Microbial desulfurization of organic sulfur compounds in petroleum

Sulfur removal from petroleum is important from the standpoint of the global environment because the combustion of sulfur compounds leads to the production of sulfur oxides, which are the source of acid rain. As the regulations for sulfur in fuels become more stringent, the existing chemical desulfurizations are coming inadequate for the "deeper desulfurization" to produce lower-sulfur fuels without new and innovative processes. Biodesulfurization is rising as one of the candidates. Several microorganisms were found to desulfurize dibenzothiophene (DBT), a representative of the organic sulfur compounds in petroleum, forming a sulfur-free compound, 2-hydroxybiphenyl. They are promising as biocatalysts in the microbial desulfurization of petroleum because without assimilation of the carbon content, they remove only sulfur from the heterocyclic compounds which is refractory to conventional chemical desulfurization. Both enzymological and molecular genetic studies are now in progress for the purpose of obtaining improved desulfurization activity of organisms. The genes involved in the sulfur-specific DBT desulfurization were identified and the corresponding enzymes have been investigated. From the practical point of view, it has been proved that the microbial desulfurization proceeds in the presence of high concentrations of hydrocarbons, and more complicated DBT analogs are also desulfurized by the microorganisms. This review outlines the progress in the studies of the microbial desulfurization from the basic and practical point of view.     

Flavin reductase coupling with two monooxygenases involved in dibenzothiophene desulfurization: purification and characterization from a non-desulfurizing bacterium,Paenibacillus polymyxa A-1

The dibenzothiophene (DBT) desulfurizing bacterium metabolizes DBT to form 2-hydroxybiphenyl without breaking the carbon skeleton. Of the DBT desulfurization enzymes, DszC and DszA catalyze monooxygenation reactions, both requiring flavin reductase. We searched for non-DBT-desulfurizing microorganisms producing a flavin reductase that couples more efficiently with DszC than that produced by the DBT desulfurizing bacterium Rhodococcus erythropolis D-1, and found Paenibacillus polymyxa A-1 to be a promising strain. The enzyme was purified to complete homogeneity. K(m) values for FMN and NADH were 2.1 microM and 0.57 mM, respectively. Flavin compounds were good substrates, some nitroaromatic compounds were also active, and regarding the electron donor, the activity for NADPH was about 1.5 times that for NADH. In the coupling assay with DszC, only FMN or riboflavin acted as the electron acceptor. The coupling reactions of P. polymyxa A-1 flavin reductase with DszC and DszA proceeded more efficiently (3.5- and 5-fold, respectively) than those of R. erythropolis D-1 flavin reductase when identical enzyme activities of each flavin reductase were added to the reaction mixture. The result of the coupling reaction suggested that, in the microbial DBT desulfurization, flavin reductase from the non-DBT-desulfurizing bacterium was superior to that from the DBT-desulfurizing bacterium.     

Purification, characterization and crystallization of enzymes for dibenzothiophene desulfurization

DszC and DszA, DBT monooxygenase and DBT sulfone monooxygenase, respectively, involved in dibenzothiophene (DBT) desulfurization, were purified to homogeneity from Rhodococcus erythropolis D-1. The two enzymes were crystallized and enzymologically characterized. We found a high activity of flavin reductase in the non-DBT-desulfurizing bacterium, Paenibacillus polymyxa A-1, which is essential for DszC and A activities, and purified to homogeneity and characterized the enzyme.     

Comparative proteomic analysis of kidney development-related proteins in the pig

The development of the kidney is a complex process that serves as a model organ system for understanding many basic developmental mechanisms, and the pig kidney provides a useful and relevant model of kidney development and function. However, the molecular cascades involved in kidney development during embryonic development in the pig have not been elucidated fully. To better understand the molecular events associated with kidney development, we evaluated changes in gene expression during kidney development (days E40, E70, and E93) and compared these expressions with adults using two-dimensional gel electrophoresis. The functionally regulated proteins were identified by comparing differentially expressed proteins in embryonic kidneys vs. adult kidney. In addition, a representative set of the proteins was subjected to liquid chromatography tandem mass spectrometry analysis. Furthermore, the identified proteins were categorized according to their biological processes and molecular functions. Interestingly, 10 of the 25 proteins identified were apoptosis and actin cytoskeleton-related proteins, such as GRP75, α-fetoprotein, ANXA2, ANXA4, DDAH2, DJ-1, SOD2, cofilin1, vil1, and calbindin1. Based on these results, the proteomic approach was applied to identify specific protein expression changes in kidney tissues during development, and the expressional changes of these embryonic kidney proteins were found to be closely associated with the regulation of kidney development.     

Enhancing the specificity of the enterokinase cleavage reaction to promote efficient cleavage of a fusion tag

In our work with designed minimalist proteins based on the bZIP motif, we have found our His-tagged proteins to be prone to inclusion body formation and aggregation; we suspect this problem is largely due to the His tag, known to promote aggregation. Using AhR6–C/EBP, a hybrid of the AhR basic region and C/EBP leucine zipper, as representative of our bZIP-like protein family, we attempted removal of the His tag with enterokinase (EK) but obtained the desired cleavage product in very small yield. EK is known for proteolysis at noncanonical sites, and most cleavage occurred at unintended sites. We manipulated experimental conditions to improve specificity of proteolysis and analyzed the cleavage products; no effect was observed after changing pH, temperature, or the amount of EK. We then suspected the accessibility of the EK site was impeded due to protein aggregation. We found that the easily implemented strategy of addition of urea (1–4 M) greatly improved EK cleavage specificity at the canonical site and reduced adventitious cleavage. We believe that this enhancement in specificity is due to a more “open” protein structure, in which the now accessible canonical target can compete effectively with adventitious cleavage sites of related sequence.     

Influence of keeping pheasants in captivity vs. nature on the biological value of meat and its use in human nutrition

The life of game birds (pheasants) in nature is coupled with a number of difficulties in all seasons of the year. This refers to finding food, breeding, laying eggs, raising the young, fleeing from their natural enemies and lack of protection from unfavorable climatic conditions. The pheasants that live in captivity--aviaries for pheasants--do not have such difficulties--they are fed regularly by quality feed for pheasants, they are protected from bad weather and natural enemies. Our research was aimed at determining the biological value of meat of pheasants grown in the two different settings--in captivity and in nature. The highest weight achieved wild pheasant males (1232.4 +/- 147.36 g). The differences between tested pheasant groups were statistically very high significant (P < 0.001). The differences between groups related to breast weight and tights with drumsticks weight were statistically very high significant (P < 0.001). Between breast parts (%) and legs parts (%) were notified very high (P < 0.001) i.e. high (P = 0.002) differences. The highest weight breast muscles and tights with drumsticks had wild pheasants (282.6 +/- 63.53 g i.e. 206.2 +/- 37.88g). Wilde pheasants had lower part (%) and lighter (g) skin with subcutaneous fatty tissue on breasts. Female pheasants cultivated on both ways had higher skin part (%) and subcutaneous fatty tissue in tights with drumsticks. Related to chemical composition of breast muscles is established statistically significant differences (P < 0.001 i.s. P = 0.040)) in part of Ca (%) and P (%). In wild pheasant tights with drumsticks muscles established statistically very significant (P < 0.001) higher part of moisture, protein and Ca, i.e. statistically very high significant (P < 0.001) lower part of fat and energetic value. Research results indicate that the quality of meat of pheasants grown in nature has higher biological value than the meat of pheasants kept in aviaries, which means it has advantages in human nutrition.