Development of novel economical methodologies for zymographic analysis of purified xylano-pectinolytic enzymes using agrowaste-based substrates.

In this study, zymographic analysis for xylanase and pectinase enzymes has been carried out using agrowaste residues, wheat bran and citrus peel as well as their extracts. Isozymic forms of xylanase as well as pectinase enzyme displayed comparable zymographic bands onto agar petriplates containing either commercial substrates (xylan and pectin), agrowaste-based substrates (wheat bran and citrus peel), or polysaccharides extracted from these agrowastes (crude xylan and pectin extracted from wheat bran and citrus peel, respectively), indicating the fact that agro residues and their extracts can be utilized as a substitute of cost-intensive commercial substrates, xylan and pectin for zymographic analysis. This is the first report revealing the zymographic analysis of xylano-pectinolytic enzymes using agro-based solid residues particles or polysaccharides extracted from agro-based residues.

A simplified and miniaturized glucometer-based assay for the detection of ß-glucosidase activity.

ß-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of ß-glucosidase activity. Single-factor experiments showed that optimum ß-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of ß-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from ß-dextranase, snailase, ß-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for ß-glucosidase activity. The easy-to-operate method was successfully used to detect a series of ß-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of ß-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.

A polygalacturonase localized in the Golgi apparatus in Pisum sativum.

Pectin is a plant cell wall constituent that is mainly composed of polygalacturonic acid (PGA), a linear α1,4-d-galacturonic acid (GalUA) backbone. Polygalacturonase (PG) hydrolyzes the α1,4-linkages in PGA. Nearly all plant PGs identified thus far are secreted as soluble proteins. Here we describe the microsomal PG activity in pea (Pisum sativum) epicotyls and present biochemical evidence that it was localized to the Golgi apparatus, where pectins are biosynthesized. The microsomal PG was purified, and it was enzymatically characterized. The purified enzyme showed maximum activity towards pyridylaminated oligogalacturonic acids with six degrees of polymerization (PA-GalUA6), with a Km value of 11 µM for PA-GalUA6. The substrate preference of the enzyme was complementary to that of PGA synthase. The main PG activity in microsomes was detected in the Golgi fraction by sucrose density gradient ultracentrifugation. The activity of the microsomal PG was lower in rapidly growing epicotyls, in contrast to the high expression of PGA synthase. The role of this PG in the regulation of pectin biosynthesis or plant growth is discussed.

A magnetic tri-enzyme nanobiocatalyst for fruit juice clarification.

The major complications in fruit juice quality improvement are the presence of polysaccharides components in the form of disrupted fruit cell wall and cell materials. Hence, breakdown of cellulose along with pectin and starch is important for the juice processing. In this context, magnetic tri-enzyme nanobiocatalyst was prepared by simultaneously co-immobilizing three enzymes; α-amylase, pectinase and cellulase onto amino-functionalized magnetic nanoparticle by 60mM glutaraldehyde concentration with 10h cross-linking time for one pot juice clarification. The prepared nanobiocatalyst was characterized by FT-IR, SEM and XRD. The thermal (50-70°C) and pH (3-6) stability studies indicated more than two folds increment in half-life and enhanced tolerance to lower pH. The immobilized enzymes retained up to 75% of residual activity even after eight consecutive cycles of reuse. Finally, the clarification of apple, grapes and pineapple juices using magnetic tri-enzyme showed 41%, 46% and 53% respective reduction in turbidity till 150min treatment.

Development of reproducible assays for polygalacturonase and pectinase.

Polygalacturonase and pectinase activities reported in the literature were measured by several different procedures. These procedures do not give comparable results, partly owing to the complexity of the substrates involved. This work was aimed at developing consistent and efficient assays for polygalacturonase and pectinase activities, using polygalacturonic acid and citrus pectin, respectively, as the substrate. Different enzyme mixtures produced by Aspergillus niger and Trichoderma reesei with different inducing carbon sources were used for the method development. A series of experiments were conducted to evaluate the incubation time, substrate concentration, and enzyme dilution. Accordingly, for both assays the recommended (optimal) hydrolysis time is 30min and substrate concentration is 5g/L. For polygalacturonase, the sample should be adjusted to have 0.3-0.8U/mL polygalacturonase activity, because in this range the assay outcomes were consistent (independent of dilution factors). Such a range did not exist for the pectinase assay. The recommended procedure is to assay the sample at multiple (at least 2) dilution factors and determine, by linear interpolation, the dilution factor that would release reducing sugar equivalent to 0.4g/L d-galacturonic acid, and then calculate the activity of the sample accordingly (dilution factor×0.687U/mL). Validation experiments showed consistent results using these assays. Effects of substrate preparation methods were also examined.

Evaluating the activity of the filamentous growth mitogen-activated protein kinase pathway in yeast.

Mitogen-activated protein kinase (MAPK) pathways are evolutionarily conserved signaling pathways that regulate diverse processes in eukaryotes. One such pathway regulates filamentous growth, a nutrient limitation response in budding yeast and other fungal species. This protocol describes three assays used to measure the activity of the filamentous growth pathway. First, western blotting for phosphorylated (activated) MAPKs (P∼MAPKs; Slt2p, Kss1p, Fus3p, and Hog1p) provides a measure of MAPK activity in yeast and other fungal species. Second, the PGU1 gene is a transcriptional target of the filamentous growth pathway. Cells that undergo filamentous growth secrete Pgu1p, an endopolygalacturonase that degrades the plant-specific polysaccharide pectin. We describe an assay that measures secreted pectinase activity, which reflects an active filamentous growth pathway. Finally, in yeast, two mucin-like glycoproteins, Msb2 and Flo11, regulate filamentous growth. Secretion of the processed and shed glycodomain of Msb2 is an indicator of MAPK activity. Flo11, the major adhesion molecule that controls filamentous growth and biofilm/mat formation, is also shed from cells. Detecting shed mucins with epitope-tagged versions of the proteins (secretion profiling) provides information about the regulation of filamentous growth across fungal species.

Effects of cold storage and 1-methylcyclopropene treatments on ripening and cell wall degrading in rabbiteye blueberry (Vaccinium ashei) fruit.

The effect of postharvest 1-methylcyclopropene and/or cold storage application on texture quality parameters during storage was determined. The changes in fruit quality (including weight loss, firmness, total soluble solids content, and ethylene production), cell wall material (including water-soluble fraction, ethylenediaminetetraacetic acid-soluble fraction, Na2CO3-soluble fraction, 4% KOH-soluble fraction, and 14% KOH-soluble fraction), and cell wall hydrolase activities (including polygalacturonase, endo-1,4-beta-D-glucanase, pectinesterase, alpha-L-arabinofuranosidase, and beta-galactosidase) were periodically measured up to 25 days after postharvest treatments. The application of cold storage reduced weight loss, ethylene production, and delayed ripening of blueberry fruit. The inhibition of senescence was associated with suppressed increase in cell wall hydrolase activities and retarded solubilization of pectins and hemicelluloses. Furthermore, no obvious differences in firmness, weight loss, ethylene production, and cell wall hydrolase activities between fruits with or without 1-methylcyclopropene application were observed, while significant lower levels of the detected parameters were found in cold storage fruit compared with fruit stored in room temperature. Thus, cold storage can be viewed as an effective means to extend the shelf life of blueberry fruit.

Optimisation of Environmental Conditions for Enhanced Production of Fungal Exopectinase Using Agro-industrial Wastes.

Management of household solid waste and agro industrial residues generated from various sources is a serious problem due to huge ever increasing population and pollution. Application of these worthless agro waste materials to generate a commercially valuable product, pectinase enzyme, using locally isolated fungal strain, Aspergillus flavipes, was the main motive of this study. Physiological characterisation and enzyme profile determination were done along with formulation of production media. Fruit skins, rags were used as C source and oil cakes were used for N source. Various combinations of these C and N sources were applied for revised production of pectinase enzyme compared to YEP basal media (29 U/ml). A huge increase in pectinase production of 40 U/ml was obtained with Citrus peel - Sesame oil cake (CS) media. The enzyme had its maximum activity at 500C, 4.5 pH. This was achieved at 45 min in 1.5% substrate concentration.

Comparison of cell wall metabolism in the pulp of three cultivars of 'Nanfeng' tangerine differing in mastication trait.

BACKGROUND: Like sweet orange (Citrus sinensis), tangerine (Citrus reticulata) is another citrus crop grown widely throughout the world. However, whether it shares a common mechanism with sweet orange in forming a given mastication trait is still unclear. In this study, three 'Nanfeng' tangerine cultivars, 'Yangxiao-26' ('YX-26') with inferior mastication trait, elite 'YX-26' with moderate mastication trait and 'Miguang' ('MG') with superior mastication trait, were selected to investigate the formation mechanism of mastication trait. RESULTS: 'MG' had the lowest contents of total pectin, protopectin and lignin and the highest gene expression levels of citrus polygalacturonase (PG) and pectin methylesterase (PME) at the end of fruit ripening, whereas 'YX-26' had the lowest water-soluble pectin (WSP) content, the highest lignin content and the lowest PG and PME expression levels. The contents of cellulose and hemicellulose were similar among the three tangerines. CONCLUSION: The fruit mastication trait of C. reticulata was determined by the proportions of WSP and protopectin as well as lignin content, not by cellulose and hemicellulose contents. Pectin content could be a major contribution to the feeling of mastication trait, while PG and PME exhibited an important role in forming a given mastication trait according to the present results as well as previous results for C. sinensis.

Constitutive and inducible pectinolytic enzymes from Aspergillus flavipes FP-500 and their modulation by pH and carbon source / Enzimas pectinolíticas constitutivas e indutíveis de Aspergillus flavipes FP-500 e sua modulação pelo pH e fonte de carbono

Growth and enzymes production by Aspergillus flavipes FP-500 were evaluated on pectin, polygalacturonic acid, galacturonic acid, arabinose, rhamnose, xylose, glycerol and glucose at different initial pH values. We found that the strain produced exopectinases, endopectinases and pectin lyases. Exopectinases and pectin lyase were found to be produced at basal levels as constitutive enzymes and their production was modulated by the available carbon source and pH of culture medium and stimulated by the presence of inducer in the culture medium. Endo-pectinase was basically inducible and was only produced when pectin was used as carbon source. Our results suggest that pectinases in A. flavipes FP-500 are produced in a concerted way. The first enzyme to be produced was exopectinase followed by Pectin Lyase and Endo-pectinase.

Avaliou-se o crescimento e a produção de enzimas por Aspergillus flavipes FP-500 em pectina, ácido poligalacturônico, ácido galacturônico, arabinose, ramnose, xilose, glicerol e glicose, em diferentes valores de pH inicial. Verificamos que a cepa produziu exopectinases, endopectinases e pectina liases. Exopectinases e pectina liases foram produzidas em níveis basais como enzimas constitutivas e sua produção foi modulada pela fonte de carbono disponível e pelo pH do meio de cultura e estimulada pela presença de indutores no meio de cultura. Endopectinase foi indutível e produzida somente quando pectina foi utilizada como fonte de carbono. Nossos resultados sugerem que as pectinases de A. flavipes FP-500 são produzidas de forma planejada. A primeira enzima a ser produzida foi expopectinase, seguida por pectina liase e endopectinase.

Turbidity-based assay for polygalacturonic acid depolymerase activity.

A technically simple and inexpensive discontinuous turbidity assay for qualitative and/or quantitative assessments of polygalacturonic acid depolymerase activity is presented. The enzyme reaction is initiated by the addition of enzyme preparation to a reaction mixture containing 0.02% polygalacturonic acid (PGA) in acetate buffer. The progress of the reaction is monitored by terminating aliquots of the reaction mixture (via heat treatment at appropriate times), subsequently adding poly(diallyldimethylammonium chloride) (PDADMAC) for turbidity development (approximately 30 min), and then measuring the turbidity (typically at 420 nm) of the resulting PGA-PDADMAC complex-containing solution. PGA depolymerase activity causes a decrease in the observed turbidity of PGA-PDADMAC-containing solutions because the stability of the interpolyelectrolyte PGA-PDADMAC complex is a function of the degree of polymerization of the PGA. The rate of turbidity change is herein shown to be proportional to a relatively wide range of enzyme concentrations. The assay is demonstrated using a commercial pectinase preparation and tomato fruit extracts.

Preparation and properties of an immobilized pectinlyase for the treatment of fruit juices.

Pectinlyase, present in different commercial pectinases used in juice technology, was immobilized on alginate beads. The optimal conditions were: 0.17 g alginate ml(-1), 1.2% (w/v or v/v) enzyme concentration and acetic-HCl/glycine-HCl buffer at pH 3.6 or tris-HCl/imidazole buffer at pH 6.4. Maximum percentage of immobilization (10.6%) was obtained with Rapidase C80. Kinetic parameters of free and immobilized pectinlyase were also determined. The pH and temperature at which activity of soluble and immobilized enzyme was maximum were 7.2 and 55 degrees C. Thermal stability was not significantly altered by immobilization, especially at 40 degrees C, showing two periods of different stability. Free and immobilized preparation reduced the viscosity of highly esterified pectin from 1.09 to 0.70 and 0.72 mm(2) s(-1), respectively, after 30 min at 40 degrees C. Furthermore, the immobilized enzyme could be re-used through 4 cycles and the efficiency loss in viscosity reduction was found to be only 9.2%.

[Study on extraction process of flavone from Ginkgo biloba leaves by enzymic treatment].

The extraction technology of flavonoids from Ginkgo biloba leaves was studied in this paper. When the crude leaves used the cellulase enzyme pretreatment before extraction process, the extraction yield of total flavone was up to 2.01%. The extraction conditions were also determined experimentally as following: concentration of enzyme in the liquor being 0.125 g/L, mixture ratio between enzyme and substrate being 1:1200, treating 2 hours, at 45 degrees C, under natural pH value.

Changes in phenol oxidase and peroxidase levels in cocoyam tubers of different postharvest ages infected by Sclerotium rolfsii sacc.

Crude aqueous extracts from the peripheral rot zone of cocoyam tubers infected by Sclerotium rolfsii sacc were shown to be inhibitory to dialysed in vivo polygalacturonase (PG) of the pathogen. The PG inhibitory action, phenol oxidase and peroxidase activities were higher in cocoyam tubers of the Xanthosoma sagittifolium varieties than in those of the Coolocasia esculenta varieties. The levels of phenol oxidase, peroxidase and PG inhibitory activities also decreased as the postharvest age of the tubers increased.

Location and characteristics of enzymes involved in the breakdown of polygalacturonic acid by Bacteroides thetaiotaomicron.

When Bacteroides thetaiotaomicron is grown in medium which contains polygalacturonic acid (PGA) as the sole carbon source, two different polygalacturonases are produced: a PGA lyase (EC 4.2.2.2) and a PGA hydrolase (EC 3.2.1.15). Both enzymes are cell associated. The PGA hydrolase appears to be an inner membrane protein. The PGA lyase is a soluble protein that associates with membranes under certain conditions. The PGA lyase was purified to apparent homogeneity. It has a molecular weight (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 74,000, a pH optimum of 8.7, a pI of 7.5, and a Km for PGA of 40 to 70 micrograms/ml. It requires calcium for maximal activity. The main product of this enzyme appears to be a disaccharide that contains a delta 4,5-unsaturated galacturonic acid residue. The PGA hydrolase can be solubilized from membranes with 2% Triton X-100 and has been partially purified. It has a pH optimum of 5.4 to 5.5, a pI of 4.7 to 4.9, and a Km for PGA of 350 to 400 micrograms/ml. The main product of this enzyme appears to be galacturonic acid. The specific activities of both PGA hydrolase and PGA lyase increase at the same rate when bacteria are exposed to PGA. The two enzymes therefore appear to be similarly regulated.

[The distribution of free and esterified carboxyl groups within the pectin molecule after the action of pectin esterase from Aspergillus niger and oranges]. / Die Verteilung der freien und veresterten Carboxylgruppen im Pektinmolekül nach Einwirkung von Pektinesterase aus Aspergillus niger und Orangen.

By reaction of pectin esterase (PE) from Aspergillus niger and oranges as well as lye, with 95% esterified citrus and apple pectin we prepared series of preparations with degrees of esterification between 35 and 77%. In these partial deesterified pectins the form of distribution of the free and esterified carboxyl groups has been determined from the activity coefficient gamma Ca2+ of the calcium counterions in the solutions of the corresponding calcium pectinates, from the electrostatic free enthalpy delta (Gel/N)KCa of the ion exchange Ca2+----2K+ in these systems as well as from the relative activity of the polygalacturonase reacting with sodium pectinate. The PE from A niger hydrolyzes the esterified carboxyl groups more or less randomly, in a manner similar to the effect of lye on pectin. On the other hand PE from oranges brings about block-like groupings of free carboxyl groups in the pectin molecule. The study revealed different reaction mechanisms of the pectin deesterification by pectin esterases from Aspergillus species and higher plants.