A purification of Helix pomatia alpa amylase involving a novel affinity-binding procedure

Shellfish glycogen was cross-linked by treatment with cyanogen bromide followed by 1,6-diaminohexane. The resulting, insoluble product efficiently adsorbed Helix pomatia alpha amylase [(1 → 4)-α-D-glucan glucanohydrolase] from crude solutions of the enzyme at 0°, but only poorly at higher temperatures. A method was developed for the purification of Helix pomatia alpha amylase involving formation of an enzyme-adsorbent complex in the cold and recovery of the alpha amylase by suspending the washed complex in buffer at 37°. After chromatography of the desorbed alpha amylase on a column of Bio-Gel P-60, the enzyme was homogenous as judged by poly(acrylamide)-gel electrophoresis. An overall purification of 360-fold was achieved with a recovery of 35%.     

Preparation of a new thermo-responsive adsorbent with maltose as a ligand and its application to affinity precipitation

A thermo-responsive polymer on which maltose was covalently immobilized as an affinity ligand was newly synthesized for purification of thermolabile proteins from the crude solution by affinity precipitation. Among the thermo-responsive polymers synthesized as carriers for adsorbent, poly(N-acryloylpiperidine)-cysteamine (pAP) has a lower critical solution temperature (LCST) of around 4 degrees C, at which its solubility exhibits a sharp change. Adsorbent for affinity precipitation was prepared by combining pAP with maltose using trimethylamine-borane as a reducing reagent. This adsorbent (pAPM) obtained showed a good solubility response: pAPM in the basal buffer (pH 7.0) became soluble below 4 degrees C and was completely insoluble above 8 degrees C. The affinity precipitation method using pAPM consisted of the following four steps: adsorption at 4 degrees C, precipitation of the complex at 10 degrees C, desorption by adding the desorption reagent at 4 degrees C, and recovery of a target protein at 10 degrees C. In the affinity precipitation of Con A from the crude extract of jack bean meal, 82% of Con A added was recovered with 80% purity by addition of 0.2 M methyl-alpha-D-mannopyranoside as a desorption reagent. In the repeated purification of Con A from the crude extract, pAPM could be satisfactorily reused without decrease in the affinity performance. Moreover, when pAPM was used for the purification of thermolabile alpha-glucosidase from the cell-free extract of Saccharomyces cerevisiae, 68% of total activity added was recovered and the specific activity per amount of protein of the purified solution was enhanced 206-fold higher than that of the cell-free extract without thermal deactivation of the enzyme.     

Enzymatic activities and protein profile of latex from Calotropis procera.

The laticifer fluid of Calotropis procera is rich in proteins and there is evidence that they are involved in the pharmacological properties of the latex. However, not much is known about how the latex-containing proteins are produced or their functions. In this study, laticifer proteins of C. procera were pooled and examined by 1D and 2D electrophoresis, masses spectrometry (MALDI-TOF) and characterized in respect of proteolytic activity and oxidative enzymes. Soluble laticifer proteins were predominantly composed of basic proteins (PI>6.0) with molecular masses varying between 5 and 95 kDa. Proteins with a molecular mass of approximately 26,000 Da were more evident. Strong anti-oxidative activity of superoxide dismutase (EC 1.15.1.1) (1007.74+/-91.89 Ug(-1)DM) and, to a lesser extent ascorbate peroxidase (EC 1.11.1.1) (0.117(d)+/-0.013 microMol H(2)O(2)g(-1)min(-1)), were detected. However, catalase (EC 1.11.1.6) was absent. The strong proteolytic activities of laticifer proteins from C. procera were shown to be shared by at least four distinct cysteine proteinases (EC 3.4.22.16) that were isolated by gel filtration chromatography. Serine and metaloproteinases were not detected and aspartic proteinase activities were barely visible. Chitinases (EC 3.2.1.14) were also isolated in a chitin column and their activities quantified. The presence of these enzymatic activities in latex from C. procera may confirm their involvement in resistance to phytopathogens and insects, mainly in its leaves where the latex circulates abundantly.     

Latex constituents from Calotropis procera (R. Br.) display toxicity upon egg hatching and larvae of Aedes aegypti (Linn.)

Calotropis procera R. Br. (Asclepiadaceae) is a well-known medicinal plant with leaves, roots, and bark being exploited by popular medicine to fight many human and animal diseases. This work deals with the fractionation of the crude latex produced by the green parts of the plant and aims to evaluate its toxic effects upon egg hatching and larval development of Aedes aegypti. The whole latex was shown to cause 100% mortality of 3rd instars within 5 min. It was fractionated into water-soluble dialyzable (DF) and non-dialyzable (NDF) rubber-free materials. Both fractions were partially effective to prevent egg hatching and most of individuals growing under experimental conditions died before reaching 2nd instars or stayed in 1st instars. Besides, the fractions were very toxic to 3rd instars causing 100% mortality within 24 h. When both fractions were submitted to heat-treatment the toxic effects were diminished considerably suggesting low thermostability of the toxic compounds. Polyacrylamide gel electrophoresis of both fractions and their newly fractionated peaks obtained through ion exchange chromatography or desalting attested the presence of proteins in both materials. When submitted to protease digestion prior to larvicidal assays NDF lost most of its toxicity but DF was still strongly active. It may be possible that the highly toxic effects of the whole latex from C. procera upon egg hatching and larvae development should be at least in part due to its protein content found in NDE However the toxicity seems also to involve non protein molecules present in DF.     

Characteristics of the amylase of Arthrobacter psychrolactophilus

Properties of the extracellular amylase produced by the psychrotrophic bacterium, Arthrobacter psychrolactophilus, were determined for crude preparations and purified enzyme. The hydrolysis of soluble starch by concentrated crude preparations was found to be a nonlinear function of time at 30 and 40 °C. Concentrates of supernatant fractions incubated without substrate exhibited poor stability at 30, 40, or 50 °C, with 87% inactivation after 21 h at 30 °C, 45% inactivation after 40 min at 40 °C and 90% inactivation after 10 min at 50 °C. Proteases known to be present in crude preparations had a temperature optimum of 50 °C, but accounted for a small fraction of thermal instability. Inactivation at 30, 40, or 50 °C was not slowed by adding 20 mg/ml bovine serum albumin or protease inhibitor cocktail to the preparations or the assays to protect against proteases. Purified amylase preparations were almost as thermally sensitive in the absence of substrate as crude preparations. The temperature optimum of the amylase in short incubations with Sigma Infinity Amylase Reagent was about 50 °C, and the amylase required Ca⁺² for activity. The optimal pH for activity was 5.0–9.0 on soluble starch (30 °C), and the amylase exhibited a K _m with 4-nitrophenyl-α-D-maltoheptaoside-4,6-O-ethylidene of 120 μM at 22 °C. The amylase in crude concentrates initially hydrolyzed raw starch at 30 °C at about the same rate as an equal number of units of barley α-amylase, but lost most of its activity after only a few hours.     

Histamine mediates the pro-inflammatory effect of latex of Calotropis procera in rats

INTRODUCTiON: Calotropis procera is known to produce contact dermatitis and the latex of this plant produces intense inflammation when injected locally. However, the precise mode of its pro-inflammatory effect is not known. In present study we have pharmacologically characterized the inflammation induced by latex of C. procera in a rat paw edema model and determined the role of histamine in latex-induced inflammation. METHODS: Inflammation was induced in the hind paw of rats by injecting different doses of dried latex (DL) of C. procera. The inhibitory effect of phenylbutazone, dexamethasone, celecoxib, cyproheptadine, chlorpheniramine and compound 48/80 on edema volume was evaluated and compared with that against carrageenan. The histamine content of DL was measured fluorometrically. RESULTS: DL produced dose-dependent inflammation of the rat paw. Cyproheptadine and chlorpheniramine effectively inhibited DL-induced inflammation (90%; p < 0.01), while anti-inflammatory drugs phenylbutazone, dexamethasone and celecoxib were more effective against carrageenan-induced inflammation. Depletion of mast cell histamine by compound 48/80 produced a significant decrease in DL-induced inflammation as compared with carrageenan (500% versus 25%). DL was also found to contain about 6 microg/g of histamine. CONCLUSIONS: Thus, our study shows that the biogenic amines play a significant role in C. procera latex-induced inflammation and antihistaminic drugs could be effectively used to inhibit inflammatory response elicited by exposure to latex.     

Three phase partitioning as a novel method for purification of ragi (Eleusine coracana) bifunctional amylase/protease inhibitor

The technique of three-phase partitioning (TPP) was used to purify a bifunctional amylase/protease inhibitor from ragi (Eleusine coracana). This process of purification is a potential method used for separation of proteins directly from large volumes of crude suspension. It involves the addition of a salt (ammonium sulphate) to the crude extract followed by the addition of an organic solvent (t-butanol). The addition of t-butanol, in the presence of ammonium sulphate pushes the protein out of the solution to form an interfacial precipitate layer between the lower aqueous and upper organic layers. The process was carried out in two steps. The various conditions required for attaining efficient purification of the protein fractions were optimized. It was seen that 30% ammonium sulphate saturation with 1:1 ratio of crude extract to tert-butanol gave 8.9- and 8.65-fold purification with 83% and 80% yield of amylase inhibitor and trypsin inhibitor, respectively, in step I. In TPP-step II, 60% ammonium sulphate saturation and ratio of aqueous phase to t-butanol of 1:2 gave maximum 20.1- and 16-fold purification with 39.5% and 32% yield of amylase inhibitor and trypsin inhibitor, respectively. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the inhibitor protein showed substantial purification and the molecular weight of the protein was found to be 14 kDa.     

Culture Conditions for Production of Thermostable Amylase by Bacillus stearothermophilus

Bacillus stearothermophilus grew better on complex and semisynthetic medium than on synthetic medium supplemented with amino acids. Amylase production on the complex medium containing beef extract or corn steep liquor was higher than on semisynthetic medium containing peptone (0.4%). The synthetic medium, however, did not provide a good yield of extracellular amylase. Among the carbohydrates which favored the production of amylase are, in order starch > dextrin > glycogen > cellobiose > maltohexaose-maltopeptaose > maltotetraose and maltotriose. The monosaccharides repressed the enzyme production, whereas inositol and d-sorbitol favored amylase production. Organic and inorganic salts increased amylase production in the order of KCI > sodium malate > potassium succinate, while the yield was comparatively lower with other organic salts of Na and K. Amino acids, in particular isoleucine, cysteine, phenylalanine, and aspartic acids, were found to be vital for amylase synthesis. Medium containing CaCl(2) 2H(2)O enhanced amylase production over that on Ca -deficient medium. The detergents Tween-80 and Triton X-100 increased biomass but significantly suppressed amylase synthesis. The amylase powder obtained from the culture filtrate by prechilled acetone treatment was stable over a wide pH range and liquefied thick starch slurries at 80 degrees C. The crude amylase, after (NH(4))(2)SO(4) fractionation, had an activity of 210.6 U mg. The optimum temperature and pH of the enzyme were found to be 82 degrees C and 6.9, respectively. Ca was required for the thermostability of the enzyme preparation.     

Inhibition of Calotropis procera latex-induced inflammatory hyperalgesia by oxytocin and melatonin

The latex of the wild growing plant Calotropis procera produces inflammation of the skin and mucous membranes upon accidental exposure. On local administration it elicits an intense inflammatory response due to the release of histamine and prostaglandins that is associated with hyperalgesia. In the present study we have evaluated the anti-inflammatory and antinociceptive activity of oxytocin and melatonin against rat paw edema induced by dried latex (DL) of C procera and compared it with that against carrageenan-induced paw edema. Aqueous extract of DL of C procera or carrageenan (1%) was injected into the subplantar surface of the rat paw and the paw volume was measured at 0, 1, 2, 3, 4, 6, 10, and 24 hours. The associated hyperalgesic response and functional impairment were also evaluated concomitantly by dorsal flexion pain test, motility test, and stair climbing ability test. The inhibitory effect of oxytocin and melatonin on edema formation and hyperalgesic response was compared with dexamethasone. DL-induced edema formation was maximum at 2 hours and was associated with decreased pain threshold and functional impairment. Treatment with melatonin significantly attenuated the edematous response while both oxytocin and melatonin increased the pain threshold and improved functional parameters. Both oxytocin and melatonin significantly inhibited the hyperalgesia associated with DL-induced paw edema. Oxytocin was found to be as effective as melatonin in ameliorating the hyperalgesic response. However, it was found to be less effective than melatonin in attenuating edema formation.     

Purification and characterization of NAD(P)H quinone reductase from the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae)

NAD(P)H quinone reductase [NAD(P)H-QR] present in the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae) was purified to homogeniety from the B-serum fraction obtained by freeze-thawing of the bottom fraction of ultracentrifuged fresh latex. The purification protocol involved acetone fractionation, heat treatment, ion exchange chromatography and affinity chromatography. The M(r) determined by SDS-PAGE for the protein subunit was 21 kDa, and the molecular mass of the native enzyme estimated by gel filtration was 83 kDa, indicating that the native enzyme is a homotetramer. The enzyme showed pH stability over a range of 6 to at least 10 (with an optimum at pH 8) and thermal stability up to 80 degrees C. High NAD(P)H-QR activity (70%) was still retained after 10 h of preincubation at 80 degrees C. A comparable substrate specificity for this enzyme was observed among menadione, p-benzoquinone, juglone, and plumbagin, with only duroquinone generating a lower activity. Positive correlations between latex NAD(P)H-QR activity and rubber yield per tapping [fresh latex (r=0.89, P<0.01), dry rubber (r=0.81, P<0.01)] together with flow time (r=0.85, P<0.01) indicated that enzyme activity could possibly be used as a marker to predict the yield potential of selected clones.     

Resolution of Rhodocyclus gelatinosus photoreceptor unit components by temperature-induced phase separation in the presence of decyltetraoxyethylene

A simple method for dissociating photoreceptor units from Rhodocyclus gelatinosus is described. Incubation of a chromatophore extract (Agalidis, I., Rivas, E. and Reiss-Husson, F. (1990) Photosynth. Res. 23, 249-255) at 4 degrees C with decyltetraoxyethylene and octyl-beta-D-thioglucopyranoside, followed by temperature-induced phase separation at 20 degrees C, led to the formation of three phases: a pellet composed of pure B875 antenna; an oily layer containing cytochrome c and other proteins; a detergent-poor supernatant containing crude reaction centers. This method provided a first step towards further purification of reaction centers and B875 antenna, respectively.     

Cycloheptaamylose as an affinity ligand of cereal alpha amylase. Characteristics and a possible mechanism of the interaction

The characteristics of alpha amylase purification on a column of cycloheptaamylose-substituted, epoxy-activated Sepharose 6B were investigated. The enzyme was recovered in high yield from crude triticale and wheat extracts. Enzyme activity assessed after elution from the column was 132% of that measured prior to chromatography. There was no evidence of beta amylase isozymes in the purified alpha amylase. Neither barley beta amylase nor sweet-potato beta amylase was retained by the column. Cycloheptaamylose did not inhibit triticale or wheat alpha amylase activity, but did inhibit barley beta amylase activity, yielding a K₁ of 4.5mm. Equilibriumdialysis experiments showed that alpha amylase did interact with cycloheptaamylose. The dissociation constant for the enzyme-ligand was 19μm. It was concluded that cycloheptaamylose bound at a non-catalytic site on the alpha amylase molecule.     

A Simple One Pot Purification of Bacterial Amylase From Fermented Broth Based on Affinity Toward Starch-Functionalized Magnetic Nanoparticle

Surface-functionalized adsorbant particles in combination with magnetic separation techniques have received considerable attention in recent years. Selective manipulation on such magnetic nanoparticles permits separation with high affinity in the presence of other suspended solids. Amylase is used extensively in food and allied industries. Purification of amylase from bacterial sources is a matter of concern because most of the industrial need for amylase is met by microbial sources. Here we report a simple, cost-effective, one-pot purification technique for bacterial amylase directly from fermented broth of Bacillus megaterium utilizing starch-coated superparamagnetic iron oxide nanoparticles (SPION). SPION was prepared by co-precipitation method and then functionalized by starch coating. The synthesized nanoparticles were characterized by transmission electron microscopy (TEM), a superconducting quantum interference device (SQUID, zeta potential, and ultraviolet–visible (UV-vis) and Fourier-transform infrared (FTIR) spectroscopy. The starch-coated nanoparticles efficiently purified amylase from bacterial fermented broth with 93.22% recovery and 12.57-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the molecular mass of the purified amylase was 67 kD, and native gel showed the retention of amylase activity even after purification. Optimum pH and temperature of the purified amylase were 7 and 50°C, respectively, and it was stable over a range of 20°C to 50°C. Hence, an improved one-pot bacterial amylase purification method was developed using starch-coated SPION.     

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.     

Production of amylase by Arthrobacter psychrolactophilus

Arthrobacter psychrolactophilus ATCC 700733 grew with a doubling time of 1.5–2.3 h (22°C) and produced up to 0.2 units/mL (soluble starch assay) of extracellular amylase in tryptic soy broth without dextrose (TSBWD) containing 0.5% or 1.0% (w/v) soluble starch or maltose as the fermentable substrate. Time-course experiments in media containing soluble starch as substrate showed that amylolytic activity appeared in cultures at 24 h (after exponential growth had ceased), reached peak levels in 72–96 h, and declined rapidly after reaching peak levels. Peak levels were highest in TSBWD containing 1.0% soluble starch. Proteolytic activity appeared at about the same time as amylolytic activity and increased during the period of amylase production. Significant amylase production was not observed in cultures in TSBWD with 0.5% glucose or in cultures grown at 28°C, but low levels of amylase were observed in TSBWD cultures grown at 19–23°C which contained no added carbohydrate. A single band of activity was observed after electrophoresis of supernatant fractions in non-denaturing gels, followed by in situ staining for amylolytic activity. The amylase possessed a raw starch-binding domain and bound to uncooked corn, wheat or potato starch granules. It was active in the Phadebas assay for -amylase. Activity was maximum on soluble starch at a temperature between 40°C and 50°C. The amylase after purification by affinity chromatography on raw starch granules exhibited two starch-binding protein bands on SDS gels of 105 kDa and 26 kDa.     

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.     

Site-directed mutagenesis of the calcium-binding site of α-amylase of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Amylases that are active under acidic conditions (pH <6), at higher temperatures (>70 degrees C) and have less reliance on Ca(2+) are required for starch hydrolysis. The alpha-amylase gene of Bacillus licheniformis MTCC 6598 was cloned and expressed in Escherichia coli BL21. The calcium-binding site spanning amino acid residues from 104 to 200 in the loop regions of domain B and D430 in domain C of amylase were changed by site-directed mutagenesis and the resultant mutant amylases were analyzed. Calcium-binding residues, N104, D161, D183, D200 and D430, were replaced with D104 and N161, N183, N200 and N430, respectively. Mutant amylase with N104D had a slightly decreased activity at 30 degrees C but a significantly improved specific activity at pH 5 and 70 degrees C, which is desirable character for a food enzyme. The amylase mutants with D183N or D200N lost all activity while the mutant amylase with D161N retained its activity at 30 degrees C but had significantly less activity at 70 degrees C. On the other hand, the activity of the mutant amylase with D430N was not changed at 30 degrees C but had an improved activity at 70 degrees C.     

Effect of amylase accumulation on hydrogen production by Clostridium beijerinckii,strain AM21B

The maximal enzymatic activity of crude amylase produced in the batch culture of Clostridium beijerinckii strain AM21B grown in PY medium with starch was obtained at 55°C and in an acidic pH range of 4.6 to 5.4. Amylase was produced in the culture medium after 4 h (46.6 units) and reached a peak (405.5 units) after 12 h cultivation at 36°C, pH 6.0. Although the most efficient production of amylase, hydrogen and cells was achieved at 36°C and pH 6.0, the maximal hydrogen evolution rate was found at 41°C and pH 7.0.     

Effect of Calotropis procera latex on isoproterenol induced myocardial infarction in albino rats.

The alcoholic extract of the latex obtained from Calotropis procera (Asclepidaceae) was evaluated for protection against isoproterenol (20 mg/100 g body wt., s.c.)-induced myocardial infarction in albino rats. The heart damage induced by isoproterenol was indicated by elevated levels of the marker enzymes such as Creatine Kinase-isoenzyme (CK-MB), Lactate dehydrogenase (LDH), Serum Glutamate Oxaloacetic Transaminase (SGOT) and Serum Glutamate Pyruvate Transaminase (SGPT) in serum with increased lipid peroxide and reduced glutathione content in heart homogenates. Microscopical examination (histopathology) was also performed on the myocardial tissue. Pretreatment with an ethanolic latex extract of Calotropis procera at a dose of 300 mg/kg body wt., administered orally thrice a day for 30 days, reduced significantly (p < 0.01) the elevated marker enzyme levels in serum and heart homogenates in isoproterenol-induced myocardial infarction. Histopathological observation revealed a marked protection by the extract in myocardial necrotic damage.