An ultraviolet-spectrophotometric method with 2-cyanoacetamide for the determination of the enzymatic degradation of reducing polysaccharides.

A rapid, facile, and sensitive uv-spectrophotometric assay has been developed for the determination of the enzymatic degradation of polysaccharides that generates reducing sugars. The assay was carried out with 2-cyanoacetamide in a single test tube. The solution was left at pH 9 by the addition of borate buffer within 5 min. Measurement of the reaction mixture at 274 nm allows a simple determination up to 600 mumol/liter of reducing sugars. The coefficient of variation was less than 2% on all measurements. The assay was developed with pectin and polygalacturonic acid from apples and has been compared with the Somogyi-Nelson method. The new assay was then exemplarily used for the determination of the enzymatic hydrolysis products of pectin from cotton.     

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 30 min and substrate concentration is 5 g/L. For polygalacturonase, the sample should be adjusted to have 0.3–0.8 U/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 0.4 g/L d-galacturonic acid, and then calculate the activity of the sample accordingly (dilution factor × 0.687 U/mL). Validation experiments showed consistent results using these assays. Effects of substrate preparation methods were also examined.     

Purified tomato polygalacturonase activity during thermal and high-pressure treatment

Extracted tomato polygalacturonase was purified by cation-exchange chromatography (and gel filtration) and characterized for molar mass, isoelectric point, as well as optimal pH for polygalacturonase activity. The enzymatic reaction of purified tomato polygalacturonase on polygalacturonic acid as substrate was investigated during a combined high-pressure/temperature treatment in a temperature range of 25 degrees to 80 degrees C and in a pressure range of 0.1 to 500 MPa at pH 4.4 (the pH of tomato-based products). The optimal temperature for initial tomato polygalacturonase activity in the presence of polygalacturonic acid at atmospheric pressure is about 55 degrees to 60 degrees C. The optimal temperature for initial tomato polygalacturonase activity during processing shifted to lower values at elevated pressure as compared with atmospheric pressure, and the catalytic activity of pure tomato polygalacturonase decreased with increasing pressure, which was mostly pronounced at higher temperatures. The elution profiles of the degradation products on high-performance anion-exchange chromatography indicated that for both thermal and high-pressure treatment all oligomers were present in very small amounts in the initial stage of polygalacturonase activity. The amounts of monomer and small oligomers increased with increasing incubation times, whereas the amount of larger oligomers decreased due to further degradation.     

Novel assay system for acidic Peptide:N-glycanase (aPNGase) activity in crude plant extract

Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials.     

Microplate quantification of plant leaf superoxide dismutases

Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals in a broad range of organisms, including plants. Quantification of SOD activity in crude plant extracts has been problematic due to the presence of compounds that interfere with the dose-response of the assay. Although strategies exist to partially purify SODs from plant extracts, the requirement for purification limits the rapidity and practical number of assays that can be conducted. In this article, we describe modification of a procedure using o-dianisidine as substrate that permits relatively rapid quantification of SOD activity in crude leaf extracts in a microplate format. The method employs the use of a commercial apparatus that permits lysis of 12 tissue samples at once and the use of Pipes buffer to reduce interference from compounds present in crude leaf extracts. The assay provided a linear response from 1 to 50 units of SOD. The utility of the assay was demonstrated using tissue extracts prepared from a group of taxonomically diverse plants. Reaction rates with tissue extracts from two grasses were linear for at least 60 min. Tissues of certain species contained interfering compounds, most of which could be removed by ultrafiltration. The presence of plant catalases, peroxidases, and ascorbate in physiological quantities did not interfere with the assay. This approach provides a means to quantify SOD activity in relatively large numbers of plant samples provided that the possibility for the presence of interfering compounds is considered. The presence of interfering compounds in certain plant tissues necessitates caution in interpreting the effects of plant stresses on SOD.     

Purification of a sugarbeet pectin modifying enzyme system from Aspergillus niger by antibody affinity column chromatography

Polyclonal antibodies were raised against polygalacturonases partially purified from culture filtrates of Aspergillus niger grown on sugarbeet pectin or polygalacturonic acid. An enzyme-linked immunosorbent assay (ELISA) for polygalacturonase was developed and the antisera were exploited in immuno-affinity chromatography to remove polygalacturonase from a mix of sugarbeet modifying enzymes. This approach has facilitated the preparation of an enzyme mix, containing a pectin deacetylase and demethoxylase together with an arabinofuranosidase, free from polygalacturonase, in a rapid, simple procedure.     

A Mass Spectrometry-Based Assay for Improved Quantitative Measurements of Efflux Pump Inhibition

Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC₅₀ value of 75 μg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC₅₀ values ranging from 19 μg/mL to 75 μg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence.     

Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras

Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or, alternatively, it has a different mechanism of substrate binding than other polygalacturonases characterized to date.     

Quantification of protopectinase SE, an endopolygalacturonase with pectin-releasing activity from Geotrichum klebahnii

Pectin releasing activity of protopectinase SE (PPase-SE) from Geotrichum klebahnii (= G. penicillatum = Trichosporon penicillatum) ATCC 42397 was determined using different batches of lemon protopectin as substrate. Results obtained showed a high degree of variability depending on the batch of protopectin used. As PPase-SE also shows polygalacturonase (PGase) activity, a method for the assay of this activity was optimised. The best assay conditions were: substrate (polygalacturonic acid) concentration of 2.0 g 1^–1, reaction time of 10 min and up to 0.17 PGase units per test tube.     

Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method

Endo-polygalacturonase (endo-PGase) activity determinations generally rely on viscosity changes or reducing sugar ends produced by this activity over polygalacturonic acid. Torres and coworkers [Enzyme Microb. Technol. 48 (2011) 123–128] showed that ruthenium red (RR) is useful for endo-PGase determination. In this article, we present a high-throughput liquid-based endo-PGase assay based on the RR method and compare it with the viscosity determination method. The reduced assay uses a small volume of enzyme solution, 40 μg of polygalacturonic acid, and 45 μg of RR for each sample determination. Furthermore, we obtained an interconversion factor for RR and viscosity activities.     

A polygalacturonase from citrus leaf explants: role in abscission

The relationship between polygalacturonase activity and abscission of citrus leaf explants was studied. Determination of polygalacturonase activity in citrus tissues requires concentration of the enzyme, use of a proper assay method, and inhibition of an oxidase present in the extracts which oxidizes the reaction products of the polygalacturonase. The polygalacturonase from citrus leaf explants is an exopolygalacturonase and appears to be a soluble enzyme.     

Behaviour of pectinases under process conditions

Hydrolysis of pectic substances by pectinases were carried out under process conditions. Polymethylgalacturonase, polygalacturonase and pectinlyase are three enzymes studies in this regard. Pectin is used as the substrate for polymethylgalacturonase and pectinlyase whereas polygalacturonic acid was used as the substrate for polygalacturonase. The initial concentration was varied between 1.0 and 5.0 kg/m³ for polymethylgalacturonase and polygalacturonase and between 4.0 and 8.0 kg/m³ for pectinylase. Hydrolysis experiments were carried out by varying initial substrate to enzyme ratio and reducing groups formed are measured. The behaviour of pectinases during hydrolysis are studied from the plot of (reducing groups formed)/initial substrate vs. initial substrate to enzyme ratio. This study is helpful in predicting the amount of substrate and enzyme required to produce the required amount of reducing groups within a stipulated time.     

Nonradioactive technique to measure protein phosphatase 2A-like activity and its inhibition by drugs in cell extracts

A nonradioactive assay has been developed that can be used to measure serine/threonine protein phosphatase (PP) activity, especially PP2A, in crude extracts from different cell lines. For this technique commercially available casein is used as an already phosphorylated substrate. The prerequisite for reliable measurements is the removal of free phosphate from cell extracts and substrate preparations with desalting columns. The use of different nonspecific or specific inhibitors as well as inhibition characteristics observed after extract dilution suggests that in the absence of magnesium, PP2A-like activity in the extracts is measured by this technique. Inclusion of magnesium allowed the detection of a protein phosphatase activity that is activated by magnesium, which is presumably PP2C. The use of structurally different as well as structurally related inhibitors of PP2A gave results comparable to those of reports from the literature that were obtained with radioactive assays. Thus, our data supported our hypothesis that this nonradioactive assay can be used for the identification of newly synthesized PP inhibitors as well as for performing structure-activity analysis within groups of such new agents.     

Chemical composition and antioxidant activities of Broussonetia papyrifera fruits

Fruits of Broussonetia papyrifera from South China were analyzed for their total chemical composition, and antioxidant activities in ethanol and aqueous extracts. In the fruit of this plant, the crude protein, crude fat and carbohydrates was 7.08%, 3.72% and 64.73% of dry weight, respectively. The crude protein, crude fat and carbohydrates were 15.71%, 20.51% and 36.09% of dry weight, respectively. Fatty acid and amino acid composition of the fruit were analyzed. Unsaturated fatty acid concentration was 70.6% of the total fatty acids. The percentage of the essential amino acids (EAAs) was 40.60% of the total amino acids. Furthermore, B. papyrifera fruit are rich in many mineral elements and vitamins. Total phenolic content was assessed using the Folin-Ciocalteau assay, whereas antioxidant activities were assessed by measuring the ability of the two extracts to scavenge DPPH radicals, inhibit peroxidation, and chelate ferric ions. Their reducing power was also assessed. Results indicated that the aqueous extract of B. papyrifera was a more potent reducing agent and radical-scavenger than the ethanol extract. GC-MS analysis of the ethanol extract showed the presence of some acid-containing compounds. The changes in total phenolic content and antioxidant capacity in B. papyrifera from four different regions grown under normal conditions were assessed. The antioxidant activity of different extracts was positively associated with their total phenolic content. These results suggest that the fruit of B. papyrifera could be used in dietary supplement preparations, or as a food additive, for nutritional gain, or to prevent oxidation in food products.     

Metabolism of cell wall polysaccharides from persimmon fruit. Pectin solubilization during fruit ripening occurs in apparent absence of polygalacturonase activity

Pectins from persimmon ( Diospyros kaki L.) fruit pericarp were sequentially extracted with 0. 05 M trans -1,2-diaminocyclohexane-N,N, N', N'-tetraacetic acid (CDTA), 0. 05 M Na₂CO₃ (1°C) and Na₂CO₃ (20°C) and the carbohydrate composition and metabolism during development determined. Young persimmon fruits contained a large proportion of pectins, 46% by dry weight, that decreased to 20% with ripening. This decrease occurred in the CDTA and Na₂CO₃ (1°C) fractions, mainly composed of uronic acids, and represents a net loss of uronic acids, arabinose and galactose. The amount of non-cellulosic neutral sugars was especially high in the Na₂CO₃ (20°C) fraction. The loss of pectins was also accompanied by a depolymerisation of the polysaccharides extracted in the three pectic fractions. However, none of these changes can be attributed to the action of polygalacturonase activity. Proteins were extracted from the pericarp tissue, but endopolygalacturonase (EC 3. 2. 1. 15) activity, determined as a decrease in viscosity of polygalacturonic acid, was not observed in the extract. Determination of exopolygalacturonase (EC 3. 2. 1. 67) activity by measuring the release of reducing groups from polygalacturonic acid was also negative. The results presented indicate that polygalacturonase is not responsible for the metabolism of pectins during persimmon fruit ripening.     

Pectinase Activity Determination: An Early Deceleration in the Release of Reducing Sugars Throws a Spanner in the Works!

Recently, it has been suggested that pectinases could be used to hydrolyze pectin in biorefineries based on pectin-rich agro-industrial wastes. However, for this to be viable, the cost of their production would need to be lowered significantly. In fact, over the last few decades, there have been many attempts to improve pectinase production by existing strains or to screen for new strains from environmental isolates. In these studies, it is necessary to measure pectinase activities. Many researchers use single-time-point assays that involve incubation of pectinolytic extracts with pectic substrates for a fixed time, followed by determination of the liberated reducing sugars. However, different researchers use quite different conditions for this assay. Furthermore, no attention has been given to the reaction profile during the assay. In the current work, we show, for the first time, that a significant deceleration of the rate of liberation of reducing sugars occurs over the first ten minutes of the reaction. As a consequence, the incubation time used in a single-time-point assay has a large effect on the value obtained for the activity. In fact, we demonstrate that, depending on the particular combination of incubation time, pectin concentration and reaction temperature, the same extract could be reported to have activities that differ by an order of magnitude. In addition, we show that the relative activities obtained with polygalacturonic acid do not correlate with those obtained with pectin. We conclude that it is currently impossible to make meaningful comparisons between pectinase activities reported in the literature by workers who have used different assay conditions. Therefore there is an urgent need for the development of a standardized assay for evaluating the saccharification potential of pectinase complexes.     

Purification and kinetic characterization of polygalacturonase - 3 from palmyrah palm (Borassus flabellifer L)

Polygalacturonase-3 was isolated and purified to homogeneity from palmyrah palm (Borassus flabellifer L.) fruit using Con A-Sepharose affinity column. The purified enzyme migrated as a single band on native and SDS–polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme was estimated to be 66 kDa by size elution chromatography. Optimum polygalacturonase activity as a function of pH and temperature was determined using polygalacturonic acid as substrate. Optimum pH and temperature values ranged between the pH?4.0–5.0 and temperature 30–40 °C. At the optimum pH and temperature, the K_m and V_max values were determined by Lineweaver–Burk method. The value K_m (0.33 mM) reveals that polygalacturonase has significant reactivity towards polygalacturonic acid. The enzyme showed varied responses towards divalent and monovalent metal ions. Ca²⁺ activated the polygalacturonase-3 enzyme protein. Both teepol and cetyltrimethylammonium bromide inhibited polygalacturonase-3 activity by 44 %, while 2-mercaptoethanol stimulated the enzyme marginally.     

Evaluation of bioactive compounds,free radical scavenging and anticancer activities of bulb extracts of Boophone disticha from Eastern Cape Province,South Africa

Boophone disticha (B. disticha) is a bulbous tropical and subtropical flowering plant widespread in Africa, which is frequently used to treat several human ailments. Until the present, there is no scientific validation on the biological activity of this plant from the Eastern Cape Province of South Africa and as a result, this study aimed to assess the bioactive compounds, free radicals scavenging and anticancer potentials of crude bulb extracts (chloroform, acetone, and ethanol) of Boophone disticha obtained from this geographical location. Standard biochemical techniques and Gas-chromatography mass spectrometry (GCMS) analysis were used to pinpoint the bioactive compounds in the crude extracts sequel to their antioxidant potentials against radicals such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydrogen peroxide and nitric oxide as well as their ferric ion reducing power. In addition, their cytotoxicity effects against Human cervix adenocarcinoma (HeLa) cells were assessed as an in vitro model for anticancer. The phytochemical evaluation of the crude extracts showed the presence of phenolics, flavonoids, and alkaloids. GCMS profiles confirmed the presence of some bioactive compounds in the crude extracts of B. disticha that could be responsible for their biological activities. The plant extracts possessed considerable antioxidant activity and exhibited dose-dependent radicals’ inhibition from all assays carried out. Furthermore, the cytotoxicity effects against HeLa cells recorded inhibition concentration (IC₅₀) of 1.5, 1.6, and 1.9 µg/mL for acetone, chloroform, and ethanolic extracts of B. disticha, respectively. Findings from the present study suggest that B. disticha could be a good prospective source of antioxidant and anticancer agents. Therefore, further research on the isolation and purification of compounds from these extracts are indispensable.     

Purification and Properties of a Polygalacturonic Acid Trans-Eliminase Produced by Bacillus pumilus

A strain of Bacillus pumilus produced an extracellular pectic enzyme with polygalacturonic acid as the substrate. This enzyme, with optimal activity at pH 8.0 to 8.5, produced reaction products that strongly absorbed light at 232 nm, indicating the presence of a pectic acid trans-eliminase (PATE). Neither pectin esterase nor polygalacturonase was detected in the cell-free culture fluid. Chromatographic examination of the end products revealed the presence of large quantities of unsaturated oligouronides unlike those found with B. polymyxa. It was found that the PATE was produced extracellularly during the negative logarithmic death phase of the organism. The filtrate from sonically treated cells did not show any activity for PATE or hydrolases for lower oligogalacturonides at any time during the growth cycle. The enzyme was inducible. Pectin, National Formulary (NF) was the best inducer, followed by polygalacturonic acid and galacturonic acid. Enzyme activity was markedly stimulated by calcium and other divalent ions. Copper and cobalt ions were inhibitory. The partially purified enzyme showed no significant activity on pectin containing a high methoxyl content (96% esterified). However, pectin NF with a lower methoxyl content (68% esterified) was attacked to a degree by the partially purified and crude enzyme preparations. The initial rate of PATE activity increased up to 60 C, about 16-fold higher than that observed at room temperature. The activation energy was calculated as 12,183 cal/mole. A protective action of calcium chloride against heat inactivation of the PATE was observed. Degradation of polygalacturonic acid by this enzyme produced several unsaturated oligouronides soon after its addition to the substrate. The major endproduct was thought to be different from that of other known PATE enzymes. Paper chromatographic studies and viscosity measurements disclosed the random cleaving nature of the enzyme an endo-PATE.