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• C-phycocyanin (C-PC) was purified by one ion exchange chromatography (IEC) step. • pH gradient elution yielded better purification results than salt gradient elution. • Different parameters were studied to purify C-PC by IEC with pH gradient elution. • C-PC with purities of 4.2 and 3.5 were obtained with overall recovery of 82.1 %. • A single purification step was used to achieve the maximum C-PC purity. C-phycocyanin (C-PC) is a phycobiliprotein that can be used as a natural blue dye in the food and cosmetic industries, as a biomarker or as an agent in medical treatments, depending on its purity grade. Here we described for the first time a single-step purification process of C-PC extracted from the wet biomass of Spirulina (Arthrospira) platensis LEB-52 using ion exchange chromatography with pH gradient elution. Different conditions varying the elution buffers and volumes, the loading pH and the addition of salt in the elution buffer were studied. The chromatographic condition that resulted in high recovery and purity consisted in equilibration and washing with 0.025 mol/L Tris-HCl buffer pH 6.5 and elution combining a step with 0.08 mol/L NaCl in 0.025 mol/L Tris-HCl buffer pH 6.5 and a pH gradient elution with 0.05 mol/L citrate buffer pH 6.2–3.0. This process resulted in C-PC with purities of 4.2 and 3.5 with recoveries of 32.6 and 49.5 %, respectively, in one purification step. [ABSTRACT FROM AUTHOR]

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: Aspergillus niger RNase belonging to RNase T2 family is purified to homogeneity using ammonium sulfate precipitation and anion-exchange chromatography from A. niger. The enzyme is glycosylated monomer with molecule mass of 28.5kDa. Optimum activity of the enzyme is achieved at pH 3.5 and temperature 55°C. The enzyme shows broad substrate specificity with poly (A), poly (C), poly (G), poly (U) and yeast RNA. The enzyme is highly stable against various metal ions and organic solvents, thus dictates its applicability in various industries. Two peptide sequences, PGGTLLQTQFWDYDP and TLDSYTALSDAGITPSEDATYK obtained by MS/MS analysis and confirmed by BLAST search, show substantial sequence homology with Aspergillus phoenicis, Aspergillus clavatus and P. merneffei. Multiple sequence analysis of the peptide sequences of purified enzyme shows 97%, 75% and 72% identity with A. phoenicis, A. clavatus and P. merneffei, respectively using Clustal W tool. Spectroscopic studies by absorbance, fluorescence, and circular dichroism reveal that the enzyme has α+β type secondary structure with approximately 29% α-helix, 24% β-sheet and 47% random coil by K2D software. [ABSTRACT FROM AUTHOR]

Abstract: Negative chromatography antibody purification (N-CAP) using the weak anion exchanger STREAMLINE™ DEAE to extract impurities while retaining the target antibody is proposed as an effective method for the recovery of antibody from rabbit serum. The effects of pH and initial protein concentration on the removal of albumin were investigated. The optimal pH and initial protein concentration for the efficient removal of albumin from rabbit serum were pH 8.0 and 0.5mg/ml, respectively. Under optimal binding conditions, DEAE successfully removed more than 90% of the albumin from rabbit serum with less than 20% IgG loss. This process offered good polyclonal IgG yield of 80% with a purity of 83% and a purification factor of 5.5. The use of a strong anion exchanger like STREAMLINE™ Q XL for albumin removal was also explored. Under similar optimized conditions, albumin removal by Q XL was as high as 90%. However, IgG recovery and purity were reduced to about 70% and 62%, respectively. Thus, N-CAP using the anion exchanger DEAE removes albumin from rabbit serum and thereby offers an efficient means of purifying polyclonal antibodies. [ABSTRACT FROM AUTHOR]

Abstract: Aminopeptidase from a solvent tolerant strain Pseudomonas aeruginosa PseA was purified and studied for its biochemical and molecular characteristics. Ion-exchange chromatography resulted in 11.9-fold purification and 38% recovery of the 56kDa enzyme. The enzyme was found to be stable over a pH range of 6.0–8.0 and appreciably thermostable up to 70°C. PseA aminopeptidase exhibited K m of 3.02mM and V max of 6.71μmol/mg/min towards l-Leu-p-nitroanilide. Remarkable stability in both hydrophilic and hydrophobic solvents makes PseA aminopeptidase unique. Partial N-terminal sequence of enzyme showed exact match with probable aminopeptidase of P. aeruginosa PAO1, coded by gene pepB. Polymerase chain reaction amplified the 1611-bp open reading frame encoding a 57.51kDa, 536 amino acid PseA PepB polypeptide. The deduced PseA PepB protein sequence contained a 24-residue signal peptide (2.57kDa) followed by a 1.28kDa propeptide and a mature product of 500 residues. Search for conserved domain in PseA aminopeptidase explored its place in zinc-metallopeptidase family. Primary sequence analysis showed the hydrophobic inclination of the protein; and the 3D structure modeling elucidated the presence of a high content of hydrophobic residues on its surface probably imparting solvent stability to it. The enzyme might find potential applications in non-aqueous enzymology due to its marked thermostability and striking solvent stability. [Copyright &y& Elsevier]

Abstract: A protease with a molecular mass of 28kDa, designated as hmsp, was isolated from fresh fruiting bodies of the edible mushroom Hypsizigus marmoreus. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, CM-cellulose, and FPLC-gel filtration on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on CM-cellulose. hmsp was thermolabile, and exhibited a temperature optimum at 50°C and a pH optimum at pH 7.5. The activity of the protease was adversely affected by PMSF, EGTA and aprotinin, indicating that it is a serine protease. Based on the N-terminal sequence, the cDNA of hmsp was cloned by using RACE combined with the TAIL-PCR method. The deduced protease sequence contained a signal peptide with 19 amino acids, a pro-region with 82 amino acids, and a mature protease with 285 amino acids and a molecular mass of 28.07kDa. It possessed the three active sites characteristic of the subtilisin family (S8A). hmsp demonstrated 63%, 57% and 44% identity in amino acid sequence respectively to Absp1, Absp2, and Gf-spr1, which are serine proteases from Agaricus bisporus and Grifola frondosa. [Copyright &y& Elsevier]