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PubMed2004

Effect of xylan and lignin removal by batch and flowthrough pretreatment on the enzymatic digestibility of corn stover cellulose.

Compared with batch systems, flowthrough and countercurrent reactors have important potential advantages for pretreating cellulosic biomass, including higher hemicellulose sugar yields, enhanced cellulose digestibility, and reduced chemical additions. Unfortunately, they suffer from high water and energy use. To better understand these trade-offs, comparative data are reported on xylan and lignin removal and enzymatic digestibility of cellulose for corn stover pretreated in batch and flowthrough reactors over a range of flow rates between 160 degrees and 220 degrees C, with water only and also with 0.1 wt% sulfuric acid. Increasing flow with just water enhanced the xylan dissolution rate, more than doubled total lignin removal, and increased cellulose digestibility. Furthermore, adding dilute sulfuric acid increased the rate of xylan removal for both batch and flowthrough systems. Interestingly, adding acid also increased the lignin removal rate with flow, but less lignin was left in solution when acid was added in batch. Although the enzymatic hydrolysis of pretreated cellulose was related to xylan removal, as others have shown, the digestibility was much better for flowthrough compared with batch systems, for the same degree of xylan removal. Cellulose digestibility for flowthrough reactors was related to lignin removal as well. These results suggest that altering lignin also affects the enzymatic digestibility of corn stover.

Bioreactors↗
PubMed2005

Flow pattern visualization in a mimic anaerobic digester using CFD.

Three-dimensional steady-state computational fluid dynamics (CFD) simulations were performed in mimic anaerobic digesters to visualize their flow pattern and obtain hydrodynamic parameters. The mixing in the digester was provided by sparging gas at three different flow rates. The gas phase was simulated with air and the liquid phase with water. The CFD results were first evaluated using experimental data obtained by computer automated radioactive particle tracking (CARPT). The simulation results in terms of overall flow pattern, location of circulation cells and stagnant regions, trends of liquid velocity profiles, and volume of dead zones agree reasonably well with the experimental data. CFD simulations were also performed on different digester configurations. The effects of changing draft tube size, clearance, and shape of the tank bottoms were calculated to evaluate the effect of digester design on its flow pattern. Changing the draft tube clearance and height had no influence on the flow pattern or dead regions volume. However, increasing the draft tube diameter or incorporating a conical bottom design helped in reducing the volume of the dead zones as compared to a flat-bottom digester. The simulations showed that the gas flow rate sparged by a single point (0.5 cm diameter) sparger does not have an appreciable effect on the flow pattern of the digesters at the range of gas flow rates used.

Bacteria, Anaerobic↗
PubMed2005

Concurrent microscopic observations and activity measurements of cellulose hydrolyzing and methanogenic populations during the batch anaerobic digestion of crystalline cellulose.

This study compares process data with microscopic observations from an anaerobic digestion of organic particles. As the first part of the study, this article presents detailed observations of microbial biofilm architecture and structure in a 1.25-L batch digester where all particles are of an equal age. Microcrystalline cellulose was used as the sole carbon and energy source. The digestions were inoculated with either leachate from a 220-L anaerobic municipal solid waste digester or strained rumen contents from a fistulated cow. The hydrolysis rate, when normalized by the amount of cellulose remaining in the reactor, was found to reach a constant value 1 day after inoculation with rumen fluid, and 3 days after inoculating with digester leachate. A constant value of a mass specific hydrolysis rate is argued to represent full colonization of the cellulose surface and first-order kinetics only apply after this point. Additionally, the first-order hydrolysis rate constant, once surfaces were saturated with biofilm, was found to be two times higher with a rumen inoculum, compared to a digester leachate inoculum. Images generated by fluorescence in situ hybridization (FISH) probing and confocal laser scanning microscopy show that the microbial communities involved in the anaerobic biodegradation process exist entirely within the biofilm. For the reactor conditions used in these experiments, the predominant methanogens exist in ball-shaped colonies within the biofilm.

Anaerobiosis↗
PubMedDate not supplied

Rapid mass spectrometric identification of proteins from two-dimensional polyacrylamide gels after in gel proteolytic digestion.

We report a rapid method for identifying proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). In-gel digestion was performed in a way such that the volume ratio of trypsin solution to gel plug was quantitatively controlled to promote reproducible digestion and to maximize the digestion yield. To make the digestion samples more compatible with MALDI-MS, the volatile salt ammonium bicarbonate in the digestion buffer was largely removed prior to peptide extraction. Samples of mixed tryptic peptides from in-gel digestion were used without purification to obtain molecular weights by MALDI-MS with alpha-cyano, 4-hydroxy-cinnamic acid as the matrix. Modifications of MALDI sample loading procedures improved the detection sensitivity by one half to one order of magnitude. The peptide mass peaks in MALDI-MS spectra were distinguished from those of impurities by using several types of controls, and masses were corrected by using trypsin autodigestion fragments as internal calibration standards. Two different peptide-matching computer programs were used to interrogate sequence databases and identify proteins. Identification was enhanced by generation of orthogonal data sets (by using different proteases) and by including experimental values of isoelectric point (pI) and molecular weight to exclude false entries in the candidate lists. Approximately 1% of the material from a spot was used in each sample loading, and nine protein spots from rat liver 2-D PAGE gels were identified correctly, as judged by comparison with identification results previously obtained from Edman sequencing. A previously identified low-abundance spot was not identified by MALDI-MS, presumably because there was insufficient material in a single gel. The sample handling procedure reported here should permit us to identify many 2-D PAGE protein spots of medium abundance.

Acrylic Resins↗
PubMed1993

Apparent digestibility of dietary fibre and other components in table olives.

The apparent digestibility of the main components of the olive (Olea europaea arolensis) has been studied using young Wistar rats. An appreciable digestibility of fibre was found (of the order of 31% Neutral Detergent Fibre) and a high digestibility of fats (higher than 60%) and sugars (100% of glucose and 76% of fructose). Some 41% of the protein was digested, a relatively low value when compared with other products. If the diet is previously desalted, the digestibility of the components diminishes. There are significant differences in the digestibility of dry matter, fibre, cellulose and lignin, but not in proteins and fat.

Animals↗
PubMed2004

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping.

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.

Alkanesulfonates↗
PubMed2003

Separation and characterization of the colloidal phases produced on digestion of common formulation lipids and assessment of their impact on the apparent solubility of selected poorly water-soluble drugs.

Colloidal mixtures containing bile salts (BS), phosphatidylcholine (PC), and medium and long-chain monoglycerides and fatty acids were prepared as model systems to represent typical intestinal contents after digestion of formulation derived lipids under both low (5 mM BS/1.25 mM PC) and high (20 mM BS/5 mM PC) BS and PC conditions. Size-exclusion chromatography of the colloidal species that formed in the medium-chain digests indicated the presence of vesicles, mixed micelles, and simple micelles, whereas the long-chain digests contained only vesicles and mixed micelles. In the long-chain digests the mixed micellar phase was the predominant drug solubilizing species for griseofulvin, danazol, and halofantrine, although for increasingly lipophilic drugs, the vesicular phase contributed an increasing proportion of the solubilization capacity. In contrast, the solubilization capacity of the vesicular phase was predominant in the medium-chain digests, and no clear trends were evident in the relationship between drug lipophilicity and proportional solubilization. These data highlight the need to consider the colloidal species that form in the small intestine during the digestion of common formulation lipids and the coincident enhancement in drug solubilization provided under these circumstances.

Chemistry, Pharmaceutical↗
PubMed2005

An in-gel digestion procedure that facilitates the identification of highly hydrophobic proteins by electrospray ionization-mass spectrometry analysis.

A procedure is described for in-gel tryptic digestion of proteins that allows the direct analysis of eluted peptides in electrospray ionization (ESI) mass spectrometers without the need of a postdigestion desalting step. It is based on the following principles: (a) a thorough desalting of the protein in-gel before digestion that takes advantage of the excellent properties of acrylamide polymers for size exclusion separations, (b) exploiting the activity of trypsin in water, in the absence of inorganic buffers, and (c) a procedure for peptide extraction using solvents of proven efficacy with highly hydrophobic peptides. Quality of spectra and sequence coverage are equivalent to those obtained after digestion in ammonium bicarbonate for hydrophilic proteins detected with Coomassie blue, mass spectrometry-compatible silver or imidazole-zinc but are significantly superior for highly hydrophobic proteins, such as membrane proteins with several transmembrane domains. ATPase subunit 9 (GRAVY 1.446) is a membrane protein channel, lipid-binding protein for which both the conventional in-gel digestion protocol and in solution digestion failed. It was identified with very high sequence coverage. Sample handling after digestion is notably simplified as peptides are directly loaded into the ESI source without postdigestion processing, increasing the chances for the identification of hydrophobic peptides.

Adenosine Triphosphatases↗
PubMed1994

Kinetic studies on phagocytosis and lysosomal digestion of rod outer segments by human retinal pigment epithelial cells in vitro.

Using novel methodology, this study describes the kinetics of rod outer segment (ROS) phagocytosis and digestion by human retinal pigment epithelial (RPE) cells in vitro and examines the effect of certain lysosomal enzyme inhibitors on ROS digestion in these cells. Human RPE cells displayed saturation of phagocytosis with respect to both ROS concentration and time. While surface-binding and ingestion phases of ROS phagocytosis saturated after 24-36 h, the rate of ROS digestion reached a maximal level within 24 h. Increasing the concentration of zinc in the culture medium from 1.9 to 100 microM had no significant effect on ROS digestion. The effects of swainsonine (an alpha-mannosidase inhibitor), pepstatin (an aspartic protease inhibitor), and leupeptin (a cysteine protease inhibitor) were also examined. At 6 h, ROS digestion was reduced 27.3 +/- 15.3% by swainsonine, 69.4 +/- 20.9% by pepstatin, and 77.0 +/- 14.4% by leupeptin. The effect of these inhibitors declined with increasing time. This study is the first to demonstrate the functional importance of cysteine and aspartic proteases in the digestion of ROS by RPE cells in vitro.

Animals↗
PubMed1992

Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion.

A new procedure is described to generate single-stranded DNA by exonuclease III (Exo III) digestion for bromodeoxyuridine (BrdU) immunocytochemistry on tissue sections. We compared this procedure with the most widely used procedure of DNA denaturation with 2 N HCl. In vivo and in vitro pulse and continuous labelling of tissues and cells were used. The specimens were fixed in formalin, ethanol, glutaraldehyde, Carnoy's, Bouin's or Zamboni's fixative and embedded in paraffin or used unfixed as cryostat sections or cytospin preparations. After Exo III digestion, BrdU substituted DNA was detected irrespective of the fixation procedure applied. The optimal protocol for nuclease digestion appeared to be simultaneous incubation, of 10 Units Exo III per ml EcoRI buffer and anti-BrdU monoclonal antibody at 37 degrees C. The advantages of Exo III digestion for BrdU immunocytochemistry compared to acid denaturation were: less non-specific nuclear background reactivity, no DNA renaturation, less DNA loss, optimal nuclear morphology, increase in antibody efficiency and the possibility for simultaneous detection of acid-sensitive tissue constituents. Disadvantages of the Exo III digestion are decreased sensitivity and the need for more rigorous pepsin pretreatment. We conclude that Exo III digestion of DNA is an appropriate alternative for acid denaturation for BrdU immunocytochemistry on sections of pulse-labelled specimens.

Animals↗
PubMed1984

Tryptic digestion of scallop S1: evidence for a complex between the two light-chains and a heavy-chain peptide.

When scallop S1(+LC) (formerly called CaMg S1) is digested by trypsin, the heavy chain degrades while the two light chains remain complexed to each other and a peptide fragment of the heavy chain. The three components of the complex comigrate during electrophoresis under nondissociating conditions and can be purified by chromatography and concentrated by precipitation with ammonium sulphate in the presence of millimolar calcium ions. The truncated regulatory light chain remains associated with the binary complex consisting of the peptide and essential light chain as long as divalent cations are present; in the presence of EDTA it dissociates. This behaviour of the light chains-peptide complex mimics that of the intact molecule. The effect of bound light chains and bound actin on the susceptibility to tryptic digestion was studied using scallop S1(+LC) and S1(-LC) (EDTA S1 according to previous nomenclature). The heavy chains of both types of S1 are labile and have two main sites susceptible to proteolysis. Tryptic digestion on site A produces an N-terminal peptide of around 70 000 and a C-terminal 24 000 fragment from S1(+LC) and a 20 000 C-terminal fragment from S1(-LC); the latter is prone to further proteolysis. Thus S1(-LC), produced in the absence of bound regulatory light chain is shorter on the C-terminal end. Proteolysis on site A abolishes actin-activated ATPase activity; the latter is prevented by digesting acto-S1. The rate of tryptic digestion on site B is somewhat slower than on site A; when either S1 is split at this site an N-terminal 63 000 peptide is produced. The corresponding C-terminal peptide can be obtained from acto-S1 when hydrolysis on site A is prevented; this is estimated as around 31 000 derived from S1(+LC) and 28 000 derived from S1(-LC). The results are compared with similar experiments where vertebrate subfragments were digested by trypsin and the possible localization of the light-chain binding peptide in the intact heavy chain is discussed.

Actins↗
PubMed1982

The effect of digestion with keratanase (Pseudomonas sp.) on certain histochemical reactions for glycosaminoglycans in cartilaginous and corneal tissues.

The effect of digestion with keratanase (Pseudomonas sp.) on the Alcian Blue (AB) pH 1.0, pH 2.5, Aldehyde Fuchsin, high iron diamine, low iron diamine and dialysed iron-ferrocyanide reactions has been tested in the costal and ear cartilage tissues of the rabbit and corneal tissues of the rat and rabbit. The effect of digestion with chondroitinases ABC and AC on the same reactions was examined in the same tissues for comparison. Digestion with keratanase diminished the intensity of all the reactions in the cartilage tissues to a variable extent; however, the diminutions in intensity of the reactions appeared to be less marked as compared with those following digestion with two chondroitinases. In the corneal stroma, all the reactions were markedly reduced in intensity following digestion with keratanase. In contrast, these reactions were only slightly or moderately diminished in intensity by digestion with the two chondroitinases. As glycosamino-glycans are known to be present in cartilage and corneal tissues and the substrate specificities of the three enzymes used are now well established, the present results are consistent with the concept that keratanase specifically degrades and releases keratan sulphates involved in the tissues.

Animals↗
PubMed1992

Proteolytic digestion of alpha-lactalbumin: physiological implications.

The kinetics of the partial digestion of bovine alpha-lactalbumin (alpha-LA) by trypsin, alpha-chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of alpha-LA to trypsin and chymotrypsin at 37 and 5 degrees C decrease in the following order: Ca(II)-alpha-LA greater than Zn(II), Ca(II)-alpha-LA greater than apo-alpha-LA. The HPLC digestion patterns of Ca(II)-alpha-LA and Zn(II), Ca(II)-alpha-LA at 5 and 37 degrees C were similar, while the corresponding digestion patterns for apo-alpha-LA were quite different, reflecting the existence of the thermally induced denaturation states of apo-alpha-LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded alpha-LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37 degrees C due to Zn(II)-induced shift of the thermal transition of alpha-LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II)-specific site does not cause any drastic changes in the overall structure of alpha-LA. The acidic form of alpha-LA (at pH 2.2 and 37 degrees C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or alpha-chymotrypsin at neutral pH. Complexation of alpha-LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to alpha-LA may have physiological significance (e.g., for nutritional transport).

Animals↗
PubMed1995

Differential digestion of the centromeric heterochromatic regions of the 5-azacytidine-decondensed human chromosomes 1, 9, 15, and 16 by NdeII and Sau3AI restriction endonucleases.

A study on the factors involved in chromosome digestion by restriction endonuclease was carried out on 5-azacytidine treated and untreated human chromosomes 1, 9, 15 and 16 by using NdeII and Sau3AI isoschizomers. After treatment with 5-azacytidine, chromosomes 1, 9, 15, and 16 showed two differentiated areas at the centromeric regions: the centromere, fully condensed, and the pericentromeric heterochromatin, decondensed. Chromosomes not treated with 5-azacytidine after digestion with Sau3AI and NdeII showed all the centromeric regions undigested, except pair number 1, digested at the pericentromeric area. Digestion of the 5-azacytidine decondensed chromosomes with Sau3AI and NdeII showed the centromeres undigested in the four chromosome pairs while the pericentromeric heterochromatin appeared largely digested. Other factors, different to target distribution, are necessary to explain the pattern of restriction endonuclease digestion observed in this communication.

Azacitidine↗
PubMed2006

Development of an on-line immobilized-enzyme reversed-phase HPLC method for protein digestion and peptide separation.

This paper describes use of a novel glass bead-based immobilized-enzyme micro column for simple and swift on-line protein digestion then peptide separation by reversed-phase HPLC. The inexpensive and easily made immobilized-enzyme micro column was prepared from aminopropyl controlled-pore glass that was reacted first with glutaraldehyde then with trypsin in the presence of phosphate buffer. Tryptic digestion of bovine serum albumin (BSA) was achieved simply by passing pretreated protein solution through the laboratory made immobilized-trypsin column; the tryptic fragments were then separated by reversed-phase HPLC. The peptide separation was found to be identical to separation of a sample which had undergone conventional enzymatic protein digestion in solution. Digestion of BSA by the immobilized-trypsin column decreased with increasing flow rate of the solution through the column, and 1.0 muL min(-1) was found to be the optimum flow rate for on-line protein digestion with our system. It was also found that the sample required pretreatment with urea before injection, because of a change in the properties of the protein in the presence of urea, and the immobilized-trypsin column lost its function in the presence of acetonitrile. This on-line proteomics system enables simple and rapid protein digestion and was successfully applied to partially micro two-dimensional (2D) chromatographic separation of proteins.

Chromatography, High Pressure Liquid↗
PubMed2006

A theoretical framework for evaluating analytical digestion methods for poorly soluble particulate beryllium.

Complete digestion of all chemical forms and sizes of particulate analytes in environmental samples is usually necessary to obtain accurate results with atomic spectroscopy. In the current study, we investigate the physicochemical properties of beryllium particles likely to be encountered in samples collected from different occupational environments and present a hypothesis that a dissolution theory can be used as a conceptual framework to guide development of strategies for digestion procedures. For monodisperse single-chemical constituent primary particles, such as those encountered when handling some types of beryllium oxide (BeO) powder, theory predicts that a digestion procedure is sufficient when it completely dissolves all primary particles, independent of cluster size. For polydisperse single-chemical constituent particles, such as those encountered during the handling of some types of beryllium metal powder, theory predicts that a digestion procedure is sufficient only when it completely dissolves the largest particle in the sample. For samples with unknown or multi-chemical constituent particles and with particles having undefined sizes, e.g., fume emissions from a copper-beryllium alloy furnace operation or dust from a beryl ore crushing operation, a surface area-limited and single-constituent-dependent dissolution theory may not predict complete dissolution, thereby requiring non-routine robust treatment procedures with post-digestion filtration, followed by examination of residual particulate material. Additionally, for beryllium, and likely other poorly soluble materials, particulate reference materials of various chemical forms and size distributions are needed to better evaluate and harmonize analytical digestion procedures. Figure Generation of aerosol particles during machining of beryllium oxide.

Aerosols↗
PubMed2001

Digestion of organic components in waste materials by high pressure ashing with infrared heating.

In respect of complete digestion of organic waste materials, a relevant potent digestion technique was developed, and a prototype of a high pressure ashing device with infrared heating (IR-HP-asher) was built. The apparatus consists of six quartz digestion vessels inside a steel autoclave. The performance of the IR-HP-asher was tested for pure organic substances, for representative wastes from the recycling industry, and for several biological samples. In all cases, complete digestion could be attained within 90 min as well as complete recovery of metal(loid)s present in the sample. Concerning the completeness of digestion and absence of analyte loss, the IR-HP-asher was found to be superior to two conventional microwave digesters also tested.

Carbon↗
PubMed2006

Phage diversity in a methanogenic digester.

It has been shown that phages are present in natural and engineered ecosystems and influence the structure and performance of prokaryotic communities. However, little has been known about phages occurring in anaerobic ecosystems, including those in methanogenic digesters for waste treatment. This study investigated phages produced in an upflow anaerobic sludge blanket methanogenic digester treating brewery wastes. Phage-like particles (PLPs) in the influent and effluent of the digester were concentrated and purified by sequential filtration and quantified and characterized by transmission electron microscopy (TEM), fluorescence assay, and field inversion gel electrophoresis (FIGE). Results indicate that numbers of PLPs in the effluent of the digester exceeded 1 x 10(9) L-1 and at least 10 times greater than those in the influent, suggesting that substantial amounts of PLPs were produced in the digester. A production rate of the PLPs was estimated at least 5.2 x 10(7) PLPs day-1 L-1. TEM and FIGE showed that a variety of phages were produced in the digester, including those affiliated with Siphoviridae, Myoviridae, and Cystoviridae.

Anaerobiosis↗