Integrating alkaline extraction of proteins with enzymatic hydrolysis of cellulose from wet distiller’s grains and solubles

Fractionation of distiller’s grains into value added products may serve to improve the economic viability of dry grind corn ethanol facilities in the wake of variable corn and ethanol prices. This research is aimed at creating a high protein, high lysine product from the grain using alkaline protein extractions in conjunction with hydrolysis of the remaining fiber to sugars which are then fermented to ethanol. Alkaline extractions improved the lysine content in protein products, although protein solubility did not exceed 45% of the total protein. In addition, oligomeric carbohydrates, starch, and other water solubles were also extracted, leading to a low purity protein product. Resulting sugar yields following ammonia fiber expansion (AFEX) pretreatment were also lower for extracted distiller’s grains. From these experiments, it does not appear likely that alkaline extraction is a useful tool for fractionation of distiller’s grains. However, pretreatment and hydrolysis can be an effective tool for further fractionation of protein.     

Extraction and partial purification of mouse uterine alkaline phosphatase.

Alkaline phosphatase in uterine homogenates from day 7 pregnant mice was solubilized using 0.2% (v/v) Triton X-100 and extracted wtih 20% (v/v) n-butanol. The procedure, which resulted in 182-fold purification, included ammonium sulfate precipitation, DEAE-cellulose anion exchange chromatography and Sephadex G200 gel filtration. Solubilization with Triton X-100 was an important step in the procedure since extraction with n-butanol alone only partially solubilized the enzyme and gave low extraction yields, much of the enzyme activity remaining in association with negatively charged residues. However, butanol extraction of Triton X-100-treated homogenates gave high yields of enzyme and eliminated p-nitrophenyl phosphatases which displayed activity in the pH range 3.0--7.5, together with a large proportion of inactive protein. The activity of the purified enzyme preparations was electrophoretically homogeneous on cellulose acetate membranes, suggesting that the alkaline phosphatase in the mouse uterus exists in a single isozymic form. Polyacrylamide-gel electrophoresis revealed that the purified preparations contained at least one protein as an impurity. Attempts to further purify the alkaline phosphatase by isoelectric focusing were unsuccessful since the enzyme was found to have an isoelectric point of about 5.0 and at this pH it was rapidly inactivated.     

Fractionation of sugar beet pulp by introducing ion-exchange groups

Sugar beet pulp (SBP) was chemically modified with the goal to utilize this method for the preparation of water-soluble polysaccharides. Yields of the trimethylammoniumhydroxypropylated (TMAHP) polysaccharide fractions prepared under vacuum in absence of NaOH or KOH, as well as their molar masses, were higher than those obtained when the samples were only extracted with hydroxide at ambient pressure. The hydroxypropylsulfonated (HPS) fractions extracted at ambient pressure with NaOH had higher yields and molar mass than the TMAHP fractions extracted under vacuum with KOH. Introduction of both ion-exchange groups in one step under vacuum with NaOH gave better yields than with KOH, but smaller than for the solely sulfonated sample. The molar masses of chemically modified fractions were smaller than those extracted only with alkali or water. According to the monosaccharide composition all the fractions are mixtures of arabinogalactans and rhamnogalacturonoans.     

Choice of extraction procedure for estimation of anterior pituitary hormone content

Searching for the best procedure for simultaneous estimation of the anterior pituitary hormones, extraction efficiencies of various media, additives such as urea and triton X-100, and physical treatments such as freezing-thawing (F-T) and sonication, were examined by measuring prolactin (PRL), growth hormone (GH), lutropin (LH), follitropin (FSH), and thyrotropin (TSH) in the extracts. Ethanolic media (60% EtOH) gave high yields of PRL at neutral to alkaline pH, but poor extraction of GH accompanied by a marked loss of its immunoreactivity during storage. Ethanolic media also gave a poor yield of LH even at high pH. Aqueous media like PBS at various pH, 0.1 M acetic acid and distilled water were considerably effective in the extraction of GH, LH, FSH and TSH if they were coupled with F-T and sonication. However, high yields of PRL could not be obtained with these aqueous media even with F-T and sonication. Hartree's 40% EtOH-6% ammonium acetate, pH 5.1, solubilized considerable amounts of glycoprotein hormones, but yielded almost no GH and only a small amount of PRL. The addition of triton X-100 to PBS (pH 7) at 0.1% resulted in the maximum extraction of glycoprotein hormones with homogenization and F-T, but further sonication was necessary for GH and PRL. When the anterior pituitaries were homogenized and frozen-thawed in PBS (pH 7) containing 1 M urea, yields of PRL, GH, LH, FSH, and TSH were maximum, and sonication did not cause any additional extraction, indicating that this procedure, i.e. homogenization and F-T in 1 M urea-PBS, would be the best for the simultaneous estimation of these anterior pituitary hormones.     

Extraction Techniques for Gastrins and Cholecystokinins in the Rat Central Nervous System

Abstract: Samples of rat striatum and synthetic sul-phated cholecystokinin octapeptide were extracted by different procedures and the solubilised cholecystokinin-like immunoreactivity analysed by gel filtration and ion-exchange chromatography. Ice-cold 90% methanol extraction gave the greatest recovery of tissue immunoreactivity without any major modification of the extracted components. The 33-amino acid form of cholecystokinin was poorly recovered by this extractant. Boiling water or a combined boiling water/acetic acid extraction gave efficient recovery of tissue immunoreactivity but chemically modified a substantial part of the octapeptide-like component. Boiling in acetic acid alone also produced this modification but in addition resulted in poor recovery of the octapeptide-like component. The combined water/ acetic acid extraction gave reasonable to good recovery of all added Cholecystokinins and gastrins, including the 33-amino acid form of cholecystokinin. Ice-cold 0.1 M HCl was less efficient than 90% methanol at solubilising tissue immunoreactivity and resulted in a substantial modification of the octapeptide component distinct from that produced by boiling extractions, possibly desulpha-tion. The results show that more than one extraction procedure is needed to study all the cholecystokinin components in brain tissue and demonstrates the necessity of using at least two chromatographic systems for such studies.     

Solubilizing water involved in protein extraction using reversed micelles

The extraction of protein using reversed micelles was investigated in relation to the amount of solubilizing water in the reversed micellar organic phase. The minimal concentration of amphiphilic molecule di-2-ethylhexyl sodium sulfosuccinate (C(20)H(37)O(7)Na) (AOT) required for 100% cytochrome c extraction was recognized. This critical AOT concentration increased with protein concentration in the aqueous phase. On this minimal AOT condition, the molar ratio of solubilizing water to extracted protein was found to be a constant of 3500 under C(KCI) = 1.0 x 10(2) mol . m(-3) in this system. This ratio means the hydrophillic surroundings required for extracting one protein molecule into the micellar organic phase under the suitable pH and salt concentration for the forward extraction. In this regard, AOT molecules seemed to take the part of water solubilizing agent in the reversed micellar extraction. This role of AOT is important to extract protein under the suitable pH and salt concentration. The amount of solubilizing water in the protein-containing system was larger than in the protein-free system. This difference shows that the water molecules accompany the extracted protein into the reversed micellar organic phase at constant ratio 2200 under C(KCI) = 1.0 x 10(2) mol . m(-3), i.e., accompanying water molecules per one extracted protein. The minimal AOT concentration increased with ionic strength. On this minimal AOT condition, the molar ratio of solubilizing water to extracted protein also increased with ionic strength, so that in higher ionic strength, more solubilizing water was required. Then more AOT was required to provide the hydrophillic surroundings for protein. The pH affected the minimal AOT concentration required for 100% protein extraction.     

Activity-based matrix metallo-protease enrichment using automated, inhibitor affinity extractions

An automated inhibitor affinity extraction method for the activity-based enrichment of matrix metallo-proteases (MMPs) is presented. Samples containing purified MMP-12 were first extracted at different flow rates in a syringe pump setup, using cartridges packed with an MMP inhibitor affinity sorbent based on an immobilized hydroxamic acid containing peptide (PLG-NHOH) with mumol/L MMP affinity. Faster extractions, a reduced number of manual manipulations, and higher extraction yields (98.9%-99.3%) were obtained over the whole flow rate range compared to batch extractions. Application of the method to synovial fluid from a rheumatoid arthritis patient followed by gelatin-zymography revealed a strong enrichment of distinct MMPs from this biological sample that were not clearly visible in the original sample. The use of an auto-sampler and a solid-phase extraction (SPE) workstation allowed full automation of the extraction procedure with the potential for on-line coupling to further sample preparation and analytical steps. MMP-12 extractions were optimized showing that ligand density is an important factor with a clear extraction yield optimum around 5 to 7.5 mmol/L. Conditioning of the stationary phase for 1 week prior to use resulted in a further slight increase in extraction yield. Under optimal conditions, an extraction yield of 99.5% was reached with a cartridge contact time of only 13 s for MMP-12. The efficacy of the extraction method for activity-based MMP profiling was further improved by the use of a broad-spectrum MMP inhibitor with nmol/L affinity (TAPI-2). This resulted in an increased extraction yield for all tested MMPs. For MMP-1, -7, -8, -10, -12, and -13 extraction yields of at least 98.8% were obtained, while for MMP-9 (full length and catalytic domain) an extraction yield of at least 96.1% was reached.     

Degradation kinetics of the main carbohydrates in birch wood during hot water extraction in a batch reactor at elevated temperatures

Hot water extraction of wood at elevated temperatures may be a suitable method to produce hemicellulose-lean pulps and to recover xylan-derived products from the water extract. In this study, water extractions of birch wood were conducted at temperatures between 180 and 240 °C in a batch reactor. Xylan was extensively removed, whereas cellulose was partly degraded only at temperatures above 180 °C. Under severe extraction conditions, acetic acid content in the water extract was higher than the corresponding amount of acetyl groups in wood. In addition to oligo- and monosaccharides, considerable amounts of furfural and 5-hydroxymethylfurfural (HMF) were recovered from the extracts. After reaching a maximum, the furfural yield remained constant with increasing extraction time. This maximum slightly decreased with increasing extraction temperature, suggesting the preferential formation of secondary degradation products from xylose. Kinetic models fitting experimental data are proposed to explain degradation and conversion reactions of xylan and glucan.     

Selective Extraction of Alkaline Phosphatase and 51 -Nucleotidase from Milk Fat Globule Membranes by a Single Phase n-Butanol Procedure

ABSTRACT

A single phase extraction procedure employing 8% (v/v) n-butanol at room temperature extracted over 90% of alkaline phosphatase activity and over 60% of 5'-nucleotidase activity from bovine milk fat globule membranes (MFGM). For 5'-nucleotidase, higher n-butanol concentrations lead to loss of activity, while lower concentrations were ineffective in extracting the enzyme. When extractions were performed at 0°C, similar yields were obtained for alkaline phosphatase extraction with 8% (v/v) n-butanol, but 5¹- nucleotidase extraction required 10% (v/v) n-butanol for similar yields. However, 5'-nucleotidase was less susceptible to denaturation during extraction at 0°C. The Km values and substrate specificities for both alkaline phosphatase and 5'-nucleotidase were unchanged by extraction with 8% (v/v) n-butanol. The 8% (v/v) n-butanol extraction procedure provides a 3-fold purification step, and an enzyme preparation suitable for further purification.     

Evaluation of metabolite extraction strategies from tissue samples using NMR metabolomics

Metabolomic analysis of tissue samples can be applied across multiple fields including medicine, toxicology, and environmental sciences. A thorough evaluation of several metabolite extraction procedures from tissues is therefore warranted. This has been achieved at two research laboratories using muscle and liver tissues from fish. Multiple replicates of homogenous tissues were extracted using the following solvent systems of varying polarities: perchloric acid, acetonitrile/water, methanol/water, and methanol/chloroform/water. Extraction of metabolites from ground wet tissue, ground dry tissue, and homogenized wet tissue was also compared. The hydrophilic metabolites were analyzed using 1-dimensional (1D) ¹H nuclear magnetic resonance (NMR) spectroscopy and projections of 2-dimensional J-resolved (p-JRES) NMR, and the spectra evaluated using principal components analysis. Yield, reproducibility, ease, and speed were the criteria for assessing the quality of an extraction protocol for metabolomics. Both laboratories observed that the yields of low molecular weight metabolites were similar among the solvent extractions; however, acetonitrile-based extractions provided poorer fractionation and extracted lipids and macromolecules into the polar solvent. Extraction using perchloric acid produced the greatest variation between replicates due to peak shifts in the spectra, while acetonitrile-based extraction produced highest reproducibility. Spectra from extraction of ground wet tissues generated more macromolecules and lower reproducibility compared with other tissue disruption methods. The p-JRES NMR approach reduced peak congestion and yielded flatter baselines, and subsequently separated the metabolic fingerprints of different samples more clearly than by 1D NMR. Overall, single organic solvent extractions are quick and easy and produce reasonable results. However, considering both yield and reproducibility of the hydrophilic metabolites as well as recovery of the hydrophobic metabolites, we conclude that the methanol/chloroform/water extraction is the preferred method. C. Y. Lin and H. Wu contributed equally.     

DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: heating under the influence of pH.

During the course of diagnostic surgical pathology, pathologists have established a large collection of formalin-fixed, paraffin-embedded tissues that form invaluable resources for translational studies of cancer and a variety of other diseases. Accessibility of macromolecules in the fixed tissue specimens is a critical issue as exemplified by heat-induced antigen retrieval (AR) immunohistochemical (IHC) staining. On the basis of observations that heating may also enhance in situ hybridization (ISH) and the similarity of formalin-induced chemical modifications that occur in protein and in DNA, we designed a study to examine the efficiency of DNA extraction from archival formalin-fixed, paraffin-embedded tissues using an adaptation of the basic principles of the AR technique, i.e., heating the tissue under the influence of different pH values. Archival paraffin blocks of lymph nodes, tonsil, and colon were randomly selected. Each paraffin block was prepared in 34 microtubes. For each paraffin block, one tube was used as a control sample, using a non-heating DNA extraction protocol. The other 33 tubes were tested using a heating protocol under 11 variable pH values (pH 2 to 12) under three different heating conditions (80, 100, and 120C). Evaluation of the results of DNA extraction was carried out by measuring yields by photometry and PCR amplification, as well as kinetic thermocycling (KTC)-PCR methods. In general, lower pH (acid) solutions gave inferior results to solutions at higher pH (alkaline). Heating tissues at a higher temperature and at pH 6-9 gave higher yields of DNA. There appeared to be a peak in terms of highest efficiency of extracted DNA at around pH 9. The average ratios 260:280 of extracted DNA also showed better values for samples heated at 120C. PCR products of three primers showed satisfactory results for DNA extracted from archival paraffin-embedded tissues by heating protocols at pH 6-12, with results that were comparable to the control sample subjected to the standard non-heating, enzymatic DNA extraction method. This study is the first to document the use of heating at an alkaline pH for DNA extraction from archival formalin-fixed, paraffin-embedded tissues, a recommendation based on the principles of AR for protein IHC. These findings may lead to a more effective protocol for DNA extraction from archival paraffin-embedded tissues and may also provide enhanced understanding of changes that occur during formalin-induced modification of nucleic acids.     

Extraction and purification of murine epidermal growth factor

We have compared six different solutions commonly used for the extraction of peptides to optimize the extraction of Epidermal Growth Factor (EGF) from mouse submandibular glands. The yield of EGF was always higher in neutral pH extractions than in acidic ones. However, the EGF extracted at neutral pH was only poorly adsorbed on to octadecylsilyl silica, whereas the EGF extracted at acidic pH was be easily adsorbed. Subsequent purification of the EGF extracts by two-stage reversed-phase High Performance Liquid Chromatography yielded highly purified EGF. Identical chromatograms were obtained from each of the different extracts. Preparative scale quantities of EGF could be purified rapidly and reproducibly at high efficiency from acidic extracts.     

Extraction of protease from Aspergillus tamarii URM 4634 in aqueous two-phase system under continuous and discontinuous process

Abstract

Aqueous two-phase systems have been studied for almost a century to separate biomolecules in harmless conditions. Proteases produced by Aspergillus tamarii URM 4634 were extracted in polyethylene glycol (PEG)/phosphate aqueous two-phase system under discontinuous and continuous (perforated rotative discs column) process. On the discontinuous process, it was evaluated the effect of operational conditions (PEG molar mass and its concentration, phosphate concentration and pH) over the partition coefficient, activity yield and purification factor. Protease partitioned to PEG-phase with partition coefficients up to 55.73. The best process parameters were 17.5% of PEG, with molar mass 8000?g·mol^?1, 15% of phosphate salt at pH 6, with 113.15% of activity yield and purification factor of 2.62. Under continuous extraction, hold up data showed that 57.1% of the discontinuous phase was available for protein extraction. Further, separation achieved 90.0% of efficiency. The yields surpassed 100% in almost all runs, and the best purification factor was 1.84, with both flows of 2?mL·min^?1. Thus, the best operational conditions reached an activity yield of 95.3% and 90.0% of separation efficiency. Hence, aqueous two-phase system PEG/phosphate extraction is an efficient process for separation of proteases produced by Aspergillus tamarii URM 4634, under continuous extraction likewise under discontinuous process.     

Ultrasound-Assisted Extraction of Arabinogalactan and Dihydroquercetin Simultaneously from Larix gmelinii as a Pretreatment for Pulping and Papermaking

An ultrasound-assisted extraction (UAE) method using ethanol was applied for extracting arabinogalactan (AG) and dihydroquercetin (DHQ) simultaneously from larch wood, as a pretreatment for pulping and papermaking. The extraction parameters were optimized by a Box-Behnken experimental design with the yields of AG and DHQ as the response values. Under optimum conditions (three extractions, each using 40% ethanol, for 50 min, 200 W ultrasound power and 1∶18 solid-liquid ratio), the yields of AG and DHQ were 183.4 and 36.76 mg/g, respectively. After UAE pretreated, the wood chips were used for Kraft pulping (KP) and high boiling solvent pulping (HBSP). The pulping yield after pretreatment was higher than that of untreated (the pulping yields of untreated HBSP and KP were 42.37% and 39.60%, and the pulping yields of HBSP and KP after UAE-pretreated were 44.23% and 41.50% respectively), as indicated by a lower kappa number (77.91 and 27.30 for untreated HBSP and KP; 77.01 and 26.83 for UAE-pretreated HBSP and KP). Furthermore, the characteristics of paper produced from pretreated wood chips were superior to those from the untreated chips: the basis weight was lower (85.67 and 82.48 g·cm⁻² for paper from untreated KP and HBSP; 79.94 and 80.25 g·cm⁻² for paper from UAE-pretreated KP and HBSP), and the tensile strengths, tearing strengths, bursting strengths, and folding strengths were higher than these of paper after UAE-pretreated, respectively.     

Influence of Soil Organic Matter on Copper Extraction from Contaminated Soil

Column experiments of copper extraction from four contaminated soils characterized by a content of Soil Organic Matter (SOM) ranging from 1% to 25% are presented and discussed. The extraction was performed by flushing the soil with an aqueous solution of a sodium salt of ethylene diamminotetraacetic acid (EDTA). Preliminary tests were performed on a soil containing 25% of organic matter, to investigate the influence of pH, concentration and volumes of EDTA on its chelant action and on the dissolution of SOM. Having selected the optimal conditions for the extraction process, a further series of tests was conducted on the four soils to evaluate the influence of organic content on copper extraction yields. EDTA solutions at 0.01 M, 0.05 M, 0.1 and 0.2 M were injected at 0.33 ml/s; copper and organic matter extraction yield were determined. At a pH of 5, 15 pore volume (PV) of a solution containing 0.05M EDTA, extracted about 99% of copper contained by the soil with the higher organic matter content. Under the same conditions, and for soil with > 6% SOM, extraction yields over 80% were achieved, while at lower organic content, copper extraction was dramatically reduced. This was attributed to the formation of highly stable copper-humate complexes and to their increasingly dissolution that occurred in the soils with higher organic matter level.

Experimental tests performed at different contamination levels (1200 mg/kg, 2400 mg/kg) showed that EDTA extraction effectiveness also depended upon initial soil Cu concentration.     

The influence of the extraction procedure on yield of indole-3-acetic acid in plant extracts

Different types of plant material, including both dry and swollen maize kernels, swollen bean seeds, bean seedlings and dry rose seeds, were extracted by different methods and the yield of IAA was determined with the indolo-α-pyrone method. Extraction of dry maize kernels during short time experiments, varying from 3 to 24 h, gave the highest IAA yield when methanol was the extractant and a significant lower yield when diethyl ether or dichloromethane were used. The duration of the extraction period increased the yield with all the extractants. Progressive extractions for several days or weeks had little effect on the yield when 100% acetone was used in contrast to methanol and ether as extractants, which increased the yield during prolonged extraction. Extractions of tissue treated to 100°C for 1 h contradicted the hypothesis that IAA is enzymatically liberated during ether extraction. Water in the extractant solvents increased the yields. This was most pronounced when aqueous acetone was used instead of 100% acetone. Increased extraction temperature augmented the IAA yields. The yield of IAA from other types of tissue extracted with methanol for periods of 3 or 24 h was, however, independent of the duration of the extraction time. This indicates that some tissues contain less not easily extractable IAA than dry maize kernels. The terms “free” and “bound” IAA are discussed; they should be replaced by “easily extractable” and “not easily extractable” IAA. The results also show that IPyA in vitro can partly be converted to IAA during extraction and fractionation.     

Extraction of chlorophylls a and b from phytoplankton using standard extraction techniques *

SUMMARY. Pigments extracted in methanol, acetone and ethanol from three cultures of green algae and one blue-green alga revealed different extraction efficiencies depending on the species, the extraction solvent used and the extraction time. Chromatographic identification and quantitative measurements of chlorophylls a and b were made from six green algae. When extraction of pigments was incomplete, chlorophyll-b was extracted faster than chlorophyll-a. This effect was more pronounced for acetone extractions, whereas methanol extractions gave the stable ratios of chlorophyll b/a after about 6–10 h. When green algae are frequent, a 6–10 h methanol extraction, without any extra manipulations, is sufficient to ensure reliable ratios of chlorophyll b/a and extraction of the major proportion of the chlorophylls without risk of induced destruction of the chlorophylls.     

Termite feeding deterrent from Japanese larch wood

Extraction of flavonoids from Japanese larch (Larix leptolepis) wood with water was carried out to prepare a termite feeding deterrent. A two-stage procedure for the extraction was adopted. The first extraction step was performed at ambient temperature (22 degrees C) and the second at elevated temperatures ranging 50-100 degrees C. The first step mainly gave a mixture of polysaccharides together with small amount of flavonoids. At the second step, the yield of extract and its chemical composition were greatly affected by the temperature. The yield of solubilised carbohydrates steadily increased with a rise in the temperature, while the overall yield of flavonoids reached its optimum at 70 degrees C. An additional increase in the temperature resulted in a decrease in the yield. Model experiments using dihydroflavonols confirmed the occurrence of oxidative dehydrogenation and/or intramolecular rearrangement during the hydrothermal treatment at higher temperatures. The crude water extracts showed strong feeding deterrent activities against the subterranean termite, Coptotermes formosanus, in a choice paper disc assay. The extracts containing flavonoids in large quantities exhibited potent termite feeding deterrent activities.     

Phytochemicals for liquid fuels and petrochemical substitutions: Extraction procedures and screening results

Various solvents were examined for their effectiveness in the extraction of nonpolar and polar compounds from plant materials for obtaining botanochemicals. Cyclohexane extraction followed by methanol gave consistently higher yields than other solvents in current use. Soxhlet extraction was found to give higher yields than shaking /decanting. Yields of nonpolar and polar extractions are reported for 80 species from the southeastern United States and southern Great Plains.