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Archaeoglobus fulgidus ferritin (AfFtn) is the only tetracosameric ferritin known to forma tetrahedral cage, a structure that remains unique in structural biology. As a result of the tetrahedral (2-3) symmetry, four openings (~45 Å in diameter) are formed in the cage. This open tetrahedral assembly contradicts the paradigm of a typical ferritin cage: a closed assembly having octahedral (4-3-2) symmetry. To investigate the molecular mechanism affecting this atypical assembly, amino acid residues Lys-150 and Arg-151 were replaced by alanine. The data presented here shed light on the role that these residues play in shaping the unique structural features and biophysical properties of the AfFtn. The x-ray crystal structure of the K150A/ R151A mutant, solved at 2.1 Å resolution, indicates that replacement of these key residues flips a "symmetry switch." The engineered molecule no longer assembles with tetrahedral symmetry but forms a typical closed octahedral ferritin cage. Small angle x-ray scattering reveals that the overall shape and size of AfFtn and AfFtn-AA in solution are consistent with those observed in their respective crystal structures. Iron binding and release kinetics of the AfFtn and AfFtn-AA were investigated to assess the contribution of cage openings to the kinetics of iron oxidation, mineralization, or reductive iron release. Identical iron binding kinetics for AfFtn and AfFtn-AA suggest that Fe2+ ions do not utilize the triangular pores for access to the catalytic site. In contrast, relatively slow reductive iron release was observed for the closed AfFtn-AA, demonstrating involvement of the large pores in the pathway for iron release. [ABSTRACT FROM AUTHOR]

Summary: The first ribonuclease (RNase) from the Cytophaga–Flavobacterium–Bacteroides phylum, dominant in the marine environment, and also from the first Bizionia species isolated from the tropics was purified and characterized. Extracellular RNase production occurred when the culture medium contained 5–7% (w/v) NaCl. The 53.0kDa enzyme was purified 29 folds with a recovery of 4% and specific activity of 630unit/mg protein. The pH and temperature optima are 6.5 and 35°C, respectively and the enzyme retains more than half of its activity (relative to optimal assay conditions) after 1h pre-incubation separately with 5% (w/v) NaCl or from pH 5.0 to 8.5 or at 50°C. Dithiothreitol and β-mercaptoethanol do not inhibit whereas human placental RNase inhibitor protein halves the RNase activity. While Mg2+, Ba2+ and Ca2+ enhanced the enzyme activity, Fe2+, Cu2+ and Hg2+ inactivated it. This RNase degrades uracil containing nucleic acids only. Our isolate could be a novel renewable source of deoxyribonuclease (DNase) -free RNase enzyme. [Copyright &y& Elsevier]

Abstract: A Bacillus sp. isolated from the Sundarbans region of the Bay of Bengal (NCBI GenBank Accession no. AY723697 ) which can tolerate 10% (w/v) NaCl, produces esterase optimally in Marine Broth 2216 medium containing 1% (w/v) NaCl. The enzyme was purified 42.7-fold with 6.4% recovery, (specific activity 569.2U/mg protein) by ammonium sulphate precipitation followed by anion and cation exchange chromatography. The serine type esterolytic enzyme has a molecular weight of 35.0kDa and is denatured into polypeptides of molecular weights 20kDa and 15kDa. The esterase was most active at pH 8.0, the pH of the seawater at the site of collection and is stable in the pH range 6.0–9.0. The optimum temperature of activity of this esterase is 45°C and the enzyme is very stable after 1h pre-incubation at 50°C. Our esterase shows about 100% activity when incubated with 1M NaCl, the activity drops to about 50% when incubated with 2.5M sodium chloride and the enzyme is completely inactivated when 4M NaCl is present during reaction. The esterase is almost inactivated by Ca2+, Hg2+ and Fe3+ ions, reducing agents and detergent. Interestingly, Co2+, a known inhibitor of many enzymes, preserved 70% of the activity of this esterase. Specific activity of the esterase increases more than twofold in the presence of water-miscible organic solvents as compared to that in aqueous buffer. When incubated for a period of 10 days in the presence of 30–70% dimethylsufoxide (DMSO), the specific activity increased by approximately two–threefold compared to the enzyme in aqueous buffer throughout the period of study. Specific activity between 1283 and 525U/mg was maintained by our enzyme when incubated with 50% DMSO for 10 days. The enzyme was most active on p-nitrophenyl acetate, ethyl acetate, alpha isomer of naphthyl acetate but shows relatively lesser activity towards triglycerides of fatty acids. Certain characteristics, such as molecular weight, effects of NaCl, metal ions (Zn2+ and Mg2+) and reactivity towards para-nitrophenyl and aliphatic esters were strikingly similar to already described marine bacterial derived esterases. Extreme stability in DMSO could make this enzyme a potential immobilized biocatalyst for application in non-aqueous based continuous bioprocesses. Higher specific activity and purification factor, better thermo tolerance and solvent stability would make our enzyme more attractive for biotechnological applications than the marine microbial derived esterases described so far. [Copyright &y& Elsevier]

Abstract: In this communication we report purification and characterization of a single salt, solvent, detergent and bleach tolerant alkaline serine protease produced by a truly marine bacterium. Based on the 16S rRNA gene sequence, absence of polyhydroxybutyrate accumulation, growth with very high NaCl levels as well as with hydrocarbons as sole carbon source, the isolate was classified as a new bacterium belonging to the gamma-Proteobacteria family. A 69-fold purification (specific activity 791.7U/mg protein, unit expressed as μmole of tyrosine liberated per minute) was achieved by a three-step purification procedure. The enzyme is active over a broad range of pH (6.0–11.0), the optimum being at 9.0. This protease enzyme shows activity from 30 to 70°C and Ca2+ increase the thermostability. This enzyme exhibits appreciable activity in presence of up to 30% NaCl and is highly stable even after 18h pre-incubation with 35% (w/v) NaCl. While Ba2+ and Ca2+ enhance the enzyme activity, heavy metals like Co2+, Zn2+, Hg2+ inactivate the enzyme. The enzyme is completely stable in presence of laboratory detergents (Tween 80 and Triton X-100), oxidizing agents, reducing agents, commercial detergents and bleaches (hydrogen peroxide and sodium perborate) after 1h of pre-incubation. Water miscible and immiscible organic solvents like ethylene glycol, ethanol, butanol, acetone, DMSO, xylene and perchloroethylene enhance as well as stabilize the enzyme activity. Reflectance measurements during wash performance studies show that our enzyme is capable of almost complete removal of recalcitrant blood and egg stains in both wet and dry wash operations. Enzymatic activity against a wide variety of substrates indicates that our enzyme can be investigated for a range of commercial applications especially for soy protein and gelatin hydrolysis in the food processing industry as well as for the dehairing process in the leather industry in addition to catalyzing hydrolysis reactions at high salt concentrations. [Copyright &y& Elsevier]