Search

[Display omitted] • PBDEs were measured in various materials from seating furniture wastes. • A single MAE-GC/MS process was adapted for both plastics, foams and textiles. • From 100 samples the sum of 8 PBDEs did not exceed the tolerated threshold. • BDE-209 was identified at higher contents mainly in hard plastics. • No direct correlation could be found between Br levels measured by XRF and PBDEs. Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants that are used in polymeric materials. Due to their adverse health effects, the use of recycled wastes has been forbidden if the total PBDE content exceeds 0.1% (w/w). The objective was to estimate the proportion of PBDEs in professional seating furnishing wastes to identify the materials in which the content of PBDEs (and particularly BDE-209) could exceed the limit to eliminate them from recycling. An analytical process (microwave extraction followed by purification and chromatographic analysis) was adapted to assess with a unique methodology the amounts of eight PBDEs in materials that result from various seating wastes, such as hard plastics, foams and accompanying textiles. X-ray fluorescence (XRF) was used to rapidly predict critical PBDE concentrations via Br. From 100 samples, the total PBDE content did not exceed the current tolerated threshold. The examined materials contained only trace levels of former PBDE formulations, and BDE-209 was identified at higher amounts, mainly in hard plastics, but these amounts were less than 312 mg kg−1. Since XRF was not reliable for quantitative measurements and was not specific, no direct correlation could be identified between Br and PBDE levels. Br was strongly associated with As in all the materials, but the presence of PBDEs was not clearly associated with the presence of other metals that are used in flame retardants. [ABSTRACT FROM AUTHOR]

Highlights • First report on the main expanded-bed chromatography parameters to purify peroxidases. • Cationic adsorbent was used to purify rice bran peroxidase. • Purification efficiency was increased by adding CaCl 2 in buffer during elution step. • Rice bran peroxidase was purified in 14.1-fold using a single technique. Abstract Peroxidases catalyze the oxidation-reduction reactions of organic substrates and present several applications in the biotechnological, clinical, environmental, and industrial areas. Among the chromatographic strategies applied to peroxidase purification to allow future uses, expanded-bed adsorption (EBA) stands out due to the large volumes of purified enzyme and potential for scale-up. In this paper, the main EBA parameters were evaluated in the purification of rice bran peroxidases. Sodium acetate buffer (pH 4.5) and expansion degree of 2.5 were the most favorable conditions to the enzyme adsorption onto Streamline® SP resin, showing an enzymatic activity at equilibrium on the solid phase (q*) of 2.68 U/mL resin, a partition coefficient (f) of 38.35 and a dynamic binding capacity (Q 10%) of 0.19 U/mL resin. Rice bran peroxidase was purified in 14.1-fold (± 1.3) with 49.8% (± 2.5) of recovery by using 25 mmol/L acetate buffer pH 5.5 in the washing step, and elution combining stepwise (0.15 mol/L NaCl) and linear gradient (0.15–1 mol/L NaCl), using acetate buffer pH 5.5 with 1 mmol/L of CaCl 2. Based on the results obtained, EBA showed to be a viable single-step strategy for peroxidase purification. [ABSTRACT FROM AUTHOR]

Various high-throughput systems and strategies are employed by the biopharmaceutical industry for early to late-stage process development for biologics manufacturing. The associated increases to experiment productivity and reduction in material consumption makes high throughput tools integral for bioprocess development. While these high-throughput systems have been successfully leveraged to generate high quality data representative of manufacturing scale processes, their data interpretation often requires complex data transformation and time-intensive system characterization. With respect to high throughput purification development, RoboColumns by Repligen operated on Tecan automated liquid handling systems offer superior performance scalability, but lack an optimized liquid delivery system that is representative of preparative chromatography. Particularly, stock Tecan liquid handling systems lack the capability to provide high-capacity continuous liquid flow and ideal linear gradient chromatography conditions. These limitations impact protein chromatography performance and hinder the application of high-throughput gradient elution experiments. In this work, we describe a Tecan Freedom EVO high-throughput purification tool that provides more continuous liquid delivery enabling continuous gradient elution capability for RoboColumn experiments as demonstrated by generation of highly linear conductivity gradients. Results demonstrate that the tool can provide RoboColumn performance and product quality data that is in agreement with larger, bench scale chromatography formats for two model purification methods. The described gradient purification method also provides more consistent performance between RoboColumns and larger column formats compared to step elution methods using the same optimized Tecan system. Lastly, new insights into the impact of discontinuous flow on RoboColumn elution performance are introduced, which may help further improve application of these data towards bioprocess development. [ABSTRACT FROM AUTHOR]

In the present study, the feasibility of immunoaffinity chromatography (IAC) as a purification technology for the analysis of bioactive components in Traditional Chinese Medicine (TCM) was evaluated. IAC was used to analyze hesperidin (HP) and narirutin (NR) in TCM preparations containing Citri reticulatae Pericarpium (CRP, Chenpi in Chinese). An IAC column for the specific extraction and enrichment of HP and NR from TCM preparations containing CRP was developed and characterized. After HP reacted with carbonyl diimidazole and coupled to protein, it was used to immune mice for the generation of antibody. Through cell fusion, cloning and screening, monoclonal antibody was obtained. The IAC column was constructed by covalently coupling specific monoclonal antibody against HP and NR to CNBr-activated Sepharose 4B and packed into a common solid phase extraction cartridge. The extraction conditions including loading, washing and eluting, as well as flow rate for the extraction of HP and NR were optimized. Under optimal conditions, the maximum capacity, extraction recovery rate and stability of IAC column was also characterized. Results revealed that the maximum capacity of IAC column for HP and NR was approximately 16 μg and the relative binding capacity per 1 mL of the column volume was 27 μg. The extraction recovery rate of IAC column for HP and NR at three spiked levels was in the range of 94.05–109.15%. After the repeated application for 5 times, no significant loss of specific recognition was observed. Using high performance liquid chromatography (HPLC) as an effective analytic tool, HP and NR could be successfully separated via IAC column without the inference from impurities, suggesting that the extraction of HP and NR using the prepared IAC column is feasible. The application of IAC can solve the problem of quantitative analysis due to severe interference or low content. Furthermore, pretreatment methods in different matrixes can be unified. The IAC purification procedure can be used as an alternative effective analytical method for the pretreatment of bioactive components in TCM. [ABSTRACT FROM AUTHOR]

Chromatographic purification of chromium (Cr), which is required for high-precision isotope analysis, is complicated by the presence of multiple Cr-species with different effective charges in the acid digested sample aliquots. The differing ion exchange selectivity and sluggish reaction rates of these species can result in incomplete Cr recovery during chromatographic purification. Because of large mass-dependent inter-species isotope fractionation, incomplete recovery can affect the accuracy of high-precision Cr isotope analysis. Here, we demonstrate widely differing cation distribution coefficients of Cr(III)-species (Cr 3+ , CrCl 2+ and CrCl 2 + ) with equilibrium mass-dependent isotope fractionation spanning a range of ∼1‰/amu and consistent with theory. The heaviest isotopes partition into Cr 3+ , intermediates in CrCl 2+ and the lightest in CrCl 2 + /CrCl 3 °. Thus, for a typical reported loss of ∼25% Cr (in the form of Cr 3+ ) through chromatographic purification, this translates into 185 ppm/amu offset in the stable Cr isotope ratio of the residual sample. Depending on the validity of the mass-bias correction during isotope analysis, this further results in artificial mass-independent effects in the mass-bias corrected 53 Cr/ 52 Cr (μ 53 Cr* of 5.2 ppm) and 54 Cr/ 52 Cr (μ 54 Cr* of 13.5 ppm) components used to infer chronometric and nucleosynthetic information in meteorites. To mitigate these fractionation effects, we developed strategic chemical sample pre-treatment procedures that ensure high and reproducible Cr recovery. This is achieved either through 1) effective promotion of Cr 3+ by >5 days exposure to HNO 3 H 2 O 2 solutions at room temperature, resulting in >∼98% Cr recovery for most types of sample matrices tested using a cationic chromatographic retention strategy, or 2) formation of Cr(III)-Cl complexes through exposure to concentrated HCl at high temperature (>120 °C) for several hours, resulting in >97.5% Cr recovery using a chromatographic elution strategy that takes advantage of the slow reaction kinetics of de-chlorination of Cr in dilute HCl at room temperature. These procedures significantly improve cation chromatographic purification of Cr over previous methods and allow for high-purity Cr isotope analysis with a total recovery of >95%. [ABSTRACT FROM AUTHOR]

The total alkaloids from Nitraria sibirica leaves have been confirmed to exhibit significant protective effects against inflammatory renal injury, hypertension and albuminuria in angiotensin II-salt hypertension. In the present study, a separation method of pH-zone-refining counter-current chromatography was established for separation of the alkaloids from N. sibirica . The separation was performed with a solvent system of MtBE- n -BuOH-H 2 O (2:2:5, v / v ) at a flow rate of 2.0 mL/min. And 15 mM triethylamine (TEA) was added to the upper organic phase, while 10 mM hydrochloric acid was added to the lower aqueous phase. As a result, a new alkaloid, schobemine (5.6 mg), and a known alkaloid, nitraramine (5.0 mg), together with fractions A and B were obtained from the total alkaloids of N. sibirica . The fractions A and B were further purified by means of pH-zone-refining counter-current chromatography with solvent systems of n -hexane- n -BuOH-H 2 O (1.5:3.5:5, v / v ) and (2:3:5, v / v ), respectively. TEA (10 mM) was added to the upper phase, and 10 mM of HCl was added to the lower phase in above two solvent systems, respectively. As a result, a known alkaloid, schoberidine (5.0 mg), and a new alkaloid, schoberimine (3.0 mg) were obtained from fractions A and B, respectively. The purities of the compounds were measured by HPLC–ELSD, and their structures were identified by ESI–MS, 1D and 2D NMR. [ABSTRACT FROM AUTHOR]

This study was carried out to investigate the purification and biochemical characterization of a new extracellular alkaliphilic and thermostable pectate lyase (Pel-20) isolated from Actinomadura keratinilytica strain Cpt20. Pure protein was obtained after sequential chromatographies on a fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC) columns. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 34125.11-Da. The enzyme had an NH 2 -terminal sequence of GFATNQGGTTGGAGGTLS, thus, sharing high homology with actinomycetes pectate lyase family. The results showed that this enzyme was completely inhibited by EDTA, which supports its belonging to the pectate lyase superfamily. It showed optimum activity at pH 10.5 and 70 °C. The thermoactivity and thermostability of Pel-20 were enhanced in the presence of 1 mM Ca 2+ . Its half-life times at 70, 80, 90, and 100 °C were 18, 12, 7, and 2 h, respectively. Its kinetic parameters, K m and V max values were 0.45 mM and 21,700 U/mg, respectively. Low-esterified pectin was the optimum substrate for the Pel-20. However, higher-esterified pectin was also weakly cleaved. Overall, the alkaliphilicity and thermostability properties of Pel-20 make it a potential candidate for future application in industrial bioprocesses. [ABSTRACT FROM AUTHOR]

Immunoglobulins G (IgG) against hepatitis B core antigen (HBcAg) was successfully purified using a purification scheme comprising ammonium sulphate precipitation and SepFast™ MM AH-1 column chromatography. Ammonium sulphate precipitation performed at 40% saturation was optimum in terms of the recovered polyclonal IgG concentration (7.8 mg/ml) and the removal of albumin (72%). The yield, purity and purification factor achieved from this simple purification method were 99%, 94% and 7.8, respectively. The IgG recovered from ammonium sulphate precipitation was subjected to SepFast™ MM AH-1 column chromatography and the purity of IgG was further increased to 98%, corresponding to a purification factor of 8.1. Protein aggregation was also reduced significantly in the purified IgG sample. Furthermore, the salt content in the purified sample was reduced by 75% and therefore the need of desalting final product was eliminated. Enzyme-linked immunosorbent assay (ELISA) showed that the antigenicity of anti-HBcAg IgG obtained after these purification processes was maintained. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) are one of the fastest growing drug molecules targeting the treatment of diseases ranging from arthritis, immune disorders, and infectious diseases to cancer. Due to its unique application principle, antibodies are commonly produced in large quantities. Plants, such as Nicotiana benthamiana , offer a unique production platform for bio-therapeutics due to their ability to produce large amounts of biomolecules in a relatively quick manner. However, purification of a target protein from plant is an arduous task due to the presence of toxic compounds in ground plant tissue and the large quantities of plant tissues to be processed. Here, a process was developed prior to the chromatographic purification of a mAb against Ebola GP1 protein expressed in N. benthamiana. The process includes a diafiltration step and a charged polyelectrolyte precipitation. The diafiltration step significantly improved the precipitation efficiency, reducing the usage of polyelectrolyte by more than 2000 fold while improving the native plant protein removed from 60% to 80%. The mAb can then be purified to near homogeneity judging from SDS-PAGE by either Protein A affinity chromatography or a tandem of hydrophobic interaction chromatography and a hydrophobic charge induction chromatography. The purified mAbs were shown to retain their binding specificity to irradiated Ebola virus. [ABSTRACT FROM AUTHOR]

Highlights: [•] We develop a microfluidic chip to purify and enrich virus by hydroxyapatite chromatography. [•] We examine purification and enrichment of the virus. [•] The viruses were isolated from the mixture of virus and protein. [•] The virus concentration was increased by hydroxyapatite chromatography. [Copyright &y& Elsevier]

• Recombinant adeno-associated virus (rAAV)-mediated gene therapy is a fast-evolving. • High throughput (HTP) chromatography significantly speed up AAV process development. • HTP chromatography using resin slurry plate and mini-column for AAV purification. • Demonstrated performance comparability between HTP and bench-scale chromatography. Recombinant adeno-associated virus (AAV) has been broadly used as a delivery tool for gene therapy applications. The development of a robust purification process is essential for delivering high purity and quality AAV products to clinic. The short clinical timelines and material limitations of early-stage development pose unique challenges to developing robust and scalable downstream purification processes. One approach to overcome these limitations is to leverage high throughput (HTP) strategies and automation technologies for purification process development, an approach that is well established in protein biologics and other areas. However, due to the unique challenges related to viral vector purification, implementing HTP approaches for gene therapy process development has not been explored extensively. In this paper, we established a HTP chromatography platform and demonstrated its capability to facilitate gene therapy purification process development using both mini-columns and self-packed resin plates. The end-to-end development workflow for AAV HTP purification is detailed in this work with the expectation of serving as an introductory for the AAV purification development field. Comparable process performance was confirmed between a bench-scale chromatography process and an HTP chromatography format. Slightly lower recovery was observed using the HTP format (62% vs 75%), as well as %full capsid enrichment (71% vs. 82%). Comparable impurity clearance capability was demonstrated between the two different systems as well. It was concluded that the established HTP chromatography formats can serve as a surrogate to bench-scale chromatography development to reduce material needs and development timelines for AAV purification development. [ABSTRACT FROM AUTHOR]

Abstract: Recently, nanofiltration techniques have been introduced by pharmaceutical industries in the downstream processes for peptides, to perform concentration, purification and salt/solvent exchange, and have been demonstrated as being suitable for integration with conventional chromatographic purification techniques, providing savings in terms of time and costs. Design of Experiments (DoE) methods have been extensively applied in the process design of the conventional techniques, under the so-called Quality by Design (QbD) concept, but applications of DoE methods to nanofiltration are still few. In this study, the nanofiltration of a model peptide, conventionally named PEP1, in trifluoroacetic acid/acetonitrile/water mixtures is studied by DoE. Statistical models for solvent flux, peptide and trifluoroacetic acid rejections are obtained by statistical Analysis of Variance and the best operating conditions for concentration are found by numerical optimization of the statistical models. The statistical models from DoE are included in the mathematical framework of the diafiltration process, to calculate the evolution of peptide, counter-ion and solvent concentrations over time, compare constant volume vs. variable volume diafiltration modes, and select the best process in terms of operating time and solvent consumption. This work demonstrates that empirical DoE models can provide phenomenological understanding of the transport mechanism through nanofiltration membranes for a specific solute of interest and successfully support the process modelling for concentration and diafiltration, providing a methodology to select the most appropriate filtration technique for a given separation problem. [Copyright &y& Elsevier]

Highlights: [•] Grapefruit coumarins were fractionated by solvent partitioning. [•] C-18 column facilitated purification of minor coumarins than Silica. [•] Nine compounds were purified using rapid hyphenated chromatography. [•] Structure elucidation of coumarins was confirmed by spectral analysis. [Copyright &y& Elsevier]

Highlights: [•] Retention behavior of monoclonal IgG1 was investigated on IEC and HIC media. [•] Dynamics of both processes was dominated by kinetic effects. [•] A short-cut method was proposed for mathematical modeling. [•] A dynamic model was used for optimization of the integrated process IEC/HIC. [ABSTRACT FROM AUTHOR]

Abstract: One of the caveats of the Mitsunobu reaction is the often difficult and laborious purification of the reaction mixture. Herein, we show that azodicarbonyl dimorpholide (ADDM) is an effective and versatile Mitsunobu reagent, which, along with its reduced form ADDM-H2, may be removed from the reaction mixture by a simple aqueous extraction. Furthermore, we demonstrate that the isolated ADDM-H2 may be re-oxidized with silver(I) oxide to regenerate ADDM. Finally, a chromatography-free Mitsunobu reaction is presented. [Copyright &y& Elsevier]

Abstract: Hydrophobic charge-induction chromatography (HCIC) is a novel downstream bioprocessing technology for antibody purification and it has several advantages over traditional purification processes. However, its separation selectivity still needs to be improved. In this work, sodium caprylate (NaCA) was used as the selective modifier to improve IgG purification from serum albumin containing feedstock with a typical HCIC resin, MEP HyperCel. The effects of NaCA on the adsorption equilibrium of bovine serum immunoglobulin G (IgG) and bovine serum albumin (BSA), as well as the dynamic binding and displacement behaviors were investigated. The binding and elution behaviors of these two proteins in the column were studied. It was found that adding 50–75mM NaCA in the liquid phase could effectively reduce the adsorption of BSA on the MEP resin, but the same treatment has little influence on the adsorption of IgG. Moreover, the mechanism of the competitive binding between caprylate and MEP ligands on the surface of BSA is discussed. It was found that by controlling NaCA addition in the loading or washing buffer, the process efficiency of IgG purification from BSA containing feedstock can be improved, and the purity of IgG could reach to over 98%. The results indicated that caprylate could be a promising albumin-selective modifier to improve the separation efficiency of antibodies with the HCIC process. [Copyright &y& Elsevier]

Abstract: Molecular imprinted polymer (MIP) techniques have been increasingly used in a variety of fields including chromatography, sample pretreatment, purification, sensors, drug delivery, and catalysts, etc. MIP is a specific artificial receptor that shows favored affinity to the template molecule. The cavities of the template are produced by carrying out polymerization of a reaction mixture followed by eliminating the template molecules by washing. Various forms of MIP materials have been prepared for diverse applications including irregularly ground particles, regular spherical particles, nanoparticles, monoliths in a stainless steel or capillary column, open tubular layers in capillaries, membranes, surface attached thin layers, and composites, etc. When an enantiomer is used as the template, then the resulting MIP can show capability of enantiomeric recognition between the pair of enantiomers. In this review, progresses in applications of enantio-selective recognition by MIPs will be critically reviewed for the recent period since 2007. [Copyright &y& Elsevier]

Abstract: Using four different chromatographic steps, β-galactosidase was purified from the ripe fruit of sweet cherry to apparent electrophoretic homogeneity with approximately 131-fold purification. The Prunus avium β-galactosidase showed an apparent molecular mass of about 100 kDa and consisted of four different active polypeptides with pIs of about 7.9, 7.4, 6.9 and 6.4 as estimated by native IEF and β-galactosidase-activity staining. The active polypeptides were individually excised from the gel and subjected to SDS-PAGE. Each of the four native enzymes showing β-galactosidase activity was composed of two polypeptides with an estimated mass of 54 and 33 kDa. Both of these polypeptides were subjected to N-terminal amino acid sequence analysis. The 54 kDa polypeptide of sweet cherry β-galactosidase showed a 43% identity with the 44 kDa subunit of persimmon and apple β-galactosidases and the 48 kDa subunit of carambola galactosidase I. The sweet cherry β-galactosidase exhibited a strict specificity towards p-nitrophenyl β-d-galactopyranoside, a pH optimum of 4.0 and K m and V max values of 0.42 mM and 4.12 mmol min−1 mg−1 of protein respectively with this substrate. The enzyme was also active towards complex glycans. Taken together the results of this study prompted a role for this class of enzymes on sweet cherry fruit ripening and softening. [Copyright &y& Elsevier]

Abstract: We have devised an efficient procedure for the synthesis of 5α-dihydrotestosterone (DHT) (1) starting from 3β-hydroxy-5α-androstan-17-one, providing the product in unprecedented 82% yield. A reported method of using toxic Jones reagent is replaced by milder oxidizing agent (NMO/TPAP) in the synthesis of a key intermediate 17β-[(tert-butyldimethylsilyl)oxy]-5α-androstan-3-one (18). This new procedure is simple, does not require special apparatus/precautions or chromatographic purification in most of the steps. [Copyright &y& Elsevier]

Abstract: In this study, resin adsorption as a means to separate and purify baicalin and wogonoside from extracts of Scutellaria baicalensis was investigated. Among the ten tested resins, the non-polar resin HPD-100 offered the best adsorption and desorption properties. Langmuir and Freundlich isotherms were used to describe the interactions between solutes and resin at different temperatures, and the equilibrium experimental data were well fitted to the two isotherms. Column packed with HPD-100 resin was used to perform dynamic adsorption and desorption tests to optimize the separation process. After one round treatment with HPD-100 resin, the contents of baicalin and wogonoside were 3.6-fold and 12.0-fold increased with recovery yields of 85.7% and 65.6%, respectively. In addition, a laboratory preparative-scale separation was carried out under the final conditions. The results showed that the preparative separation of baicalin and wogonoside can be easily and efficiently achieved via adsorption and desorption on HPD-100 resin. The developed method is a promising basis for large-scale preparation of baicalin and wogonoside from S. baicalensis extracts. [Copyright &y& Elsevier]

Abstract: A thermoalkaline protease with a molecular weight of 22kDa was purified from the Bacillus cereus SIU1 strain using a combination of Q-Sepharose and Sephadex G-75 chromatography. The kinetic analyses revealed the K m, V max and k cat to be 1.09mgml−1, 0.909mgml−1 min−1 and 3.11s−1, respectively, towards a casein substrate. The protease was most active and stable at pH 9.0 and between a temperature range of 45–55°C. It was fully stable at 0.0–2.0% and moderately stable at 2.5–10.0% (w/v) sodium chloride. Phenyl methyl sulfonyl fluoride, ethylene diamine tetra acetic acid and ascorbic acid were inhibitory with regard to enzyme activity, whereas cysteine, β-mercaptoethanol, calcium, magnesium, manganese and copper at concentration of 1.0mM increased enzyme activity. Sodium dodecyl sulfate, Triton X-100, Tween 80, hydrogen peroxide and sodium perborate significantly enhanced protease activity at 0.1 and 1.0% concentrations. In the presence of 0.1 and 1.0% (w/v) detergents, the protease was fairly stable and retained 50–76% activity. Therefore, it may have a possible application in laundry formulations. An initial analysis of the circular dichroism (CD) spectrum in the ultraviolet range revealed that the protease is predominantly a β-pleated structure and a detailed structural composition showed ∼50% β-sheets. The CD-based conformational evaluation of the protease after incubation with modulators, metal ions, detergents and at different pH values, revealed that the change in the β-content directly corresponded to the altered enzyme activity. The protease combined with detergent was able to destain blood stained cloth within 30min. [Copyright &y& Elsevier]

Abstract: Fusion M13 phage with disulfide constrained heptapeptide, C-WSFFSNI-C, inserted into the minor coat protein (gpIII), has been selected in the current study as ligand in direct purification of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli (E. coli) feedstock. The selected fusion phage showed strong association with the surface of the core particle. In the present study, this fusion M13 phage was immobilized onto Streamline base matrix via epoxy activation and used as adsorbent to capture HBcAg from crude E. coli homogenate. The maximum binding capacity for the adsorbent was 3.76mg/mL with equilibrium coefficient of 1.83mg/mL. Due to the slow uptake rate of HBcAg by M13 phage-immobilized adsorbents, a modified EBAC operation with recirculation of feedstock into the expanded bed has been investigated in this study. The introduction of feedstock recirculation has led to an 18% increase in yield; however, the purity of the eluted product was reduced by 15% compared with typical EBAC operation. The level of antigenicity exhibited by the core particles purified by both EBAC operations employed in the present study was comparable to that purified using sucrose ultracentrifugation. [Copyright &y& Elsevier]

Abstract: An extracellular aspartyl proteinase from Mucor mucedo DSM 809 submerged cultures was purified by a two-steps chromatographic procedure. The enzyme had a molecular weight (MW) of 32.7kDa, and an isoelectric point (pI) value of 4.29; no evidence of N-linked glycosylation was found. As judged by mass spectrometry, the primary structure of the M. mucedo enzyme presented homology with Rhizopus spp. proteinases. The secondary structure showed 4% α-helix, 48% β-sheet and 48% random coil structure in 20mM phosphate buffer (pH 5.8), as evidenced by circular dichroism spectroscopy. When acting on milk to provoke curd formation, the proteinase showed maximum potency at pH 5.0 and at 40°C. The enzyme was heat-sensitive and became completely inactivated after incubation at 55°C for 10min. These results indicate that the milk-clotting enzyme from M. mucedo can be considered as a potential substitute for bovine chymosin in cheese manufacturing. [Copyright &y& Elsevier]

Abstract: Recombinant human serum albumin (rHSA) is considered as an alternative of human serum albumin and used to treat patients with severe burn, shock or blood loss. However, separation and purification of rHSA are difficult and have become the bottle neck in industrial production. In this study, ethanol/K2HPO4 aqueous two-phase system (ATPS) and hydrophobic interaction chromatography (HIC) were integrated to provide a new approach for the extraction and purification of rHSA from high density fermentation broth. Using a 0.01–73L ATPS scale, the extraction of rHSA from the fermentation broth attained an average recovery of 100.4%. At the same time, 99.8% of cells and 87.2% of polysaccharides as well as some other protein impurities were also removed. The activity of proteinase A in the broth was also remarkably decreased. The purified rHSA appeared as a single band on reduced SDS–PAGE gel, and it had a purity of 99.1% as determined by HPLC. It was essentially identical to the plasma-derived HSA in terms of molecular weight and circular dichroism spectrum. The total recovery of rHSA was 75.2%, which was 1.1–2.0 times higher than that obtained from conventional processes. Residual polysaccharide was reduced to 1.61μg/mg rHSA and the degree of coloring was lower than that of plasma-derived HSA. The procedure employed in this study has the advantages of simple operation, shorter time, low energy consumption and high yield, and it could produce rHSA with high purity. It is therefore suitable in the production of rHSA and other biological products produced by high-density fermentation. [Copyright &y& Elsevier]

Abstract: The crude polysaccharides (GFP) were isolated from the fruiting bodies of Grifola frondosa and purified by DEAE cellulose-52 chromatography and Sephadex G-100 size-exclusion chromatography in that order. Three main fractions, GFP-1, GFP-2 and GFP-3, were obtained through the isolation and purification steps. Then the antioxidant activities of these three fractions were investigated in vitro. The results showed that GFP-1, GFP-2 and GFP-3 possessed significant inhibitory effects on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical and superoxide radical; their reducing power, ferrous ions chelating effect and the inhibition ability of the rat liver lipid oxidation where also strong. These results suggest that G. frondosa polysaccharides could be a suitable natural antioxidant and may be the functional foods for humans. [Copyright &y& Elsevier]

Abstract: A novel neutral heteropolysaccharide (DDP-1-D) was purified from hot water extracts of dried stem of Dendrobium densiflorum by DEAE-52 and Sephacryl S-200 High-Resolution Chromatography. The heteropolysaccharide had an average molecular weight about 9440Da. It was composed mainly of glucose and mannose in the ratio of 3.01:1. Structural features of DDP-1-D were elucidated by a combination of chemical and instrumental techniques, including FT-IR, GC–MS, periodate oxidation–Smith degradation, 1H and 13C NMR spectroscopies (including COSY, TOCSY, HSQC, and HMBC spectra). The results indicated that DDP-1-D is a mannoglucan and has a backbone consisting of (1→4)-linked α-d-Glcp, (1→6)-linked α-d-Glcp, (1→2)-linked α-d-Manp and (1→4)-linked β-Manp. This is the first study to provide clear evidence for the structure of the polysaccharide in D. densiflorum. [Copyright &y& Elsevier]

Abstract: Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be performed in ion-exchange, affinity and hydrophobic-interaction mode. It has also been shown that SDC can be performed on monoliths and membrane-based supports in both analytical and preparative scale. Recently, SDC in ion-exchange and hydrophobic interaction mode was also employed successfully for the removal of trace proteins from monoclonal antibody preparations and for the enrichment of low abundance proteins from human plasma. In this review, the principals of SDC are introduced, and the potential for separation of proteins and peptides in micro-analytical, analytical and preparative scale is discussed. [Copyright &y& Elsevier]

Abstract: Functionalized magnetic particles are emerging as a reliable and convenient technique in the purification of biomacromolecules (proteins and nucleic acids) and cell separation. In this study, we used novel solid nickel ferromagnetic particles coated with Protein A for the affinity purification of antibody. The study demonstrated that IgG can be purified from undiluted mouse serum in as few as 5min using Protein A-coated nickel particles. Further, protein crosslinking was shown to stabilize the Protein A on the nickel particle surfaces to minimize Protein A leaching during the affinity purification and elution of IgG. The separation procedure is gentle, scalable, automatable, efficient and economical. By modifying the functional groups of amino acids in the protein coating, crosslinked nickel particles can be used not only for protein affinity purification but for other biological sample preparation and chromatographic applications as well. Methods proposed and tested in this study can be easily modified for small and medium scale antibody purification in lab and pre-clinical research. [Copyright &y& Elsevier]

Abstract: A novel heme peroxidase from Jatropha curcas, an important source of bio-diesel, was purified to homogeneity using ammonium sulfate fractionation, desalting chromatography and ion exchange chromatography. Molecular mass of this purified enzyme was around 48kDa as detected by SDS-PAGE. Gel filtration analysis revealed that the enzyme was a monomer under native conditions. The purified enzyme had broad substrate specificity with the ideal substrates of guaiacol and o-phenylenediamine. The optimum temperature, pH and K m value of this peroxidase for guaiacol was 60°C, 5.0 and 0.17mM, respectively. In addition, NaCl (2.5M) significantly enhanced the activity of this peroxidase. The purified enzyme was stable under high temperature (70% activity retained after 1h incubation at 70°C), extreme pH environment (93% or more activity retained after 2h incubation under pH 3–12), high NaCl concentration (88% or more activity retained after 2h incubation with 1–4M NaCl) and organic solvents (95% or more activity retained after 54h incubation with various organic solvents). Moreover, this peroxidase was resistant against 20mM hydrogen peroxide, 8M urea, 3M guanidine hydrochloride and 20mM EDTA. However, the peroxidase activity was significantly inhibited by sodium azide, dithiothreitol, CTAB, β-mercaptoethanol, DMSO, toluene and ferrous ion. The enzyme had long shelf life with 180 days at 4°C and 14 days at room temperature. This new robust peroxidase may bring a better understanding for the high anti-adversity property of J. curcas. Meanwhile, the broad substrate specificity, wide stability against high temperature, extreme pH, organic solvent and hydrogen peroxide suggested that the enzyme could be a potential candidate peroxidase source for industrial and biomedical applications. [Copyright &y& Elsevier]

Abstract: Integration of supercritical fluid/mass spectrometry (SFC/MS) and reversed phase liquid (HPLC/MS) chromatographic screening techniques into a single chromatographic system and utilized in “walk up” mode, enabled us to produce an orthogonal data set for selecting purification conditions for medicinal chemistry compounds. To streamline the overall workflow, we also demonstrate the use of automated batch data processing of individual data files to identify suitable separation conditions without user intervention. We have addressed the chromatographic challenges that hinder the identification of the intended target and thus the selection of ideal purification conditions. For instance, multiple component-of-interest (COI) peaks, co-elution of impurities with the COI, and chromatographic suitability factors such as retention times and peak shapes are all important considerations when selecting appropriate methods for purification and, therefore, are bottlenecks to an automated approach. Since SFC and HPLC data were collected in parallel from separate instruments in our workflow, the time required for the separation scientist to analyze acquired data from both systems was a time-limiting factor. To reduce data processing time and accelerate or “FastTrack” samples to purification, two unique and automated solutions were introduced. We describe the implementation of an integrated, multi-column, walk-up HPLC/SFC/MS system, and the implementation of an intelligent, automated method selection application which uses advanced data evaluation criteria to selectively score and identify the most practical separation conditions for SFC/MS and HPLC/MS methodologies. [Copyright &y& Elsevier]

Abstract: This paper demonstrates a combined approach for separating an active pharmaceutical ingredient (API) from a heavily contaminated waste stream. The approach uses organic solvent nanofiltration (OSN) to improve the application of counter-current chromatography (CCC) in an industrial process. OSN provides an efficient route for exchanging solutes from the process solvent into the desired mobile phase for CCC, generating a CCC feed containing less than 0.01% (area % by GC) of the original process solvents. The high solvent burden of CCC was additionally reduced through recovery of mobile phase using OSN, with the recovered solvent containing less than 1% (area % by HPLC) impurities. The recovered solvent was then successfully recycled into a subsequent CCC run with no indication of impurity build-up. Coupling OSN with CCC improved the mass-intensity of the CCC process, reducing the solvent use by 56%. OSN can be a useful tool in facilitating the application of CCC to pharmaceutical process streams. [Copyright &y& Elsevier]

Abstract: A PID controller able to support the operator in the operation of the Multi-column Countercurrent Solvent Gradient Purification (MCSGP) process which is a continuous, countercurrent chromatographic process has been developed. As measurement, only the online UV signals at each column outlet are used. This guarantees a simple and cheap control implementation and a fast control action. Accordingly, the controller does not guarantee any purity or yield value, but simply that the withdrawn window of the product is centered in a specific region of the UV chromatogram where the purity specifications are expected to be satisfied. This can be determined by the operator based on the batch chromatogram selected for designing the MCSGP operating conditions. Thus the controller provides a reliable and efficient tool for the operator to run properly a MCSGP unit in combination with suitable offline analytics for the quantification of purity and yield. The applications are discussed involving the purification of a model protein and a peptide. It is shown that the developed controller is effective in driving the unit to steady state during start up and in keeping a stable steady state while rejecting external disturbances. [Copyright &y& Elsevier]

Abstract: A novel glycosaminoglycan-like sulfated polysaccharide (AAP) from the pleopods of Haliotis discus hannai Ino was purified by DEAE ion exchange chromatography followed with S-300 HR geltrion chromatography. Chemical composition analysis indicated that AAP was composed of galactosamine, glucuronic acid, fucose, galactose with a ratio of 2.14:2.37:2.94:1, the content of sulfate was 15.5%. The average molecular weight (M w) was 56.2kDa in a TSK G4000 column. Further IR, 1D, 2D NMR spectroscopic analysis of the correlation signals of different sugar units gave a proposal repeating structure, as follows: Display Omitted In vitro anticoagulant assay indicated AAP prolonged both the activated partial thromboplastin time (APTT) and thrombin time (TT), with a 22.5U/mg and 72.0U/mg compared with standard heparin, respectively. The anticoagulant property of AAP was mainly attributed to powerful potentiation thrombin by anti-thrombin III. [Copyright &y& Elsevier]

Abstract: Today more and more attentions had been attracted by many nutritionists and pharmacologists on polysaccharides from natural plants or animals due to their significant biological activities. In this research three polysaccharides (BRR-W1, BRR-WA1 and BRR-WA2) were isolated and purified from the water extract of Boschniakia rossica by DEAE Sepharose Fast Flow and Sepharose 6 Fast Flow column chromatography. Chemical and physical characteristics of three polysaccharides were investigated by a combination of chemical and instrumental analysis methods. The assays of their effect on macrophages activation were also investigated in vitro, including phagocytosis of macrophages, detections for NO production and TNF-α secretion. The results indicated that the effect of polysaccharides on macrophages activation was influenced by their respective physicochemical properties. [Copyright &y& Elsevier]

Abstract: A family of linear hexamer peptide ligands HWRGWV, HYFKFD and HFRRHL, initially identified for their affinity to the Fc portion of human immunoglobulin G (hIgG), also have potential for use in the purification of human immunoglobulins A (hIgA) and M (hIgM). HWRGWV demonstrated the strongest binding affinity to hIgM, followed by hIgA and hIgG respectively. The effects of N-terminal acetylation of the peptide, as well as elution buffer pH, on the chromatographic elution of human IgG, IgA and IgM from HWRGWV resins at various peptide densities (0.04–0.55meq/g) were investigated. Over 80% recovery and 90% purity were achieved for human IgG and IgA isolation from complete minimum essential medium (cMEM) using HWRGWV resin at optimum peptide densities. For human IgM, 75.7% recovery and 86.0% purity were achieved by using HWRGWV at a low peptide density of 0.04meq/g. Although HYFKFD and HFRRHL exhibited their ability for isolation of human IgG, IgA and IgM from cMEM as well, HWRGWV is the best option among them for large-scale purification of human IgG, IgA and IgM based on conditions tested. [Copyright &y& Elsevier]

Abstract: The growing demand on plasmid DNA (pDNA) manufacture for therapeutic applications requires a final product with higher quality and quantity, spending the least time. Most of the current processes for pDNA production use at least one chromatographic step, which often constitutes a key-step in the purification sequence. Monolithic stationary phases are new alternatives to the conventional matrices, which offer fast separation of pDNA due to their excellent mass transfer properties and their high binding capacity for large molecules, as pDNA. However, the efficient recovery of pure pDNA focuses on a suitable balance of the feedstock, adsorbent and mobile phase properties. To satisfy the increasing demand for pharmaceutical grade plasmids, we developed a novel downstream process which overcomes the bottlenecks of common lab-scale techniques while complying with all regulatory requirements. This work reports an integrative approach using the carbonyldiimidazole monolith to efficiently purify the supercoiled (sc) pDNA active conformation from other plasmid topologies and Escherichia coli impurities present in a clarified lysate. The monolith specificity and selectivity was also assessed by performing experiments with plasmids of several sizes of 2.7, 6.05 and 7.4 kilo base pairs (kbp), verifying the applicability to purify different plasmids. Hence, the process yield of the pDNA purification step using the CDI monolith was 89%, with an extremely reduced level of impurities (endotoxins and gDNA), which was reflected in good transfection experiments of the sc plasmid DNA sample. Overall, the analytical results and transfection studies performed with the pDNA sample purified with this monolithic enabling technology, confirmed the suitability of this pDNA to be used in pharmaceutical applications. [Copyright &y& Elsevier]

Abstract: A microwave-assisted extraction (MAE) method is presented for the extraction of xanthones, α-mangostin and γ-mangostin from Garcinia mangostana. The MAE conditions including extraction temperature, liquid/solid ratio, extraction time and concentration of ethanol were optimized with an orthogonal test, and 5g sample was extracted with the optimized conditions. The crude extraction of MAE was successfully isolated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (0.8:0.8:1:0.6, v/v) in one-step separation. The separation yielded 75mg of α-mangostin at 98.5% purity, and 16mg of γ-mangostin at 98.1% purity from 360mg crude extract of G. mangostana in less than 7h. The purity of the two xanthones was determined by HPLC. Their structures were further identified by ESI-MS, 1H NMR and 13C NMR. [Copyright &y& Elsevier]

Abstract: The design of gradient simulated moving bed (SMB) chromatographic processes requires an appropriate selection of the chromatographic system followed by the determination of adsorption isotherm parameters in the relevant range of mobile phase conditions. The determination of these parameters can be quite difficult for recombinant target proteins present in complex protein mixtures. The first part of this work includes the estimation of adsorption isotherm parameters for streptokinase and a lumped impurity fraction present in an Escherichia coli cell lysate for a hydrophobic interaction chromatography (HIC) matrix. Perturbation experiments were carried out using a Butyl Sepharose matrix with purified recombinant protein on buffer equilibrated columns as well as with crude cell lysate saturated columns. The Henry constants estimated for streptokinase were found to exhibit in a wide range a linear dependence on the salt concentration in the mobile phase. These parameters were applied in subsequent investigations to design a simulated moving bed (SMB) process capable to purify in a continuous manner recombinant streptokinase from the E. coli cell lysate. [Copyright &y& Elsevier]

Abstract: Acid/base mobile phase modifiers affect enantioseparations in ways that are not fully understood yet, for the lack of systematic studies. This makes chiral analysis of some pharmaceuticals difficult to reproduce. Once a column has been exposed to a modifier, the selectivity of certain pairs of enantiomers may change, for the better or the worse. We study the behavior of five enantiomeric pairs, three which are highly sensitive to the addition of certain modifiers and two that have little sensitivity to these modifiers. Their use permits the determination of the extent of the memory effect response on individual columns. The selectivity of 4-chlorophenylalanine methyl and ethyl ester, and of ketoprofen improve as a solution of ethanesulfonic acid is percolated through the column. As a result, these pairs are most useful for the determination of the extent of acid memory effect on a column. The selectivity of propranolol HCl and, to a lesser degree, Tröger''s base increases as a solution of diisopropylethylamine is percolated through the column. The separation of each one of these five pairs is inversely affected by the percolation of the opposite acid/base solution. We used trans-stilbene oxide (TSO) as a ‘standard’ to determine the column stability because no memory effect is observed for it (its retention, enantioselectivity, and resolution remain constant). Understanding whether a column is under the influence of the memory effect is critical to both the analysis of pharmaceutical ingredients and to the development of preparative purification techniques for racemic mixtures. Thus, columns that were unreliable for method development and method transfer, due to the memory effect and a lack of proper solvent exposure records, can now be used. [Copyright &y& Elsevier]

Abstract: In the first part of this work adsorption isotherm parameters were acquired to describe the migration of recombinant streptokinase in Butyl Sepharose columns at different salt concentrations. Based on these results, a simulated moving bed (SMB) chromatographic process was designed and realised, which exploits a two-step salt gradient and allows the continuous separation of streptokinase from contaminants present in a clarified Escherichia coli cell lysate solution. This second part describes the design of the three-zone open-loop gradient SMB process applying both equilibrium theory and an equilibrium stage model and presents results of a series of experiments aiming to obtain pure streptokinase. Moreover, the potential of the SMB process and the design approach are evaluated. [Copyright &y& Elsevier]

Abstract: Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin''s biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor. [Copyright &y& Elsevier]

Abstract: A water-soluble polysaccharide obtained from Acanthopanax senticosus leaves (ASL), was fractionated by DEAE–Sepharose fast-flow column chromatography, and purified by Sephadex G-75 gel-permeation column chromatography. The characteristics of ASP-2-1 were determined by chemical analysis, high-performance capillary electrophoresis (HPCE), high-performance gel-permeation chromatography (HPGPC). The results show that ASP-2-1 contained 89.47% carbohydrate, 7.45% uronic acid, 2.16% protein and seven kinds of monosaccharides including rhamnose, xylose, glucose, mannose, arabinose, galactose and glucuronic acid in a molar ratio of 7.45:18.63:25.15:0.93:8.35:2.79:5.69, with an average molecular weight of about 14,573Da. Furthermore, the immunobiological and antioxidant activities, in vitro, of ASP-2-1 were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and ferric-reducing antioxidant power assay (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH ), superoxide radical and hydroxyl radical ( OH) free radical-scavenging assay, respectively. The results showed that ASP-2-1 exhibited significantly higher immunomodulatory activities against the lymphocyte proliferation in vitro, pronounced reductive power (FRAP value: 785.1μM at 0.2mg/ml), strong hydroxyl radical (89.56% at 1mg/ml) scavenging activity, moderate superoxide radicals (65.32% at 1mg/ml) and DPPH radicals (68.9% at 1mg/ml) scavenging activities. ASP-2-1 should be explored as a novel and potential natural antioxidant and immunostimulating agent for use in functional foods or medicine. [Copyright &y& Elsevier]

Abstract: Ureases are used in beverage, fertilizer and pharmaceutical industries. We report purification, characterization and a simple method for immobilization of urease form Momordica charantia seeds. The purification of the enzyme is a two steps process involving anion exchange chromatography using DEAE–Sepharose matrix followed by size exclusion chromatography. The fold purification of the enzyme was found to be 6.5. The estimated molecular mass and K m of the enzyme were 174.5kDa and 34±0.5mM, respectively. The purified urease was immobilized on chitosan beads activated with glutaraldehyde. The activation of chitosan beads and immobilization were confirmed by Fourier Transform Infrared (FTIR) spectrometry and Scanning Electron Microscope (SEM), respectively. Moreover, Energy Dispersive X-ray (EDX) analysis shows increased percentage of nitrogen which further confirms immobilization. The efficiency of immobilization was found to be approximately 97%. The immobilized enzyme retains 60% activity till fourteen successive batches of enzymatic reactions which makes it a highly stable product. Comprehensively, the enzyme shows desirable properties for various applications. [Copyright &y& Elsevier]

Abstract: Proteins conjugated to neutral biopolymers are of keen interest to the food and pharmaceutical industries. Conjugated proteins are larger and more charge shielded than un-reacted proteins, making purification difficult using conventional beaded chromatographic supports because of slow mass transfer rates, weak binding, and viscous solutions. Past methods developed for pharmaceuticals are unsuitable for foods. In this work, a food-grade whey protein–dextran conjugate was purified from a feed solution also containing un-reacted protein and dextran using either a column packed with 800mL of a beaded support that was specifically designed for purification of conjugated proteins or an 8mL tube monolith. The monolith gave a similar dynamic binding capacity as the beaded support (4–6g/L), at a 42-fold greater mass productivity, and 48-fold higher flow rate, albeit at somewhat lower conjugate purity. Performance of the monolith did not depend on flow rate. In conclusion, monoliths were found to be well suited for the purification of whey protein–dextran conjugates. [Copyright &y& Elsevier]

Abstract: An extracellular thermostable lipase from Amycolatopsis mediterranei DSM 43304 has been purified to homogeneity using ammonium sulphate precipitation followed by anion exchange chromatography and hydrophobic interaction chromatography. This protocol resulted in a 398-fold purification with 36% final recovery. The purified A. mediterranei DSM 43304 lipase (AML) has an apparent molecular mass of 33kDa. The N-terminal sequence, AANPYERGPDPTTASIEATR, showed highest similarity to a lipase from Streptomyces exfoliatus. The values of and for p-nitrophenyl palmitate (p-NPP) at the optimal temperature (60°C) and pH (8.0) were 0.099±0.010mM and 2.53±0.06mmol/minmg, respectively. The purified AML displayed significant activity towards a range of short and long chain triglyceride substrates and p-nitrophenyl esters. Hydrolysis of glycerol ester bonds occurred non-specifically. The purified AML displayed significant stability in the presence of organic solvents (40%, v/v) and catalyzed the synthesis of the flavour ester isoamyl acetate in free and immobilized states. [ABSTRACT FROM AUTHOR]

Abstract: A novel NADH-dependent dehydrogenases/reductases (SDRs) superfamily reductase (PsCRII) was isolated from Pichia stipitis. It produced ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] in greater than 99% enantiomeric excess. This enzyme was purified to homogeneity by ammonium sulfate precipitation followed by Q-Sepharose chromatography. Compared to similar known reductases producing (S)-CHBE, PsCR II was more suitable for production since the purified PsCRII preferred the inexpensive cofactor NADH to NADPH as the electron donor. Furthermore, the K m of PsCRII for ethyl 4-chloro-3-oxobutanoate (COBE) was 3.3mM, and the corresponding V max was 224μmol/mg protein/min. The catalytic efficiency is the highest value ever reported for NADH-dependent reductases from yeasts that produce CHBE with high enantioselectivity. In addition, this enzyme exhibited broad substrate specificity for several β-keto esters using NADH as the coenzyme. The properties of PsCRII with those of other carbonyl reductases from yeasts were also compared in this study. [ABSTRACT FROM AUTHOR]

Abstract: Histidine-tagged proteins have been purified with immobilized nickel affinity chromatography. This method, however, bears some serious disadvantages, including carcinogenicity and potential leakage of the nickel and the handling and disposal costs. We developed a novel purification process for water-soluble histidine-tagged green fluorescent protein from the cocoons of transgenic silkworms, unlike conventional E. coli expression system which sometimes produced insoluble and inactive inclusion bodies. 60.3% extraction efficiency was achieved with 50mM Tris containing 150mM NaCl, pH 7.5. The extract was purified in one step on zinc-immobilized hydroxyapatite eluted with phosphate buffer. Purity was 91.2% with a recovery of 30.0μg from 160mg of silk fiber. This purification method may provide a simple non-toxic alternative to nickel-immobilized metal affinity chromatography for purification of other histidine-tagged proteins. [Copyright &y& Elsevier]

Abstract: Dityrosine (DY) can be used as a biomarker to detect oxidative protein damage and selective proteolysis. It is generally prepared by horseradish peroxidase (HRP)-catalyzed oxidation of l-tyrosine (Y) followed by multistep chromatographic separations. In this study, we present an alternative method for the preparation of DY by HRP-catalyzed synthesis of N,N′-diBoc-dityrosine (DBDY) from N-Boc-l-tyrosine (BY). The presence of the tert-butoxycarbonyl (Boc) group ensured that the fraction of further oxidized by-products (e.g., trimers and pulcherosine) was quite low. The yield of DBDY (37.5%) was comparable to that reported for DY (> 26%). DBDY could be purified by a simple one-step silica column chromatography procedure that resulted in a purity of 89.7%. DBDY is considered to be better than DY for subsequent chemical reactions (for binding to polymers, amino acids, drugs, antibodies, etc.) because such reactions can be selectively performed by using the carboxylic acid and amine groups in the following sequence: first, the carboxylic acid groups are used; next, the Boc groups are removed; and finally, the amino groups are used. To prepare DY, the Boc groups in DBDY were simply and completely removed by treatment with trifluoroacetic acid. [ABSTRACT FROM AUTHOR]

Abstract: A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of four prenylflavanones from the crud extract after microbial biotransformation of kurarinone was developed successfully using a stepwise elution with two-phase solvent system composed n-hexane–ethyl acetate–methanol–water at the volume ratios of 1:1:0.7:1 (v/v) and 1:1:1.2:1 (v/v). A total of 7mg of 4a,5a-dihydroxy norkurarinone (1), 11mg of 7-methoxyl-4a,5a-dihydroxy norkurarinone (2), 14mg of 6a-hydroxykurarinone (3) and 10mg of norkurarinone (4) were obtained in one-step separation from 520mg of the crude biotransformation sample with purities of 94.0%, 97.8%, 99.5% and 98.7%, respectively. Among them, compounds 1–3 are novel compounds, and their chemical structures were identified on the basis of the extensive spectral methods such as UV, IR, HR-MS and NMR. [ABSTRACT FROM AUTHOR]