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Large amounts of 10 Be are produced at the PSI muon production facility by high-energy proton spallation in polycrystalline graphite. For the extraction of 10 Be out of large amounts of carbon, pyrolysis followed by chemical purification has been performed. Approx. 270 g of graphite from Target E92, which had received a total proton charge of 29 Ah between 2002 and 2005, have been burned at 1000 °C in a stream of oxygen. The volatile radioactive oxidation product 3 H 2 O was trapped in 3 water bubblers connected in series. The remainder, a white hygroscopic solid material mainly consisting of 7 Li 2 O, 9/10 BeO and 10/11 B 2 O 3 , was dissolved in HF and subsequently purified by ion exchange chromatography. Radioactive impurities such as 22 Na, 44 Ti, 54 Mn, 60 Co, 101 Rh, 133 Ba and 172 Hf have been separated from the final product. The purified material represents a mixture of approx. 6.5 mg 9 Be and 3.5 mg (3.3 MBq) 10 Be. It is ready to be used for scientific investigations requiring large amounts of this precious isotope. [ABSTRACT FROM AUTHOR]

Three kinds of polysaccharides, namely, BSP1A, BSP2A, and BSP3B, were isolated from raw bamboo shoot (Dendrocalamus latiflorus) after purification and classification by DEAE cellulose-52 (ion-exchange chromatography) and Sephadex G-50. The molecular weights of BSP1A, BSP2A, and BSP3B were 10.2, 17.0 and 20.0 kDa, respectively, which were measured through GPC (gel performance chromtatography) methods. BSP1A contained arabinose, glucose, and galactose in a molar ratio of 1.0:40.6:8.7. BSP2A and BSP3B contained arabinose, xylose, glucose, and galactose in molar ratios of 6.6:1.0:5.2:10.4 and 8.5:1.0:5.1:11.1, respectively. The existence of the O-glycopeptide bond in BSP1A, BSP2A, and BSP3B was demonstrated by β-elimination reaction. FTIR spectra of the three polysaccharides showed that both BSP2A and BSP3B contained β-D-pyranose sugar rings. However, BSP1A exhibited both β-D-pyranose and α-D-pyranose sugar rings. Congo red test indicated that BSP1A and BSP2A displayed triple helix structures, but BSP3B did not. NMR spectroscopy revealed that BSP1A may exhibit a β-1,6-Glucan pyran type as the main link, and few 1,6-glycosidic galactose pyranose and arabinose bonds were connected; BSP2A mainly demonstrated →5)β-Ara(1→and→3)β-Gal(1→connection. Furthermore, BSP3B mainly presented →3)β-Glu(1→and→3)β-Gal(1→connection and may also contain few other glycosidic bonds. [ABSTRACT FROM AUTHOR]

This paper describes the purification and characterization of a novel acid stable and pH tolerant α-amylase from a Aspergillusoryzae JGI 21 isolated from Mangalore. The enzyme displayed a molecular weight of 22 kDa and it was stable over a broad range of acidic and alkaline pH with maximum activity and stability at 6.5. The optimum temperature of enzyme stability was found to be around 24+/-2°C. The purification of α-amylase by ammonium sulphate precipitation and ion-exchange chromatography resulted in 23.56 fold increase in its activity (100.38 U/mg protein). Considering its promising properties, this enzyme can find potential applications in the food industry as well as in laundry detergents. [ABSTRACT FROM AUTHOR]

Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria from samples collected from hyper saline soda lakes (Wadi Natrun Valley, Egypt), resulted in isolation of potent CGTase producing alkaliphilic bacterium, termed NPST-10. 16S rDNA sequence analysis identified the isolate as Amphibacillus sp. CGTase was purified to homogeneity up to 22.1 fold by starch adsorption and anion exchange chromatography with a yield of 44.7%. The purified enzyme was a monomeric protein with an estimated molecular weight of 92 kDa using SDS-PAGE. Catalytic activities of the enzyme were found to be 88.8 U mg-1 protein, 20.0 U mg-1 protein and 11.0 U mg-1 protein for cyclization, coupling and hydrolytic activities, respectively. The enzyme was stable over a wide pH range from pH 5.0 to 11.0, with a maximal activity at pH 8.0. CGTase exhibited activity over a wide temperature range from 45 °C to 70 °C, with maximal activity at 50 °C and was stable at 30 °C to 55 °C for at least 1 h. Thermal stability of the purified enzyme could be significantly improved in the presence of CaCl2. Km and Vmax values were estimated using soluble starch as a substrate to be 1.7 ± 0.15 mg/mL and 100 ± 2.0 μmol/min, respectively. CGTase was significantly inhibited in the presence of Co2+, Zn2+, Cu2+, Hg2+, Ba2+, Cd2+, and 2-mercaptoethanol. To the best of our knowledge, this is the first report of CGTase production by Amphibacillus sp. The achieved high conversion of insoluble raw corn starch into cyclodextrins (67.2%) with production of mainly β-CD (86.4%), makes Amphibacillus sp. NPST-10 desirable for the cyclodextrin production industry. [ABSTRACT FROM AUTHOR]

Abstract: Trypsin was purified from the viscera of barbel by precipitation using ammonium sulphate (0–80%), Sephadex G-100, and Mono Q-Sepharose ion exchange chromatography. The trypsin was purified 27-fold, with 79U/mg specific activity and 31% recovery. The enzyme had a molecular weight of 24kDa; purified trypsin appeared as a single band on native-PAGE. The optimum pH and temperature for enzyme activity were pH 10.0 and 55°C with BAPNA used as a substrate. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECTPYSQ. The Michaelis–Menten constant (K m) and catalytic constant (k cat) values of the enzyme were 0.018mM and 1.21s−1, respectively. The study also investigated the effects of purified trypsin on the recovery of carotenoproteins from shrimp (Parapenaeus longirostris) shells through hydrolysis using 1.0U barbel trypsin/g shrimp shells for 1h at 30°C. The freeze-dried carotenoproteins recovered contained 71.09% protein, 16.47% lipid, 7.78% ash, and 1.79% chitin. [Copyright &y& Elsevier]

Abstract: An extracellular protease from Pseudomonas aeruginosa A2 grown in media containing shrimp shell powder as a unique source of nutriments was purified and characterized. The enzyme was purified to homogeneity from culture supernatant by ultrafiltration, Sephadex G-100 gel filtration and Sepharose Mono Q anion exchange chromatography, with a 2.23-fold increase in specific activity and 64.3% recovery. The molecular mass of the enzyme was estimated to be 34kDa. Temperature and pH with highest activity were 60°C and 8.0, respectively. The protease activity was inhibited by EDTA suggesting that the purified enzyme is a metalloprotease. The enzyme is stable in the presence of organic solvents mainly diethyl ether and DMSO. The lasB gene, encoding the A2 elastase, was isolated and its DNA sequence was determined. The A2 protease was tested for shrimp waste deproteinization in the process of chitin preparation. The percent of protein removal after 3h hydrolysis at 40°C with an enzyme/substrate (E/S) ratio of 5U/mg protein was about 75%. Additionally, A2 proteolytic preparation demonstrated powerful depilating capabilities of hair removal from bovine skin. Considering its promising properties, P. aeruginosa A2 protease may be considered a potential candidate for future use in several biotechnological processes. [Copyright &y& Elsevier]

Abstract: A novel laccase from a white rot fungus Abortiporus biennis was purified following a purification scheme involving ammonium sulfate saturation, ion exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose, and fast protein liquid chromatography-gel filtration on Superdex 75. The molecular mass of the enzyme was 56kDa estimated by SDS-PAGE. The protein was monomeric with an N-terminal amino acid sequence of AIGPVADLTISNG, which exhibited partial homology to the sequence of laccases from family Polyporaceae, but was different from other families. Its optimal pH was 3.0–4.0, and the optimal temperature was 60°C. The laccase displayed a Km value of 33.5μM with ABTS as a substrate at pH 3.5 and 37°C. It demonstrated anti-proliferative activity against Hep G2 and MCF-7 cells with IC50 values of 12.5μM and 6.7μM, respectively, and exhibited inhibitory activity against HIV-1 reverse transcriptase with an IC50 value of 9.2μM. [Copyright &y& Elsevier]

Abstract: An extracellular alkaline protease (Prot-2) selectively secreted by Botrytis cinerea growing in medium containing Spirulina algae as inducer was purified to homogeneity by a combination of ammonium sulfate precipitation, gel filtration and ion-exchange chromatography, followed by size-exclusion chromatography. Prot-2 presented a single 30-kDa band on SDS-PAGE, which showed proteolytic activity following renaturation. Prot-2 has a monomeric structure, is active in the pH range 5.0–9.0 and shows an optimal temperature of activity at 50°C. Prot-2 is thermostable, and activated by Ca2+. The inhibitory action of reducing agents (dithiothreitol or β-mercaptoethanol) was suppressed by dithiobis-nitrobenzoic acid (DTNB) addition, indicating the role played by disulfide in enzyme activity and/or stability. Prot-2 showed extreme stability towards non-ionic surfactants (5% Tween 20, 5% Triton X-100 and Nonidet P-40). It was relatively stable in 25% aqueous/organic solvent mixtures and was activated by oxidizing agents (H2O2 and sodium perborate). A broad specificity of Prot-2 was tested using a range of natural and synthetic oligopeptide substrates. It was further supported by the hydrolysis profile of the insulin B chain by Prot-2. [Copyright &y& Elsevier]

Abstract: A bacterial strain LL1 producing κ-carrageenase was isolated from the decayed seaweed collected from Yellow Sea, China and identified as Pseudoalteromonas porphyrae. The extracellular κ-carrageenase in the supernatant of cell culture of the marine bacterium P. porphyrae LL1 was purified to homogeneity with a 202.6-fold increase in specific κ-carrageenase activity as compared to that in the supernatant by ultrafiltration, gel filtration chromatography, and anion-exchange chromatography. According to the data from sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 40.0kDa. The purified enzyme could actively convert κ-carrageenan into tetrasaccharides, but poorly convert λ-carrageenan. The optimal pH and temperature of the purified enzyme were 8.0 and 55°C, respectively. The enzyme was significantly stimulated by Mg2+ and Ba2+. The enzyme was inhibited by phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid, EDTA, EGTA and 1,10-phenanthroline. The K m and V max values of the purified enzyme for κ-carrageenan were 4.4mg/ml and 0.1mg/minml, respectively. The amino acid sequence (NPQPHIAKPGQTWILQEKRS) of N-terminus of the purified enzyme was identical to that of N-terminus of the deduced protein encoded by the gene encoding κ-carrageenase cloned from the marine bacterium. [ABSTRACT FROM AUTHOR]

Abstract: An organic solvent-tolerant lipase from newly isolated Pseudomonas aeruginosa LX1 has been purified by ammonium sulfate precipitation and ion-exchange chromatography leading to 4.3-fold purification and 41.1% recovery. The purified lipase from P. aeruginosa LX1 was homogeneous as determined by SDS-PAGE, and the molecular mass was estimated to be 56kDa. The optimum pH and temperature for lipase activity were found to be 7.0 and 40°C, respectively. The lipase was stable in the pH range 4.5–12.0 and at temperatures below 50°C. Its hydrolytic activity was found to be highest towards p-nitrophenyl palmitate (C16) among the various p-nitrophenol esters investigated. The lipase displayed higher stability in the presence of various organic solvents, such as n-hexadecane, isooctane, n-hexane, DMSO, and DMF, than in the absence of an organic solvent. The immobilized lipase was more stable in the presence of n-hexadecane, tert-butanol, and acetonitrile. The transesterification activity of the lipase from P. aeruginosa LX1 indicated that it is a potential biocatalyst for biodiesel production. [ABSTRACT FROM AUTHOR]

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: Intrinsically disordered proteins (IDPs) is a term used to describe proteins that do not have a well-defined tertiary structure. IDPs have many roles such as in cell cycle control (p53), neuronal signal transmission (myelin basic protein), and protein stability (dehydrins). Producing recombinant IDPs in bacteria for nuclear magnetic resonance (NMR) studies is problematic because the lack of stable tertiary structure makes them excellent substrates for bacterial proteases, which will cause loss in yield. We have developed a two-step method to produce the grape dehydrin K2 and YSK2 using Escherichia coli. Dehydrins are expressed by certain plants in response to dehydration, increased salinity, or low temperatures. Purification of 10mg/L (K2) and 15mg/L (YSK2) was performed by boiling bacterial pellets to lyse the cells, remove most of the contaminating proteins, and denature bacterial proteases. This resulted in protein purity comparable to that produced by sonication and nickel affinity chromatography. Boiling was followed by cation exchange chromatography to remove the remaining trace contaminants. The sample was shown to be more than 95% pure by reversed-phase high-performance liquid chromatography. The method presented here can easily be adapted to the purification of other IDPs and heat-stable proteins without requiring multiple chromatography steps or the use of protease inhibitors. [Copyright &y& Elsevier]

Abstract: A platelet aggregation inhibitor, named snake venom platelet aggregation dissociator (SV-PAD)-1, with a dissociative reaction of ADP-induced platelet aggregation, was purified from the venom of Protobothrops elegans (Sakishima-habu) by gel-filtration employing Sephadex G-100, and ion-exchange chromatographies using DEAE-Sepharose Fast Flow, CM-Sepharose Fast Flow, and Mono S. By this procedure, about 1.5mg of purified protein was obtained from 1.0g of P. elegans venom. The purified protein showed a single protein band and the molecular weight was about 110kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The pI of purified protein showed four-bands of 7.7, 7.8, 7.95, and 8.15. This protein strongly inhibited ADP-induced platelet aggregation in rabbit platelet-rich plasma (PRP), and its IC50 was about 58nM. It inhibited ristocetin-induced platelet aggregation in rabbit PRP (IC50: 100nM), but hardly blocked collagen-induced platelet aggregation. This protein promptly dissociated platelet aggregation in rabbit PRP stimulated by high-concentration ADP. [Copyright &y& Elsevier]

Abstract: Invertase is a major protein of grape juice and wine. Accordingly, in order to study the biochemical and structural characteristics of this protein and for a better understanding of its physico-chemical properties, large amounts of the pure protein are needed. A simple method for the purification of the grape vacuolar invertase in a preparative-scale is described in this work. The grape protein was isolated and purified from must by ultrafiltration and anion exchange chromatography. The identification and purity determination of the grape invertase fraction were assessed by SDS-PAGE, and were then confirmed using nanoLC-chip-MS/MS analysis. The laboratory fractionation procedure presented in this work generated large quantities of pure grape vacuolar invertase from must. [Copyright &y& Elsevier]

Abstract: A procedure based on BIA Separations CIM DEAE anion-exchange chromatography was developed to separate double-stranded (ds) RNA of hypovirus infecting phytopathogenic fungus Cryphonectria parasitica. Using a linear gradient of 25mM 4-morpholinepropanesulfonic acid (MOPS), pH 7.0 as a binding buffer, and 25mM MOPS, 1.5M NaCl, 0.1mM EDTA, 15% isopropanol (v/v), pH 7.0 as an elution buffer, hypoviral dsRNA was additionally purified from nucleic acid species present in preparations partially purified by standard CF-11 cellulose chromatography. Moreover, crude phenol/chloroform extracts of the fungal tissue were also applied to monolithic supports and CIM DEAE chromatograms revealed clear evidence for hypoviral presence without CF-11 chromatography, nucleic acid precipitation, and electrophoresis. [Copyright &y& Elsevier]

Abstract: The present work deals with the purification and drying of bromelain, one of the vegetal proteases. The purification of the enzyme from fruit Ananas comosus was carried out by precipitation and by chromatography. Further, the drying of chromatographically purified fraction was carried out in freeze dryer and spray dryer. The activity was expressed in terms of casein digesting units (CDU). The bromelain extract of higher purity was obtained from the fruit using ion exchange chromatography. The elution efficiency in chromatographic purification was obtained in the range of 80–90%. The fold purity obtained by chromatography was 3.3 times as compared to that obtained by precipitation. Bromelain thus obtained was subjected to spray and freeze drying. It was observed that the yield obtained in spray dryer at an outlet temperature of 40–50°C was almost 50–70% and in case of freeze dryer the yield obtained was 96%. The resulting residual activity and the specific activity was compared between spray-dried and freeze-dried powder. The purity of enzyme obtained in the present study was found to be 2.8 times more than that of a commercial sample. [Copyright &y& Elsevier]

Polygalacturonase produced by Streptomyces lydicus was purified to homogeneity by ultrafiltration and a combination of ion exchange and gel filtration chromatographic procedures. The purified enzyme was an exo-polygalacturonase with a molecular weight of 43kDa. It was optimally active at 50°C and pH 6.0. The enzyme was stable from pH 4.0 to 7.0 and at or below 45°C for 90min. K m value for polygalacturonic acid was 1.63mg/mL and the corresponding V max was 677.8μM min−1 mg−1. The inhibition constant (K i) for gluconic acid d-lactone was 20.75mM. Purified enzyme had been inhibited by N-bromosuccinimide, while l-tryptophan could induce enzyme activity, indicating the involvement of tryptophan at the active site. [Copyright &y& Elsevier]

The purification and characterization of xylanase from Aspergillus ficuum AF-98 were investigated in this work. The extracellular xylanase from this fungal was purified 32.6-fold to homogeneity throughout the precipitation with 50–80% (NH4)2SO4, DEAE-Sephadex A-50 ion exchange chromatography and Sephadex G-100 chromatography. The purified xylanase (specific activity at 288.7U/ mg protein) was a monomeric protein with a molecular mass of 35.0kDa as determined by SDS-PAGE. The optimal temperature and pH for the action of the enzyme were at 45°C and 5.0, respectively. The xylanase was activated by Cu2+ up to 115.8% of activity, and was strongly inhibited by Hg2+, Pb2+ up to 52.8% and 89%, respectively. The xylanase exhibited Km and V max values of 3.267mg/mL, 18.38M/min/mg for beechwood xylan and 3.747mg/mL, 11.1M/min/mg for birchwood xylan, respectively. [Copyright &y& Elsevier]

A manganese peroxidase (MnP) from a wood-degrading fungus Trichophyton rubrum LSK-27 was purified to homogeneity by anion-exchange chromatography followed by gel filtration. Molecular mass of the purified enzyme was determined to be about 42 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Spectrophotometric analysis of the enzyme revealed one Soret maximum at 407 nm, and two visible peaks at 502 and 644 nm, which are consistent with photometric spectra of other MnPs. Mass spectrometric analysis of the digested protein revealed that it had a very high homology to a unique peroxidase (a hybrid of MnP and lignin peroxidase) from Bjerkandera sp. B33/3. Bjerkandera MnP was able to oxidize veratryl alcohol, whereas T. rubrum LSK-27 MnP could not. T. rubrum LSK-27 MnP had the highest pI of 8.2 among MnPs reported so far. The enzyme was stable at rather high temperatures, and when compared with other MnPs, this MnP was more stable in the presence of high concentrations of H2O2. [Copyright &y& Elsevier]