercuribenzoate could be reversed by Fe2+ ions and by reduced glutathione, respectively.

Discovery of oxidative enzymes for food engineering : Tyrosinase and sulfhydryl oxidase

Enzymes offer many advantages in industrial processes, such as high specificity, mild treatment conditions and low energy requirements. Therefore, the industry has exploited them in many sectors including food processing. Enzymes can modify food properties by acting on small molecules or on polymers such as carbohydrates or proteins. Crosslinking enzymes such as tyrosinases and sulfhydryl oxidases catalyse the formation of novel covalent bonds between specific residues in proteins and/or peptides, thus forming or modifying the protein network of food. In this study, novel secreted fungal proteins with sequence features typical of tyrosinases and sulfhydryl oxidases were iden-tified through a genome mining study. Representatives of both of these enzyme families were selected for heterologous produc-tion in the filamentous fungus Trichoderma reesei and biochemical characterisation. Firstly, a novel family of putative tyrosinases carrying a shorter sequence than the previously characterised tyrosinases was discovered. These proteins lacked the whole linker and C-terminal domain that possibly play a role in cofactor incorporation, folding or protein activity. One of these proteins, AoCO4 from Aspergillus oryzae, was produced in T. reesei with a production level of about 1.5 g/l. The enzyme AoCO4 was correctly folded and bound the copper cofactors with a type-3 copper centre. However, the enzyme had only a low level of activity with the phenolic substrates tested. Highest activity was obtained with 4-tert-butylcatechol. Since tyrosine was not a substrate for AoCO4, the enzyme was classified as catechol oxidase. Secondly, the genome analysis for secreted proteins with sequence features typical of flavin-dependent sulfhydryl oxidases pinpointed two previously uncharacterised proteins AoSOX1 and AoSOX2 from A. oryzae. These two novel sulfhydryl oxidases were produced in T. reesei with production levels of 70 and 180 mg/l, respectively, in shake flask cultivations. AoSOX1 and AoSOX2 were FAD-dependent enzymes with a dimeric tertiary structure and they both showed activity on small sulfhydryl compounds such as glutathione and dithiothreitol, and were drastically inhibited by zinc sulphate. AoSOX2 showed good stabil-ity to thermal and chemical denaturation, being superior to AoSOX1 in this respect. Thirdly, the suitability of AoSOX1 as a possible baking improver was elucidated. The effect of AoSOX1, alone and in combi-nation with the widely used improver ascorbic acid was tested on yeasted wheat dough, both fresh and frozen, and on fresh water-flour dough. In all cases, AoSOX1 had no effect on the fermentation properties of fresh yeasted dough. AoSOX1 nega-tively affected the fermentation properties of frozen doughs and accelerated the damaging effects of the frozen storage, i.e. giving a softer dough with poorer gas retention abilities than the control. In combination with ascorbic acid, AoSOX1 gave harder doughs. In accordance, rheological studies in yeast-free dough showed that the presence of only AoSOX1 resulted in weaker and more extensible dough whereas a dough with opposite properties was obtained if ascorbic acid was also used. Doughs containing ascorbic acid and increasing amounts of AoSOX1 were harder in a dose-dependent manner. Sulfhydryl oxidase AoSOX1 had an enhancing effect on the dough hardening mechanism of ascorbic acid. This was ascribed mainly to the produc-tion of hydrogen peroxide in the SOX reaction which is able to convert the ascorbic acid to the actual improver dehydroascorbic acid. In addition, AoSOX1 could possibly oxidise the free glutathione in the dough and thus prevent the loss of dough strength caused by the spontaneous reduction of the disulfide bonds constituting the dough protein network. Sulfhydryl oxidase AoSOX1 is therefore able to enhance the action of ascorbic acid in wheat dough and could potentially be applied in wheat dough baking.

Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia

2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dicholorocatechol. NADPH was preferred over NADH. The enzyme had Km value of 14 μImage for 2,4-dichlorophenol, and 100 μImage for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg2+ and Zn2+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity.

Indole oxygenase from the leaves of Jasminum grandiflorum

An indole oxygenase from the leaves of Jasminum grandiflorum was isolated and purified to near homogeneity. The purified enzyme system catalyses the conversion of indole to anthranilic acid. It is optimally active at pH 4.8 and at 30°C. Apart from indole, the oxygenase also attacks 5-hydroxy indole and 5-bromoindole. Both sulfhydryl reagents and sulfhydryl compounds inhibited the enzyme activity. Copper specific metal chelators such as salicylaldoxime, diethyl dithiocarbamate and neocuproine, inhibited the enzyme activity drastically. Inhibition caused by atebrine, could be reversed by FAD. Dialysis resulted in complete loss of enzyme activity. Inactive enzyme could be reactivated only by the addition of both FAD and Cu2+, suggesting that indole oxygenase is a cuproflavoprotein.

Substrate-mediated purification and characterization of a 3-hydroxybenzoic acid-6-hydroxylase from Micrococcus

3-Hydroxybenzoic acid-6-hydroxylase from Micrococcus sp. was purified to homogeneity in a single step using the substrate-mediated interaction of the enzyme with blue-Sepharose. The enzyme was bound to the affinity matrix in the presence of 3-hydroxybenzoic acid and was eluted in its absence. The molecular weight of the purified enzyme is 70,000 with no subunit structure. The flavoenzyme required the exogenous addition of FAD for its complete activity and had a strict preference for NADH over NADPH. The activity of the enzyme was drastically inhibited by Cu2+ and Hg2+ and the inhibition was reversed by thiol reagents.

p-Hydroxyphenylacetate-3-hydroxylase. A two-protein component enzyme

p-Hydroxyphenylacetate-3-hydroxylase, an inducible enzyme isolated from the soil bacterium Pseudomonas putida, catalyzes the conversion of p-hydroxyphenylacetate to 3,4-dihydroxyphenylacetate. The enzyme requires two protein components: a flavoprotein and a colorless protein referred to as the coupling protein. The flavoprotein alone in the presence of p-hydroxyphenylacetate and substrate analogs catalyzes the wasteful oxidation of NADH with the stoichiometric generation of H2O2. A 1:1 complex of the flavoprotein and coupling protein is required for stoichiometric product formation. Such complex formation also eliminates the nonproductive NADH oxidase activity of the flavoprotein. A new assay measuring the product formation activity of the enzyme was developed using homoprotocatechuate-2,3-dioxygenase, as monitoring the oxidation of NADH was not sufficient to demonstrate enzyme activity. The coupling protein does not seem to have any redox center in it. Thus, this 2-component flavin hydroxylase resembles the other aromatic hydroxylases in that the only redox chromophore present is FAD.

In vitro and in vivo regulation of assimilatory nitrite reductase from Candida utilis

The nitrate assimilation pathway in Candida utilis, as in other assimilatory organisms, is mediated by two enzymes: nitrate reductase and nitrite reductase. Purified nitrite reductase has been shown to be a heterodimer consisting of 58- and 66-kDa subunits. In the present study, nitrite reductase was found to be capable of utilising both NADH and NADPH as electron donors. FAD, which is an essential coenzyme, stabilised the enzyme during the purification process. The enzyme was modified by cysteine modifiers, and the inactivation could be reversed by thiol reagents. One cysteine was demonstrated to be essential for the enzymatic activity. In vitro, the enzyme was inactivated by ammonium salts, the end product of the path way, proving that the enzyme is assimilatory in function. In vivo, the enzyme was induced by nitrate and repressed by ammonium ions. During induction and repression, the levels of nitrite reductase mRNA, protein, and enzyme activity were modulated together, which indicated that the primary level of regulation of this enzyme was at the transcriptional level. When the enzyme was incubated with ammonium salts in vitro or when the enzyme was assayed in cells grown with the same salts as the source of nitrogen, the residual enzymatic activities were similar. Thus, a study of the in vitro inactivation can give a clue to understanding the mechanism of in vivo regulation of nitrite reductase in Candida utilis.

Composición quimica y digestibilidad in situ de los pastos angleton (Dichantium aristatum, poir.), Colonial (Panicum maximum, Jacq.) y Taiwan (Pennisetum purpureum, Schum)

Se determinó la digestibilidad de los pastos Angleton, Colonial y Taiwan mediante el método in situ, disponiendo para ello de tres novillos de la raza criolla Reyna cuya edad oscilaba entre 12 y 18 meses y con un peso promedio de 261 kg, los cuales estuvieron provistos de una fístula ruminal. El objetivo propuesto fue obtener y comparar los estimados cuantitativos relativos a la degradación ruminal a diferentes tiempos de incubación (24,48 y 72 horas} tanto de materia seca como de proteína bruta. Los pastos fueron cosechados a los 35 días después del rebrote y se analizaron químicamente según procedimientos de la A.O.A.C (1984) para materia seca (MS), proteína bruta (PB), extracto etéreo (EE), extracto libre de nitrógeno (ELN), fibra bruta (FB) y cenizas (C) (Weende), y según el método de Van Soest (CATIE, 1987) para fibra neutro detergente (FND)y fibra ácido detergente (FAD), así como Hemicelulosa (HC). Se incubaron 10 gr de las muestras de cada uno de los pastos en bolsas de nylon. Para analizar estadísticamente los valores de degradación obtenidos, se utilizaron análisis de varianza dentro de un DCA para determinar la significancia entre pastos en los tiempos medidos y prueba de rango múltiple de Duncan para comparar medias de los pastos dentro de cada tiempo, obteniéndose diferencias altamente significativas entre ellos (P <0.01), y al observar la separación de medias se manifestó la superioridad del Taiwan en todos los tiempos de incubación, sin embargo el Colonial, no presentó diferencias significativas con el Taiwan y el Angleton en el tiempo de 72 horas. Se concluye como resultado de este estudio, que a una edad de rebrote de 35 días, el. Taiwán es superior al Angleton y al Colonial en lo que respecta a solubilidad de materia seca v proteína bruta al mismo tiempo el Colonial. mostró superioridad ante el Angl.eton debido a su mayor solubilidad de materia seca. Las mayores degradaciones de materia seca se presentaron en el. período de 0 a 24 horas de fermentación para los tres pastos; en cambio para proteína bruta ocurrieron para el. Angleton y el Taiwán entra 1as 24 y 48 horas y para e1 Colonia1 entre O y 24 horas. En qenera1, a través de la dinámica de digestión de 1os pastos se observó la influencia negativa que ejerce proporcionalmente a su contenido, la fracción de fibra (fibra neutro detergente y fibra acido detergente).

Composición química de la biomasa verde y amonificada con diferentes niveles de urea del pasto Gamba (Andropogon gayanus), cv CIAT-621 en etapa fonológica de pansoneo, Santa Rosa, Sabana Grande, Managua

Se llevó a cabo un estudio con el objetivo de determinar el mejoramiento de la composición química de la biomasa verde y tratada con Urea como proceso de amonificación. El pasto utilizado fue el Gamba (Andropogon gayanus Kunth), cv CIAT- 621 el cual se encontraba en etapa fenológica de pansoneo. El muestreo se realizó durante el mes de noviembre del año 2009 en la Finca Santa Rosa, Universidad Nacional Agraria. Managua, Nicaragua. Los tratamiento consistieron en cuatro niveles de aplicación de Urea; 0, 1, 3 y 5 % en base al forraje verde a tratar, diluido en 0.5 lt de agua, y almacenados en bolsas de polietileno durante 21 días a temperatura ambiente. El diseño utilizado fue un DCA (Diseño completo al Azar) con tres repeticiones. Las variables de estudio para cada tratamiento fueron, porcentajes de; materia seca, proteína cruda, fibra ácido detergente (FAD), fibra neutro detergente (FND), calcio y fósforo. Se realizaron análisis de varianza (ANDEVA) y separaciones de medias, usando Duncan (P<0.05). Para el análisis estadístico las variables codificadas en porcentajes se transformaron, según, 2 arco seno p (Dos veces Arco seno de la raíz cuadrada de la proporción). Se encontró diferencias significativas (P ≤ 0.05) para las variables Proteína Cruda (PC), Fibra Ácido Detergente (FAD), Calcio y Fósforo. La PC varío de 5.24 % a 11.48 % para 0 % y 3 % de Urea respectivamente, mientras la FAD disminuyo de 56.06 % a 43.64 % para los mismos tratamientos. Los minerales evaluados presentaron una tendencia inversa con los tratamientos de Urea, incrementándose el Ca y disminuyendo el Fósforo a medida que aumentaba la dosis de Urea. La Fibra Neutro Detergente aunque no presentó diferencias estadísticas entre los distintos tratamiento fue mejorada (disminución del contenido fibroso) con el tratamiento 3 % de Urea. Se concluye que el tratamiento de 3 % de Urea es el más recomendado para la amonificación de forraje verde en etapa fenologica de pansoneo en Andropogon gayanus Kunth cv CIAT 621. Estos resultados son halagadores ya que con el uso de esta tecnología se evidencia la transformación de materiales maduros de baja o nula calidad en alimentos que provean nutrientes (Proteína – Energía y Minerales) al animal durante la época seca

Composición química de la biomasa verde y amonificada pasto Gamba (Andropogon gayanus, Kunth), cv CIAT-612, en inicio de floración, Santa Rosa, Sabana Grande, Managua, Nicaragua

Se llevó a cabo un estudio con el objetivo de determinar el mejoramiento de la composición química de la biomasa verde y tratada con Urea como proceso de amonificación. El pasto utilizado fue el Gamba (Andropogon gayanus Kunth), cv CIAT-621 el cual se encontraba en etapa fenológica de pansoneo. El muestreo se realizó durante el mes de noviembre del año 2009 en la Finca Santa Rosa, Universidad Nacional Agraria. Managua, Nicaragua. Los tratamiento consistieron en cuatro niveles de aplicación de Urea; 0, 1, 3 y 5 % en base al forraje verde a tratar, diluido en 0.5 lt de agua, y almacenados en bolsas de polietileno durante 21 días a temperatura ambiente. El diseño utilizado fue un DCA (Diseño completo al Azar) con tres repeticiones. Las variables de estudio para cada tratamiento fueron, porcentajes de; materia seca, proteína cruda, fibra ácido detergente (FAD), fibra neutro detergente (FND), calcio y fósforo. Se realizaron análisis de varianza (ANDEVA) y separaciones de medias, usando Duncan (P<0.05). Para el análisis estadístico las variables codificadas en porcentajes se transformaron, según, arco seno 2 p (Arco seno de dos veces la raíz cuadrada de la proporción). Los resultados encontrados demuestran diferencias significativas (P ≤ 0.05) para las variables Proteína Cruda (PC), Fibra Neutro y Ácido Detergente (FND, FAD), Calcio y Fósforo. La PC varío de 2.22 % a 6.07 % para 0 % y 5 % de Urea respectivamente, mientras la FND disminuyó de 78.47 % a 73.16 % para 0 y 3 % respectivamente. La FAD disminuyo de 52.46 % a 47.72 % para 0 y 3 % de inclusión de Urea. Los minerales evaluados presentaron una tendencia inversa con los tratamientos de Urea, Incrementándose el Ca desde el tratamiento testigo(0.65 %) hasta 1.24 % para 0 y 5 % de Urea. El Fósforo disminuyo de 0.21 % para el testigo a 0.14 % para 1 % de Urea.. Se concluye que el tratamiento de 3 % de Urea es el más recomendado para la amonificación de forraje verde en etapa fonológica de inicio de floración del Andropogon gayanus Kunth y que la tecnología de amonificación en verde ejerce un efecto positivo en el mejoramiento de la calidad del forraje.

Composición química de la biomasa verde del pasto Guinea (Panicum maximum, Jack), CV Colonial, con diferentes niveles de inclusión de urea. Finca Santa Rosa, sabana Grande, Managua

Se llevó a cabo un estudio con el objetivo de determinar el mejoramiento de la composición química de la biomasa verde y tratada con Urea como proceso de amonificación. El pasto utilizado fue el Guinea (Panicum maximum, Jacq) CV Colonial el cual se encontraba en etapa de plena maduración (75 días). Durante el mes de septiembre del 2011 se realizó un corte de uniformidad para proceder al muestreo durante el mes de noviembre en la Finca Santa Rosa, Universidad Nacional Agraria. Managua, Nicaragua.Los tratamiento consistieron en cuatro niveles de plicación de Urea; 0, 1, 3 y 5 % en base al forraje verde a tratar, diluido en 0.5 lt de agua, y almacenados en bolsas de polietileno durante 21 días a temperatura ambiente.El diseño utilizado fue un DCA (Diseño completo al Azar) con tres repeticiones. Las variables de estudio para cada tratamiento fueron,porcentajes de; materia seca, proteína cruda, fibra ácido detergente (FAD), fibra neutro detergente (FND) y cenizas. Se realizaron análisis de varianza (ANDEVA) y separaciones de medias, usando Tukey (P<0.05). Para el análisis estadístico las variables codificadas en porcentajes se transformaron, según, 2 arco seno p (Dos veces Arco seno de la raíz cuadrada de la proporción).Se encontró diferencias significativas (P≤ 0.05) para las variables Materia seca (MS), Proteína Cruda (PC)y, Cenizas (CEN), no encontrando diferencias estadísticas ( P > 0.05) para las variables Fibra Neutro Detergente (FND) y Fibra Ácido Detergente FAD).El porcentaje de materia seca vario desde 48.04 % hasta 24.30 % para 0 y 5 % de Urea respectivamente. La PC pasó de 3.36 % para el tratamiento sin Urea a 8.37 % cuando se aplicó 5 % de Urea mientras la CEN aumento de 7.85 % a 8.60 % para 0 y 3 % de urea. La FDN y FAD aunque no presentaron diferencias estadísticas para los tratamientos evaluados, fue mejorado (disminución del contenido fibroso) con el tratamiento 5% de Urea. Se concluye que el tratamiento de 5 % de Urea es el más recomendado para la amonificación de forraje verde del pasto guinea (Panicum maximum, Jacq) CV Colonial. Con estos resultados se confirma que con el uso de esta tecnología se evidencia la transformación de materiales maduros de baja o nula calidad en alimentos que provean nutrientes al animal durante las épocas críticas

Evaluación bromatológica del ensilaje de Guácimo de Ternero (Guázuma ulmifolia Lam.) y el pasto Cubano CT-115 (Pennisetum purpureum x P. tiphoides), bajo diferentes niveles de proporción de hojas, más melaza en la alimentación animal

El presente estudio se realizó con el objetivo de determinar la calidad bromatológica (materia seca MS, proteína bruta PB, fibra neutro detergente FND, fibra acido detergente FAD, nitrógeno amoniacal N-NH· y pH del ensilaje de Guácimo de Ternero (Guázuma ulmifolia Lam.) y el Pasto Cubano CT-115 (Pennisetum purpureum x P. tiphoides), bajo diferentes niveles de proporción de hojas, más melaza en la alimentación animal. El muestreo se realizó en la granja porcina y en los laboratorios de bromatología y microbiología de la Facultad de Ciencia Animal de la UNA. Los micros silos fueron conservados por 30 días. Los tratamientos evaluados fueron tres niveles de proporción del Guácimo de ternero y el pasto cubano CT-115, más melaza. Los tratamientos se distribuyeron en un diseño completamente al azar (DCA) con tres repeticiones. El tratamiento uno (T1) consistió 50 % de Guácimo de ternero y 50 % de pasto cubano CT- 115; el tratamiento dos (T2) en 60 % de Guácimo de ternero y 40 % de pasto cubano CT- 115; y el tratamiento tres (T3) en 70 % de Guácimo de ternero y 30 % de pasto cubano CT- 115. Los resultados de la calidad bromatológica para MS por tratamiento fueron para T3:31.02%, T2: 32.03 % y para T1: 28.01 %. La PB alcanzó valores de: 15.03 %, 13.07 %, 15.02 % para T3, T1 y T2, respectivamente. La FND presentó valores por tratamiento de T1: 63.18 %, T3: 64.80 % y T2: 70.02 %. Para los FAD, los resultados fueronT2 con 51.14 %, T3 con 52.15 % y T1 con 51.36 %; el N-NH3: T1 con 6.3 %, T2 con 6.7 % y T3 con 4.0 %; .Los resultados para el pH fueron para T1: 4.89 %, para T3: 5.22 % y para T2: 5.37 %. Con base en estos resultados, el T1 (MS) y T3 (PB) presentó mejor resultado en la calidad bromatológica y pH (T1), superando al resto de los tratamientos, aunque obtuvo mejor resultado el T1 (FND) y para el caso de FAD el (T2). El uso del Guácimo de ternero y el pasto cubano CT-115 y melaza en el ensilaje mejora su calidad bromatológica.

Pipeline Corrosion Analyzed by Fuzzy Reliability Methods

Fuzzy reliability methods are used to study the corrosion of pipelines. Three methods are used. They consist of using fracture failure modes, failure assessment diagram (FAD) and residual strength for establishing fuzzy reliability. Calculations are made by application of JC, improved GA-JC and Mente-carlo methods. Examples for oilfield injecting water pipeline show the residual strength well agree with field data. Mente-carlo methods appear to yield results that have better agreement with field data.