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PubMed2005

Effect of pepsin digestion on the antivenom activity of equine immunoglobulins.

Enzyme digestion of animal-derived sera followed by antibody purification is a classical process used to prepare snake antivenoms worldwide. In this work, we have studied the effect of the harsh conditions prevailing during the digestion step on the activity of the final product, F(ab')(2). To this purpose, the recovery of the activity of anti-Bothrops hyperimmune equine plasma was determined after pepsin digestion under different sets of processing conditions. The balance between pH level and reaction time was found to be critical, reflecting a compromise between complete cleavage of immunoglobulins and strong denaturation of the F(ab')(2) fragments. For pH in the range 2.8-3.2, 30-65% of the initial activity was lost depending mainly on the processing time, as determined by a competition ELISA technique. Pepsin digestion was also carried out with purified immunoglobulins from the same plasma. SDS PAGE run on the digested immunoglobulins allowed us to verify that the lightest isotypes were more resistant to digestion than the heavier ones. In conclusion, for equine F(ab')(2) antivenom production, it seems convenient to carry out digestion at pH values sufficiently low to ensure that total IgG breakdown is achieved in the shortest time compatible with precise operation in the production scale.

Animals↗
PubMed2004

Anaerobic sewage treatment in a one-stage UASB reactor and a combined UASB-Digester system.

The treatment of sewage at 15 degrees C was investigated in a one-stage upflow anaerobic sludge blanket (UASB) reactor and a UASB-Digester system. The latter consists of a UASB reactor complemented with a digester for mutual sewage treatment and sludge stabilisation. The UASB reactor was operated at a hydraulic retention time of 6h and a controlled temperature of 15 degrees C, the average sewage temperature during wintertime of some Middle East countries. The digester was operated at 35 degrees C. The UASB-Digester system provided significantly (significance level 5%) higher COD removal efficiencies than the one-stage UASB reactor. The achieved removal efficiencies in the UASB-Digester system and the one-stage UASB reactor for total, suspended, colloidal and dissolved COD were 66%, 87%, 44% and 30%, and 44%, 73%, 3% and 5% for both systems, respectively. The stability values of the wasted sludge from the one-stage UASB reactor and the UASB-Digester system were, respectively, 0.47 and 0.36g CH(4)-COD/g COD. Therefore, the anaerobic sewage treatment at low temperature in a UASB-Digester system is promising.

Bacteria, Anaerobic↗
PubMed2004

An automated spectrophotometric system for monitoring buffer capacity in anaerobic digestion processes.

Anaerobic digestion is a suitable method for the treatment of wastewater and organic wastes, yielding biogas as a useful by-product. A common way of preventing instability problems and avoiding acidification in anaerobic digesters is to keep the organic load to the digester far below its maximum capacity. An improved way of operating digesters would be to use monitoring and control systems for increased organic load under controlled conditions such that the digester performance is improved. The partial alkalinity, which indicates the bicarbonate concentration, has in many cases been found to be a suitable parameter to monitor. Here, an automated monitoring system for alkalinity measurements is described. It is shown to be applicable for measuring a wide range of bicarbonate concentrations. The system shows potential for monitoring anaerobic digesters as it responds to the alkalinity of digester effluent, as well as being stable over a relatively long time span with few maintenance requirements.

Automation↗
PubMed2005

Quantitative measurement of protein digestion in simulated gastric fluid.

The digestibility of novel proteins in simulated gastric fluid is considered to be an indicator of reduced risk of allergenic potential in food, and estimates of digestibility for transgenic proteins expressed in crops are required for making a human-health risk assessment by regulatory authorities. The estimation of first-order rate constants for digestion under conditions of low substrate concentration was explored for two protein substrates (azocoll and DQ-ovalbumin). Data conformed to first-order kinetics, and half-lives were relatively insensitive to significant variations in both substrate and pepsin concentration when high purity pepsin preparations were used. Estimation of digestion efficiency using densitometric measurements of relative protein concentration based on SDS-PAGE corroborated digestion estimates based on measurements of dye or fluorescence release from the labeled substrates. The suitability of first-order rate constants for estimating the efficiency of the pepsin digestion of novel proteins is discussed. Results further support a kinetic approach as appropriate for comparing the digestibility of proteins in simulated gastric fluid.

Electrophoresis, Polyacrylamide Gel↗
PubMed1997

Digestion of Pollen Components by Larvae of the Flower-Specialist Bee Chelostoma florisomne (Hymenoptera: Megachilidae).

Pollen digestion in larvae of the solitary bee Chelostoma florisomne (Megachilidae), a pollen-specialist (oligolectic) species on Ranunculus spp. (Ranunculaceae), was investigated using histological techniques. Serial sections from the anterior-, median-, and posterior-midgut/hindgut regions of the larval alimentary canal were selectively stained and examined for disappearance of lipids, proteins, and carbohydrates from ingested Ranunculus pollen grains. During digestion, pollen contents were observed to gradually extrude through the grain apertures. Most of the digestion of lipids and proteins occurred in the anterior- and median-midgut, whereas carbohydrates disappeared more slowly and mainly in the median- and posterior-midgut. In the hindgut, pollen comprised mainly empty exine shells, which were crushed and often broken, suggesting that some components of the wall are degraded during digestion, causing collapse of the outer, chemically resistant wall layer (exine). Lipids were completely digested, but small quantities of proteins and carbohydrates were often still present in grains in the posterior midgut/hindgut. The pollenkitt appeared to be completely absorbed, with none evident in the posterior midgut or in larval feces. The findings indicate that this oligolectic bee has the digestive equipment necessary to effectively remove and absorb the nutrients present in the pollen of its restricted food source. Comparison with similar studies in adult honey bees suggests that differing digestive abilities might be an underlying factor in pollen specialization in bees.

Journal Article↗
PubMed2003

Toxicity of di-(2-ethylhexyl) phthalate on the anaerobic digestion of wastewater sludge.

Previous studies on the microbial degradation of individual phthalic acid esters (PAEs) have demonstrated that the compounds with short ester hydrocarbon chains are easily biodegraded and mineralized, but PAEs with long ester chains are less susceptible to degradation and some of them are considered recalcitrant. Moreover, they inhibit methanogenesis. However, studies have not been made on the effect of feeding a combination of recalcitrant and biodegradable PAEs into anaerobic digesters treating wastewater sludge. The present study was conducted with wastewater sludge from the Los Angeles Bureau of Sanitation's Hyperion Treatment Plant. Di (2-ethylhexyl) phthalate (DEHP), the most common persistent PAE found in wastewater, and di-n-butyl phthalate (DBP), a common PAE with short ester chains, were sorbed into the sludge fed to a bench-scale digester for a period of 12 weeks. DEHP degradation was always poor, and accumulation of DEHP was correlated with inhibition of the microbial degradation of DBP and with process instability of the test digester. Inhibition of the DBP removal was completely reversed after DEHP addition was discontinued, but biogas production never recovered to the level observed in a control digester. Other process parameters of digester performance were not affected by DEHP accumulation. These results are similar to the toxic effects of long chain fatty acids on sludge digestion, suggesting that DEHP or its degradation products affect all the microbial populations in the anaerobic bioreactor. Our results imply that high levels of DEHP or other recalcitrant PAEs in wastewater sludge are likely to compromise methanogenesis and removal of biodegradable PAEs in sludge digesters.

Bacteria, Anaerobic↗
PubMed2003

Mechanisms of floc destruction during anaerobic and aerobic digestion and the effect on conditioning and dewatering of biosolids.

Laboratory anaerobic and aerobic digestion studies were conducted using waste activated sludges from two municipal wastewater treatment plants in order to gain insight into the mechanisms of floc destruction that account for changes in sludge conditioning and dewatering properties when sludges undergo anaerobic and aerobic digestion. Batch digestion studies were conducted at 20 degrees C and the dewatering properties, solution biopolymer concentration and conditioning dose requirements measured. The data indicated that release of biopolymer from sludges occurred under both anaerobic and aerobic conditions but that the release was much greater under anaerobic conditions. In particular, the release of protein into solution was 4-5 times higher under anaerobic than under aerobic conditions. Both the dewatering rate, as characterized by the specific resistance to filtration and the amount of polymer conditioning chemicals required was found to depend directly on the amount of biopolymer (protein + polysaccharide) in solution. Little difference in dewatering properties and conditioning doses was seen between the two activated sludges from different plants. Differences in the cations released between anaerobic and aerobic digestion suggest that the digestion mechanisms differ for the two types of processes. Enzyme activity data showed that during aerobic digestion, polysaccharide degradation activity decreased to near zero and this was consistent with the accumulation of polysaccharides in aerobic digesters.

Bacteria, Aerobic↗
PubMed2002

Nitrogen transformations during aerobic/anoxic sludge digestion.

Laboratory experiments were conducted to study and compare nitrogen transformations occurring under both aerobic digestion and aerobic/anoxic (A/A) digestion. The process performance was examined at different sludge residence times (SRTs), temperatures and anoxic cycles. Both modes of operation gave comparable solids reduction results. However, introduction of anoxic periods to aerobic sludge digestion appears to be a promising alternative to control pH during digestion through endogenous nitrate respiration (ENR). Operating an aerobic digester with an anoxic phase to achieve complete denitrification would also improve supernatant quality over that achieved solely by aerobic digestion. Alternating A/A operation can conserve most of the influent alkalinity and maintain near neutral pH condition over prolonged periods. The A/A digestion of mixed primary/waste-activated sludge achieved up to 43.7% reductions in volatile suspended solids, 33.7% removal of total nitrogen, and a specific ENR rate of 5.75 x 10(-2) mg NO3-N/mg VSSd. Optimum results were obtained at 10 d SRT, 30 degrees C temperature, and 50% anoxic cycle length.

Ammonia↗
PubMed1995

Inhibition of proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells by bovine milk caseins and their digests.

The modulating effect of bovine milk casein components and their digests on the proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells induced or not induced by mitogens has been studied with a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. All the casein components and their digests tested had little mitogenic effect on the proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells. Intact kappa-casein significantly inhibited the proliferative responses of mouse spleen lymphocytes and Peyer's patch cells induced by mitogens such as lipopolysaccharide from Salmonella typhimurium, concanavalin A, phytohaemagglutinin and pokeweed mitogen. In contrast, intact alpha s1-casein and beta-casein had little effect. kappa-Casein had an inhibitory effect after digestion by pancreatin or trypsin, but not after pepsin or chymotrypsin digestion. Both pancreatin and trypsin digests of alpha s1-casein and beta-casein significantly inhibited the proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells induced by mitogens, whereas pepsin and chymotrypsin digests of both caseins were without effect. Moreover, the trypsin digest of each casein component had an inhibitory effect on mouse spleen lymphocyte proliferation in the absence of mitogen. Since trypsin is a major proteinase in pancreatin, the substrate specificity of trypsin seems to be important for the formation of the inhibitory peptides from casein components. These observations suggest that intact kappa-casein and some peptides formed from milk casein components by the action of trypsin may suppress the immune responsiveness of neonates.

Animals↗
PubMed2004

Distribution and estimation of anaerobic zoosporic fungi along the digestive tracts of sheep.

The status of anaerobic zoosporic (Chytridiomycota) fungi along the entire digestive tract of sheep was assessed both analytically and microscopically. Digest samples were taken from different segments of the digestive tracts of three newly killed sheep that previously had been used in experimental dietary studies. These digest samples were tested for the presence of rumen fungi by assessing the recovery of live fungi from the samples, direct observation of digested plant remains under the scanning electron microscope (SEM), and using a chitin assay as an estimation of fungal biomass. Live anaerobic fungi were recovered from the abomasum, small and large intestine, caecum and faeces of sheep, but not from the digest samples of rumen and omasum. However, SEM examination of the samples confirmed the presence of fungal structures from all of these organs. In the large intestine and caecum samples the observed sporangial structures were rounded and showed conspicuous surface pitting. Results of the chitin assay indicated that the anaerobic fungi might account for up to 20% of the total microbial biomass in the rumen of sheep. The results of this study support the view that anaerobic fungi may be present as a resistant stage in the lower reaches of the digestive tract.

Animals↗
PubMed2002

Identification and relative quantitation of protein mixtures by enzymatic digestion followed by capillary reversed-phase liquid chromatography-tandem mass spectrometry.

In this report, we describe an approach for identification and relative quantitation of individual proteins within mixtures using LC/MS/MS analysis of protein digests. First, the proteins are automatically identified by correlating the tandem mass spectra with peptide sequences from a database. Then, peak areas of identified peptides from one protein are added together to define the total reconstructed peak area of the protein digest. The total reconstructed peak area is further normalized to the peak area of an internal standard protein digest present in the mixture at a constant level. The method was illustrated using digested mixtures of five standard proteins as follows. One protein was gradually diluted while the other four components were present in the mixtures at constant level. This study revealed that relative peak area of the variable protein increased linearly (trend line R2 = 0.9978) with increasing amount from 10 to 1000 fmol, while relative peak areas of four constant proteins remained approximately the same (within 20% relative standard deviation). To further evaluate the applicability of this method for the quantitation of proteins from complex mixtures, human plasma protein digest was spiked with 200 and 400 fmol of myoglobin digest. Total peak area of myoglobin peptides was normalized to the total peak area of apolipoprotein A-I peptides from human plasma, which played the role of an internal standard. The myoglobin/apolipoprotein A-I peak area ratio was 2 times larger for the human plasma digest spiked with a double amount of myoglobin. After several repetitions, the error of the relative peak area measurements remained below 11%, suggesting that the method described here can be used for relative concentration measurements of proteins in the complex biological mixtures. In the presented method, chemical derivatization steps are not needed to create an internal standard, as in isotope-coded affinity tag or similar methods.

Amino Acid Sequence↗
PubMed2004

MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports.

Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacrylamide gel electrophoresis. In an effort to minimize sample handling and increase sample throughput, we have developed a novel in-gel digestion protocol where sample preparation is performed directly on a MALDI probe with prestructured sample support. The protocol consists of few sample-handling steps and has minimal consumption of reagents, making the protocol sensitive, timesaving, and cost-efficient. Performance of the on-probe sample preparation protocol was demonstrated by analysis of a set of rat liver proteins obtained from a fluorescently stained (Cy3 and SyproRuby) two-dimensional polyacrylamide gel. The success rate of protein identification by on-probe tryptic digestion and MALDI peptide mass mapping was 89%. The on-probe in-gel digestion procedure provided superior sensitivity and peptide mass mapping performance as compared to our standard in-gel digestion protocol. The on-probe digestion technique resulted in significantly improved amino acid sequence coverage of proteins, mainly due to efficient recovery and detection of large (>1.5 kDa) hydrophobic peptides. These observations indicate that numerous tryptic peptides are lost when using the standard in-gel digestion methods and sample preparation techniques for MALDI MS. This study also demonstrates that the on-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research, including protein identification and determination of posttranslational modifications.

Animals↗
PubMed1993

Comparison of sequence preference of tomaymycin- and anthramycin-DNA bonding by exonuclease III and lambda exonuclease digestion and UvrABC nuclease incision analysis.

The DNA bonding sites of two pyrrolo[1,4]benzodiazepine derivatives--tomaymycin (Tma) and anthramycin (Atm)--were identified by exonuclease III (exo III) digestion, lambda exonuclease (lambda exo) digestion, and UvrABC nuclease incision analysis. exo III digestion stalls 4-5 bases 3' to a drug-DNA adduct. While this method can recognize most of the Atm-and Tma-DNA modification sites, it is complicated in that exo III digestion is also stalled by certain unmodified sequences and by drug bound to the opposite strand. lambda exo digestion stalls 1-2 bases 5' to a drug-DNA adduct. The lambda exo method also recognizes most of the drug-DNA bonding sites and renders a cleaner background; however, it is also affected by opposite-strand drug bonding. Due to their intrinsic digestion polarities, these two exonucleases tend to be stalled by the drug-DNA adduct at one end of the DNA molecule. Purified UvrA, UvrB, and UvrC proteins acting together make dual incisions 6-8 bases 5' and 4 bases 3' to a Atm- or Tma-DNA adduct. This nuclease complex recognizes all the Tma- and Atm-DNA bonding sites identified by exonuclease digestion methods, and all the UvrABC incisions can be attributed to drug modifications in the incised DNA strand. The degree of UvrABC nuclease incision increases with increasing drug concentrations for DNA modification. Using the UvrABC incision method, we have identified the sequence preference of Tma- and Atm-DNA adduct formation in three DNA fragments, and we have found that these two drugs have different preferred sites for adduction. Both Tma- and Atm-DNA bonding is strongly influenced by the 5' and 3' neighboring bases; the orders of preferred 5' and 3' bases for Tma are A > G, T > C, and A, C > G, T, and for Atm the orders are A > G > T > C and A > G > T, C. The preferred triplets for Tma bonding are -AGA- > -GGC-, -TGC-, and AGC- and for Atm are -AGA-, -AGG- > -GGA-, -GGG-.

Anthramycin↗
PubMed2004

Enzymatic high digestion of soybean milk residue (okara).

The objective of this study was to digest okara in high yield by food-processing enzymes. Autoclaving of okara was effective in increasing cellulase digestion for the primary cell wall, and the digestion was accelerated by the formation of single cells by stirring. Most of the residual okara after autoclaving and cellulase digestion was found to be the secondary cell walls compared with the cellulase-treated soybean single cells. The secondary cell wall was found to be composed of galacturonic acid, neutral sugars, and protein and was considered to be a complex of these compositions. Many cellulolytic and proteolytic enzymes could not digest the secondary cell wall; however, it was found that two pectinases could digest the secondary cell wall. A series of digestions resulted in yields of 83-85% from the raw okara, and the final residues were identified as oil body complexes in the soybean cells and fiber-like organ between the cells.

Cell Wall↗
PubMedDate not supplied

Chemically modified, immobilized trypsin reactor with improved digestion efficiency.

Tryptic digestion followed by identification using mass spectrometry is an important step in many proteomic studies. Here, we describe the preparation of immobilized, acetylated trypsin for enhanced digestion efficacy in integrated protein analysis platforms. Complete digestion of cytochrome c was obtained with two types of modified-trypsin beads with a contact time of only 4 s, while corresponding unmodified-trypsin beads gave only incomplete digestion. The digestion rate of myoglobin, a protein known to be rather resistant to proteolysis, was not altered by acetylating trypsin and required a buffer containing 35% acetonitrile to obtain complete digestion. The use of acetylated-trypsin beads led to fewer interfering tryptic autolysis products, indicating an increased stability of this modified enzyme. Importantly, the modification did not affect trypsin's substrate specificity, as the peptide map of myoglobin was not altered upon acetylation of immobilized trypsin. Kinetic digestion experiments in solution with low-molecular-weight substrates and cytochrome c confirmed the increased catalytic efficiency (lower K(M) and higher k(cat)) and increased resistance to autolysis of trypsin upon acetylation. Enhancement of catalytic efficiency was correlated with the number of acetylations per molecule. The favorable properties of the new chemically modified trypsin reactor should make it a valuable tool in automated protein analysis systems.

Acetonitriles↗
PubMed1993

Rapid partial digestion of biological tissues with nitric acid for the determination of trace elements by atomic spectrometry.

Determination of Cu, Fe, Mn and Zn in fresh bovine liver and BCR bovine liver and pig kidney reference materials, after digestion at 105 degrees C with nitric acid for various times, showed that the trace elements were completely released after heating for only 20 min. Centrifugation of the samples after digestion improved the separation from undigested fat. This partial-digestion method, based on heating about 0.2 g of sample with 2 ml of nitric acid for 20 min, was tested on a range of biological reference materials and on fresh bovine liver for the four elements. Comparison was made with results obtained after digestion by a recommended hydrogen peroxide--sulfuric acid digestion procedure. Results obtained by inductively coupled plasma atomic emission spectrometry or by flame and graphite-furnace atomic absorption spectrometry showed good agreement between the two digestion procedures, and results for the reference materials agreed well with the certified values. Between-batch precision was better than 4% for Cu, Fe and Zn at levels of 30 to 190, 210 to 320 and 100 to 140 micrograms g-1, respectively. For Mn, the precision varied between 7 and 14% for measured concentrations of 8 to 12 micrograms g-1. The partial-digestion procedure offers simplicity, speed, low cost and the ability to handle a large number of samples at the same time.

Animals↗
PubMed2002

Pressure assisted chelating extraction: a novel technique for digesting metals in solid matrices.

This work describes a novel technique for the digestion of metals in solid matrices. The technique is called pressure assisted chelating extraction (PACE). In a typical procedure, a solid sample is placed in a stainless steel cell and is mixed with appropriate chelating agents. Using a programmed sequence of temperature, static time, pressure and thermal equilibration available in ASE 200, the metal is removed under moderate temperature (up to 200 degrees C) and pressure (up to 3000 psi). PACE achieves metal recovery that is equivalent to that of wet digestion techniques and also provides for a clean and safe operation by substituting the strong acids commonly used during wet digestion with chelating agents. It uses less solvents and significantly less time (minutes vs. hours) for metal digestion. PACE has been validated using certified standard reference materials (SRMs) including industrial sludge, buffalo river sediments and coal fly ash. The total time required to remove metals was approximately 20 min. Results show that the PACE system provides an ideal platform for efficient, rapid, and safe metal digestion. Good agreement between measured and reference values for Pb, Mn, and Cu were found with recoveries averaging between 80 and 101% and a relative standard deviation of less than 5%. This approach may provide an alternative digestion technique for environmental samples, alloys, biological materials and samples of geological importance. The potential advantage offered lies in non-destruction of the sample, automation and the exclusion of concentrated mineral acids during the digestion procedure.

Chelating Agents↗
PubMed1996

Cleavage of human complement component C5 by cysteine proteinases from Porphyromonas (Bacteroides) gingivalis. Prior oxidation of C5 augments proteinase digestion of C5.

Since severe periodontitis is characterized by an acute inflammatory response with cellular infiltration and microbial overgrowth, plasma proteins could be exposed to both proteinases and oxidants released from the granulocytes, as well as to proteinases from the microorganisms. When human complement component C5 was digested by cysteine proteinases (i.e. gingipain-R and gingipain-K) from Porphyromonas gingivalis, limited cleavage of the C5 molecule was observed. If C5 was first oxidized by hydroxyl radicals, these gingipains converted modified C5 to fragments that exhibited significantly greater pro-inflammatory activity than did digests of unmodified C5. After cleavage of oxidized C5 by gingipain-R, the digest exhibited measurably greater neutrophil enzyme release and chemotaxis of human polymorphonuclear leukocytes (PMNs) compared with the activities of unoxidized C5 digests. Gingipain-K generates virtually no polarization or chemotactic activity of human PMNs from C5, nor is enzyme release stimulated by these C5 digests. However, when oxidized C5 was digested by gingipain-K, human PMNs were stimulated for polarization, chemotaxis and enzyme release indicating that an active fragment had been generated. Proteolysis of oxidized C5 evokes greater neutrophil activation than does proteolysis of unoxidized protein, a fact which supports the hypothesis that oxidation and proteolysis may be coupled to enhance the destructive effects of the inflammatory process. These results, in which digests of both oxidized and unmodified complement component C5 were evaluated, support the general concept that oxidation and proteolysis may participate cooperatively in amplifying both the severity and duration of the inflammatory reaction.

Adhesins, Bacterial↗