[Show abstract][Hide abstract] ABSTRACT: Proteolytic enzymes have a wide range of applications in different industrial fields therefore the development of strategies focused in the optimization of their catalytic performance is a topic of great interest. Enzyme immobilization onto solid supports induces changes at structural level in the immobilized systems, and consequently some reaction characteristics can be enhanced; furthermore, immobilized enzymes can be reused in the same process. Due to the unique physicochemical and functional properties that chitin and its derivate chitosan possess, these biopolymers are an excellent option as support for enzyme immobilization. This review discusses the effects on the biochemical and operational performance of proteases immobilized in chitin and chitosan based materials. Some aspects related with the industrial applications of the immobilization systems are also mentioned.
[Show abstract][Hide abstract] ABSTRACT: Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5kDa according to SDS-PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40°C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe(2+)>Hg(2+)>Mn(2+)>K(+)>Mg(2+)>Li(+)>Cu(2+)). Trypsin activity decreased continuously as NaCl concentration increased (0-30%). Km and kcat values were 0.13mM and 1.46s(-1), respectively. Results suggest the enzyme have a potential application where room processing temperatures (25-35°C) or high salt (30%) concentration are needed, such as in fish sauce production.
[Show abstract][Hide abstract] ABSTRACT: Solid wastes generated from the seafood industry represent an important environmental pollutant; therefore, utilization of those wastes for the development of processing biochemical tools could be an attractive and clean solution for the seafood industry. This study reports the immobilization of semi-purified acidic proteases from Monterey sardine stomachs onto chitin and chitosan materials extracted from shrimp head waste. Several supports (chitosan beads, chitosan flakes, and partially deacetylated flakes) were activated either with genipin or Na-tripolyphosphate and evaluated as a mean to immobilize acidic proteases. The protein load varied within the 67-91 % range on different supports. The immobilization systems based on chitosan beads achieved the highest protein loads but showed the lowest retained catalytic activities. The best catalytic behavior was obtained using partially deacetylated chitin flakes activated either with genipin or Na-tripolyphosphate. According to results, the immobilization matrix structure, as well as acetylation degree of chitin-chitosan used, has considerable influence on the catalytic behavior of immobilized proteases. Partially deacetylated chitin flakes represent a suitable option as support for enzyme immobilization because its preparation requires fewer steps than other supports. Two abundant seafood by-products were used to obtain a catalytic system with enough proteolytic activity to be considered for biotechnological applications in diverse fields.
[Show abstract][Hide abstract] ABSTRACT: Lentinula edodes is a mushroom of commercial interest which can be grown on several lignocellulosic substrates, such as vineyard pruning and wheat straw. In this study we obtained methanolic extracts from both of these substrates, and crude enzymatic extract from L. edodes. The methanolic extract were analyzed and characterized by thin layer chromatography and spectrophotometry finding that vineyard pruning extracts contained at least sixs UV light-active fraction; the three most polar fraction (Rf<0.5) were easy oxidized by the gungal extract with or without ABTS intermediary. In contrast, the methanolic extract of whest straw showed only three fraction dimly revealed to UV light, which were not oxidized by the enzymatic extrac even using ABTS. The presence of a higher number or subfractions in vineyard pruning methanolic extract, their possible higher number or subfractions in vineyard pruning methanolic extract, their possible higher concentration and the easily with which some of them were transformed by the fungus extracellular enzymes would explain in part because vineyard pruning is an excellent substrate for mushroom cultivation regarding to other substrates
[Show abstract][Hide abstract] ABSTRACT: Pterygoplichthys disjunctivus viscera chymotrypsin was purified by fractionation with ammonium sulfate (30-70 % saturation), gel filtration, affinity, and ion exchange chromatography. Chymotrypsin molecular weight was approximately 29 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), shown a single band in zymogram. Electrofocusing study suggested being an anionic enzyme (pI ≈ 3.9), exhibiting maximal activity at pH 9 and 50 °C, using Suc-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulfonyl fluoride (PMSF) (99 %), and N-tosyl-L: -phenylalanine chloromethyl ketone (TPCK) (94 %). Enzyme activity was affected by the following ions in decreasing order: Hg(2+), Fe(2+), Cu(2+), Li(1+), Mg(2+), K(1+), Mn(2+), while Ca(2+) had no effect. Chymotrypsin activity decreased continuously as NaCl concentration increased (from 0 to 30 %). K (m) and V (max) values were 0.72 ± 1.4 mM and 1.15 ± 0.06 μmol/min/mg of protein, respectively (SAAPNA as substrate). Results suggest the enzyme has a potential application where low processing temperatures are needed, such as in fish sauce production.
Fish Physiology and Biochemistry 07/2012; 39(2). DOI:10.1007/s10695-012-9684-3 · 1.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plant volatiles have complex intra- and interspecific effects in the environment that include plant/herbivore interactions. Identifying the quantity and quality of volatiles produced by a plant is needed to aid the process of determining which chemicals are exerting what effects and then examining whether these effects can be manipulated to benefit society. The qualitative characterization of volatile compounds emitted by pecan, Carya illinoinensis (Wang.) K. Koch, was begun in order to establish a database for investigating how these volatiles affect Acrobasis nuxvorella Nuenzig, a monophagous pest of pecan. Headspace solid-phase microextraction combined with gas chromatography-mass spectrometry was used for the analysis of the volatile constituents of pecan during three phenological stages (dormant buds, intact new shoot growth and intact nutlets) of the Western Schley and Wichita cultivars.
About 111 distinct compounds were identified from the two cultivars, accounting for ∼99% of the headspace volatiles. The chromatographic profiles of both varieties revealed variations in the volatile composition and proportion between cultivars, with a predominance of terpene hydrocarbons, of the sesquiterpenes class, as well as monoterpenes.
The significantly higher responsiveness recorded for the larvae of A. nuxvorella to C. illinoinensis shoots indicates that the larvae may be activated by terpenes emanating from the new shoot growth. This is the first study that has examined volatiles of pecan in Mexico.
Pest Management Science 12/2011; 67(12):1522-7. DOI:10.1002/ps.2205 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Arbuscular mycorrhizal fungi (AMF) are used as biofertilizers because when colonizing the roots they can improve growth, production and plant water status. The potential benefit of host is higher with native AMF. In this study the effect of a mixed inoculum and a consortium of seven species of AMF native of the Sonoran Desert associated with Cucurbita pepo var. pepo planted with drought, and high and low salinity was evaluated. The response of these inocula was compared with AMF from a temperate region and the control. The experimental design was a completely random design and a factorial analysis was performed: 4 (three inocula and the control) × 2 (with and without stress) with low salinity or drought; for high saline stress data an ANOVA was applied. The variables analyzed were shoot and root dry weight, percentage of leaf moisture, water and osmotic potential, percentage of mycorrhized roots, mycelium in soil and total number of spores. It was found that mixed inoculum of native AMF decreased physiological stress in C. pepo var. pepo caused by drought and low salinity, while the consortium of seven species of native AMF improved response to high saline stress, compared with other treatments (p<0.05).
[Show abstract][Hide abstract] ABSTRACT: Vermiculated sailfin catfish (Pterygoplichthys disjunctivus, Weber, 1991), a member of the Loricariidae family and an invasive species of several inland waters around the world, possess
an enormous digestive tract representing about 10% of fish weight. Thus, the aim of this study was to partially characterize
proteases from their digestive tracts. Azocasein digestion of the crude extract of intestine at different pH values and temperatures
revealed the presence of alkaline proteases with optimum activities at pH 9.0 and 50°C. Incubation assays of the crude extract
with inhibitors such as phenyl methyl sulfonyl fluoride, N-α-p-tosyl-l-lysine chloromethyl ketone, N-tosyl-phenyalanine chloromethyl ketone, benzamidine, pepstatin A and ethylenediamine tetra-acetic acid showed that trypsin
and chymotrypsin are the main alkaline proteinases present. Zymography showed that the crude extract of Pterygoplichthys
disjunctivus viscera contained proteases with molecular masses ranging from 21.5 to 116kDa. Trypsin and chymotrypsin were inhibited by
the following ions in decreasing order: Hg2+, Fe2+, Cu2+, Li+, Mg2+, K+, while Mn2+, and Ca2+ had no effect. Activities decreased continuously as the NaCl concentration increased from 0 to 30%. These results constitute
important background information for future studies and for the potential biotechnological use of the crude digestive extract
from this invasive species.
[Show abstract][Hide abstract] ABSTRACT: Lysyl oxidase (LOX) was detected and partially purified from jumbo squid (Dosidicus gigas) muscle, for the first time. A procedure for the purification of LOX from jumbo squid muscle which consisted of urea extraction, Sephadex G-75 and anion exchange chromatography was developed. Activity of partially purified LOX was 390-fold higher than the original extract. Two protein fractions with 32 and 24 kDa were detected by SDS-PAGE. The enzyme was strongly inhibited by β-aminopropionitrile fumarate, a specific LOX inhibitor. LOX was purified with 3.8% yield, showing a specific activity of 0.078 IU mg−1 protein. This knowledge will help understand the behaviour of jumbo squid protein during cool storage or manufacture.
International Journal of Food Science & Technology 06/2011; 46(8):1711 - 1715. DOI:10.1111/j.1365-2621.2011.02676.x · 1.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Concentrated urine formation in the kidney is accompanied by conditions that favor the accumulation of reactive oxygen species (ROS). Under hyperosmotic conditions, medulla cells accumulate glycine betaine, which is an osmolyte synthesized by betaine aldehyde dehydrogenase (BADH, EC 126.96.36.199). All BADHs identified to date have a highly reactive cysteine residue at the active site, and this cysteine is susceptible to oxidation by hydrogen peroxide. Porcine kidney BADH incubated with H(2)O(2) (0-500 μM) lost 25% of its activity. However, pkBADH inactivation by hydrogen peroxide was limited, even after 120 min of incubation. The presence of coenzyme NAD(+) (10-50 μM) increased the extent of inactivation (60%) at 120 min of reaction, but the ligands betaine aldehyde (50 and 500 μM) and glycine betaine (100 mM) did not change the rate or extent of inactivation as compared to the reaction without ligand. 2-Mercaptoethanol and dithiothreitol, but not reduced glutathione, were able to restore enzyme activity. Mass spectrometry analysis of hydrogen peroxide inactivated BADH revealed oxidation of M278, M243, M241 and H335 in the absence and oxidation of M94, M327 and M278 in the presence of NAD(+). Molecular modeling of BADH revealed that the oxidized methionine and histidine residues are near the NAD(+) binding site. In the presence of the coenzyme, these oxidized residues are proximal to the betaine aldehyde binding site. None of the oxidized amino acid residues participates directly in catalysis. We suggest that pkBADH inactivation by hydrogen peroxide occurs via disulfide bond formation between vicinal catalytic cysteines (C288 and C289).
[Show abstract][Hide abstract] ABSTRACT: Renal hyperosmotic conditions may produce reactive oxygen species, which could have a deleterious effect on the enzymes involved in osmoregulation. Hydrogen peroxide was used to provoke oxidative stress in the environment of betaine aldehyde dehydrogenase in vitro. Enzyme activity was reduced as hydrogen peroxide concentration was increased. Over 50% of the enzyme activity was lost at 100 μM hydrogen peroxide at two temperatures tested. At pH 8.0, under physiological ionic strength conditions, peroxide inhibited the enzyme. Initial velocity assays of betaine aldehyde dehydrogenase in the presence of hydrogen peroxide (0-200 μM) showed noncompetitive inhibition with respect to NAD(+) or to betaine aldehyde at saturating concentrations of the other substrate at pH 7.0 or 8.0. Inhibition data showed that apparent V(max) decreased 40% and 26% under betaine aldehyde and NAD(+) saturating concentrations at pH 8.0, while at pH 7.0 V(max) decreased 40% and 29% at betaine aldehyde and NAD(+) saturating concentrations. There was little change in apparent Km(NAD) at either pH, while Km(BA) increased at pH 7.0. K(i) values at pH 8 and 7 were calculated. Our results suggest that porcine kidney betaine aldehyde dehydrogenase could be inhibited by hydrogen peroxide in vivo, thus compromising the synthesis of glycine betaine.
Redox report: communications in free radical research 12/2010; 15(6):282-7. DOI:10.1179/135100010X12826446921941 · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Kidney medulla cells are exposed to a wide range of changes in the ionic and osmotic composition of their environment as a consequence of the urine concentrating mechanism. During antidiuresis NaCl and urea concentrations increase and an efficient urinary concentrating mechanism is accompanied by medullar hypoxia. Medullar hypotonicity increases reactive oxygen species, a byproduct of mitochondria during ATP production. High intracellular ionic strength, hypoxia and elevated ROS concentration would have deleterious effects on medulla cell function. Medulla cells respond to hypertonicity by accumulating organic osmolytes, such as glycine betaine, glycerophosphorylcholine, sorbitol, inositol, and taurine, the main functions of which are osmoregulation and osmoprotection. The accumulation of compatible osmolytes is thus crucial for the viability of renal medulla cells. Studies about the effects of reactive oxygen species (ROS) on the enzymes involved in the synthesis of osmolytes are scarce. In this review we summarize the information available on the effects of ROS on the enzymes involved in osmolyte synthesis in kidney.
Life sciences 10/2010; 87(17-18):515-20. DOI:10.1016/j.lfs.2010.08.003 · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The enzyme 5′-nucleotidase of jumbo squid (Dosidicus gigas) mantle was purified and its SDS–PAGE showed a single band of 33kDa, whereas a protein with a molecular mass of 107kDa was detected by gel filtration suggesting a homotrimeric nature of this enzyme. Subunits of the named enzyme were not linked by covalent bonds. Isoelectric focusing of this enzyme showed a pI of 3.6–3.8 and presented a hyperbolic kinetics with Vmax of 1.16μM/min/mg of protein, Km of 1.49mM, Kcat of 3.48μM of Pιs−1 and Kcat/Km relation of 356.52 ((mol/L)−1s−1). Purified enzyme preferred AMP as substrate (by 6.7-folds) than IMP, showing a Km of 6.34mM, Vmax of 0.19μM/min/mg of protein a Kcat of 0.3388mol of Pιs−1 and Kcat/Km relation of 53.44 ((mol/L)−1s−1). The low Km in relation to purified AMP deaminase of the same organism suggested a high contribution of 5′-nucleotidase in AMP degradation in jumbo squid mantle.
[Show abstract][Hide abstract] ABSTRACT: The NAD+-dependent animal betaine aldehyde dehydrogenases participate in the biosynthesis of glycine betaine and carnitine, as well as in polyamines catabolism. We studied the kinetics of inactivation of the porcine kidney enzyme (pkBADH) by the drug disulfiram, a thiol-reagent, with the double aim of exploring the enzyme dynamics and investigating whether it could be an in vivo target of disulfiram. Both inactivation by disulfiram and reactivation by reductants were biphasic processes with equal limiting amplitudes. Under certain conditions half of the enzyme activity became resistant to disulfiram inactivation. NAD+ protected almost 100% at 10 microM but only 50% at 5mM, and vice versa if the enzyme was pre-incubated with NAD+ before the chemical modification. NADH, betaine aldehyde, and glycine betaine also afforded greater protection after pre-incubation with the enzyme than without pre-incubation. Together, these findings suggest two kinds of active sites in this seemingly homotetrameric enzyme, and complex, unusual ligand-induced conformational changes. In addition, they indicate that, in vivo, pkBADH is most likely protected against disulfiram inactivation.
Archives of Biochemistry and Biophysics 01/2008; 468(2):167-73. DOI:10.1016/j.abb.2007.09.022 · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lentinula edodes is considered an alternative recycling agent for agricultural wastes, and there have been several studies to understand the relationship between its growth and ligninolytic activity. We tested the effect of wood from viticulture pruning, extracted with solvents of differing polarity, on the biomass production and activity pattern of ligninolytic enzymes. The analysis was done by measuring the mycelial dry mass and enzyme activity of liquid growth medium during the culture of L. edodes, adding either single extracts or a combination of extracts. Polar extracts enhanced mycelial production, and the activity patterns of lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, and laccase were comparable to their activities predicted by ligninolysis models proposed for other fungi. We conclude that the polar extracts could be useful for enhancing fungal biomass production and for modifying lignin degradation because the regulation of ligninolytic enzyme activity is differentially influenced by the polarity of the extract.
Canadian Journal of Microbiology 11/2007; 53(10):1150-7. DOI:10.1139/W07-080 · 1.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Trehalose 6-phosphate synthase was purified from Selaginella lepidophylla plants and three aggregates of the enzyme were found by molecular exclusion chromatography, ion exchange chromatography and electrophoresis. Molecular exclusion chromatography showed four activity peaks with molecular weights of 624, 434, 224 and 115 kDa. Ion exchange chromatography allowed three fractions to be separated with TPS activity which eluted at 0.35, 0.7 and 1 M KCl. Native PAGE of each pool had three protein bands with apparent M(r) 660, 440 and 200 kDa. Western blot results showed that anti-TPS antibody interacted with 115 and 67 kDa polypeptides; these polypeptides share peptide sequences as indicated by internal sequence data. The effects of pH and temperature on enzyme stability and activity were studied. For fractions eluted at 0.35 and 1.0 M KCl, the optimum pH is 5.5, while an optimum pH of 7.5 for 0.7 M fraction was found. The three fractions eluted from ion exchange chromatography were stable in a pH 5-11 range. Optimal temperatures were 25, 45 and 55 degrees C for 0.7, 0.35 and 1.0 M fractions, respectively. The 0.7 M KCl fraction showed highest stability in a temperature range of 25-60 degrees C, whereas the 0.35 M KCl fraction had the lowest in the same temperature range.
[Show abstract][Hide abstract] ABSTRACT: Isolation of high-quality RNA is a necessary step in gene expression analysis. Although many methods can be used to isolate
RNA from plants where contamination of preparations with complex carbohydrates or phenolic compounds is a problem, the application
of these methods toSelaginella lepidophylla tissues has failed to obtain good-quality RNA. Here we introduce 2 modifications to the method developed by Chomczynski and
Sacchi (1987), generating a simple and rapid method that allows the isolation of intact RNA fromS. lepidophylla-dehydrated tissues. Although the introduced modifications are not new, their addition proved to be decisive for success in
RNA isolation. Quality of the RNA obtained was evaluated by electrophoresis in agarose and by 3 different PCR-based techniques—RT-PCR,
RNA differential display, and synthesis of a cDNA library.