Purification and characterization of phytase with a wide pH adaptation from common edible mushroom Volvariella volvacea (Straw mushroom).

A novel phytase with a molecular mass of 14 kDa was isolated from fresh fruiting bodies of the common edible mushroom Volvariella volvacea (Straw mushroom). The isolation procedure involved successive chromatography on DEAE-cellulose, CM-cellulose, Affi-gel blue gel, Q-Sepharose and Superdex-75. The enzyme was a monomeric protein and was unadsorbed on DEAE-cellulose, CM-cellulose and Affi-gel blue gel, but was adsorbed on Q-Sepharose. The enzyme was purified 51.6-fold from the crude extract with 25.9% yield. Its N-terminal amino acid sequence GEDNEHDTQA exhibited low homology to the other reported phytases. The optimal pH and temperature of the purified enzyme was 5 and 45oC, respectively. The enzyme was quite stable over the pH range of 3.0 to 9.0 with less than 30% change in its activity, suggesting that it can be used in a very wide pH range. The enzyme exhibited broad substrate selectivity towards various phosphorylated compounds, but lacked antifungal activity against tested plant pathogens.

Purification and characterization of Mn-peroxidase from Musa paradisiaca (banana) stem juice.

Mn-peroxidase (MnP), a biotechnologically important enzyme was purified for the first time from a plant source Musa paradisiaca (banana) stem, which is an agro-waste easily available after harvest of banana fruits. MnP was earlier purified only from the fungal sources. The enzyme was purified from stem juice by ultrafiltration and anion-exchange column chromatography on diethylamino ethylcellulose with 8-fold purification and purification yield of 65%. The enzyme gave a single protein band in SDS-PAGE corresponding to molecular mass 43 kDa. The Native-PAGE of the enzyme also gave a single protein band, confirming the purity of the enzyme. The UV/VIS spectrum of the purified enzyme differed from the other heme peroxidases, as the Soret band was shifted towards lower wavelength and the enzyme had an intense absorption band around 250 nm. The Km values using MnSO4 and H2O2 as the substrates of the purified enzyme were 21.0 and 9.5 μM, respectively. The calculated kcat value of the purified enzyme using Mn(II) as the substrate in 50 mM lactate buffer (pH 4.5) at 25°C was 6.7s-1, giving a kcat/Km value of 0.32 μM-1s-1. The kcat value for the MnP-catalyzed reaction was found to be dependent of the Mn(III) chelator molecules malonate, lactate and oxalate, indicating that the enzyme oxidized chelated Mn(II) to Mn(III). The pH and temperature optima of the enzyme were 4.5 and 25°C, respectively. The enzyme in combination with H2O2 liberated bromine and iodine in presence of KBr and KI respectively. All these enzymatic characteristics were similar to those of fungal MnP. The enzyme has the potential as a green brominating and iodinating agent in combination with KBr/KI and H2O2.

Extraction and purification of acidic polysaccharide from Moerella iridescens / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To optimize extraction and purification methods of acidic polysaccharide from Moerella iridescens (MIAP).</p><p><b>METHODS</b>With alkali extraction process and orthogonal experiment,the time consumption,temperature,pH value of the solution and alcohol concentration during the extraction were optimized. The crude products were deprived of protein,pigment and ion,then were purified with DEAE-cellulose ion-exchange chromatography and verified with Sephadex G-100 and cellulose acetate membrane electrophoresis,and examined with infrared spectrum.</p><p><b>RESULTS</b>The optimized extraction conditions were as follows: extraction time 6 h,extraction temperature 70 degree,the solution pH 8.0 and the concentration of alcohol precipitation 70%. Intuitive features showed that the MIAP was pure white crystalline granular with slight dark brown color. The purification results demonstrated that the target MIAP was eluted and identified as a homogeneous components by DEAE-cellulose ion exchange column,Sephadex G-100 and cellulose acetate membrane electrophoresis. Infrared spectral scanning suggested that MIAP was α-D-type terminated glucopyranose. Intuitive features showed that MIAP was soft and cottony white.</p><p><b>CONCLUSION</b>The extraction process with orthogonal test has been optimized and the acidic polysaccharide from Moerella iridescens is successfully isolated.</p>

Purification and Characterization of Intracellular Cellulase from Aspergillus oryzae ITCC-4857.01

Purification and characterization of intracellular cellulase produced by A. oryzae ITCC-4857.01 are reported. The enzyme was purified by ion-exchange chromatography using DEAE-cellulose followed by Gel filtration. The purification achieved was 41 fold from the crude extract with yield of 27%. The purified enzyme showed single band on poly acrylamide gel. The molecular weight as determined by SDS-PAGE and gel filtration was 38 KDa and 38.6 KDa respectively and contained only one subunit. The enzyme is glycoprotien as nature and contained 0.67% neutral sugar. The apparent Km value of the enzyme against cellulose was 0.83%. The enzyme showed the highest relative ativities on CMC followed by avicel, salicin and filter paper. The optimum pH of activity was 5.5 and very slight activity was observed at or above pH 7.5 as well as bellow pH 3.5. The optimum tempreture of the activity was 45degrees C and the highest activity was exhibited in 35 to 45degrees C. The enzyme lost their activities almost completely (95~100%) at 80 degrees C or above and as well as bellow 25degrees C.

Purification and characterization of two alpha-amylases from wheat

Using chromatography on DEAE-cellulose, four forms, A1, A2, A3 and A4 of germinated wheat Sakha 8 alpha-amylases were demonstrated. alpha-Amylases A2 and A3 were purified to homogencity by Sephacryl S-200 column and the homogeneity proved by polyacrylamide gel electrophoresis. The molecular masses of alpha-amylases A2 and A3 were 29,000 and 31,000, respectively. The Km values of alpha-amylases A2 and A3 were 7.8 and 5.0 mg starch/ml, respectively. The affinity between substrate and the two enzymes decreased in the order of potato starch > glycogen > starch > corn starch for alpha-amylase A2 and glycogen > potato starch > starch > corn starch for alpha-amylase A3. The two enzymes were found to have the same pH and temperature optima of 7.5 and 50°C, and were heat stable up to 50°C. The effect of metal ions showed that Ca[+2], Co[+2] and Mg[+2] caused activating effects on alpha-amylases A2 and A3, while Cd[+2], Fe[+2], Cu[+2], Ni[+2] and Hg[+2] caused partial or strong inhibition effects. The three metal chelators, EDTA, citric and oxalic acids, have the same pattern of inhibition toward alpha-amylases A2 and A3, where 1 mM of these chelators caused 50% inhbiition of amylolytic activity and most enzyme activity was lost at 20 mM. This information will have a bearing on their potential use in the food industries

Catalytic and regulatory properties of sulphur metabolizing enzymes in cyanobacterium Synechococcus elongatus PCC 7942.

Synechococcus elongatus PCC 7942 was able to grow with several S sources. The sulphur metabolizing enzymes viz. ATP sulphurylase, cysteine synthase, thiosulphate reductase and L- and D-cysteine desulphydrases were regulated by sulphur sources, particularly by sulphur amino acids and organic sulphate esters. Sulphur starvation reduced ATP sulphurylase and cysteine synthase whereas reduced glutathione appreciated Cys degradation activity. With partially purified enzymes apparent Km values for sulphate, ATP, D- and L-Cys, thiosulphate, sulphide and O-acetyl serine were in a range of 12-50 microM. p-Nitrophenyl sulphate inhibited ATP sulphurylase competitively. Met was a feedback inhibitor of several key enzymes.

Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L) root membranes

In mammals, hexokinase (HK) is strategically located at the outer membrane of mitochondria bound to the porin protein. The mitochondrial HK is a crucial modulator of apoptosis and reactive oxygen species generation. In plants, these properties related to HK are unknown. In order to better understand the physiological role of non-cytosolic hexokinase (NC-HK) in plants, we developed a purification strategy here described. Crude extract of 400 g of maize roots (230 mg protein) contained a specific activity of 0.042 æmol G6P min-1 mg PTN-1. After solubilization with detergent two fractions were obtained by DEAE column chromatography, NC-HK 1 (specific activity = 3.6 æmol G6P min-1 mg PTN-1 and protein recovered = 0.7 mg) and NC-HK 2. A major purification (yield = 500-fold) was obtained after passage of NC-HK 1 through the hydrophobic phenyl-Sepharose column. The total amount of protein and activity recovered were 0.04 and 18 percent, respectively. The NC-HK 1 binds to the hydrophobic phenyl-Sepharose matrix, as observed for rat brain HK. Mild chymotrypsin digestion did not affect adsorption of NC-HK 1 to the hydrophobic column as it does for rat HK I. In contrast to mammal mitochondrial HK, glucose-6-phosphate, clotrimazole or thiopental did not dissociate NC-HK from maize (Zea mays) or rice (Oryza sativa) mitochondrial membranes. These data show that the interaction between maize or rice NC-HK to mitochondria differs from that reported in mammals, where the mitochondrial enzyme can be displaced by modulators or pharmacological agents known to interfere with the enzyme binding properties with the mitochondrial porin protein.

Purification and characteristics of creatininase from Arthrobacter sp / 生物工程学报

A creatininase produced from a Arthrobacter sp. was purified 145-fold by a series of steps including heat treatment, ammonium sulfate precipitation, DEAE-Cellulose ion-exchange and hydrophobic chromatography. The specific activity of the pure enzyme was 209u/mg. The subunit molecular mass of creatininase was estimated to be 33 700D by SDS-PAGE. The creatininase was stable in the pH range between 6.0 - 9.0 and below 60 degrees C . Its Km value for creatinine was estimated to be 21.14 mmol/L. The enzyme was markedly inactivated by incubation with 1 mmol/L of Hg2+, Ag2+, Li+, Cu2+ and 20 mmol/L of 1, 11-Phananthroline respectively. Activation was observed when the enzyme was incubated with 1 mmol/L of Co2+ and Mn2+.

Purification and characterization of bacillus cereus cellulase using semi-solid state fermentation

Cell free extract of Bacillus cereus was fractionated by 50% ammonium sulphate resulting in 22.5% protein reduction, and purified by 133.3 fold using chromatography on DEAE-Sephadex A50 and CM-Sephadex C[50]. The pure enzyme consists of one peptide chain with molecular weight of 58,000 Dalton. The enzyme was found to be rich in glycine, glutamic, serine and alanine. The apparent K[m] and V[max] values of the purified enzyme were 2.56 mg/ml and 91.74U/ml respectively. Maximum cellulase activity was observed when incubated with CMC [carboxymethyl cellulose] at pH 7 for 30 min. at 45°C

Expression of cre gene in Escherichia coli and bioassay its expression product / 生物工程学报

The Cre recombinase from bacteriophage P1 can recognize specific DNA sequences, cleave DNA at specific target sites, and then ligate it to the cleaved DNA of a second site. In this study, cre gene was cloned into the pGEM-T Easy vector via PCR procedure. Then the cre gene was inserted into an expression vector pET-29a and expressed in E. coli BL21 (DE3). A 38 kD soluble protein was expressed and named CRE. CRE was purified by DEAE-52 chromatography. Bioassay of the partially purified product showed that CRE can cleave the plasmid pGLGFP which contains two loxP site with the same direction.

Purification and characterization of bacterial CM-cellulase from camel rumen fluid [Camelus dromedarius]

Bacterial CM-cellulase [1,4-beta-D-glucanohydrolase, [EC 3.2.1.4]] from camelrumen fluid, an enzyme that causes a random secession of cellulose chainyielding glucose and cellotriose, was purified and characterized. Thepurification procedure included ammonium sulfate precipitation andchromatographies on DEAE-cellulose, and Sepharose 6B columns. By ion exchangeon DEAE-cellulose, five isoenzymes of CM-cellulase were obtained. CM-cellulase CII, CIV and CV were purified 11.2, 13.9 and 25.9 fold with 17.3%, 12.5% and 13.7% recovery in activities, respectively. The molecularweights of CM-cellulase CII, CIV and CV were estimated to be 12000, 13000 and 14600, respectively, for the native and 13000 for the denatured enzymes,respectively, suggesting that isoforms are monomeric. Amino acid compositionof the three CM-cellulases were detected. CM-cellulases CII, CIV and CV hadisoelectric points at 5.1, 6.6 and 5.1 and km values of 8.3, 6.25 and 5 mgCM-cellulase/ml, respectively, with more affinity toward CM-cellulase. CM-cellulases CII and CV had similar temperature optima at 40C, while themaximum activity for cellulase CIV was at 50C and had identical pH optimaat 5.5. The effect of divalent metal cations and different inhibitor on thethree isoenzyme activities was examined

Isolation and characterization of NADP+-linked isocitrate dehydrogenase of germinating pea seeds (Pisum sativum).

NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective.

Purification and properties of antiviral proteins from the leaves of Bougainvillea xbuttiana.

A non-phytotoxic, resistance inducing, proteinaceous antiviral principle was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration from the leaves of Bougainvillea xbuttiana. It imparted resistance against tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV) in their respective test hosts viz. Nicotiana glutinosa, N. tabacum var. Samsun NN, and Cyamopsis tetragonoloba, respectively. The purified principle eluted as a single peak upon gel filtration, but exhibited two polypeptides on SDS-PAGE with Mr 28,000 and 24,000. The two polypeptides were found to be highly basic, rich in lysine with pI around 10.0 and 10.5, respectively. Since this principle effected local lesion inhibition in both treated and untreated top leaves of test host, it might be acting in the initial stages of virus infection as a systemic inducer.

Purificación del componente c1s del sistema complemento y obtención de un antisuero específico / Purification of the c1s component of the complement system and obtention of a specific antiserum

Se describe un método de purificación del componente C1s del sistema complemento a partir de una mezcla de sueros normales mediante cromatografías secuenciales en DEAE-celulosa, TEAE-celulosa y Sephacryl S-200. La presencia de C1s en las fracciones eluídas en las diferentes corridas cromatográficas se detectó por nefelometría. La pureza del C1s obtenido se determinó por inmunoelectroforesis contra un suero antiproteínas séricas humanas y un antisuero específico. A partir del C1s purificado se produjo en conejos un antisuero de buena calidad, útil para la cuantificación del C1s sérico

Immunological assay after vaccination of goats with brucella melitensis rev.I and brucella abortus 45/20 vaccines at Saudi Arabia

The living smooth Brucella melitensis Rev. I vaccine given in the normal dose [7x10[8] organism] to goats 3 to 5 months of age stimulated a marked increase in agglutinin titer. Fractionation of pools of serum by gel filtration by anion exchange chromatography, using diethylaminoethyl [DEAE] cellulose showed that both IgM and IgG agglutinins were present from 12 to 47 days after goats were vaccinated. Only mercaptoethanol [ME]- sensitive agglutinins were detected in most goats 4 months after vaccination, but 2 of 30 goats retained ME- resistant agglutinins for the 13 1/2 - month observation period. Fractionation of serums from goats representative of these 2 types of serologic response indicated that most goats had only IgM agglutinins whereas the 2 given goats had activity in the IgG fraction. Adult goats given Brucella abortus 42/20 adjuvant vaccine and revaccinated 5 months later developed low agglutination titers to smooth antigen, and all became test Positive to the antiglobulin test. Fractionation of serum of pools taken 1 week and 8 months after revaccination indicated that antibody activity was restricted to the IgG fraction. Sera from 5 vaccinated and nonvaccinated goats which were Positive by bacteriological culture examination at necropsy 31 to 47 days after goats were given conjunctival inoculation of virulent B. melitensis had antibody activity in both IgG and IgM fractions Test Positive reaction to the ME, complement-fixation [CF], and antiglobulin [AG] test were restricted to the IgG-containing fractions of serum whereas reactions to the agglutination [STT] and the card tests appeared with either or both fractions. The effect of these findings on the choice of tests for the differentiation of vaccination response is discussed

Purification, characterisation and steady state kinetic properties of cytosolic pyruvate kinase free of phosphoenol pyruvate phosphatase activity from germinating mung beans (Vigna radiata L.)

Mung bean pyruvate kinase (PK) practically free from PEP-phosphatase has been purified about 36 fold. The enzyme is irreversibly inactivated on desalting by gel filtration or dialysis (without EDTA). The inactivation is also observed in the presence of ATP, Mg2+ or thiols but is prevented by a non-proteinous, heat stable, small molecular mass factor present in the mung bean extract. Mung bean PK has a molecular mass of 210 kDa. It shows single exponential decay of activity at various temperatures (-4 to 60 degrees C). The Km of PEP and ADP are found to be 0.12 and 0.24 mM, respectively at pH 6.5, when the enzyme is saturated with the second substrate. The Km values for PEP and ADP are 0.05 and 0.16 mM, at pH 8.5 and 0.09 and 0.17 mM, respectively at pH 7.5. The optimum pH is 7.5. The enzyme shows an absolute requirement for Mg2+ (Km 0.43 mM) or Mn2+ ions (Km 0.125 mM). Potassium ions are not essential but activate the enzyme in the presence of Mg2+ or Mn2+ ions. ATP shows competitive inhibition with ADP and non-competitive with PEP. Kinetic studies at different pHs and effects of ATP suggest the formation of a ternary complex (E.ADP.PEP) by a combination of random and compulsory ordered pathways depending on the experimental conditions.

Detección de aciduria metilmalónica mediante pruebas de tamizaje metabólico / Detection of methylmalonic aciduria by means of metabolic screening

La búsqueda de aciduria metilmalónica es uno de los procedimientos en el Programa de Detección de Errores Congénitos del Metabolismo que se realiza en varias unidades del IMSS en Monterrey, Nuevo León, para estudiar muestras procedentes de personas con sospecha de enfermedad metabólica genética. De un total de 2100 exámenes realizados en pacientes pediátricos, cinco resultaron positivos mediante la prueba del color y en todos ellos se corroboró la presencia del ácido metilmalónico mediante cromatografía A1 de celusosa. Aunque no se obtuvo diagnóstico de precisión, es probable que más de una forma del padecimiento exista en nuestro medio. Debido a la gravedad de este defecto, a la posibilidad del manejo médico y la conveniencia del asesoramiento genético en las familias portadoras, se justifica este tipo de detección en las unidades pediátricas o de atención materno-infantil.

Obtención, purificación y caracterización de dos formas de hormona luteinizante de la adenohipófisis caprina (gLH) / Obtention, purification and characterization of two forms of caprine luteinizing hormone from adenohypophsis (gLH)

Se describe la obtención, purificación y caracterización de dos proteínas hipofisiarias de origen caprino con carga eléctrica diferente y con características de hormona luteinizante (LH). La fracción no retenida en la cromatografía de intercambio catiónico (CM-celulosa) designada CM-lab y la correspondiente al segundo pico de dicha cromatografía (CM-2ab), se repurificaron en una cromatografía de intercambio aniónico (DEAE-celulosa) en condiciones idénticas, para obtener LH-I (CM-lab-DEAE-la) con un rendimiento de 76 mg/kg de adenohipófisis, y a la LH-II (CM2ab-DEAE-lb) con 15 mg/kg. La diferencia de carga de cada proteína se determinó por su movilidad relativa (Rf) mediante una electroforesis en geles de poliacrilamida (PAGE) en condiciones nativas. La LH-I mostró un Rf de 0.09 (banda mayoritaria) y 0.415, mientras la LH-II presentó 2 bandas de tipo catiónico con un Rf de 0.14, 0.19 (banda mayoritaria). La determinación del peso molecular relativo de LH-I y LH-II, se llevo a cabo por medio de una electroforesis en geles de poliacilamida-dodecilsulfato de sodio (SDS-PAGE). En condiciones no reductoras (NR), ambas proteínas presentaron un peso molecular relativo de 36.5 kilodaltones (KDa) correspondiente a la forma monomérica, semejante a los estándares NIDDK-oLH-26 y USDA-bLH5, mientras que en presencia de 2ß-mercaptoetanol (condiciones reducidas), las proteínas presentaron un peso molecular de 22.4 y 20.2 KDa. El análisis por inmunotransferencia (Western-blot) en condiciones NR utilizando el anti-oLH (CSU-204), permitió identificar bandas inmunorreactivas de peso molecular de 50,43, 36.5 (mayoritaria), y 22.4 KDa, tanto en los estándares como en las fracciones obtenidas en este estudio. La potencia biológica realizada en un bioensayo in vivo 3 + 3 balanceado fue de 1.03 UI/mg para la LH-I. y de 0.65 UI/mg para la LH-II con intervalos de confianza de 95 por ciento. Su actividad inmunológica se determinó con un radioinmunoensayo (RIA) homólogo de LH caprina, a la dosis esperada del 50 por ciento (ED 50). Los resultados fueron de 1.7 ng/ml y 3.5 ng/ml para LH-I y LH-II respectivamante. Se concluye que esta técnica permite la obtención de dos proteínas con carga eléctrica diferente, con actividad biológica e inmunológica de LH, y con peso molecular idéntico

Purification of Inositol Triphosphate Kinase from Bovine Brain / 영남의대학술지

Inositol 1,4,5-triphosphate(InsP,) is a second messenger for obilizing intracellular Cal'. It can be dephosphorylated by soluble and particulate forms on InsP, 5-phosphatase, or phosphorylated to produce inositol 1,3,4,5-tetrakisphosphate(InsP,) by InsP, 3-kinase. These enzymes represent possible targets for the regulation of the InsP,AnsP. signal. InsP, 3-kinase which catalyses th ATP-dependent phosphorylation of InsP, was purified from bovine brain tissue. All operation were carried out at 41C. Fresh tissure was homogenized and centrifuged. The supernatant was pooled. Proteins were precipitated from 10% polyethylene glycol, and suspended solution was applied to DEAE cellulose column for chromatography. As the result of above procedure, two isozymes of InsP, 3-kinase, I and U were obtained. Each isozyme was applied to Matriz green gel, Calmodulin-Affigel 15 column and subsequent phenyl-TSK HPLC column. Specific activites(SA) and fold of puriety were observed at each purification step of chromatography. At DEAE cellulose chromatography, SA were 1, 0.6 and 11, 4.8 nM/min/mg, and folds

Porphyrinogen carboxylyase. Studies on existence of isoenzymes

Porphyrinogen carboxylyase from normal rat liver was subjected to purification methods. Two different purification protocols were used. In both cases, the inital steps consisted in obtaining a liver homogenate, followed by centrifugation, salt precipitation and phosphate gel absorption. Scheme I consisted in then submiting the preparation to DEAE-cellulose, followed by Sephacryl S-200 and Phenyl-sepharose sequential column chromatographies. Scheme II involved an affinity column followed by a Sephadex G-75 gel filtration column. In both cases, the enzyme was stored at-20 degrees Celsius until its assay. The addition of 2mM dithiotreytol to the incubation media or to the enzyme extract before storage, did not help improve the activity nor the stability of the enzyme. Those fractions containing the maximal enzyme activity, detected using Uroporphyrinogen III or Pentacarboxy-porphyrinogen III as substrate, were not alawys present in the same tubes for the different columns employed. In addition, the degree of purification obtained in some steps was different according to the substrate employed. The results suggest the existence of at least two isoenzymes for rat liver porphyrinogen carboxy-lyase.