Search

• Magnetic graphitized carbon black based on crystal growth method was first applied to a high-throughput screen of multi-pesticide residues in traditional Chinese medicine (TCM). • The proposed clean-up material coupled with anhydrous CaCl 2 which forms chelate precipitates with polyphenols, flavonoids, pigments and alkaloids in the TCM, can effectively remove interference and improve the purification efficiency. • This work compared to other reported methods for determining pesticides in TCM. • The liquid chromatography-quadrupole time-of-flight mass spectrometry method coupled with a self-built database enables the simultaneous determination of multi-component pesticide residues. In this study, a high-throughput method for analyzing 300 pesticide residues in Radix Codonopsis and Angelica sinensis was established by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) using iron tetroxide loaded graphitized carbon black magnetic nanomaterial (GCB/Fe 3 O 4) as the purification material. It was optimized that saturated salt water and 1 % acetate acetonitrile were used as the extraction solution, then the supernatant was purified with 2 g anhydrous CaCl 2 and 300 mg GCB/Fe 3 O 4. As a result, 300 pesticides in Radix Codonopsis and 260 in Angelica sinensis achieved satisfactory results. The limits of quantification of 91 % and 84 % of the pesticides in Radix Codonopsis and Angelica sinensis reached 10 μg/kg, respectively. The matrix-matched standard curves ranging from 10 to 200 μg/kg were established with correlation coefficients (R) above 0.99. The pesticides meeting SANTE/12682/2021 accounted for 91.3 %, 98.3 %, 100.0 % and 83.8 %, 97.3, 100.0 % of the total pesticides added in Radix Codonopsis and Angelica sinensis respectively, which were spiked at 10, 20,100 μg/kg. The technique was applied to screen 20 batches of Radix Codonopsis and Angelica sinensis. Five pesticides were detected, three of which were prohibited according to the Chinese Pharmacopoeia (2020 Edition). The experimental results showed that GCB/Fe 3 O 4 coupled with anhydrous CaCl 2 exhibited good adsorption performance and could be used for sample pretreatment of various pesticide residues in Radix Codonopsis and Angelica sinensis. Compared with the reported methods for determining pesticides in traditional Chinese medicine (TCM), the proposed method has the advantage of less time-consuming in the clean-up procedure. Furthermore, as a case study on root TCM, this approach may serve as a reference for other TCM. [ABSTRACT FROM AUTHOR]

Recent studies of magnetic carrier technology have focused on its applications in separation and purification technologies, due to easy separation of the target from the reaction medium by applying an external magnetic field. In the present study, Fe 3 O 4 superparamagnetic nanoparticles were prepared to utilize a chemical co-precipitation method, then the surfaces of the nanoparticles were modified with arsanilic acid derivatives which were used as the specific nanocarriers for the affinity purification of alkaline phosphatase from the hen's egg yolk. The six different types of magnetic nanocarriers with varied lengths of the linkers were obtained. All samples were characterized step by step and validated using FTIR, SEM, EDX, VSM and XRD analysis methods As the results were shown, the use of inflexible tags with long linkers on the surface of the nanocarrier could lead to better results for separation of alkaline phosphatase from the hen's egg yolk with 76.2% recovery and 1361.7-fold purification. The molecular weight of the purified alkaline phosphatase was estimated to be 68 kDa by SDS–PAGE. The results of this study showed that the novel magnetic nanocarriers were capable of purifying alkaline phosphatase in a practically time and cost effective way. [ABSTRACT FROM AUTHOR]

A depth filter is widely used in biopharmaceutical manufacturing process. In this study, we found that buffer exchange to reduce conductivity dramatically improved the removal of impurities in depth filtration. The host cell protein clearance was comparable to that of protein A affinity chromatography, which is generally used as a first capture step in monoclonal antibody purification. In addition, a combination of different depth filters showed enhanced purification. Additional flow-through purification is possible without any sample preparation, and a biopharmaceutical-quality purity level (<100 ppm for host cell protein and <5% for high molecular weight species) can be attained. These results represent the possibility of an antibody manufacturing process using an entirely flow-through mode. [ABSTRACT FROM AUTHOR]

Immobilized metal affinity chromatography (IMAC) technique is used for fast and reliable purification of histidine(His)-tagged recombinant proteins. The technique provides purification under native and denaturing conditions. The aim of this study is to evaluate three commercially available IMAC kits (Thermo Scientific, GE Healthcare and Qiagen) for the purification of a 6xHis-tagged recombinant CagA (cytotoxin-associated gene A) protein from IPTG-induced Escherichia coli BL21(DE3) culture. The kits were tested according to the manufacturer instructions and the protein was purified with only GE Healthcare and Qiagen kits under denaturing conditions. 1% (w/v) SDS was used as denaturing agent in PBS instead of extraction reagent of Thermo Scientific kit to lyse bacterial cells from 100 ml culture. The 6xHis-tagged recombinant protein was purified by the three kits equally. [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]

Egg yolk immunoglobulin (IgY) is a superior functional equivalent to mammalian IgG. However, the preparation of refined and highly purified IgY is still attributed as difficult task. Protein M (a transmembrane protein from human mycoplasma) has been newly demonstrated as an ideal affinity regent for mammalian antibody purification. This study aimed to evaluate the interaction between protein M and IgY. The results showed protein M could be a superior affinity reagent for IgY, scFv as well as IgYΔFc, based on pull down and western blot investigations; in addition, it was found that ∼125 times increase of effective IgY in the elutent was obtained using protein M affinity chromatography column compared with traditional IgY extraction methods. This indicates, the purification strategy of protein M is entirely different to traditional IBPs and the salient purification feature of protein M would be a breakthrough for purifying not only non-mammalian antibodies, but also monoclonal antibodies and engineered antibodies based on variable region. [ABSTRACT FROM AUTHOR]

A novel pretreatment method involving microwave-assisted extraction and solid-phase purification combined with dispersive liquid–liquid microextraction (MAE-SPP-DLLME) followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) was established for the simultaneous determination of six antibacterial pharmaceuticals including metronidazole, tinidazole, chloramphenicol, thiamphenicol, malachite green and crystal violet. The conditions of MAE were optimized using an orthogonal design and the optimal conditions were found to be 8 mL for acetonitrile, 50 °C for 5 min. Then, neutral alumina column was employed in the solid-phase purification. Finally, the critical parameters affecting DLLME, including selection of extraction and dispersive solvent, adjustment of pH, salt concentration, extraction time, were investigated by single factor study. Under optimum conditions, good linearities ( r > 0.9991) and satisfied recoveries (Recoveries > 87.0%, relative standard deviation (RSD) < 6.3%) were observed for all of the target analytes. The limits of detection and quantification were 4.54–101.3 pg kg −1 and 18.02–349.1 pg kg −1 , respectively. Intra-day and inter-day RSDs were all lower than 3.6%. An obvious reduction in matrix effect was observed by this method compared with microwave assisted extraction followed by purification. The established method was sensitive, rapid, accurate and employable to simultaneously determine target analytes in farmed fish, river fish and marine fish. [ABSTRACT FROM AUTHOR]

The distribution of IgG in different salting-out extraction systems (SOESs), including ethanol/K 2 HPO 4 , Na 2 CO 3 , and trisodium citrate, was investigated. The SOESs that were composed of 20% K 2 HPO 4 (w/w)–14% ethanol (w/w), 19% Na 2 CO 3 (w/w)–13% ethanol (w/w) and 25% trisodium citrate (w/w)–19% ethanol (w/w) showed satisfactory results, with IgG recovery rates as high as 97.4%, 93.1%, and 90.2%, respectively. ELISA, CD and IR analyses confirmed the active retention and structural constant of IgG in the K 2 HPO 4 –ethanol system. An optimized system that consisted of 14% ethanol (w/w)–20% K 2 HPO 4 (w/w) (pH 7.0) was selected for extracting active plasma proteins directly from pig plasma. As a result, recovery rates as high as 95.7% for IgG and 93.0% for albumin were achieved. In addition, some contaminating proteins were also removed by this system, which may provide a new alternative method for the separation and purification of immunoglobulin. [ABSTRACT FROM AUTHOR]

Lately, researchers have made several efforts to improve vaccine production to fight highly contagious respiratory diseases like influenza. One of the most promising options for reducing the impact of this virus is DNA vaccination. However, a large quantity of highly pure plasmid DNA (pDNA) is necessary to attain this goal. The present work describes the production and purification of the plasmid NTC7482-41H-VA2HA expressing influenza virus hemagglutinin using an agmatine monolith. This ligand was chosen to purify supercoiled (sc) pDNA from complex lysates because of its versatile multimodal character. Its natural intervention in several biological systems together with its similarity with the highly studied arginine ligand allowed the development of a simpler and more specific purification process. Agmatine works under two strategies: descending ammonium sulfate gradient and ascending sodium chloride gradient. Furthermore, pH manipulation revealed an important role in pDNA isoforms selectivity. Dynamic binding capacity (DBC) experiments were performed varying different parameters and showed an increase with pDNA concentration, while high flow rate and high pH had the opposite effect. Sc pDNA was purified with high yield and was efficient with respect to cell transfection and cell viability. This monolith showed to be appropriate to purify the plasmid NTC7482-41H-VA2HA, providing a valuable tool for pDNA influenza vaccines preparation. [ABSTRACT FROM AUTHOR]

Glycocalicin (GC) is a large extracellular proteolytic fragment of glycoprotein Ib, a membrane platelet component playing an essential role in the physiological processes of platelet adhesion and aggregation. GC contains the binding sites for thrombin and von Willebrand factor. GC circulates normally in vivo in significant concentrations and the plasma level of this protein reflects a complex function of factors including platelet count or platelet turnover. It can therefore serve as a good indicator for many diseases like hypoplastic thrombocytopenia and idiopathic thrombocytopenic purpura. For this reason, several purification assays have been previously described. In this work, we describe a novel analytical method for GC purification from human platelets based on preparative HPLC gel filtration followed by immuno-affinity chromatography on NHS activated column conjugated with specific antibody. Pure GC was obtained from tiny amount of starting material. Our protocol of GC purification is simple, fast and provides a pure end product. [ABSTRACT FROM AUTHOR]

Inhibition of DPP-IV may improve glycemic control in diabetics by preventing the rapid breakdown and there by prolonging the physiological action of incretin hormones. Barbel muscle protein hydrolysate (BMPH) was noted to exhibit DPP-IV inhibitory activity, with an IC 50 value of 1.94 mg/mL. It was fractionated into five major fractions ( F I – F V ) by size exclusion chromatography using a Superdex peptide. The F III fraction was noted to display the highest inhibitory activity, with an IC 50 value of 1.23 mg/mL, and was, therefore, further fractionated by RP-HPLC. Four major peptide sub-fractions were selected. The results revealed that the SF 4 sub-fraction showed the highest DPP-IV inhibitory activity, with an IC 50 value of 0.21 mg/mL. This sub-fraction was submitted to RP-HPLC, ESI–MS, and ESI–MS/MS analyses. The findings indicated that SF4 consisted of two peptides (IC 50 = 96 μg/mL), namely PP1 and PP2, whose structures were identified as Trp-Ser-Gly (330 Da) and Phe-Ser-Asp (349 Da), respectively. This is the first report of these sequences from barbel proteins. The structural modelling through docking simulations results with DPP-IV showed that the Trp-Ser-Gly peptide bound to DPP-IV with high affinity. Overall, the results suggested that BMPH can be considered as a promising natural source of DPP-IV inhibitory peptides. [ABSTRACT FROM AUTHOR]

The oligonucleotide binding fold (OB-fold) is a small structural motif present in many proteins. It is originally named for its oligonucleotide or oligosaccharide binding properties. These proteins have been identified as essential for replication, recombination and repair of DNA. We have successfully purified a protein contains OB-fold from the stem of Tinospora cordifolia , a medicinal plants of north India. Stems were crushed and centrifuged, and fraction obtained at 60% ammonium sulphate was extensively dialyzed and applied to the weak anion exchange chromatography on Hi-Trap DEAE-FF in 50 mM Tris–HCl buffer at pH 8.0. Eluted fractions were concentrated and applied to gel filtration column to get pure protein. We observed a single band of 20-kDa on SDS-PAGE. Finally, the protein was identified as OB-fold by MALDI-TOF. The purified OB-fold protein was characterized for its secondary structural elements using circular dichroism (CD) in the far-UV region. Generally the OB-fold has a characteristic feature as five-stranded beta-sheet coiled to form a closed beta- barrel. To estimate its chemical stability, guanidinium chloride-induced denaturation curve was followed by observing changes in the far-UV CD as a function of the denaturant concentration. Analysis of this denaturation curve gave values of 8.90 ± 0.25 kcal mol −1 and 3.78 ± 0.18 M for Δ G D ° (Gibbs free energy change at 25 °C) and C m (midpoint of denaturation), respectively. To determine heat stability parameters of OB-fold protein, differential scanning calorimetry was performed. Calorimetric values of Δ G D °, T m (midpoint of denaturation), Δ H m (enthalpy change at T m ), and Δ C p (constant-pressure heat capacity change) are 9.05 ± 0.27 kcal mol −1 , 85.2 ± 0,3 °C, 105 ± 4 kcal mol −1 and 1.6 ± 0.08 kcal mol −1 K −1 . This is the first report on the isolation, purification and characterization of OB-fold protein from a medicinal plant T. cordifolia. [ABSTRACT FROM AUTHOR]

The developments in the use of plasmid DNA (pDNA) in gene therapy and vaccines have motivated the search and improvement of optimized purification processes. In this context, dipeptides l -tyrosine- l -tyrosine and l -tyrosine- l -arginine are synthetized to explore their application as affinity ligands for supercoiled (sc) plasmid DNA (pDNA) purification. The synthesis is based on the protection of N -Boc- l -tyrosine, followed by condensation with l -tyrosine or l -arginine methyl esters in the presence of dicyclohexylcarbodiimide (DCC), which after hydrolysis and acidification give the afforded dipeptides. The supports are then obtained by coupling l -tyrosine, l -tyrosine- l -tyrosine and l -tyrosine- l -arginine to epoxy-activated Sepharose and are characterized by high resolution magic angle spinning (HR-MAS) NMR and Fourier transform infrared spectroscopy (FTIR). Surface plasmon resonance (SPR) biosensor is used to establish the promising ligand to be used in the chromatographic experiments and ascertain experimental conditions. Sc isoform showed the highest affinity to the dipeptides, followed by linear (ln) pDNA, being the open circular (oc) the one that promoted the lowest affinity to l -tyrosine- l -arginine. Saturation transfer difference (STD)-NMR experiments show that the interaction is mainly hydrophobic with the majority of the 5′-mononucleotides, except for 5′-GMP with l -tyrosine- l -arginine Sepharose that is mainly electrostatic. The support l -tyrosine Sepharose used in chromatographic experiments promotes the separation of native pVAX1- LacZ and pcDNA3-FLAG-p53 samples (oc+sc) by decreasing the salt concentration. The results suggest that it is possible to purify different plasmids with the l -tyrosine Sepharose, with slight adjustments in the gradient conditions. [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]

A method of using high-speed counter-current chromatography (HSCCC) for preparative isolation and purification of oligostilbenes from the ethanol extracts of seed kernel of Iris lactea Pall. var. chinensis (Fisch.) Koidz was established in this study. Four oligostilbenes were successfully separated and purified by HSCCC with two sets of two-phase solvent system, n -hexane-ethyl acetate-methanol-water (3:6:4.2:5.5, v/v/v/v) in the head-to-tail elution mode for the first separation to mainly isolate vitisin A (58 mg), ɛ-viniferin (76 mg) and peak II (43 mg) from 300 mg of the crude ethanol extracts, and then light petroleum–ethyl acetate–methanol–water (5:5:3:6, v/v/v/v) in the tail-to-head elution mode for the second separation to isolate vitisin B (52 mg) and vitisin C (11 mg) from 100 mg of peak II. The purities of the isolated four oligostilbenes were all over 95.0% as determined by HPLC. Vitisin A, vitisin B and vitisin C, resveratrol tetramers, were isolated from Iris lactea for the first time . The preparation of crude sample was simple and the HSCCC method for the isolation and purification of four oligostilbenes was rapid, efficient and economical. [ABSTRACT FROM AUTHOR]

The application of antibodies to small molecules in the field of bioanalytics requires antibodies with stable biological activity and high purity; thus, there is a growing interest in developing rapid, inexpensive and effective procedures to obtain such antibodies. In this work, a ractopamine (RAC) derivative, N -4-aminobutyl ractopamine (ABR), was synthesized for preparing new specific affinity chromatography to purify a murine monoclonal antibody (mAb) against RAC from ascites. The performance of the new specific chromatography was compared with four other purification methods in terms of recovery, purity and biological activity of mAb. These four purification methods were prepared by using specific ligands (RAC and RAC-ovalbumin) and commercial ligands (protein G and protein A), respectively. The results showed that the highest recovery (88.1%) was achieved using the new chromatography; in comparison, the recoveries from the other methods were all below 70%. The purity of the mAbs from the new chromatography was 88.3%, while, the highest purity of 97.6% was from protein G chromatography and the lowest purity of 84.7% was from protein A chromatography. The biological activity of the purified mAb from all of the chromatography methods was comparable in enzyme-linked immunosorbent immunoassay (ELISA). [ABSTRACT FROM AUTHOR]

An efficient high-performance countercurrent chromatography (HPCCC) based method has been developed for the purification of chemically synthesized 1-adenosin-5′-yl 3-(3-methylbut-3-enyl)triphosphoric acid diester (ApppI). ApppI is an adenosine triphosphate (ATP) analogue with biological significance due to its varied actions in the body. ApppI was synthesized and purified as its tetrabutylammonium (TBA) salt and converted successfully into its more practical sodium salt form after purification. The amount of TBA hydroxide (2.0, 2.5 and 3.0 eq) used in the synthesis of ApppI was shown to exert an effect on the purification process with HPCCC and on the overall yield (8%, 16% and 22%, respectively). 1-Adenosin-5′-yl 3-(3-methylbut-3-enyl)diphosphoric acid diester (AppI) was also isolated as a side product. [ABSTRACT FROM AUTHOR]

Highlights: [•] Efficient and highly selective green solvent extraction method for membrane protein FhuA. [•] Purity 95% (2-methyltetrahydrofuran) and 80% (cyclopentylmethylether), yield 70mg FhuA per liter fermenter broth. [•] Alternative to chloroform extraction of membrane proteins. [ABSTRACT FROM AUTHOR]

Highlights: [•] A simple and efficient method for large-scale preparative separation of phenylethanoid glycosides from Cistanche deserticola. [•] Eight kinds of macroporous resins were tested and HPD300 was found best for phenylethanoid glycosides purification. [•] The purity of total phenylethanoid glycosides in the product was 76.58%. [•] The equilibrium adsorption data of HPD300 were fitted to the Freundlich isotherm. [•] The breakthrough curves showed that acteoside had a higher affinity to the resin than echinacoside. [ABSTRACT FROM AUTHOR]

Highlights: [•] Pectinase has many important applications in various industrial processes. [•] Alcohol/salt based ATPS was used to purify pectinase from mango peel. [•] Purification factor, yield, partition coefficient and selectivity were optimised. [•] Pectinase from mango peel was recovered with a high purification factor (11.7) and a high yield (97.1%). [Copyright &y& Elsevier]

Highlights: [•] We develop an efficient, simple and economic process for purification of porcine pancreas lipase using aqueous two-phase system (ATPS). [•] Phase diagrams were determined to find an operating region for forming ATPS firstly. [•] We used stability ratio instead of TLL and without NaCl in the system. [•] This system obtained an enzyme partition coefficient of 12.7, the extraction efficiency of 94.7%, and the purification factor of approximately 4. [Copyright &y& Elsevier]

Abstract: An affinity purification method that isolates T-2 toxin-specific IgY utilizing a T-2-toxin-immobilized column was developed. The T-2 toxin was covalently coupled via a carbonyldiimidazole-activated hydroxyl functional group to amine-activated sepharose beads. The affinity-purified IgY was characterized by gel electrophoresis, fast protein liquid chromatography, enzyme-linked immunosorbant assay, surface plasmon resonance and mass spectrometry. A competitive inhibition ELISA (CI-ELISA) was performed using affinity-purified IgY with a T-2 toxin detection sensitivity of 30ng/mL, which falls within the maximum permissible limit of 100ng/mL. The cross reactivity of IgY towards deoxynivalenol, zearalenone, fumonisin B1 and HT-2 was significantly reduced after affinity purification. A surface plasmon resonance (SPR)-based inhibition assay was also applied for quantitative determination of T-2 toxin in spiked wheat samples. The results obtained indicate the feasibility of utilizing this IgY-based assay for the detection of T-2 toxin in food samples. [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: In this study, berenil was tested as a ligand, specifically to purify plasmids of different sizes pVAX1-LacZ (6.05Kbp) and pCAMBIA-1303 (12.361Kbp) from clarified Escherichia coli alkaline lysates. For this purpose, chromatographic experiments were performed using Sepharose derivatized with berenil. The results showed that both pDNA molecules are completely purified using lower amounts of salt in the eluent than those previously reported for other pseudo-affinity and hydrophobic interaction chromatography based processes. Total retention of all lysate components was achieved with 1.3M ammonium sulphate in the eluent buffer and pDNA elution was obtained by decreasing the salt concentration to 0.55M. All impurities were eluted after decreasing the concentration to 0M. The recovery yield for pCAMBIA-1303 (45%) was lower than that obtained for pVAX1-LacZ (85%), however the larger pDNA showed a higher purity level. Purification of pVAX1-LacZ was also performed using non-clarified E. coli process streams, replacing the clarification step with a second chromatographic run on the berenil-Sepharose. Using the same binding and elution conditions as before, a pure plasmid sample was obtained with a 33% yield and with all host impurity levels in accordance with the requirements established by the regulatory agencies. These results suggest that this chromatographic method is a promising alternative to purify pDNA for therapeutic use. [Copyright &y& Elsevier]

Abstract: Huperzine A (HupA) and Huperzine B (HupB) are natural alkaloids existed in Lycopodium plants. They both have potential clinical application for treating Alzheimer''s Disease (AD). For the purpose of better utilizing the limited plant resources, a quick and low cost method to separate and purify HupA and HupB from Huperzia serrata (Thunb. ex Murray) was established in this paper. Low polarity macroporous resin SP850 was selected from eight kinds of resins during initial purification. Trifluoroacetic acid (TFA) was proved to be the best acid modifier reagent among all acids used in our experiment for improving separation. HupA and HupB were baseline separated on a C18 column by preparative high performance liquid chromatography (Preparative HPLC), the optimal gradient mobile phase system contained methanol increasing from 15% (v/v) to 35% (v/v) and 0.1% (v/v) TFA within the water. The purity of HupA and HupB obtained was 99.1% and 98.6%, respectively, and the total recovery for them was 83.0% and 81.8%, respectively. [Copyright &y& Elsevier]

Abstract: This study describes an optimization of [6]-, [8]- and [10]-gingerol isolation and purification in semi-preparative HPLC scale and their anti-proliferative activity. The gingerols purification was carried out in HPLC system using a Luna-C18 and the best mobile phase evaluated was MeOH/H2O (75:25, v/v). This new methodology for the gingerols isolation was very effective, since considerable amounts (in the range of milligrams) with a good purity degree (∼98%) were achieved in 30min of chromatographic run. [6]-, [8]- and [10]-Gingerol purified by this methodology inhibited the proliferation of MDA-MB-231 tumor cell line with IC50 of 666.2±134.6μM, 135.6±22.6μM and 12.1±0.3μM, respectively. These substances also inhibited human fibroblasts (HF) cell proliferation, however in concentrations starting from 500μM. In conclusion, our results demonstrate an optimization of gingerols isolation and their specific anti-proliferative activities against tumor cells, suggesting their use as important models for drug design in an attempt to develop new compounds with fewer side effects when compared to conventional chemotherapy. [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: 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: Recovery and purification of stem cells are determining steps in order to obtain the purity and viability required for transplantation. In this context, immunochemical techniques have been widely preferred due to their high selectivity. CD133, a glycoprotein expressed by stem cells, is a well-used marker for isolation of neural stem cells. Transplantation of neural stem cells into patients can promote neural growth and improve neuronal functions. In this study, a new method for site-specific PEGylation of CD133-Biotin antibody is performed through streptavidin–biotin conjugation. Purification was carried out by ion-exchange chromatography. The characterization of the single PEGylated CD133-Biotin antibody was confirmed using electrophoresis with silver staining and I2–BaCl2 for PEG detection. Moreover, online PEG quantification directly after the chromatographic step was conducted (in each fraction) to detect exact elution times of PEG. In conclusion, the novel CD133-Biotin antibody PEGylation strategy conducted in this study could be used as a process step in route to neural stem cell recovery and purification via the modification of existing techniques such as aqueous two phase systems, PEGylated affinity columns or fluidized chromatography. [Copyright &y& Elsevier]

Abstract: Monodispersed and functional immobilized metal affinity magnetic chondroitin sodium sulfate nanoparticles (short as IMAN @ Fe (III)) were prepared and employed in extracting of Phosvitin Phosphopeptides (short as PPPs) from egg yolk. It was found that the diameter of the magnetic CS nanoparticles was about 20nm, and they could easily be aggregated by a magnet when suspending in the aqueous solution. The adsorption equilibrium of PPPs onto the obtained nanocarriers fitted well with the Langmuir model. The adsorption capacity of PPPs onto the superparamagnetic nanoparticles was influenced by pH and the initial concentration of the peptides solution. The final nitrogen/phosphorus molar ratios (short as N/P) of PPPs from crude egg yolk peptides and phosvitin peptides were low to 5.78 and 5.23, respectively. Compared with traditional methods, the need for preparation of phosvitin before purification is obviated and the higher purity of PPPs were obtained. In conclusion, this type of IMAN @ Fe (III) would bring advantages to the conventional separation techniques of PPPs from chicken egg yolk. [Copyright &y& Elsevier]

Abstract: Chrysophanol has high pharmaceutical values. However, it was difficult to use the traditional extraction method to extract high-concentration chrysophanol. Therefore, the purpose of this study is to purify and separate chrysophanol in traditional herb, Rheum Palmatum LINN, by using supercritical fluid extraction (SFE) and preparative high-performance liquid chromatography (P-HPLC) for rapid and large-scale isolation. The method is efficient for selective extraction of chrysophanol from the herbs, which have complex compositions. The extraction efficiency of chrysophanol with SFE is 25× higher than that of boiled water extraction under the same extraction time. The optimal conditions for SFE were 210atm and 85°C for 30min; for P-HPLC, a C18 column was used with a gradient elution of methanol and 1% acetic acid at a flow rate of 10mL/min. According to 1H NMR and LC–MS analyses, the purity of the isolated chrysophanol was as high as 99%. The recovery for chrysophanol in Rheum after SPE/PHPLC processing was in the range of 88–91.5%. Compared with other extraction and purification methods, the sequential system (SFE/P-HPLC) achieved the highest amount of extracted chrysophanol from Rheum Palmatum LINN (0.38mg/g) and the shortest run time (3h). Hence, this rapid and environmentally friendly method can separate compounds based on polarity with high efficiencies and, coupled with P-HPLC, it may be applicable in the large-scale production of foods and medicines in the future. [Copyright &y& Elsevier]

Abstract: In the present study, we have focused our attention to prepare molecular imprinted composite cryogel membranes for purification of hepatitis B surface antibody (anti-HBs) by fast protein liquid chromatography. Before the preparation of the molecular imprinted composite cryogel membranes (MI-CMs) by free radical polymerization at sub-zero temperature, we have synthesized and characterized the anti-HBs imprinted particles. Then, the cryogel membranes (CMs) were characterized by swelling test, scanning electron microscopy and Fourier transform infrared spectroscopy. Prior to chromatographic purification studies, the effective parameters on the anti-HBs adsorption process were evaluated by investigating the dependency of the adsorption capacity on flow-rate, anti-HBs concentration, contact time and ionic strength. The maximum anti-HBs adsorption capacity was calculated as 701.4mIU/g CM. The selectivity of the MI-CMs was shown by competitive adsorption of anti-HBs, total anti-hepatitis A antibody (anti-HAV) and total immunoglobulin E (IgE) adsorption studies. The MI-CMs have relative selectivity coefficients as 5.45 for anti-HBs/total anti-HAV and 9.05 for anti-HBs/total IgE, respectively. The phosphate buffer solution (pH 7.4) containing 1.0M NaCl was used for elution, almost completely, of adsorbed anti-HBs molecules. The MI-CMs could be used many times without any significant decrease in the adsorption capacity. The chromatographic purification performances of the MI-CMs were also investigated. The chromatographic parameters such as capacity and separation factors, the theoretical plate number and resolution of the MI-CMs were calculated as 5.48, 6.02, 1153.9, and 1.72 for anti-HBs molecules, respectively. As a conclusion, we can say that the MI-CMs could be used for specific purification of anti-HBs from anti-HBs positive human plasma. [Copyright &y& Elsevier]

Abstract: An alkaline protease from salt tolerant alkaliphilic actinomycetes, Nocardiopsis alba strain OK-5 was purified to homogeneity by 27 and 13 fold with a yield of 35 and 13% using two-steps and one-step method, respectively. The purification methods involved hydrophobic interaction on phenyl sapharose matrix. The apparent molecular mass was 20kDa. The temperature optimum shifted from 70 to 80°C in 4M NaCl and 30% Na-glutamate, with significant stability at 60–80°C in Na-glutamate. Deactivation rate constant (K d) increased and half life (t 1/2) decreased with the increasing temperatures from 37 to 80°C. The order of stability was: 30% Na-glutamate>4M NaCl>2M NaCl>0M NaCl. The enzyme was stable even at 80°C in 30% Na-glutamate with K d 4.11 and t 1/2 168.64min. The activation energies (E), enthalpy (ΔH*) and entropy (ΔS*) for protease deactivation in with Na-glutamate were 31.97kJ/mole, 29.23kJ/mole and −211.83J/mole, respectively. The change in free energy (ΔG*) for protease deactivation at 60°C in 30% Na-glutamate was 101.70kJ/mole. Protease had the highest activity and stability at pH 10–11. While the enzyme was highly resistant against chemical denaturation, it had varied responses to metal ions. Complete inhibition by PMSF confirmed serine nature of the protease. Na-glutamate, H2O2, β-mercaptoethanol and different surfactants enhanced the activity. [Copyright &y& Elsevier]

Abstract: This paper describes a new strategy, which aims to make on-column poly-histidine tag removal more useful in the production of recombinant proteins by improving the yield and efficiency of on-column exopeptidase cleavage. This involves improvement of the on-column cleavage condition by using imidazole concentrations in the range of 100–500mM in the cleavage buffer. At 300mM imidazole, maximum on-column cleavage yield (in excess of 99%) was achieved in 3h of incubation. However, as a result of the increased imidazole concentration, this new strategy of on-column cleavage results in some residual uncleaved poly-histidine tagged proteins (∼0.1%) and the production of cleaved dipeptides, both of which need to be further removed in a subsequent step. A method involving the recirculation of recovered proteins and peptides through the immobilized metal affinity chromatography (IMAC) column (same-column recirculation) was found to be superior to subtractive IMAC for the purpose of contaminant clearance. Recovery of the detagged target proteins was achieved using 10 column volumes of recovery buffer, which had the effect of diluting the imidazole concentration to a suitably low level for contaminant removal by same-column recirculation. This strategy was also applicable at a higher adsorbent loading of 10mg protein/mL adsorbent with an optimal ratio of 200mU of DAPase per mg of adsorbed tagged maltose binding protein (MBP), giving a cleavage yield of 99.1% in 3h. Finally, on-column cleavage conditions including the effect of protease concentration and incubation time on the new strategy have been investigated and comparisons are made for different tag removal strategies. [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 objective of this study was to adapt and improve an environmentally friendly and fast routine method for the analysis of ferulic and p-coumaric acids released from grass cell-walls by alkaline hydrolysis. This methodological development was performed on maize samples selected for their contrasted contents in ferulic and p-coumaric acids as a consequence of their different maturity stages (from stage of 7th leaf with visible ligule to stage of silage harvest). We demonstrate that the Carrez method is an efficient substitute to the common solvent-consuming extraction by ethyl acetate for the preparation of samples suitable for HPLC–ESI-MS analysis. We prove that it is possible to replace methanol by ethanol in the Carrez step and at last we propose a scale reduction of this procedure that offer a first step towards high throughput determinations. The new method leads to a solvent consumption reduced by a factor 100 and only requires ethanol as organic solvent. [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: Agarose coated gigaporous polystyrene microspheres were evaluated as a novel matrix for immobilized-metal affinity chromatography (IMAC). With four steps, nickel ions were successfully immobilized on the microspheres. The gigaporous structure and chromatographic properties of IMAC medium were characterized. A column packed with the matrix showed low column backpressure and high column efficiency at high flow velocity. Furthermore, this matrix was used for purifying superoxide dismutase (SOD), which was expressed in Escherichia coli (E. coli) in submerged fermentation, on an Äkta purifier 100 system under different flow velocities. The purity of the SOD from this one-step purification was 79% and the recovery yield was about 89.6% under the superficial flow velocity of 3251cm/h. In conclusion, all the results suggested that the gigaporous matrix has considerable advantages for high-speed immobilized-metal affinity chromatography. [Copyright &y& Elsevier]

Abstract: Rotavirus VP6 self-assembles into high order macrostructures useful as novel scaffolds for the construction of multifunctional hybrid nanobiomaterials. This application requires large quantities of high quality pure material with strict structural consistency. Strategies for obtaining high quality recombinant VP6 and different characterization techniques are explored and compared in this work. VP6 was expressed in the insect cell–baculovirus system. VP6 assemblies were selectively purified utilizing an ion exchange and size exclusion (SE) chromatography. Purification steps were monitored and characterized by dynamic light scattering (DLS), ELISA, SDS-PAGE, HPLC and Western blot. DLS showed that the initial ultrafiltration step removed small particles, the intermediate anion exchange chromatographic step completely removed the baculovirus, whereas the final size exclusion chromatography permitted the selective recovery of correctly assembled VP6 nanotubes and discrimination of non-assembled VP6, as confirmed by transmission electron microscopy. VP6 assembled into tubular structures with diameter of 75nm and several nanometers in length. The purification yield was 20% of multimeric assemblies with a purity >98%. The resulting material was suitable for the production of functionalized hybrid nanobiomaterials through in situ synthesis of metallic nanoparticles. [Copyright &y& Elsevier]

Abstract: Monacolin K from red yeast rice was extracted by accelerated solvent extraction (ASE). The effects of various extraction parameters including extraction temperature, static extraction time and cycle index on yield were investigated using a DIONEX ASE 300 system to select the optimal conditions by an orthogonal test design L9 (3)3. The optimum extraction conditions were determined as follows: extraction temperature 120°C, static extraction time 7min, and cycle index 3. Under the optimal conditions, the yield of ASE extract and monacolin K was 5.35% and 9.26mg/g of dry red yeast rice, respectively. A separation and purification method of monacolin K was then established using high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (8:2:5:5, v/v/v/v). From 300mg of crude extract, 51.2mg of monacolin K was obtained with the purity of 98.7%. The chemical structure of isolated compound was identified by UV, ESI-MS and 1H NMR. [ABSTRACT FROM AUTHOR]

Abstract: The adsorbent ω-aminodecyl-agarose was evaluated as to its feasibility for the adsorption of human serum and plasma proteins, aiming at the purification of immunoglobulin G (IgG). The contribution of electrostatic and hydrophobic interactions (mixed-mode) and the effects of buffer system on the adsorption of serum proteins were also studied. The adsorption isotherm parameters of human serum albumin (HSA) and IgG were evaluated, pointing to the existence of cooperative effects in the process. A positive (n =2.30±0.38) and negative cooperativity (n =0.63±0.12) were observed for IgG and HSA binding, respectively. High purity IgG was obtained (based on total protein concentration and nephelometric analysis of HSA, transferrin, and immunoglobulins A, G, and M) with a 75% recovery in Hepes 25mmolL−1 pH 6.8 feeding human serum. These results indicate that the use of ω-aminodecyl-agarose is a potential technique for purification of IgG from human serum. [Copyright &y& Elsevier]

Abstract: Purification of target proteins from a crude biological mixture containing proteins, peptides and other biomolecules is the chromatographic challenge. Mixed mode chromatography offers additional selectivities to improve the overall productivity of commercial bioprocesses with novel chromatographic sorbents being introduced to overcome the problem. HEA HyperCel™ (n-hexyl amine) and PPA HyperCel™ (phenyl propyl amine) are industry scalable mixed mode chromatography sorbents where both hydrophobic and electrostatic interactions are predominant. Our study focuses on understanding the underlying mechanism of interaction of protein with the sorbent. Parameters like buffer conditions, pH and temperature were tuned to study the adsorption and desorption conditions of the protein. Dynamic binding capacity of HEA HyperCel™ and PPA HyperCel™ sorbents was studied with human IgG as a model protein. Our study shows that, in HEA the interaction of IgG to the sorbent is predominantly hydrophobic as the binding is enhanced (50–60mg/ml of sorbent) by presence of salt in buffer and increase in temperature. Binding capacity of PPA is 50–60mg/ml of sorbent irrespective of temperature effect and/or the presence of salt. The chromatographic experiments show that the interaction could be hydrophobic or ionic or some charge transfer mechanism depending upon the buffer conditions. [Copyright &y& Elsevier]

Abstract: In this study, thermo-sensitive N-alkyl substituted polyacrylamide polymer PNNB was synthesized by using N-hydroxymethyl acrylamide(NHAM), N-isopropyl acrylamide (NIPA) and butyl acrylate (BA) as monomers, and its low critical solution temperature (LCST) was controlled to be 28°C. The recovery of the thermo-sensitive polymer was over 98%. Butanol as a hydrophobic ligand was covalently attached onto polymer PNNB and butyl ligand density was 80μmolg−1 polymer. The affinity polymer was used for purification of lipase from crude material. Optimized condition was pH 7.0, 35°C adsorption temperature, 120min adsorption time and 0.5mgml−1 initial concentration of lipase. The adsorption isotherm accords with a typical Langmuir isotherm. The maximum adsorption capacity (Q m) of the affinity polymer for lipase was 24.8mgg−1polymer. The affinity copolymer could be recycled by temperature-inducing precipitation and there was only about 6% loss of adsorption capacity after five recyclings. Specific activity of lipase was improved from 14IUmg−1 to 506IUmg−1 protein, and its recovery achieved 82%. The affinity polymer is suitable for the purification of target proteins from the crude material with large volume and dilute solution. [Copyright &y& Elsevier]

Abstract: Lactoferrin is an important nutriceutical with various physiological functions. It is present in whey at very low concentrations. This work describes a mixed-mode (hydroxyapatite) chromatography method for one-column fractionation of lactoferrin from whey. Lactoperoxidase, a protein with similar molecular weight and isoelectric point, was initially desorbed from the matrix under isocratic conditions. Lactoferrin was obtained in homogeneity without lactoperoxidase activity and free from other major whey proteins such as alpha lactoalbumin and beta lactoglobulin. [Copyright &y& Elsevier]

Abstract: Polyamine precipitation conditions for removing host cell protein impurities from the cell culture fluid containing monoclonal antibody were studied. We examined the impact of polyamine concentration, size, structure, cell culture fluid pH and ionic strength. A 96-well microtiter plate based high throughput screening method was developed and used for evaluating different polyamines. Polyallylamine, polyvinylamine, branched polyethyleneimine and poly(dimethylamine-co-epichlorohydrin-ethylenediamine) were identified as efficient precipitants in removing host cell protein impurities. Leveraging from the screening results, we incorporated a polyamine precipitation step into a monoclonal antibody purification process to replace the Protein A chromatography step. The optimization of the overall purification process was performed by taking the mechanisms of both precipitation and chromatographic separation into account. The precipitation-containing process removed a similar amount of process-related impurities, including host cell proteins, DNA, insulin and gentamicin and maintained similar product quality in respect of size and charge variants to chromatography based purification. Overall recovery yield was comparable to the typical Protein A affinity chromatography based antibody purification process. [Copyright &y& Elsevier]

Abstract: A new thermostable Cu/Zn SOD from a thermotolerant yeast strain Kluyveromyces marxianus NBIMCC 1984 has been purified and characterized. The purification procedure comprises thermal treatment and dialysis, ion-exchange chromatography and chromatofocusing. The methodology is a rapid, efficient and highly specific, generating pure preparation (specific activity 996Umg of protein−1) with a yield of 53%. The purified enzyme is a homodimer with Mw of 34034Da and has high N-terminal homology with other yeasts’ Cu/Zn SOD enzymes. The protein is characterized with some unique features such as—thermostability (t 1/2 at 70°C=30min), pH stability in the alkaline range (7.5–8.5) and resistance to inhibitors and variety of chemicals. These characteristics reveal possibilities for wide practical application of K. marxianus Cu/Zn SOD enzyme. [Copyright &y& Elsevier]

Abstract: Countercurrent chromatographic (CCC) separation and purification of various ribonucleases (RNases) was performed using the small-scale cross-axis coil planet centrifuge (X-axis CPC) with aqueous–aqueous polymer phase systems. RNases B and A were well resolved from each other with an aqueous–aqueous polymer phase system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate (pH 9.2) as the mobile lower phase. The commercial RNase A samples obtained from three different companies were also highly purified using the 16.0% (w/w) PEG 1000–6.3% (w/w) dibasic potassium phosphate–6.3% (w/w) monobasic potassium phosphate system (pH 6.6) using the upper phase as the mobile phase. Recombinant RNase Po1, an RNase T1 family enzyme, was further successfully separated from the crude extract using the same solvent system with the lower phase used as the mobile phase. The RNase activities were well preserved during the CCC separation. The overall results demonstrate that the small-scale X-axis CPC is useful for a simple and rapid purification of various RNases in a preparative-scale. [Copyright &y& Elsevier]

Abstract: Water-soluble salvianolic acid A (Sal A) and salvianolic acid B (Sal B) were successfully isolated and purified from the crude extract of Salvia miltiorrhiza by high-speed counter-current chromatography (HSCCC). The solvent system was n-hexane–ethyl acetate–methanol–water (3:6:6:10, v/v/v/v). 4.27mg of Sal A and 32.09mg of Sal B were obtained from 260mg of the crude sample. The purities of Sal A and Sal B were 96.67% and 97.43%, respectively. Their structures were identified by 1H NMR and 13C NMR. Antioxidant activities of Sal A and Sal B were also evaluated and compared by the methods of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS +) radical cation decolourisation assay. Both Sal A and Sal B showed high radical scavenging activities with their EC50 values being 1.43±0.09 and 1.81±0.01μg/ml in DPPH radical method. The ABTS results showed that Sal A and Sal B exhibited high total antioxidant activities, their EC50 values were 1.35±0.00 and 1.43±0.01μg/ml, respectively. [Copyright &y& Elsevier]

Abstract: High-speed counter-current chromatography methods, combined with resin chromatography were applied to the separation and purification of flavonoid glycosides from the Chinese medicinal herb, Radix Astragali. Five flavonoid glycosides, namely calycosin-7-O-β-d-glucoside, ononin, (6aR, 11aR)-9,10-dimethoxypterocarpan-3-O-β-d-glucoside, (3R)-2′-hydroxy-3′,4′-dimethoxyisoflavan-7-O-β-d-glucoside and calycosin-7-O-β-d-glucoside-6′′-O-acetate, were obtained. Among them, calycosin-7-O-β-d-glucoside-6′′-O-acetate was preparatively separated from Radix Astragali for the first time. Their structures were identified by ESI–MS, 1H NMR, 13C NMR, and 2D NMR. [Copyright &y& Elsevier]

Abstract: A highly efficient and low-cost affinity chromatography strategy for lysozyme (LZM) purification is reported. Using tris(hydroxymethyl)aminomethane (Tris) as ligand and macroporous silica spheres as matrix, a novel affinity column was prepared. The high specificity, stability and repeatability of this Tris immobilized affinity column were proved by LZM separations from protein mixture solutions for 20 circles and 6 months test. LZM purified from chicken egg white on the Tris affinity column had even higher purity than the commercial standard and well-maintained activity of 8287U/mg (activity of commercial LZM was 8171U/mg). The efficient affinity process avoiding expensive or fragile ligand would bring advantages to the routine production of LZM from chicken egg white. [Copyright &y& Elsevier]