Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein binds the ribonucleotide substrates while the R2 protein contains a binuclear iron center and a tyrosyl free radical, essential for activity. The crystal structures of the corresponding Escherichia coli proteins suggest that the distance from the active site in R1 to the tyrosyl radical buried in R2 is about 35 A. Therefore, an electron pathway was suggested between the active site and the tyrosyl radical. Such a pathway could include a conserved tryptophan on the suggested R1 interaction surface of R2 and a conserved aspartic acid hydrogen bonded both to the tryptophan and to a histidine iron ligand. To find experimental support for such an electron pathway, we have replaced the conserved tryptophan in mouse R2 with phenylalanine or tyrosine and the aspartic acid with alanine. All the mutated R2 proteins were shown to bind metal with the same affinity as native R2 and to form the binuclear iron center. In addition, the W103Y and D266A proteins formed a normal tyrosyl free radical while only low amounts of radical were observed in the W103F protein. Neither the kinetic rate constants nor the equilibrium dissociation constant of the R1/R2 complex was affected by the mutations as shown by BIAcore biosensor technique. However, all mutant R2 proteins were completely inactive in the enzymatic assay, supporting the hypothesis that the tryptophan and aspartic acid residues are important links in an amino acid residue specific long-range electron transfer.
Microorganisms are known to be natural oil producers in their cellular compartments. Microorganisms that accumulate more than 20% w/w of lipids on a cell dry weight basis are considered as oleaginous microorganisms. These are capable of synthesizing vast majority of fatty acids from short hydrocarbonated chain (C6) to long hydrocarbonated chain (C36), which may be saturated (SFA), monounsaturated (MUFA), or polyunsaturated fatty acids (PUFA), depending on the presence and number of double bonds in hydrocarbonated chains. Depending on the fatty acid profile, the oils obtained from oleaginous microorganisms are utilized as feedstock for either biodiesel production or as nutraceuticals. Mainly microalgae, bacteria, and yeasts are involved in the production of biodiesel, whereas thraustochytrids, fungi, and some of the microalgae are well known to be producers of very long-chain PUFA (omega-3 fatty acids). In this review article, the type of oleaginous microorganisms and their expertise in the field of biodiesel or omega-3 fatty acids, advances in metabolic engineering tools for enhanced lipid accumulation, upstream and downstream processing of lipids, including purification of biodiesel and concentration of omega-3 fatty acids are reviewed.
Isolation of lignins from hardwood and softwood biomass samples, containing 26.1% and 28.1% lignin, respectively, has been performed with the use of alkaline and organosolv pretreatment methods. The effect of catalyst loading, ethanol content, particle size, and pretreatment time on the yields and properties of the isolated lignins were investigated. Alkaline lignins had higher carbohydrate contentup to 30% and exhibited higher molecular weights in the range of 3000 Da, with a maximum phenolic hydroxyl content of 1 mmol g −1 for birch and 2 mmol g −1 for spruce. Organosolv lignins, on the other hand, showed high purity93% or higherdespite the more extensive biomass dissolution into the pretreatment medium; they also exhibited a lower range of molecular weights between 600 and 1600 Da depending on the source and pretreatment conditions. Due to the lower molecular weight, phenolic hydroxyl content was also increased, reaching as high as 4 mmol g −1 with a simultaneous decrease in aliphatic hydroxyl content as low as 0.6 mmol g −1 . Efficient lignin dissolution of 62% for spruce and 69% for birch, achieved at optimal pretreatment conditions, was combined with extensive hemicellulose removal.
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