Search

Silver-111 (111Ag, t 1/2 = 7.47 d) is a β − emitter suitable for targeted cancer therapy due to favourable decay properties. The production of no-carrier added 111Ag via Isotope Separation On-Line (ISOL) technique is being investigated at the Legnaro National Laboratories of the Italian Institute of Nuclear Physics (ISOLPHARM project). Stable Cadmium-111 (111Cd) is co-produced as isobaric contaminant, hence a chemical separation process must be developed to selectively harvest 111Ag. In this study, a chromatographic procedure employing the commercially available CL resin was investigated by using stable Ag+ and Cd2+. Results indicate that CL resin allows to efficiently separate Ag+ from Cd2+ and recover the former with high yields. • Silver-111 (t 1/2 = 7.47 days) is a β − emitting radionuclide suitable for cancer therapy. • During 111Ag production via ISOL method, 111Cd is co-produced. • Chromatographic method using commercially available CL resin was developed to separate silver from cadmium. [ABSTRACT FROM AUTHOR]

The extraction condition of curcumin from Curcuma longa L was optimized through four factors and three levels orthogonal experiment based on the results of single factor tests. Under the optimal conditions: the concentration of ethanol 80%, extraction temperature 70°C, the ratio of liquid to material 20, and extraction time 3 h, a crude extract with the yield of curcumin 56.8 mg/g could be obtained. The isolation and purification of curcuminoids from the crude extract was performed on high performance counter current chromatography employing an optimized solvent system n‐hexane/ethyl acetate/methanol/water (2/3/3/1, v/v/v/v). From 97 mg crude sample (in which the purity of curmumin was 68.56%), 67 mg curmumin, 18 mg demethoxycurcumin, and 9.7 mg bisdemethoxycurcumin with a high‐performance liquid chromatography purity of 98.26, 97.39, and 98.67%, respectively, were obtained within 70 min. The antioxidant activities and cytotoxicity of purified curcumin was comparable to that of the commercial product, indicating that the biological activity of curcumin could be maintained by this method. [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]

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]

With the increasing global demand for precise and efficient pharmaceuticals and the biopharma industry moving towards Industry 4.0, the need for advanced process integration, automation, and modeling has increased as well. In this work, a method for automatic modeling and calibration of an integrated preparative chromatographic system for pharmaceutical development and production is presented. Based on a user-defined system description, a system model was automatically generated and then calibrated using a sequence of experiments. The system description and model was implemented in the Python-based preparative chromatography control software Orbit. [ABSTRACT FROM AUTHOR]

A chromatographic procedure for Rb, K, Ba and Sr one-by-one separation from geological materials has been developed by using a single column with packing Sr-spec resin, followed by high-precision Rb isotope measurement by multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS). The main matrix elements (Ti, Al, Mg, Fe, Mn, Na and Ca) were removed with 4.4 mL of 3 M HNO3 first; Rb and K were then sequentially eluted in different volumes with the same acid. After that, Ba was eluted with 8 M HNO3, and finally Sr was eluted with Milli-Q water. This procedure enables us to collect pure Rb, K, Ba and Sr one by one in a single column with recovery close to 100%. The purified Rb was measured by using a standard-sample bracketing method via MC-ICP-MS. The short-term precision for δ87Rb was better than ±0.03‰ (2SD, for 15 hours), and the long-term (more than 12 months) external precision was better than ±0.06‰ (2SD) based on repeated analysis of Rb standard solution NIST SRM 984. Matrix effects on Rb isotope measurement by MC-ICP-MS were tested by doping pure Rb standard with various amounts of matrix elements and were found to be insignificant when Na/Rb ＜ 2, Ca/Rb ＜ 4 and K/Rb ＜ 25. We measured a set of geological reference standards by this method and found significant Rb isotopic variation. Our study suggests that Rb isotope geochemistry may be a promising tracer of various cosmochemical and geological processes. [ABSTRACT FROM AUTHOR]

A new protease was isolated from the intestine of small red scorpionfish. After ammonium sulfate precipitation, the enzyme was purified to homogeneity by a two-step chromatography with 6.69% recovery and fivefold increase in specific activity. The molecular weight of the protease was about 24 kDa as estimated by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme optimum pH and temperature were pH 10.0 and 40°C, respectively. The protease was stable at temperatures below 40°C and over a broad pH range (8.0-11.0). The Km and Kcat values were 0.36 mmol L-1 and 1.61 s-1, respectively. Interestingly, the enzyme presented specificity for casein, egg albumin, bovine serum albumin, and gelatin with obtained degrees of hydrolysis ranging from 3.14% to 6.58%, after 2 h of hydrolysis. Moreover, gluten hydrolysate analysis confirmed protein hydrolysis and solubilization. These results suggested the potential use of the protease in the preparation of food protein hydrolysates with interesting properties. [ABSTRACT FROM AUTHOR]

In this paper, sample preparation protocols based on common cation-exchange and Sr-specific extraction chromatographic resin were evaluated and investigated for natural geological samples prior to 87Sr/86Sr ratio measurement using MC-ICP-MS or TIMS. Several CRMs and real geological samples were digested using HF, HNO3 and HClO4 in closed vessels prior to sequential chemical purifications and then Sr isotopic ratios were determined by MC-ICP-MS or TIMS. Because HREEs reside in the Sr fraction when common cation-exhange resin is used, this purification technique is unsuitable prior to MC-ICP-MS analysis, as doubly loaded HREE interfere on Sr masses. We observe an obviously positive relationship between the radiogenic 87Sr/86Sr ratio and the content of HREEs in the Sr fractions, as well as a negative relationship between un-radiogenic 84Sr/86Sr or 84Sr/88Sr ratios and the content of HREEs in the Sr fractions during MC-ICP-MS Sr isotopic measurement. Such effects are insignificant for TIMS measurement, because ionization temperatures are generally lower and can be well controlled during the analyses. In contrast to the traditional Sr purification method (cation exchange resin), Sr-specific extraction chromatographic resin produces high purity Sr fractions, making it feasible for both MC-ICP-MS and TIMS. [ABSTRACT FROM AUTHOR]

Dual flow chromatography (DFC) separations are performed with back and forth flow for rapid method development, design of experiments (DOE), quality-by-design (QbD), or high-throughput chromatographic purification. Although different than conventional unidirectional flow through chromatography, chromatographic principles still control the separations. Selectivity coefficients and Langmuir adsorption isotherms control the separation chemistry properties of the column and dictate the mobile phase conditions needed to achieve separation. However, the kinetic rates of diffusion and interaction of mobile phase molecules with the stationary phase, column channeling, and other column properties are not germane to the practice of DFC. Chromatographic conditions developed with DFC can be scaled to any size, including laboratory and industrial preparative columns. One practical use of DFC is in the pharmaceutical industry where multidimensional method development and high-throughput, parallel processing has been used to improve R&D productivity and decrease the time to manufacturing of biopharmaceutical drugs. [ABSTRACT FROM AUTHOR]

Given recent advances in automated oligosaccharide synthesis, analytical techniques that can be coupled to a synthetic framework are needed to not just identify but also purify to homogeneity protected carbohydrate compounds at levels of ≥99.5% purity. Herein, an alternate-pump recycling high-performance liquid chromatography (R-HPLC) method has been developed to allow purification of protected carbohydrates at levels of ≥99.5% purity. [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]

A novel method for Fmoc/ tBu solution-phase peptide synthesis and the development of a new benzyl-type group-assisted purification (GAP) protecting group is reported. This GAP protecting group is utilized in place of a polymer support, facilitating C→N Fmoc peptide synthesis without chromatography or recrystallization. The GAP group can be added and removed in high yield, and was used to synthesize over 1 gram of the immunostimulant, thymopentin, in high overall yield (83 %) and purity (99 %). [ABSTRACT FROM AUTHOR]

The interest of pharmaceutical companies for complementary high-performance chromatographic tools to assess a product's purity or enhance this purity is on the rise. The high-throughput capability and economic benefits of supercritical fluid chromatography, but also the 'green' aspect of CO2 as the principal solvent, render supercritical fluid chromatography very attractive for a wide range of pharmaceutical applications. The recent reintroduction of new robust instruments dedicated to supercritical fluid chromatography and the progress in stationary phase technology have also greatly benefited supercritical fluid chromatography. Additionally, it was shown several times that supercritical fluid chromatography could be orthogonal to reversed-phase high-performance liquid chromatography and could efficiently compete with it. Supercritical fluid chromatography is an adequate tool for small molecules of pharmaceutical interest: synthetic intermediates, active pharmaceutical ingredients, impurities, or degradation products. In this review, we first discuss about general chromatographic conditions for supercritical fluid chromatography analysis to better suit compounds of pharmaceutical interest. We also discuss about the use of achiral and chiral supercritical fluid chromatography for analytical purposes and the recent applications in these areas. The use of preparative supercritical fluid chromatography by pharmaceutical companies is also covered. [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]

Pulmonary surfactant preparations extracted from natural sources have been used to treat millions of newborn babies with respiratory distress syndrome (RDS) and can possibly also be used to treat other lung diseases. Due to costly production and limited supply of animal-derived surfactants, synthetic alternatives are attractive. The water insolubility and aggregation-prone nature of the proteins present in animal-derived surfactant preparations have complicated development of artificial surfactant. A non-aggregating analog of lung surfactant protein C, SP-C33Leu is used in synthetic surfactant and we recently described an efficient method to produce rSP-C33Leu in bacteria. Here rSP-C33Leu obtained by salt precipitation of bacterial extracts was purified by two-step liquid gel chromatography and analyzed using mass spectrometry and RP-HPLC, showing that it is void of modifications and adducts. Premature New Zealand White rabbit fetuses instilled with 200mg/kg of 2% of rSP-C33Leu in phospholipids and ventilated with a positive end expiratory pressure showed increased tidal volumes and lung gas volumes compared to animals treated with phospholipids only. This shows that rSP-C33Leu can be purified from bacterial lipids and that rSP-C33Leu surfactant is active against experimental RDS. [ABSTRACT FROM AUTHOR]

A methodology to purify a Ho solution has been developed by a combination of activity and mass concentration measurements in order to further determine the Ho half-life. The isobaric interference at m/ q ≃ 166 requires Ho purification from non-natural Er with a high purification degree due to the large amount of Ho as opposed to Er. The Ho/Er separation was achieved using high-performance liquid chromatography on a semi-preparative column followed by purification on gravitational chromatography. The efficiency of the separation was evaluated after precise determination of the Er isotopic composition. The purification methodology enabled to separate Ho from Er. [ABSTRACT FROM AUTHOR]

Flavonoids are the main chemical constituents ofSmilax chinaL. and thought to be responsible for the anti-inflammatory activity ofSmilax chinaL. In this study, a suitable HSCCC method was developed to separate the three main flavonoids in a one-separation from the anti-chronic pelvic inflammation disease (CPID) effective fraction ofSmilax chinaL. with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:10:1:10, v/v/v/v). 42 milligrams of astilbin, 10 mg neoisoastilbin, and 6 mg quercetin-3-O-α-L-rhamnoside were separated from 200 mg of the anti-CPID effective fraction ofSmilax chinaL. with purity of 98.3%, 98.7%, 98.5%, respectively, by HPLC analysis. Orthogonal test L9(34) was also applied to investigate the optimum extracting conditions of the three flavonoids. HSCCC was successfully used for the isolation and purification of the three flavonoids from the anti-CPID effective fraction ofSmilax chinaL. and the neoisoastilbin was first separated fromSmilax chinaL. [ABSTRACT FROM AUTHOR]

Forsythoside A and suspensaside A were isolated and purified fromForsythia suspensaby one step of high-speed counter-current chromatography for the first time using a solvent system of ethyl acetate-n-butanol-methanol-water (4:0.5:0.5:5,v/v). The purities of the two compounds were 98.19% and 98.05%, respectively, as determined by HPLC. Their structures were identified by MS, UV, and NMR analysis. Such a simple and effective method was fairly useful to prepare pure compounds as reference substances for related study onForsythia suspensa. [ABSTRACT FROM PUBLISHER]

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]

An effective high–speed counter–current chromatography (HSCCC) method was established for semi-preparative isolation and purification of bioactive chemical constituents from the Tibetan medicinal plantPedicularis longifloravar.tubiformis(Klotzsch) P. C. Tsoong. With a two-phase solvent system composed of chloroform–methanol–water (8:4:5, v/v), 40 mg of extract was separated to yield luteolin (12.5 mg), apigenin (9.6 mg), and chrysoeriol (4.8 mg), with purities of 99.3, 98.2, and 98.6%, respectively, as determined by high-performance liquid chromatography (HPLC). The chemical structures of these three components were identified by1H NMR and13C NMR. [ABSTRACT FROM PUBLISHER]

Perchloric acid was found to be a suitable medium for the quantitative leaching of Sr(II) from homogeneous and calcined (Th,Sr)O particularly with respect to the contamination from Th(IV). Sr is a cause of major concern to the environment due to its long half life (28.6 years), significant abundance in large inventory of spent nuclear fuels (~350 thousand tons) awaiting geological disposal and its chemical similarity to Ca(II), an essential element for the living beings. Application of Sr as a parent radionuclide for Y (used in therapy radiopharmaceuticals) is possible provided it can be made available at desired high purity. In this context, the distribution coefficients of Sr(II), Th(IV), Zr(IV), Y(III), Eu(III) and Cs(I) were determined using Sr selective crown ether 4,4′(5′)-di -tert-butyl -dicyclohexano -18 -crown -6 by solid-liquid extraction in perchloric acid medium. Feasibility of employing extraction chromatography using Sr selective resin for the recovery and purification of Sr(II) from leached perchloric acid medium was explored. Perchloric acid medium is better than nitric acid medium for the uptake of Sr by Sr selective chromatographic resin under varying loading conditions of Sr(II). Similarly pH 2 solution appears better eluent of Sr(II) than distilled water. Present work offers a novel approach for setting up a Sr-Y generator. [ABSTRACT FROM AUTHOR]

The present paper discusses a comparative study concerning performance efficiency and sustainable impact of three purification processes for degenotoxification of Active Pharmaceutical Ingredients (APIs) post reaction streams: recrystallization, flash chromatography and organic solvent nanofiltration (OSN). Two case studies in each process were selected for evaluation of the separation technologies featuring the same model API being Mometasone furoate (Meta) glucocorticoid and two genotoxic impurities (GTIs). Methyl mesylate (MeMS) and 4-dimethylaminopyridine (DMAP) were chosen as model impurities due to their incidental appearance in glucocorticoids based on a common methanesulfonylation manufacturing step. Successful degenotoxification was achieved in all cases concerning DMAP and MeMS reaching final GTI levels below the regulatory thresholds with the exception of DMAP using recrystallization. API losses were 5% and 6.4% for OSN, 6.4% and 11.9% for flash chromatography and 14.9% and 16.4% for recrystallization during the removal of DMAP and MeMS, respectively. The API loss occurring during the purification processes has a significant impact on the outcome of cost analysis. Mass and carbon intensity values are highest for OSN and lowest for recrystallization, while flash chromatography has intermediate values. Although recrystallization is more time consuming, it should not be discarded without careful analysis since API loss often declines at larger scales and it generally gives the API in the desired crystallographic form. Solvent recycling has a significant impact on the sustainability of all the processes by the reduction of mass intensity by two orders of magnitude (from 400–1300 to 14–63 kg/kg-API, depending on the process) and narrowing down carbon intensity to the range of 100–200 kg-CO2/kg-API. OSN requires the use of 7 diavolumes, therefore its high performance is achieved at the cost of high solvent usage. Hence, solvent recycling also has a higher positive environmental impact on OSN. [ABSTRACT FROM AUTHOR]

In this study; sheep carbonic anhydrase-II (SCA-II) (E.C: 4.2.1.1) was purified from sheep liver and in vitro effects of heavy metals on the enzyme was examined. SCA-II isozyme was purified with about 203 purification fold, a specific activity of 2320 EU/mg-protein and a yield of 72 by using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. Purity of the SCA-II enzyme was verified by SDS-PAGE technique and subunit molecular mass of the enzyme was found as 29 kDa. In addition to this, inhibitory effects of some metal ions on the enzyme were examined. In this study, sheep liver tissue was chosen; because the liver is an organ in which metal wastes of air, water and food are collected and it is easy to obtain the liver tissue. Because of the very important duties of CA enzyme on living beings, the effect of metals on the CA enzyme was investigated. [ABSTRACT FROM AUTHOR]

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: 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: 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: 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: 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: 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: 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: 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: 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]

Semi-preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of oridonin from Isodon rubescens by using a two-phase-solvent system composed of n-hexane-ethyl acetate-methanol-water (2.8:5:2.8:5, v/v/v/v). The targeted compound isolated, collected and purified by HSCCC was analyzed by high performance liquid chromatography (HPLC). A total of 40.6 mg of oridonin with the purity of 73.5% was obtained in less than 100 min from 100 mg of crude Isodon rubescens extract. The chemical structure of the compound was identified by IR, ¹H-NMR and 13C-NMR. [ABSTRACT FROM AUTHOR]

New regio- and chemoselective [4+2+1] domino cyclization consisting of the formation of two spiro rings and five σ bonds has been developed for the synthesis of spiro-substituted benzo[b]furo[3,4-e][1,4]diazepine derivatives. The reaction is a multicomponent domino green process and can be readily performed by reacting inexpensive starting materials of benzene-1,2-diamines, tetronic acid and 2,2-dihydroxy-2H-indene-1,3-dione in aqueous solution under microwave irradiation. The present synthesis shows attractive characteristics, such as the use of water as reaction media, convenient one-pot operation, short reaction periods of 10–18 min and reduced waste production without the use of any strong acids or metal promoters. This synthesis serves as a nice addition to GAP (Group-Assistant-Purification) chemistry in which purification viachromatography and recrystallization can be avoided, and the pure products were obtained simply by washing the crude products with 95% EtOH. [ABSTRACT FROM AUTHOR]

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: 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]

Three flavonoid glycosides including quercetin-3-O-[2''-O-(6'''-O-E-sinapoyl)-β-D-glucopyranosyl]-β-D-glucopyranoside(I), quercetin-3-O-[2''-O-(6'''-O-E-feruloyl)-β-D-glucopyranosyl]-β-D-glucopyranoside(II) and quercetin-3-O-[2''-O-(6'''-O-E-feruloyl)-β-D-glucopyranosyl]-β-D-galactopyranoside(III) were isolated and purified from Hedyotis diffusa Willd by high-speed counter-current chromatography (HSCCC). This run was carried out with a two-phase solvent system composed of n-hexane-ethyl acetate-n-butanol-methanol-1.0% acetic acid (1:1:3.5:1:4.5, v/v) by eluting the lower phase as the mobile phase with a flow-rate at 2.0 ml/min. Consequently, 29.6 mg of I, 35.1 mg of II, 41.3 mg of III with purities of over 95% were obtained from 200 mg of the crude extracts in a single run in less than 130 min. The structure of the isolated compounds was confirmed by MS, 1H NMR, and 13C NMR analysis. [ABSTRACT FROM AUTHOR]

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: 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]

Synthetic oligodeoxynucleotides are purified with use of a catching by polymerization, washing, and releasing approach. The method does not require any chromatography, and purification is achieved by simple operations such as shaking, washing, and extraction. It is therefore useful for large-scale purification of synthetic oligonucleotide drugs. In addition to purification of oligonucleotides, this catching by polymerization concept is expected to be equally useful for purification of other synthetic oligomers such as peptides and oligosaccharides. [ABSTRACT FROM AUTHOR]

Abstract: We report an improved procedure for purification of the ω subunit of Escherichia coli RNA polymerase. In contrast to the original procedure, the revised procedure: (i) allows purification of ω entirely from the soluble fraction, obviating the need for denaturation/renaturation, (ii) results in >99% pure ω in only two chromatographic steps, and (iii) improves the yield of purified ω by at least 5-fold. Reconstitution of E. coli RNAP from ω purified by this procedure, as well as purified sigma and core RNAP lacking ω, produces active holoenzyme in vitro, and co-overexpression of ω from a plasmid containing rpoZ and an additional plasmid encoding the other RNAP core subunits results in production of active core enzyme in vivo. [Copyright &y& Elsevier]

Purification tags are robust tools that can be used to purify a wide selection of target proteins, which makes them attractive candidates for implementation into platform processes. However, tag removal remains an expensive and significant issue that must be resolved before these tags can become widely used. One alternative is self-cleaving purification tags, which can provide the purity and versatility of conventional tags but eliminate the need for proteolytic tag removal. Many of these self-cleaving tags are based on inteins, but other emerging technologies, such as the FrpC and SrtAc proteins, have also been reported. In this review, we cover affinity and non-chromatographic self-cleaving purification tags and their potential industrial applications. [Copyright &y& Elsevier]

Abstract: We developed streamlined, automated purification protocols for the production of milligram quantities of untagged recombinant human cyclophilin-A (hCypA) and untagged human proliferating cell nuclear antigen (hPCNA) from Escherichia coli, using the ÄKTAxpress™ chromatography system. The automated 2-step (cation exchange and size exclusion) purification protocol for untagged hCypA results in final purity and yields of ⩾93% and ∼5mgL−1 of original cell culture, respectively, in under 12h, including all primary sample processing and column equilibration steps. The novel automated 4-step (anion exchange, desalt, heparin-affinity and size exclusion, in linear sequence) purification protocol for untagged hPCNA results in final purity and yields of ⩾87% and ∼4mgL−1 of original cell culture, respectively, in under 24h, including all primary sample processing and column equilibration steps. This saves in excess of four full working days when compared to the traditional protocol, producing protein with similar final yield, purity and activity. Furthermore, it limits a time-dependent protein aggregation, a problem with the traditional protocol that results in a loss of final yield. Both automated protocols were developed to use generic commercially available pre-packed columns and automatically prepared minimal buffers, designed to eliminate user and system variations, maximize run reproducibility, standardize yield and purity between batches, increase throughput and reduce user input to a minimum. Both protocols represent robust generic methods for the automated production of untagged hCypA and hPCNA. [Copyright &y& Elsevier]

Black currant is an important material in the food industry, but little research has been reported on the isolation of phenolic acids because of their low content. In the present study, high-speed countercurrent chromatography (HSCCC) has been successfully used for the preparative isolation of the minor phenolic compounds from the ethyl acetate extracts of black currant fruit. The HSCCC separation was performed with a two phase solvent system composed of n-hexane/EtOAc/MeOH/H2O (5:15:4:7 v/v) at a flow rate of 1.5 mL/min. From a 500 mg crude sample 0.8 mg of protocatechuic acid, 1.0 mg of caffeic acid, 0.5 mg of 4-hydroxybenzoic acid, and 2.5 mg of myricetin were purified by a one step HSCCC operation,. Their chemical structures were confirmed by MS and NMR. [ABSTRACT FROM AUTHOR]

Abstract: HSCCC technique in a semi-preparative scale was successfully applied in isolation and purification of nootkatone from the essential oil of fruits of Alpinia oxyphylla Miquel. Twelve kinds of two-phase solvent systems, consisting of seven non-aqueous and five organic-aqueous solvent systems, were selected with not only suitable partition coefficients of nootkatone but also suitable separation factors between nootkatone and valencene, the dominant impurity in the essential oil. Further on HSCCC, n-hexane–chloroform–acetonitrile (10:1:10, v/v) amongst the non-aqueous solvent systems and n-hexane–methanol–water (5:4:1, v/v) amongst the organic-aqueous solvent systems were separately screened out. However, n-hexane–methanol–water (5:4:1, v/v) was thought optimal due to quite shorter elution time and better HSCCC peak form. By eluting the lower phase of this solvent system in head–tail mode, 3.1mg of nootkatone was obtained at a purity of 92.30% by GC–MS from 80mg of crude essential oil in one step operation in less than 4h. The chemical structure of nootkatone fraction was confirmed by EI-MS and 1H NMR. [Copyright &y& Elsevier]

Abstract: Two chymotrypsins (chymotrypsin A and B) have been purified to homogeneity from the hepatopancreas of crucian carp (Carassius auratus) by ammonium sulphate fractionation and chromatographies on DEAE-Sepharose, Sephacryl S-200 HR, Phenyl-Sepharose and SP-Sepharose. The molecular masses of chymotrypsin A and B were approximately 28 and 27kDa, respectively, by SDS–PAGE. Purified chymotrypsins also revealed single bands by native-PAGE. Optimum temperatures of chymotrypsin A and B were 40 and 50°C, and optimal pHs were 7.5 and 8.0 using Suc-Leu-Leu-Val-Tyr-AMC as substrate. Both enzymes were effectively inhibited by serine proteinase inhibitors and slightly activated by metal ions such as Ca2+ and Mg2+, while inactivated by Mn2+, Cd2+, Cu2+, Fe2+ to different degrees. Apparent K ms of chymotrypsin A and B were 1.4 and 0.5μM, and K cats were 2.7S−1 and 3.4S−1, respectively. Immunoblotting analysis using anti-chymotrypsin B weakly cross reacted with chymotrypsin A. [Copyright &y& Elsevier]