Purification and characterization of three phytases from germinated lupine seeds (Lupinus albus var. amiga)

Three phytases were purified about 14200-fold (LP11), 16000-fold (LP12), and 13100-fold (LP2) from germinated 4-day-old lupine seedlings to apparent homogeneity with recoveries of 13% (LP11), 8% (LP12), and 9% (LP2) referred to the phytase activity in the crude extract. They behave as monomeric proteins of a molecular mass of about 57 kDa (LP11 and LP12) and 64 kDa (LP2), respectively. The purified proteins belong to the acid phytases. They exhibit a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 80 microM (LP11), 300 microM (LP12), and 130 microM (LP2) and k(cat) = 523 s(-1) (LP11), 589 s(-1) (LP12), and 533 s(-1) (LP2) at pH 5.0 and 35 degrees C. The phytases from lupine seeds exhibit a broad affinity for various phosphorylated compounds and hydrolyze phytate in a stepwise manner.     

Purification and characterization of a hexanol-degrading enzyme extracted from apple

An enzyme having activity toward n-hexanol was purified from apple, and its biochemical characteristics were analyzed. The purification steps consisted of sedimentation with ammonium sulfate, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephadex G-100 column. The obtained enzyme had a yield of 16.00% with a specific activity of 18879.20 U/mg protein and overall purification of 142.77-fold. The enzyme showed activity to isoamylol, 1-propanol, n-hexanol, and isobutanol but not toward methanol and ethanol. With n-hexanol as a substrate, the optimum conditions were pH 4.0 and 30 °C for enzyme activity and pH 3.0-4.0 and temperatures below 40 °C for enzyme stability. The enzyme activity was increased significantly by adding l-cysteine and Fe(2+) at all tested concentrations and slightly by Zn(2+) at a high concentration but decreased by additions of EDTA, Ga(2+), K(+), Mg(2+), sodium dodecyl sulfate (SDS), sodium aluminum sulfate (SAS), dithiothreitol (DTT), and glutathione (GSH). The enzyme activities toward n-hexanol and n-hexanal were increased by NADH but decreased by NAD(+), in contrast to a decrease toward n-hexane by addition of both NAD(+) and NADH.     

Mechanical weeding effects on soil structure under field carrots (Daucus carota L.) and beans (Vicia faba L.)

The successful production of organic vegetables relies heavily on mechanical weeding, flame weeding and stale seedbeds. These operations involve repeated passes by tractors. Mechanical weeding also involves regular tillage. This combination of repeated tillage and compaction changes soil structure. We studied these structural changes in two fields of organic carrots and one field of beans in eastern Scotland. Structure was described by measuring soil strength with a vane shear tester and a cone penetrometer, by measuring bulk density and by visual assessment. Under beans, vane shear strength below the growing root zone was highly variable and in some areas was high enough to restrict root growth (>50 kPa). The carrots were grown in beds containing crop rows separated by bare soil. The bare soil was regularly weeded mechanically. The structure of this weeded soil in the top 10 cm layer of a loam eventually became disrupted and compacted enough to deter root growth (vane shear strength of 70 kPa). In addition the topsoil and subsoil in the wheel-tracks between the beds became very compact with little distinguishable structure. This compaction extended to the subsoil and persisted into the next cropping season (cone resistance >3 MPa at 35–50 cm depth). Reduced tillage by discing without ploughing was used to incorporate the straw used to protect the carrots overwinter and prepare the soil for the next crop. The resulting topsoil quality was poor leading to anaerobic growing conditions which restricted growth of the following crop and led to losses of the greenhouse gas nitrous oxide. The greatest threat to soil quality posed by mechanical weeding was subsoil compaction by tractor wheeling.     

Purification and characterization of a methylene urea-hydrolyzing enzyme from Rhizobium radiobacter (Agrobacterium tumefaciens)

Slow-release fertilizers are gaining acceptance to increase fertilizer use efficiency and reduce environmental impact. The release of nitrogen from methylene urea, a common slow release N fertilizer, is controlled by microbial decomposition. An enzyme hydrolyzing slow-release nitrogen fertilizer, methylene urea, was purified from Rhizobium radiobacter (Agrobacterium tumefaciens) to homogeneity using a four-step purification procedure with an overall yield of 3%. The active enzyme has a molecular mass of approximately 180 kDa determined by size exclusion chromatography, and the SDS page of the purified protein indicated three subunits of different sizes (62, 34 and 32 kDa). The N-terminal amino acid sequence of the 62 kDa fragment indicates identity with urease subunits from Mycobacterium tuberculosis (73%) and Helicobacter pylori (71%). However, for the internal amino acid sequences of the 62 kDa fragment no matches with known proteins were found. Some internal peptides in the smaller subunits (32 and 34 kDa) are homologous to urease subunits and unknown proteins in Agrobacterium tumefaciens. Based on the kinetic properties, substrate selectivity, and inhibition characteristics, the novel enzyme (MUase) is an intracellular enzyme complex with urease activity. The enzymatic mechanism of methylene urea breakdown was studied using a novel LC-MS method for MU analysis, which indicates that all cold-water soluble nitrogen forms of methylene urea are subjected to hydrolysis, and the hydrolysis proceeds via methylurea, urea and other yet unidentified hydrolysis-products, suggesting that the isolated enzyme complex performs a multistep hydrolysis. The microbiological and molecular data is useful in determining the soil factors affecting the efficacy of methylene urea as a slow release fertilizer in agricultural production systems.     

Characterization of Greek populations of faba bean (Vicia faba L.) and their evaluation using a new parameter

Using data from four environments of a two-year experiment, a collection of 55 Greek faba bean populations wascharacterized on the basis of ten morphological and ten agronomical traits. The collection can be described ashaving flowers with medium intensity of streaks on standard petal, two to three flowers per inflorescence, leaveswith six leaflets per leaf, sub-elliptic leaflet shape and small or medium leaflet size. The stems had weak ormedium pigmentation at flowering time, were of low or medium thickness, had medium height and mediumbranching from basal nodes and high resistance to lodging. Pods were mainly basal with pendent attitude, flattenedin shape, had dark colour at maturity and were small with few ovules and seeds. Most seeds had testa of light greencolour. This characterization makes the present collection similar to other South European faba bean populations.Apart from characterization, collections should also be evaluated from the breeding point of view. Since themost desirable populations within a collection are those with valuable but 'low frequency' levels of traits, theparameter R (rareness) was proposed. For each trait, a partial rareness (R ) parameter is calculated using only ithose trait levels that can be considered as having 'low frequency'. The parameter R is the sum of the 'partialrarenesses'. Populations with high frequencies of the 'lowest frequency' levels of traits in the collection arecharacterized by high values of R and can, therefore, be more easily identified.     

Collection, evaluation and classification of Greek populations of faba bean (Vicia faba L.)

Fifty-five Greek Vicia faba L. populations, collected from diverse areas, were planted at two dry and low fertility sites for evaluation and classification. Yield evaluation, which was carried out by Principal Component Analysis (PCA) on the basis of seven yield traits, showed the number of pods per plant, number of ovules and seeds per pod, and branching from the basal nodes to be the most important traits for population evaluation regarding yield. For population classification, four dissimilarity coefficients (Manhattan, Average Taxonomic Distance, Euclidean distance and squared Euclidean distance) and four multivariate methods (PCA, UPGMA, Neighbor-joining and Principal Coordinate Analysis) were evaluated using fifteen morphological and seven yield traits. Neighbor-joining was chosen as the most suitable multivariate method. This method combined with PCA for the seven yield traits, placed the populations into six groups. As revealed by the application of PCA on all twenty-two traits the grouping was based mainly on pod characteristics, stem thickness, plant height, 1000 seed weight and branching from basal nodes. Based on the results of the present study, a model is proposed for conserving cross-pollinated species, such as faba bean.     

Uptake and distribution of manganese applied to leaves of Vicia faba (cv. Herzfreya) and Zea mays (cv. Regent) plants

Uptake and transport of Mn applied to leaves was studied in maize (Zea mays cv. Regent) and horse bean (Vicia faba cv. Herzfreya), two important crops in Egypt. Under controlled conditions in a growth chamber, maize and horse bean plants were grown in solution culture without Mn. After 20 days, Mn was applied to one older leaf by submerging part of the leaf blade into 0.1 mM MnSO₄ or MnEDTA solution for 48 h. At harvest (24 h later), plants were divided into fractions and Mn concentrations and contents determined. Plants without Mn application served as control. Only 1% of the Mn supply was taken up. Most of it remained at the application zone. However, part of the Mn moved out of the leaf of application and was preferentially transported to the shoot apex. This was indicated by a up to 2 times higher Mn concentration of the youngest leaf. When Mn was applied as MnEDTA, Mn uptake was lower but translocation enhanced compared to MnSO₄. There were no consistent differences between the plant species although mobility of Mn seemed to be higher in maize. Although the amounts of Mn taken up and translocated were low, the results suggest that in these plant species, leaf-applied Mn may contribute to the Mn nutrition of new growth.     

Effects of intercropping and Rhizobium inoculation on yield and rhizosphere bacterial community of faba bean (Vicia faba L.)

The effects of intercropping with maize and Rhizobium inoculation on the yield of faba bean and rhizosphere bacterial diversity were analyzed by terminal restriction fragment length polymorphism, amplified 16S rDNA restriction analysis (ARDRA), and 16S rDNA sequencing. The results showed that intercropping but not Rhizobium inoculation significantly increased the faba bean yield. Probably the relatively high level of native rhizobia in soil annulled the effect of rhizobia inoculation. ARDRA results showed that intercropping did not affect bacterial diversity whereas Rhizobium inoculation decreased bacterial diversity. The canonical correspondence analysis showed that the composition of bacterial community was changed apparently by intercropping, and there was a positive correlation (P = 0.724) between faba bean yields and intercropping and an apparent correlation (P = 0.648) between intercropping and total N. The available content of K and P had a lower effect on the bacterial community composition than did the total N content, Rhizobium inoculation, and microbial biomass C. Rhizobium inoculation negatively correlated with microbial biomass C (P = −0.827). These results revealed a complex interaction among the intercropped crops, inoculation with rhizobia, and indigenous bacteria and implied that the increase of faba bean production in intercropping might be related to the modification of rhizosphere bacterial community.     

Purification and characterization of amylases from small abalone (Sulculus diversicolor aquatilis)

Amylases II-1 and II-2 with molecular weights of 55.7 and 65 kDa, respectively, were purified to electrophoretical homogeneity from small abalone (Sulculus diversicolor aquatilis) by ammonium sulfate fractionation, Sepharose CL-6B, CM-Sepharose CL-6B, and Sephacryl S-100 chromatographs. They had optimal temperatures of 45 and 50 degrees C and an optimal pH of 6.0. The purified amylases were stable at pH 5.0-8.0 and 6.0-8.0, respectively. They were completely or partially inhibited by Hg(2+), Cu(2+), Cd(2+), Zn(2+), iodoacetamide, phenylmethanesulfonyl fluoride, and N-ethylmaleimide, suggesting the existence of cysteine at their active sites. Digestion tests against various polysaccharides suggested that the purified amylases II-1 and II-2 are neoamylases which can hydrolyze both alpha-1,4 and alpha-1,6 glucosidic bonds. Amylase II-2 might be an exo- and II-1 an endo-/exo-amylase.     

Purification and characterization of trimethylamine-N-oxide demethylase from jumbo squid (Dosidicus gigas)

Trimethylamine-N-oxide demethylase (TMAOase) was purified from Jumbo squid (Dosidicus gigas) and characterized in detail herein. The TMAOase was extracted from squid with 20 mM Tris-acetate buffer (pH 7.0) containing 1.0 M NaCl, followed by acid treatment and heat treatment. Then it was purified by deithylaminoethyl-cellulose and Sephacryl S-300 chromatography, subsequently resulting in an 839-fold purification. The molecular mass of the TMAOase was defined to be 17.5 kDa. The optimum pH of the purified TMAOase was 7.0, and its optimum temperature was confirmed to be 55 degrees C. The TMAOase was stable to heat treatment up to 50 degrees C and stable at pH 7.0-9.0. Reducing agents such as DTT, Na2SO3, and NADH were effective at activating TMAOase, and ethylenediaminetetraacetic acid, as well as Mg2+ and Ca2+, could also enhance the activity of TMAOase remarkably, whereas the TMAOase could be significantly inhibited by tea polyphenol, phytic acid and acetic acid. In addition, the TMAOase converted TMAO to dimethylamine and formaldehyde stoichiometrically with a K(m) of 26.2 mM.     

Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. nov. SK006 isolated from an Asian traditional fermented shrimp paste

Bacillus sp. nov. SK006 producing four extracellular fibrinolytic enzymes was isolated from fermented shrimp paste, a traditional and popular Asian seasoning. One fibrinolytic enzyme was purified to homogeneity with a molecular mass of 43-46 kDa by SDS-PAGE and gel filtration chromatography. The specific activity was determined to be 11.2 units/mg using plasmin as a standard. The enzyme displayed optimal activity at 30 degrees C and pH 7.2. It was stable below 40 degrees C for 4 h between pH 5.0 and pH 11.0. Zinc ion stimulated the enzyme activity whereas Cu2+, Ca2+, Fe3+, and Hg2+ caused its inhibition. The fibrinolytic activity was strongly inhibited by PMSF and moderately inhibited by EDTA as well as PCMB. The enzyme exhibited a higher affinity toward N-Succ-Ala-Ala-Pro-Phe-pNA and was able to degrade fibrin clots either by forming active plasmin from plasminogen or by direct fibrinolysis. The N-terminal amino acid sequence was found to be AQSVPYEQPHLSQ, which is different from that of other known fibrinolytic enzymes.     

Purification and characterization of a polyphenol oxidase from the seeds of field bean (Dolichos lablab)

The polyphenol oxidase from field bean (Dolichos lablab) seeds has been purified to apparent homogeneity by a combination of ammonium sulfate precipitation, DEAE-Sephacel chromatography, phenyl agarose chromatography, and Sephadex G-200 gel filtration. The purified enzyme has a molecular weight of 120 +/- 3 kDa and is a tetramer of 30 +/- 1.5 kDa. Native polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of a single isoform with an observed pH optimum of 4.0. 4-Methyl catechol is the best substrate, followed by catechol, and L-3,4-dihydroxyphenylalanine, all of which exhibited a phenomenon of inhibition by excess substrate. No activity was detected toward chlorogenic acid, catechin, caffeic acid, gallic acid, and monophenols. Tropolone, both a substrate analogue and metal chelator, proved to be the most effective competitive inhibitor with an apparent K(i) of 5.8 x 10(-)(7) M. Ascorbic acid, metabisulfite, and cysteine were also competitive inhibitors.     

Purification and biochemical characterization of insoluble acid invertase (INAC-INV) from pea seedlings

Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the β-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.     

Purification of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide with an antihypertensive effect from loach (Misgurnus anguillicaudatus)

To isolate and characterize novel angiotensin I-converting enzyme (ACE) inhibitory peptide from loach (Misgurnus anguillicaudatus), six proteases, pepsin, α-chymotrypsin, bromelain, papain, alcalase, and Neutrase, were used to hydrolyze loach protein. The hydrolysate (LPH) generated by bromelain [ratio of enzyme to substrate, 3:1000 (w/w)] was found to have the highest ACE inhibitory activity (IC(50), 613.2 ± 8.3 μg/mL). Therefore, it was treated by ultrafiltration to afford fraction of LPH-IV (MW < 2.5 kDa) with an IC(50) of 231.2 ± 3.8 μg/mL, having higher activity than the other fractions. Then, LPH-IV was isolated and purified by consecutive purification steps of gel filtration chromatography and reverse-phase high-performance liquid chromatography to afford a purified peptide with an IC(50) of 18.2 ± 0.9 μg/mL, an increase of 33.7-fold in ACE inhibitory activity as compared with that of LPH. The purified peptide was identified as Ala-His-Leu-Leu (452 Da) by Q-TOF mass spectrometry and amino acid analyzer. An antihypertensive effect in spontaneously hypertensive rats revealed that oral administration of LPH-IV could decrease systolic blood pressure significantly.     

Influence of Rhizobium/Azotobacter and Rhizobium/Azospirillum combined inoculation on mineral composition of faba bean (Vicia faba L.)

 Mixed inoculation of Vicia faba L. with four different Rhizobium/Azospirillum and Rhizobium/Azotobacter combinations led to changes in total content, concentration and/or distribution of the mineral macro- and micronutrients, K, P, Ca, Mg, Fe, B, Mn, Zn and Cu, when compared with plants inoculated with Rhizobium only. The effects varied to a great extent among the Azotobacter and Azospirillum strains selected for combined inoculation. Received: 6 June 1998     

The role of potassium fertilizer in nodulation and nitrogen fixation of faba bean (Vicia faba L.) plants under drought stress

Three-week-old nodulated faba bean plants were subjected to different levels of drought stress (onehalf, one-quarter, or one-eighth field capacity) for 5 weeks. Half the stressed plants were treated with KCl at 10 mg kg^-1 soil or 150 mg kg^-1 soil at the beginning of the drought stress. Nodulation and nitrogenase activity were significantly decreased by increasing drought stress. Leghaemoglobin and protein contents of nodule cytosol were also severely inhibited by drought sttess. This decline was attributed to the induction of protease activity. However, carbohydrate contents of the nodule cytosol increased significantly. This accumulation was attributed to a sharp decline in invertase activity and low use of sugar by the bacteroids We conclude that harmful effects of water deficits can be alleviated by increasing K⁺ supplementation.     

Purification and characterization of trypsin from the spleen of tongol tuna (Thunnus tonggol)

Trypsin from tongol tuna (Thunnus tonggol) spleen was purified to 402-fold by ammonium sulfate precipitation, followed by a series of chromatographic separations. The molecular mass of trypsin was estimated to be 24 kDa by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin appearing as a single band on native PAGE showed the maximal activity at pH 8.5 and 65 degrees C. It was stable in a wide pH range of 6-11 but unstable at the temperatures greater than 50 degrees C. The enzyme required calcium ion for thermal stability. The activity was strongly inhibited by 1.0 g/L soybean trypsin inhibitor and 5 mM TLCK and partially inhibited by 2 mM ethylenediaminetetraacetic acid. Activity was lowered with an increasing NaCl concentration (0-30%). The enzyme had a Km for Nalpha-p-tosyl-L-arginine methyl ester hydrochloride of 0.25 mM and a Kcat of 200 s-1. The N-terminal amino acid sequence of trypsin was determined as IVGGYECQAHSQPHQVSLNA and was very homologous to other trypsins.     

Purification and characterization of the 7S vicilin from Korean pine (Pinus koraiensis)

Pine nuts are economically important as a source of human food. They are also of medical importance because numerous pine nut allergy cases have been recently reported. However, little is known about the proteins in pine nuts. The purpose of this study was to purify and characterize pine nut storage proteins. Reported here is the first detailed purification protocol of the 7S vicilin-type globulin from Korean pine (Pinus koraiensis) by gel filtration, anion exchange, and hydrophobic interaction chromatography. Reducing SDS-PAGE analysis indicated that purified vicilin consists of four major bands, reminiscent of post-translational protease cleavage of storage proteins during protein body packing in other species. The N-terminal ends of vicilin peptides were sequenced by Edman degradation. Circular dichroism (CD) and differential scanning calorimetry (DSC) analyses revealed that pine nut vicilin is stable up to 80 degrees C and its folding-unfolding equilibrium monitored by intrinsic fluorescence can be interpreted in terms of a two-state model.