Kinetics of the acid precipitation of soya protein in a continuous-flow tubular reactor

The acid precipitation of soya protein was studied in a continuous-flow tubular reactor under conditions of turbulent flow. Preliminary batchwise experiments of a semiquantitative nature were also carried out on a bench-scale reactor to better define the parameters affecting precipitate growth. The experiments indicated the dominant growth mechanism to be the aggregation of primary precipitate particles produced by the contacting of the protein and acid streams. The rate of particle growth was observed to rise with an increase in the protein concentration as well as with greater intensity of turbulence. The final mean particle size decreased with increased intensity of turbulence. A theoretical model was set up to simulate the growth of the precipitate particles.     

Recovery of canola meal proteins by precipitation

Recovery of rapeseed proteins from defatted canola meal by precipitation was investigated. The ability of different precipitating agents, such as sodium hexametaphosphate (HMP), carboxymethylcellulose (CMC), ammonium sulphate, and isoelectric precipitation using HCl, were evaluated based on the yield and mean size of protein aggregates. Almost 94% of dissolved protein was precipitated in the presence of 2.7M ammonium sulphate, while the largest mean protein particle size (32 mum) was obtained in the presence of HMP at pH 3.3.     

Concentration of Rift Valley Fever and Chikungunya Viruses by Precipitation

Simple and efficient methods for concentrating Rift Valley fever (RVF) virus and chikungunya (CHIK) virus are described. Ammonium sulfate, potassium sulfate, or alcohol was used as a precipitating agent and the precipitate was resuspended to volumes suitable for further processing and purification. The methods permitted concentration of live RVF virus and CHIK virus about 100-fold with negligible losses of virus. RVF virus retained a high level of infectivity with potassium aluminum sulfate and alcohol, but CHIK virus retained a higher infectivity level with ammonium sulfate than with potassium aluminum sulfate. The data indicate that serum plays an important role in the concentration of both viruses, at least when the sulfate methods are used.     

A novel Method for the selective recovery and purification of γ‐polyglutamic acid from Bacillus licheniformis fermentation broth

Microbially produced gamma‐polyglutamic acid (γ‐PGA) is a commercially important biopolymer with many applications in biopharmaceutical, food, cosmetic and waste‐water treatment industries. Owing to its increasing demand in various industries, production of γ‐PGA is well documented in the literature, however very few methods have been reported for its recovery. In this paper, we report a novel method for the selective recovery and purification of γ‐PGA from cell‐free fermentation broth of Bacillus licheniformis. The cell‐free fermentation broth was treated with divalent copper ions, resulting in the precipitation of γ‐PGA, which was collected as a pellet by centrifugation. The pellet was resolubilized and dialyzed against de‐ionized water to obtain the purified γ‐PGA biopolymer. The efficiency and selectivity of γ‐PGA recovery was compared with ethanol precipitation method. We found that 85% of the original γ‐PGA content in the broth was recovered by copper sulfate‐induced precipitation, compared to 82% recovery by ethanol precipitation method. Since ethanol is a commonly used solvent for protein precipitation, the purity of γ‐PGA precipitate was analyzed by measuring proteins that co‐precipitated with γ‐PGA. Of the total proteins present in the broth, 48% proteins were found to be co‐precipitated with γ‐PGA by ethanol precipitation, whereas in copper sulfate‐induced precipitation, only 3% of proteins were detected in the final purified γ‐PGA, suggesting that copper sulfate‐induced precipitation offers better selectivity than ethanol precipitation method. Total metal content analysis of the purified γ‐PGA revealed the undetectable amount of copper ions, whereas other metal ions detected were in low concentration range. The purified γ‐PGA was characterized using infrared spectroscopy. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Selective precipitation and purification of monovalent proteins using oligovalent ligands and ammonium sulfate

This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: (i) the removal of high-molecular-weight impurities through the addition of ammonium sulfate to the crude cell lysate; (ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and (iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins--for which appropriate oligovalent ligands can be synthesized--and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation.     

二氯苯磺酸沉淀毛发水解液中亮氨酸和精氨酸的新方法

本文介绍了一种以二氯苯磺酸为沉淀剂从毛发水解液中分步沉淀亮氨酸和精氨酸的方法。利用两种沉淀形成速度的不同,通过控制反应条件,实现了亮氨酸和精氨酸的分步沉淀,确定了沉淀条件对目标氨基酸沉淀效率的影响,得到合适的工艺条件:200g人发,水解得400mL水解液;加50g二氯苯磺酸沉淀剂,在5℃加晶种,间歇搅拌12h,过滤得亮氨酸复合物沉淀;在沉淀亮氨酸之后的母液中再加50g二氯苯磺酸沉淀剂,于相同的温度条件下加晶种,连续搅拌至生成稠厚的沉淀,再静置沉淀12h,过滤得精氨酸复合物沉淀。亮氨酸的沉淀率为71.0%,母液中残留亮氨酸浓度为7.6g/L;精氨酸的沉淀率为76.6%,母液中残留精氨酸浓度为8.9g/L。     

Crystalline pneumococcus antibody

1. The immune precipitate formed by antipneumococcus horse serum and the specific polysaccharide is not hydrolyzed by trypsin as is the diphtheria toxin-antitoxin complex, and purified pneumococcus antibody cannot be isolated by the method used for the isolation and crystallization of diphtheria antitoxin. 2. Type I pneumococcus antibody, completely precipitable by Type I polysaccharide, may be obtained from immune horse serum globulin by precipitation of the inert proteins with acid potassium phthalate. 3. The antibody obtained in this way may be fractionated by precipitation with ammonium sulfate into three main parts. One is insoluble in neutral salts but soluble from pH 4.5 to 3.0 and from pH 9.5 to 10.5. This is the largest fraction. A second fraction is soluble in 0.05 to 0.2 saturated ammonium sulfate and the third fraction is soluble in 0.2 saturated ammonium sulfate and precipitated by 0.35 saturated ammonium sulfate. The second fraction can be further separated by precipitation with 0.17 saturated ammonium sulfate to yield a small amount of protein which is soluble in 0.17 saturated ammonium sulfate but insoluble in 0.25 saturated ammonium sulfate. This fraction crystallizes in poorly formed, rounded rosettes. 4. The crystallization does not improve the purity of the antibody and is accompanied by the formation of an insoluble protein as in the case of diphtheria antitoxin. 5. None of the fractions obtained is even approximately homogeneous as determined by solubility measurements. 6. Purified antibody has also been obtained by dissociating the antigen-antibody complex. 7. The protective value of the fractions is quite different; that of the dissociated antibody being the highest and that of the insoluble fraction, the lowest. 8. All the fractions are immunologically specific since they do not precipitate with Type II polysaccharide nor protect against Type II pneumococci. 9. All the fractions give a positive precipitin reaction with antihorse rabbit serum. The dissociated antibody gives the least reaction. 10. Comparison of the various fractions, either by their solubility in salt solution or through immunological reactions, indicates that there are a large number of proteins present in immune horse serum, all of which precipitate with the specific polysaccharide but which have very different protective values, different reactions with antihorse rabbit serum, and different solubility in salt solutions.     

Rapid and high-yield purification of porcine heart tissue-type plasminogen activator by heparin-sepharose choromatography

A rapid and high-yield procedure for the purification of single polypeptide tissue-type plasminogen activator (t-PA) from porcine heart tissue has been developed. Delipidated heart tissue was extracted with 0.45 M potassium acetate. The extract was fractionated with ammonium sulfate and purified by a combination of affinity chromatography on heparin-Sepharose CL-6B and gel filtration on Toyopearl HW-55S. The final product had a specific activity of 220,000 IU/mg protein and gave a single protein band (apparent molecular weight; 67,000) in SDS-polyacrylamide gels in the presence or absence of a reducing agent. The increase in specific activity was 3,200-fold, most of which was achieved in the step of heparin-Sepharose chromatography. The yield calculated from the active ammonium sulfate precipitate was about 90% and 500 micrograms or more of the purified enzyme was obtained from 1 kg wet tissue. This procedure may also be useful for the large-scale production of highly purified t-PA from other tissues or tissue culture cells.     

Strategy for a protein purification design using C-phycocyanin extract

A variety of techniques have been developed for the separation and recovery of proteins. The cost of purifying the product is frequently determined by the desired quality of the final product, which is evaluated by measuring the purity. In this work the design of a protein purification process for C-phycocyanin, a phycobiliprotein that can be used in the food and medical industries, was established. The study evaluated the use of ammonium sulfate precipitation, ion exchange chromatography and gel filtration to purify C-phycocyanin in a variety of sequences. The final design included the C-phycocyanin extraction step, precipitation with ammonium sulfate and ion exchange chromatography. When the elution step was studied, the kind of elution and pH were considered in order to obtain a product with a final purity of 4.0 with a purification factor of 6.35, so that, at the end of the strategy, C-phycocyanin of analytical grade would be obtained.     

Purification of single-strand DNA binding protein from an Escherichia coli lysate using counter-current chromatography,partition and precipitation

Single-strand DNA binding protein (SSB) from Escherichia coli lysate was purified by counter-current chromatography (CCC) using the ammonium sulfate precipitation method in a coiled column. About 5 ml of E. coli lysate was separated by CCC using a polymer phase system composed of 16% (w/w) polyethylene glycol (PEG) 1000 and 17% (w/w) ammonium sulfate aqueous polymer two-phase solvent system. The precipitation of proteins in the lysate took place in the CCC column, and the SSB protein was eluted in the fraction 51-56. Many other impurities were either eluted immediately after the solvent front or precipitated in the column. The identities of the proteins in the fractions and in the precipitate were confirmed by SDS-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.     

Phosphoprotein Phosphatase of Soybean Hypocotyls: PURIFICATION, PROPERTIES, AND SUBSTRATE SPECIFICITIES

A soybean histone-type protein kinase was used to prepare ³²P-labeled histone H1 as substrate for purification and characterization of a phosphoprotein phosphatase (EC 3.1.3.16) from soybean hypocotyls. The phosphatase has been purified 169-fold by ammonium sulfate fractionation, ethanol precipitation, and chromatography on Sephadex G-150, DEAE-Sephadex A-25 and Sephadex G-100. The activity of the phosphoprotein phosphatase is distinct from that of acid and alkaline phosphatases (EC 3.1.3.1) as well as from that of nucleotidases. The final enzyme preparation does not contain histone protease activity, although it can be detected during the early stages of purification. The protease(s) apparently can attack phosphorylated histone H1, indicating that phosphorylation does not protect the protein against proteolytic degradation.     

Determination of meso-alanopine and D-strombine by high pressure liquid chromatography in extracts from marine invertebrates

meso-Alanopine and D-strombine are separated by high pressure liquid chromatography using a cation exchange resin and 2.5 X 10(-5) M sulfuric acid as eluant, at a flow rate of 1.0 ml/min, 20 degrees C column temperature and a pressure of 4 500 kPa. Both opines were detected by conductivity. Separation and quantitation was possible in the range of 0.05 to 25 nmol of meso-alanopine and D-strombine. Chemically or enzymatically synthesized opines were quantitated using alanopine/strombine dehydrogenase from Crassostrea angulata. The enzyme was purified by ammonium sulfate precipitation, Sephadex G-100 filtration and fast-protein-liquid chromatography. Specific activity of the final preparation was 500 U/mg protein with glycine as substrate. The formation of meso-alanopine and D-strombine was demonstrated in neutralized perchloric acid extracts from muscle tissue of Arenicola marina L. following enhanced muscular activity and in Mytilus edulis L., Nucula nitida and Crassostrea angulata after 24 h of anoxia.     

Nonidentity of Some Simian Virus 40-induced Enzymes with Tumor Antigen

The complement-fixing tumor (T) antigen induced by simian virus 40 (SV40) has been prepared from SV40-infected cell cultures, from infected cell cultures treated at the time of infection with 1-beta-d-arabinofuranosylcytosine (ara-C), and from SV40-transformed cells. Upon partial purification, the T antigen exhibited the following properties: it was tightly adsorbed by calcium phosphate gel, it was precipitated by acetic acid at pH 5 or by ammonium sulfate at about 20 to 32% saturation, and it had a molecular weight greater than 250,000, as estimated by Sephadex G-200 gel chromatography. In contrast, deoxycytidylate (dCMP) deaminase, thymidylate (dTMP) kinase, and thymidine (dT) kinase were less strongly bound to calcium phosphate and were not precipitated at pH 5; these enzymes also had much lower molecular weights than the T antigen, as did dihydrofolic (FH(2)) reductase. Furthermore, higher ammonium sulfate concentrations were required to precipitate dCMP deaminase, dTMP kinase, and FH(2) reductase activities than to precipitate the T antigen. Another difference was that the T antigen was not stabilized, but dCMP deaminase, dTMP kinase, and dT kinase, were stabilized, respectively, by dCTP, dTMP, and dT or dTTP. Deoxyribonucleic acid (DNA) polymerase activity resembled the T antigen in adsorption to calcium phosphate, in precipitation by ammonium sulfate or at pH 5, and in the rate of inactivation when incubated at 38 C. However, the polymerase activity could be partly separated from the T antigen by Sephadex G-200 gel chromatography. The cell fraction containing partially purified T antigen also contained a soluble complement-fixing antigen (presumably a subunit of the viral capsid) which reacted with hyperimmune monkey sera. The latter antigen was present in very low titers or absent from cell extracts prepared from SV40-infected monkey kidney cell cultures which had been treated with ara-C at the time of infection, or from SV40-transformed mouse kidney (mKS) or hamster tumor (H-50) cells. The T antigen, however, was present in usual amounts in SV40-transformed cells or ara-C treated, infected cells.     

Lead removal through biological sulfate reduction process

The feasibility of lead removal through biological sulfate reduction process with ethanol as electron donor was investigated. Sulfide-rich effluent from biological process was used to remove lead as lead sulfide precipitate. The experiments were divided into two stages; Stage I startup and operation of sulfidogenic process in a UASB reactor and Stage II lead sulfide precipitation. In Stage I, the COD:S ratio was gradually reduced from 15:1 to 2:1. At the COD:S ratio of 2:1, sulfidogenic condition was achieved as identified by 80-85% of electron flow by sulfate reducing bacteria (SRB). COD and sulfate removal efficiency were approximately 78% and 50%, respectively. In Stage II, the effluent from UASB reactor containing sulfide in the range of 30-50 mg/L and lead-containing solution of 45-50 mg/L were fed continuously into the precipitation chamber in which the optimum pH for lead sulfide precipitation of 7.5-8.5 was maintained. It was found that lead removal of 85-95% was attained.     

Precipitation and recovery of metal sulfides from metal containing acidic wastewater in a sulfidogenic down-flow fluidized bed reactor

This study reports the feasibility of recovering metal precipitates from a synthetic acidic wastewater containing ethanol, Fe, Zn, and Cd at an organic loading rate of 2.5 g COD/L-day and a COD to sulfate ratio of 0.8 in a sulfate reducing down-flow fluidized bed reactor. The metals were added at increasing loading rates: Fe from 104 to 320 mg/L-day, Zn from 20 to 220 mg/L-day, and Cd from 5 to 20 mg/L-day. The maximum COD and sulfate removals attained were 54% and 41%, respectively. The biofilm reactor was operated at pH as low as 5.0 with stable performance, and no adverse effect over COD consumption or sulfide production was observed. The metals precipitation efficiencies obtained for Fe, Zn, and Cd exceeded 99.7%, 99.3%, and 99.4%, respectively. The total recovered precipitate was estimated to be 90% of the theoretical mass expected as metal sulfides. The precipitate was mainly recovered from the bottom of the reactor and the equalizer. The analysis of the precipitates showed the presence of pyrite (FeS2), sphalerite (ZnS) and greenockite (CdS); no metal hydroxides or carbonates in crystalline phases were identified. This study is the first in reporting the feasibility to recover metal sulfides separated from the biomass in a sulfate reducing process in one stage.     

Polyacrylic acid based plasma fractionation for the production of albumin and IgG: Compatibility with existing commercial downstream processes

Commercial fractionation of human plasma into immunoglobulin- and albumin-rich fractions is often initiated with sequential cold ethanol-based precipitation methods, which have changed little over the past 70 years. The required low temperature (−4 to −8°C) and high concentrations of ethanol 8–40%) necessitate large-scale fixed processing lines, and major capital investment and operating costs. The resulting fractions are then further purified by ethanol based precipitation or chromatographic procedures to obtain the purified final product. Aqueous polyacrylic acid (PAA) based precipitation, which readily interfaces with existing downstream processing, could offer advantages with respect to cost, safety, environmental impact, and flexibility. Sequential precipitation with 7%, 12%, and 20% (w/v) solutions of PAA 8000 in the presence of a kosmotropic salt (sodium citrate) gave fibrinogen-, immunoglobulin-, and albumin-rich fractions with 80–90% yield and 64%, 55%, and 82% purity, respectively. Further purification of the IgG-rich precipitate by caprylic acid precipitation and anion exchange chromatography, achieved a target purity of >99%. This was also achieved for the downstream processing of the albumin-rich precipitate using a two-step ion exchange chromatographic procedure. This work shows that PAA precipitation can be used in place of cold ethanol precipitation to generate crude IgG and albumin fractions which can be purified to final products of acceptable purity.     

Purification and characterization of protein phosphatase 2A from petals of the tulip Tulipa gesnerina

The holoenzyme of protein phosphatase (PP) from tulip petals was purified by using hydrophobic interaction, anion exchange and microcystin affinity chromatography to analyze activity towards p-nitrophenyl phosphate (p-NPP). The catalytic subunit of PP was released from its endogenous regulatory subunits by ethanol precipitation and further purified. Both preparations were characterized by immunological and biochemical approaches to be PP2A. On SDS-PAGE, the final purified holoenzyme preparation showed three protein bands estimated at 38, 65, and 75 kDa while the free catalytic subunit preparation showed only the 38 kDa protein. In both preparations, the 38 kDa protein was identified immunologically as the catalytic subunit of PP2A by using a monoclonal antibody against the PP2A catalytic subunit. The final 623- and 748- fold purified holoenzyme and the free catalytic preparations, respectively, exhibited high sensitivity to inhibition by 1 nM okadaic acid when activity was measured with p-NPP. The holoenzyme displayed higher stimulation in the presence of ammonium sulfate than the free catalytic subunit did by protamine, thereby suggesting different enzymatic behaviors.     

Precipitation of dilute chromatographic samples (ng/ml) containing interfering substances for SDS-PAGE

SDS-PAGE of chromatographic fractions requires prior removal of salts, detergents, denaturants, or organic solvents which may perturb the electrophoretic separation. Likewise, to successfully visualize minute amounts of protein present in chromatographic fractions, they must often be concentrated before analysis by SDS-PAGE. In this study, we used a dye precipitation procedure for simultaneous removal of interfering substances and concentration of dilute samples (ng/ml) before analysis by SDS-PAGE. Nanogram amounts of protein (143 ng) were effectively precipitated with a pyrogallol red-molybdate reagent from commonly used chromatographic buffers containing various interfering solutes or solvents. Proteins were successfully precipitated from solution in the presence of organic solvents (acetonitrile, methanol, 2-propanol), chaotropic agents (6 M urea, 6 M guanidine-HCl), a protein stabilizer (40% sucrose), metal chelators (30 mM EDTA and 30 mM EGTA), or high salt (1.0 M NaCl). Detergents, at concentrations up to twice their critical micelle concentrations, from the nonionic class (Triton X-100, Tween 20) or from the zwitterionic class (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) did not inhibit protein precipitation. Some interference was observed when proteins were precipitated in the presence of ammonium sulfate (0. 5-2.0 M). Proteins did not precipitate in the presence of ionic detergents (SDS and cetyltrimethylammonium bromide). The sensitivity of the combined pyrogallol red-molybdate precipitation/SDS-PAGE procedure is approximately 7 ng. Two other methods of precipitating proteins (trichloroacetic acid and phenol-ether) both exhibited varying degrees of effectiveness, ranging from 714 to 7 ng/ml, in the precipitation of individual proteins. In summary, the pyrogallol red-molybdate protein precipitation procedure facilitates the SDS-PAGE analysis of dilute protein samples (ng/ml) from chromatographic fractions of various compositions. The method is useful for rapid pilot-scale protein fractionation and facilitates the ongoing propensity of researchers to work with minuscule amounts of protein.     

Cholecalciferol sulfate identification in human milk by HPLC

Synthetic vitamin D3 sulfate was prepared by reacting cholecalciferol with sulfamic acid in pyridine. Vitamin D3 sulfate ammonium salt was purified by crystallisation and transformed in sulfate sodium salt. Homogeneity was controlled by reverse phase high pressure liquid chromatography (HPLC). Purified synthetic vitamin D3 sulfate sodium salt was used as a reference. Milk whey was obtained after protein precipitation by adding ethanol. Vitamin D3 sulfoconjugate was identified in supernatant (lyophylized) after purification by Sephadex LH 20 and HPLC. Milk whey purified fraction obtained exhibited the same ultra-violet absorption (UV) as synthetic vitamin D3 sulfate; after solvolysis, cholecalciferol was liberated from natural and synthetic sulfoconjugate. The results confirmed that vitamin D3 sulfate was present in human milk.     

Solubilization and partial purification of the adenosine triphosphatase from a corn root plasma membrane fraction

The K(+)-stimulated ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 x Mo 17) by solubilization with 30 millimolar octyl-beta-d-glucopyranoside followed by precipitation with dilute ammonium sulfate. The specific activity of the enzyme was increased about five times by this procedure. The molecular weight of the detergent-extracted ATPase complex was estimated to be at least 500,000 daltons by chromatography on a Bio-Gel A-5m column. Negative staining electron microscopy indicated that the detergent-extracted material consisted of amorphous particles, while the ammonium sulfate precipitate was composed of uniform vesicles with an average diameter of 100 nanometers. The protein composition of the ammonium sulfate precipitate was significantly different from that of the plasma membrane fraction when compared by sodium dodecyl sulfate gel electrophoresis. The characteristics of the partially purified ATPase resembled those of the plasma membrane associated enzyme. The ATPase required Mg(2+), was further stimulated by K(+), was almost completely inhibited by 0.1 millimolar diethylstilbestrol, and was not affected by 5.0 micrograms per milliliter oligomycin. Although the detergents sodium cholate, deoxycholate, Triton X-100 and Lubrol WX also solubilized some membrane protein, none solubilized the K(+)-stimulated ATPase activity. Low concentrations of each detergent, including octyl-beta-d-glucopyranoside, activated the ATPase and higher concentrations inactivated the enzyme. These results suggest that the plasma membrane ATPase is a large, integral membrane protein or protein complex that requires lipids to maintain its activity.