Beta-glucosidase from Chalara paradoxa CH32: purification and properties

The hyphomycete Chalara paradoxa CH32 produced an extracellular beta-glucosidase during the trophophase. The enzyme was purified to homogeneity by ion-exchange and size-exclusion chromatography. The purified enzyme had an estimated molecular mass of 170 kDa by size-exclusion chromatography and 167 kDa by SDS-PAGE. The enzyme had maximum activity at pH 4.0-5.0 and 45 degrees C. The enzyme was inactivated at 60 degrees C. At room temperature, it was unstable at acidic pH, but it was stable to alkaline pH. The purified enzyme was inhibited markedly by Hg(2+) and Ag(2+) and also to some extent by the detergents SDS, Tween 80, and Triton X-100 at 0.1%. Enzyme activity increased by 3-fold in the presence of 20% ethanol and to a lesser extent by other organic solvents. Purified beta-glucosidase was active against cellobiose and p-nitrophenyl-beta-D-glucopyranoside but did not hydrolyze lactose, maltose, sucrose, cellulosic substrates, or galactopyranoside, mannopyranoside, or xyloside derivatives of p-nitrophenol. The V(max) of the enzyme for p-NPG (K(m) = 0.52 mM) and cellobiose (K(m) = 0.58 mM) were 294 and 288.7 units/mg, respectively. Hydrolysis of pNPG was inhibited competitively by glucose (K(i) = 11.02 mM). Release of reducing sugars from carboxymethylcellulose by a purified endoglucanase produced by the same organism increased markedly in the presence of beta-glucosidase.     

Comparison of marker- and pedigree-based methods for estimating heritability in an agroforestry population of Vitellaria paradoxa C.F. Gaertn. (shea tree)

We implemented a regression-based method between pairwise relatedness estimated from markers and phenotypic similarity to estimate heritability of traits related to leaf size and morphology in a wild tree population (Vitellaria paradoxa C.F. Gaertn.: shea tree). We then compared the results with heritability estimated with a classical pedigree-based method. We tested both approaches in an agroforestry population of this tree species, a very important one and abundant in the Sudano-Sahelian zone of Africa. Twelve microsatellite loci were used to estimate pairwise relatedness after selection of estimator coefficients based on Monte Carlo simulation. The regression-based method applied to 200 individuals did not display a significant trend with physical distance between trees for relatedness as well as for actual variance of relatedness. In consequence, estimates of narrow-sense heritability of traits related to leaf size were not significant. The pedigree-based method using a progeny test with 39 families and 15 individuals per family from the same population showed high and significant estimates of narrow-sense heritability for the same traits (h² = 0.36–0.95), demonstrating a marked genetic variation within the population. This discrepancy between methods stresses the poor performance of the molecular marker-based method. This can be explained by the absence of fine-scale structure within the agroforestry population of shea trees, other parameters being consistent with recommended values. The regression-based method does not seem well adapted to the agroforestry tree population. New experiments in tree populations and theoretical approaches are needed to evaluate the real potential of the marker-based methods.     

Latent polyphenol oxidases from sago log (Metroxylon sagu): partial purification, activation, and some properties

Latent polyphenol oxidase (LPPO), an enzyme responsible for the browning reaction of sago starches during processing and storage, was investigated. The enzyme was effectively extracted and partially purified from the pith using combinations of nonionic detergents. With Triton X-114 and a temperature-induced phase partitioning method, the enzyme showed a recovery of 70% and purification of 4. 1-fold. Native PAGE analysis of the partially purified LPPO revealed three activity bands when stained with catechol and two bands with pyrogallol. The molecular masses of the enzymes were estimated by SDS-PAGE to be 37, 45, and 53 kDa. The enzyme showed optimum pH values of 4.5 with 4-methylcatechol as a substrate and 7.5 with pyrogallol. The LPPO was highly reactive toward diphenols and triphenols. The activity of the enzyme was greatly enhanced in the presence of trypsin, SDS, ethanol, and linoleic acid.     

A stable serine protease, wrightin, from the latex of the plant Wrightia tinctoria (Roxb.) R. Br.: purification and biochemical properties

Today proteases have become an integral part of the food and feed industry, and plant latex could be a potential source of novel proteases with unique substrate specificities and biochemical properties. A new protease named "wrightin" is purified from the latex of the plant Wrightia tinctoria (Family Apocynaceae) by cation-exchange chromatography. The enzyme is a monomer having a molecular mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0, and an extinction coefficient (epsilon1%280) of 36.4. Optimum activity is achieved at a pH of 7.5-10 and a temperature of 70 degrees C. Wrightin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity; for example, the Km value is 50 microM for casein as substrate. Wrightin showed weak amidolytic activity toward L-Ala-Ala-p-nitroanilide but completely failed to hydrolyze N-alpha-benzoyl- DL-arginine-p-nitroanilide (BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably thermostable, retaining complete activity at 70 degrees C after 60 min of incubation and 74% of activity after 30 min of incubation at 80 degrees. Besides, the enzyme is very stable over a broad range of pH from 5.0 to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents, and metal ions. Thus, wrightin might be a potential candidate for various applications in the food and biotechnological industries, especially in operations requiring high temperatures.     

Tissue-Specific Variations of Esterase Activities in the Tadpoles and Adults of Pseudis paradoxa (Anura: Hylidae)

Water, Air, &; Soil Pollution - We determined basal levels of cholinesterase (ChE) and carboxylesterase (CbEs; two substrates: α-naphthyl acetate and 4-nitrophenylvalerate) in different...     

Structural characterization and biological activities of an exopolysaccharide kefiran produced by Lactobacillus kefiranofaciens WT-2B(T)

Lactobacillus kefiranofaciens, isolated from kefir grains, produces an extracellular polysaccharide when cultured, not only in PYG10 medium but also in a liquid medium containing a rice hydrolysate that had been previously degraded by treatment with a glucoamylase. The maximum yield of the polysaccharide, using the rice hydrolysate as the medium, was 2.5 g/L after a 7-day culture period at pH 5.0 and 33 degrees C. Compositional analysis, methylation analysis, specific rotation, and (1)H and (13)C NMR spectroscopy revealed that the structures of polysaccharides obtained from these two different culture media are essentially identical. The polysaccharide is composed of a hexasaccharide repeating unit and, thus, is known as kefiran. The weight-average molecular weight and the z-average radius of gyration of a sample, purified from the rice hydrolysate medium, were determined to be 7.6 x 10(5) g/mol and 39.9 nm, respectively, by gel permeation chromatography equipped with a multiangle laser-light-scattering photometer. Changes in blood pressure and serum components were examined in SHRSP/Hos rats, using doses of 100 and 300 mg of kefiran/kg of rat. A suppression in the increase in blood pressure was observed in these rats after 30 days. This activity is discussed in terms of the concentration of serum components of the rat, with emphasis on lipid components such as cholesterols, triglycerides, and free fatty acids.     

Study of the synergistic effects of salinity, pH, and temperature on the surface-active properties of biosurfactants produced by Lactobacillus pentosus

Many studies have investigated the effects of pH, temperature, and salinity on the surface-active properties of various surfactants, although in most cases the variables have been studied separately, without considering the effects of any interactions between them. In the present study, a Box-Behnken factorial design was applied to study the effects of pH, temperature, and salinity on the surface-active properties of a biosurfactant produced by Lactobacillus pentosus. The data obtained enabled development of a second-order model describing the interrelationships between operational and experimental variables, by equations including linear, interaction, and quadratic terms. The variable that had the greatest effect on the surface-active properties of the biosurfactant was pH. Moreover, at pH 3-5.5, decreases in salinity and temperature acted synergistically, reducing the surface tension of the biosurfactant; at pH 8, the same effect was observed with increasing salinity and temperature.     

Isolation, antifungal activity, and structure elucidation of the glutarimide antibiotic, streptimidone, produced by Micromonospora coerulea.

The antibiotic Ao58A,which showed strong antifungal activity against some plant pathogenic fungi, was purified from the culture broth and mycelial mats of Micromonospora coerulea strain Ao58 using various chromatographic procedures. The molecular formula of the antibiotic Ao58A was deduced to be C(16)H(23)NO(4) (M + H, m/z 294.1707) by high-resolution FAB mass spectroscopy. Analyses of (1)H NMR, (13)C NMR, and 2D NMR spectral data revealed that the antibiotic Ao58A is the glutarimide antibiotic streptimidone, 4-(2-hydroxy-5, 7-dimethyl-4-oxo-6,8-nonadienyl)-2,6-piperidinedione. The antibiotic Ao58A was very effective in inhibiting growth of Phytophthora capsici,Didymella bryoniae, Magnaporthe grisea, and Botrytis cinerea in the range approximately 3-10 microg mL(-)(1) of MICs. In vivo evaluation of the antibiotic Ao58A under greenhouse condition showed strong control efficacies against the development of P. capsici, B. cinerea, and M. grisea on pepper, cucumber, and rice plants, respectively. The antibiotic Ao58A was equally as effective as metalaxyl, vinclozolin, and tricyclazole in the control of these plant diseases. However, it did not show any phytotoxicity on the plants even when treated with 500 microg mL(-)(1).     

Influence of Common Reed (Phragmites australis) on CH4 Production and Transport in Wetlands: Results from Single-Plant Laboratory Experiments

Laboratory culture experiments were conducted with common reed (Phragmites australis) to elucidate the role of root exudates on CH₄ production in wetland soils as well as the importance of different plant organs as routes of CH₄ to the atmosphere. In the 50 d experiment period, root exudates ranged from 0.03 to 1.53 µmolg^?1d^?1, which increased with reed growth. CH₄ production rate of soil was stimulated as root exudates collected was added. CH₄ transport capacity rate also increased with plant growth and influenced by light intensity. Root tips were the most important part of controlling diffusion of CH₄ into reed shoots, and leave transport accounted for 45.34% of total emissions into the atmosphere.     

Simple purification (desalting) procedure to facilitate structural analysis of an alkali-solubilized/neutralized starch solution by intermediate-pressure size-exclusion chromatography

A technique was established to remove impurities (e.g., salts) from starch dissolved in strong alkali and neutralized with acid to accommodate starch structural analysis via intermediate-pressure size-exclusion chromatography (IPSEC). Starch (corn and wheat) subjected to an alkaline-microwave dissolution scheme (35 s microwave heating in a mixture of 6 M urea and 1 M KOH) was either treated with ion-exchange resin or passed through a desalting column to remove salt/urea contaminants. Control (untreated) starch solution analyzed by IPSEC displayed a significant interfering peak (attributable to salt/urea), which coeluted with the starch amylose peak. The interfering peak was most efficiently eliminated by first passing the starch solution through a desalting column, which process effectively removed impurities (e.g., salts/urea) without appearing to adversely impact the starch structural analysis. This simple technique coupled with the rapid alkaline-microwave starch dissolution procedure greatly expedites structural investigation of starch by facilitating analysis by IPSEC.     

Hemicelluloses of ragi (finger millet, Eleusine coracana, Indaf-15): isolation and purification of an alkali-extractable arabinoxylan from native and malted hemicellulose B

Hemicelluloses (A and B) were isolated from an Indo-African hybrid variety of finger millet (ragi, Eleusine coracana) by extracting the starch-free residue with 10% sodium hydroxide under a continuous stream of nitrogen, and changes in their sugar composition during malting for 96 h were studied. Hemicellulose B, obtained in higher yield from both native (N) and malted (M) flours, was found to be completely soluble in water, richer in uronic acid, and more viscogenic than hemicelullose A. Fractional precipitation of hemicellulose B by ammonium sulfate resulted in four precipitable fractions (F-60, F-70, F-80, and F-100) and a nonprecipitable (NP) fraction varying in their yield and arabinose, xylose, galactose, and glucose contents. A progressive increase in the pentose-to-hexose ratio (P:H) from 0.42:1.0 in F-60 to 1.94:1.0 in NP was observed in native hemicellulose B fractions; however, in malted hemicellulose B the P:H ratio increased from 0.43:1.0 in F-60 to 1.56:1.0 in F-80 and then decreased to 1.13:1.0 in NP. The major fraction, F-70 (N, 44.5%; M, 38.5%), was separated into eight subfractions on DEAE-cellulose by successive elution with water, ammonium carbonate (AC) (0.1, 0.2, and 0.3 M AC), and sodium hydroxide (0.1 and 0.2 M) differing in their yield and neutral sugar composition. The purity of the major glucuronoarabinoxylan fraction (0.1 M AC eluted) was ascertained by Sepharose CL-4B, HPSEC, cellulose acetate, and capillary electrophoresis methods. A significant decrease in the molecular mass of arabinoxylan from 1200 to 1120 kDa upon malting for 96 h is an indication of cell wall degradation by the inducible cell wall degrading enzymes.     

Production of Bioflocculant by Staphylococcus cohnii ssp. from Palm Oil Mill Effluent (POME)

Staphylococcus cohnii ssp. was found to produce a bioflocculant with high flocculating activity for kaolin suspension. Using optimized culture conditions, a flocculating activity of 70.3?% at a bioflocculation concentration of 0.3?mg/L at pH 7 with Ca²⁺ as the cation and 88.9?% in 1.2?mg/L of bioflocculant concentration for Al³⁺ as the cation was obtained for bacteria that were incubated at temperature of 40?°C for 2-day incubation period. It was found to be effective for flocculation of a kaolin suspension over weakly acidic pH (6?C8); divalent and trivalent (Ca²⁺ and Al³⁺) enhanced the flocculating activity. This bioflocculant possesses a good flocculating activity, which can be promoted by the addition of trivalent and bivalent cations in kaolin suspension.     

Analysis of the main components of the aguamiel produced by the maguey-pulquero (Agave mapisaga) throughout the harvest period

The main characteristics of the aguamiel (maguey-pulquero sap) during the harvest period of the Agave mapisaga plants were assessed to establish its stability through time and the industrial potential of its components. Only minor differences in aguamiel composition were detected among samples collected at different time points of the harvest period. The aguamiel analyzed contained 11.5 wt % of dry matter, which was composed mainly of sugars (75 wt %). Among these sugars, 10 wt % were fructo-oligosaccharides (FOS), which are known to be important in the food industry for their prebiotic properties. Other components include 0.3 wt % of free amino acids (with most essential amino acids and four neurotransmitters: GABA, GLY, GLX, and ASX), 3 wt % of proteins, and 3 wt % of ashes.