Proteoliposomes as matrix vesicles' biomimetics to study the initiation of skeletal mineralization

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite (HA) seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Ion transporters control the availability of phosphate and calcium needed for HA deposition. The lipidic microenvironment in which MV-associated enzymes and transporters function plays a crucial physiological role and must be taken into account when attempting to elucidate their interplay during the initiation of biomineralization. In this short mini-review, we discuss the potential use of proteoliposome systems as chondrocyte- and osteoblast-derived MVs biomimetics, as a means of reconstituting a phospholipid microenvironment in a manner that recapitulates the native functional MV microenvironment. Such a system can be used to elucidate the interplay of MV enzymes during catalysis of biomineralization substrates and in modulating in vitro calcification. As such, the enzymatic defects associated with disease-causing mutations in MV enzymes could be studied in an artificial vesicular environment that better mimics their in vivo biological milieu. These artificial systems could also be used for the screening of small molecule compounds able to modulate the activity of MV enzymes for potential therapeutic uses. Such a nanovesicular system could also prove useful for the repair/treatment of craniofacial and other skeletal defects and to facilitate the mineralization of titanium-based tooth implants.

Increased expression of keratinase and other peptidases by Candida parapsilosis mutants

Keratinases are enzymes of great importance involved in pathogenic processes of some fungi. They also have a widespread ecological role since they are responsible for the degradation and recycling of keratin. On the one hand, studying them furthers our knowledge of pathogenicity mechanisms, which has important implications for human health, and on the other hand, understanding their ecological role in keratin recycling has biotechnological potential. Here, a wild-type keratinolytic Candida parapsilosis strain isolated from a poultry farm was treated with ethyl methanesulfonate in order to generate mutants with increased keratinase activity. Mutants were then cultured on media with keratin extracted from chicken feathers as the sole source of nitrogen and carbon. Approximately 500 mutants were screened and compared with the described keratinolytic wild type. Three strains, H36, I7 and J5, showed enhanced keratinase activity. The wild-type strain produced 80 U/mL of keratinolytic activity, strain H36 produced 110 U/mL, strain I7, 130 U/mL, and strain J5, 140 U/mL. A 70% increase in enzyme activity was recorded for strain J5. Enzymatic activity was evaluated by zymograms with proteic substrates. A peptidase migrating at 100 kDa was detected with keratin, bovine serum albumin and casein. In addition, a peptidase with a molecular mass of 50 kDa was observed with casein in the wild-type strain and in mutants H36 and J5. Gelatinase activity was detected at 60 kDa. A single band of 35 kDa was found in wild-type C. parapsilosis and in mutants with hemoglobin substrate.

Placental hydroxymethylation vsmethylation at the imprinting control region 2 on chromosome 11p15.5

In addition to methylated cytosines (5-mCs), hydroxymethylcytosines (5-hmCs) are present in CpG dinucleotide-enriched regions and some transcription regulator binding sites. Unlike methylation, hydroxymethylation does not result in silencing of gene expression, and the most commonly used methods to study methylation, such as techniques based on restriction enzymatic digestion and/or bisulfite modification, are unable to distinguish between them. Genomic imprinting is a process of gene regulation where only one member of an allelic pair is expressed depending on the parental origin. Chromosome 11p15.5 has an imprinting control region (ICR2) that includes a differentially methylated region (KvDMR1) that guarantees parent-specific gene expression. The objective of the present study was to determine the presence of 5-hmC at the KvDMR1 in human placentas. We analyzed 16 third-trimester normal human placentas (chorionic villi). We compared two different methods based on real-time PCR after enzymatic digestion. The first method distinguished methylation from hydroxymethylation, while the other method did not. Unlike other methylation studies, subtle variations of methylation in ICRs could represent a drastic deregulation of the expression of imprinted genes, leading to important phenotypic consequences, and the presence of hydroxymethylation could interfere with the results of many studies. We observed agreement between the results of both methods, indicating the absence of hydroxymethylation at the KvDMR1 in third-trimester placentas. To the best of our knowledge, this is the first study describing the investigation of hydroxymethylation in human placenta using a genomic imprinting model.

From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones

The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia.

Cell wall polysaccharides of common beans (Phaseolus vulgaris L.)

The soluble and insoluble cotyledon (SPF-Co and IPF-Co) and tegument (SPF-Te and IPF-Te) cell wall polymer fractions of common beans (Phaseolus vulgaris) were isolated using a chemical-enzymatic method. The sugar composition showed that SPF-Co was constituted of 38.6% arabinose, 23.4% uronic acids, 12.7% galactose, 11.2% xylose, 6.4% mannose and 6.1% glucose, probably derived from slightly branched and weakly bound polymers. The IPF-Co was fractionated with chelating agent (CDTA) and with increasing concentrations of NaOH. The bulk of the cell wall polymers (29.4%) were extracted with 4.0M NaOH and this fraction contained mainly arabinose (55.0%), uronic acid (18.9%), glucose (10.7%), xylose (10.3%) and galactose (3.4%). About 8.7% and 10.6% of the polymers were solubilised with CDTA and 0.01M NaOH respectively and were constituted of arabinose (52.0 and 45.9%), uronic acids (25.8 and 29.8%), xylose (9.6 and 10.2%), galactose (6.1 and 3.9%) and glucose (6.5 and 3.8%). The cell wall polymers were also constituted of small amounts (5.6 and 7.2%) of cellulose (CEL) and of non-extractable cell wall polymers (NECW). About 16.8% and 17.2% of the polymers were solubilised with 0.5 and 1.0M NaOH and contained, respectively, 92.1 and 90.7% of glucose derived from starch (IST). The neutral sugar and polymers solubilization profiles showed that weakly bound pectins are present mainly in SPF-Co (water-soluble), CDTA and 0.01-0.1M NaOH soluble fractions. Less soluble, highly cross-linked pectins were solubilised with 4.0M NaOH. This pectin is arabinose-rich, probably highly branched and has a higher molecular weight than the pectin present in SPF-Co, CDTA and 0.01-0.1M NaOH fractions.

Influence of depectinization in the ultrafiltration of West Indian cherry (Malpighia glabra L.) and pineapple (Ananas comosus (L.) Meer) juices

West Indian cherry (Malpighia glabra L.) and pineapple (Ananas comosus (L.) Meer)juice clarification by cross-flow UF, using polysulphone hollow fiber and ceramic tubular membranes with, respectively, nominal molecular weight cut off values of 100kDaltons and average pore diameters of 0.01mm, were studied. The influence of enzymatic treatment using enzyme concentrations of 20, 100 and 300mg/L, a time of 90min and a temperature of 40ºC for depectinization was verified. The juices were then clarified in a laboratory scale filtration unit, with an effective filtration area of 0.12m² for the polysulphone hollow fiber membrane and of 0.005m² for the ceramic tubular membranes. The influence of enzymatic treatment on viscosity, turbidity and total pectin of the juice, before ultrafiltration, is reported. Membrane performance was evaluated in terms of flow rate and clarity of the permeate. The permeate flow rate of depectinized pineapple juice was higher (30 - 60%) for both membranes. Depectinized West Indian cherry juice presented a lower permeate flow rate for the polysulphone hollow fiber membrane. The increase in permeate flow rate, with the use of the 300mg/L and 100mg/L enzyme concentration was not significant, so it is economically advantageous to ultrafilter depectinized juice, treated with an enzyme concentration of 20mg/L.

The influence of the broccoli (Brassica oleracea var. itálica)fill weigth on postharvest quality

Broccoli is a vegetable of important economic value as well as a source of vitamin C and anticarcinogenic property substances. Although it can be minimally processed, this vegetable presents fast senescence, characterized by yellowing, loss of turgescence, development of off-odors, increase in enzymatic activity and reduction of nutritional value. Among the frequently used techniques that can extend the shelf-life of minimally processed broccoli, we have the use of cool storage and modified atmosphere packaging (MAP). Several factors interfere in the efficiency of MAP, influencing the speed and intensity of the changes on atmosphere composition. This work aimed to study the influence of the amount packaged broccoli on its postharvest quality, when stored at 1ºC. Results demonstrated strong influence of the use of MAP compared to control treatment. It was observed that for all packaged treatments there was better vitamin C retention, lower loss of weight, lower peroxidase activity and better turgidity score of broccoli. This study demonstrated that the treatments with higher amounts of product showed the worst performance on vitamin C and aroma evaluation. The peroxidase activity was also influenced, and the best results were found in the treatment with higher amounts, which presented lowest values. However there is a tendency of increase in the activity along the time, especially at the end of the experiment. It was not detected the influence of the fill weight about color evaluation.

Chemical and nutritional evaluation of kernels of bocaiuva, Acrocomia aculeata (Jacq.) Lodd.

Protein characterization and results of proximate composition and mineral analyses of fruit kernels of bocaiuva, Acrocomia aculeata (Jacq.) Lodd., are reported. The kernels presented high contents of oil (51.7%), protein (17.6%) and fiber (15.8%). The seeds´ soluble proteins were isolated according to their solubility. The main separated proteins were globulins (53.5%) and glutelins (40.0%). Moreover, the presence of low molecular mass proteases in these two fractions was shown by the SDS-PAGE method. The assays of protease-inhibitory and hemagglutinating activities showed that bocaiuva´s protein fractions were not resistant to trypsin or chymotrypsin activities and that both had low lectin content. The globulin in vitro digestibility assay resembled a casein standard. Neither globulin nor glutelin enzymatic hydrolyses increased significantly (p < 0.05) after heat treatment. Threonine and lysine are the most limiting amino acids, respectively from two major protein fractions of the bocaiuva kernel, globulin (47.1% amino acid score) and glutelin (49.5% amino acid score), in terms of the theoretical profiles for children in the age range of 2 to 5 years recommended by the FAO/WHO. Bocaiuva kernels are found to be rich in calcium, phosphorus and manganese compared to some fruit nuts such as cashew and coconut.

Production and partial characterization of lipase from Penicillium verrucosum obtained by submerged fermentation of conventional and industrial media

The growing interest in lipase production is related to the potential biotechnological applications that these enzymes present. Current studies on lipase production by submerged fermentation involve the use of agro-industrial residues aiming at increasing economic attractiveness. Based on these aspects, the objective of this work was to investigate lipase production by Penicillium verrucosum in submerged fermentation using a conventional medium based on peptone, yeast extract, NaCl and olive oil, and an industrial medium based on corn steep liquor, Prodex Lac (yeast hydrolysate), NaCl and olive oil, as well as to characterize the crude enzymatic extracts obtained. Kinetics of lipase production was evaluated and the highest enzymatic activities, of 3.15 and 2.22 U.mL-1, were observed when conventional and industrial media were used, respectively. The enzymatic extract showed optimal activity in the range from 30 to 40 °C and at pH 7.0. Although the industrial medium presents economical advantages over the conventional medium, the presence of agro-industrial residues rich in nitrogen and other important nutrients seemed to contribute to a reduction in lipase activity.

In vitro protein digestibility of enzymatically pre-treated bean (Phaseolus vulgaris L.) flour using commercial protease and Bacillus sp. protease

The common bean (Phaseolus vulgaris L.) is a staple food in the Brazilian diet and represents the major source of dietary protein and other micronutrients and minerals. Despite the considerable protein concentration in beans, the food is considered of low biological value when compared to animal proteins and other plant protein sources. To improve the availability of protein in beans, enzymatic treatments were performed in four cultivars (ON, OPNS, TAL and VC3). The approach was a completely randomized design with four replicates. We used a 4 × 3 factorial arrangement (four cultivars and three treatments: treatment 1-addition of commercial protease (Trypsin 250, Difco), treatment 2-addition of protease from Bacillus sp., and treatment 3:-control without enzyme addition). The enzyme: substrate ratio was 5% w/w (amount of enzyme per total protein in bean flour). The approach was a completely randomized design with four replicates. A 4 × 3 factorial arrangement (four cultivars and three treatments, the same as those mentioned above) was used. The concentration of total protein (g.100 g-1 of dry matter) in the samples ranged from 16.94 to 18.06%, while the concentration of total phenolics was between 0.78 and 1.12% (g Eq. tannic acid.100 g-1 dry matter). The in vitro protein digestibility of enzymatically untreated bean flour (control) ranged from 47.30 to 56.17% based on the digestibility of casein. Concentrations of P, K, Ca, Mg, and Zn observed in the four cultivars tested were within the average values available in the literature. Treatment 2 with protease from Bacillus sp. induced decreases in the levels of Cu and Mn. The average Fe content increased in all bean flour samples when treated with proteases, reaching a maximum increase of 102% in the TAL flour treated with protease from Bacillus sp. The digestibility of all beans tested was significantly increased (p < 0.05) after the enzyme treatment. The greatest change was observed in the OPNS cultivar treated with protease from Bacillus sp., which increased its digestibility from 54.4% (control treatment) to 81.6%.

Production of glucose and fructose syrups from cassava (Manihot esculenta Crantz) starch using enzymes produced by microorganisms isolated from Brazilian Cerrado soil

The high demands for sugars and the development of enzymatic technology have increased the production of sweeteners, especially for glucose and fructose syrups. This work describe a technology for glucose and fructose syrups from Brazilian cassava starch using enzymes produced by soil microrganisms isolated from the Brazilian Cerrado soil. Firstly, Aspergillus niger and Streptomyces sp. were isolated from the soil and used as glucoamylase (GA) and glucose isomerase (GI) producer sources. After characterization, GA and GI exhibited optimum pH 4.5 and 8.0, respectively. GA showed maximum activity at 60 ºC and GI at 85 ºC. GA and GI retained 65 and 80%, respectively, of initial activity after 180 minutes of incubation at 60 ºC. The kinetic parameters Km and Vmáx were 0.476 (mg.mL-1) and 8.58 (µmol/minute) for GA and 0.082 (M) and 48.20 (µmol/minute) for GI. The maximum glucose syrups production occurred after 24 hours of reaction with a 98% yield. The production of fructose syrups with 42% (w/v) was reached after 96 hours of reaction.

Polyphenoloxidase and peroxidase in avocado pulp (Persea americana Mill.)

The aim of the present investigation was to evaluate the enzymatic activity of polyphenoloxidase and peroxidase in avocado pulps, from the Northwest area of Paraná-Brazil, in order to compare the varieties on their enzymatic activity for both, minimum and industrial processing. Enzymatic extracts were prepared from avocado pulp of Choquete, Fortuna and Quintal varieties, in green and ripe maturation stage. Thermal treatment was applied with temperatures 60, 65, 70, 75 and 80 °C. The enzymatic activities were determined by using spectrophotometer. A decline of polyphenoloxidase activity was observed in all of the varieties when both, temperature and time increased. Total inactivation of enzymes was not observed in the largest temperature. Fortuna and Choquete variety showed the lowest polyphenoloxidase activity in the ripe stage. Soluble peroxidase showed activity in the green stage, whereas, ionically bound peroxidase activity increased with the change from green to ripe maturation stage in Choquete variety.

Clarification of pineapple juice by microfiltration

In the present work, pineapple juice was first hydrolyzed with a commercial pectinase (Ultrazym 100 G) and then clarified by microfiltration. A tubular polyethersulfone membrane with an average cut-off of 0.3 µm and a total effective filtration area of 0.05 m² was applied. The transmembrane pressures were 1.5 and 3.0 bar, respectively, and the processes was conducted at room temperature. The results showed that the pineapple juice permeate fluxes were of 57.77 L/m²/hours (1.5 bar) and 46.85 L/m²/hours (3.0 bar). Concentration polarization and possibly fouling occurred during the processes. The best clarified juice fluxes were obtained when low transmembrane pressures (1.5 bar) were applied.

Influence of temperature on the respiration rate of minimally processed organic carrots (Daucus Carota L. cv. Brasília)

The objective of this work was to study the influence of temperature on the respiration rate of minimally processed organic carrots (Daucus Carota L. cv. Brasília) with and without the application of a gelatin film. The samples were packed in flexible bags and stored at 1, 5 and 10 °C. During the five days of storage, the CO2 and O2 concentrations in the headspace of the package were monitored by gas chromatography, and the mathematical model based on enzymatic kinetics was used to estimate the respiration rate of minimally processed organic carrots. The effect of temperature on the respiration rate was evaluated by the Arrhenius equation. The results showed that the O2 concentration decreased during the storage period and the CO2 concentration increased. The lowest O2 concentrations of 2.59 and 2.66% were found for the samples stored at 10 °C with and without the film, respectively. For the CO2 concentration, the highest concentrations of 16.25 and 16.32% were again found for the temperature of 10 °C with and without the application of the film, respectively. At the temperature of 1 °C, the maximum respiratory rates for the samples without and with the film were 10.82 and 10.44 mL CO2.kg-1/hour, respectively, after 72 hours of storage. The greatest respiratory rate was obtained at 10 °C, the maximum peak being reached after 50 hours. Activation energy values were of 50.59 kJ.mol-1, for the samples with the film, and 51.88 kJ.mol-1 for the samples without the film.

Production of isomaltulose obtained by Erwinia sp. cells submitted to different treatments and immobilized in calcium alginate

In recent decades, there has been an increase in the studies of isomaltulose obtainment, due to its physicochemical properties and physiological health benefits. These properties, which include low cariogenicity, low glycemic index and greater stability, allow the use of this sweetener as a substitute for sucrose in foods; besides the fact that it can be converted to isomalt, a dietary non-cariogenic sugar alcohol used in pharmaceuticals as well as in the food industry. Isomaltulose (6-O-α-D-glucopyronosyl-1-6-D-fructofuranose) is a disaccharide reducer obtained by the enzymatic conversion of sucrose - the α-glucosyltransferase enzyme. Different treatments were performed for the preparation of whole cells; lysed cells; and crude enzyme extract of Erwinia sp. D12 strain immobilized in calcium alginate. The packed bed column of granules, containing Erwinia sp. cells sonicated and immobilized in calcium alginate (CSI), reached a maximum conversion of 53-59% sucrose into isomaltulose and it presented activity for 480 hours. The converted syrup was purified and the isomaltulose crystallization was performed through the lowering of temperature. The isomaltulose crystals presented purity of 96.5%.