Effect of enzyme dehydration on alcalase‐catalyzed dipeptide synthesis in near‐anhydrous organic media

The effect of enzyme dehydration by molecular sieves on the coupling of phenylalanine amide and the carbamoylmethyl ester of N‐protected phenylalanine in near‐anhydrous tetrahydrofuran was investigated. This coupling was catalyzed by Alcalase covalently immobilized onto macroporous acrylic beads (Cov); these immobilized enzymes were hydrated prior to use. The dehydration kinetics of Cov by molecular sieve powder were determined by incubating Cov with different amounts of molecular sieve powder for different periods of time (0–80 h). Subsequently, the remaining coupling activity of Cov was measured. Dehydration‐induced inactivation of Cov by molecular sieve powder was found to occur in three phases: (1) an initial, rapid, major dehydration‐induced inactivation that takes place during the first activity measurement, (2) a phase of first‐order inactivation, and (3) a plateau phase in activity. These dehydration kinetics were incorporated into a previously found reaction kinetics model. The resulting model was then used to fit progress curve data of the coupling in the presence of different amounts of molecular sieve powder. Upon establishment of parameter values, the model was used to predict independent data sets and found to work well. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:870–875, 2013     

Anaerobic ammonium-oxidizing bacteria in marine environments: widespread occurrence but low diversity

Laboratory and field studies have indicated that anaerobic ammonium oxidation (anammox) is an important process in the marine nitrogen cycle. In this study 11 additional anoxic marine sediment and water column samples were studied to substantiate this claim. In a combined approach using the molecular methods, polymerase chain reaction (PCR), qualitative and quantitative fluorescence in situ hybridization (FISH), as well as (15)N stable isotope activity measurements, it was shown that anammox bacteria were present and active in all samples investigated. The anammox activity measured in the sediment samples ranged from 0.08 fmol cell(-1) day(-1) N(2) in the Golfo Dulce (Pacific Ocean, Costa Rica) sediment to 0.98 fmol cell(-1) day(-1) N(2) in the Gullmarsfjorden (North Sea, Sweden) sediment. The percentage of anammox cell of the total population (stained with DAPI) as assessed by quantitative FISH was highest in the Barents Sea (9% +/- 4%) and in most of the samples well over 2%. Fluorescence in situ hybridization and phylogenetic analysis of the PCR products derived from the marine samples indicated the exclusive presence of members of the Candidatus'Scalindua' genus. This study showed the ubiquitous presence of anammox bacteria in anoxic marine ecosystems, supporting previous observations on the importance of anammox for N cycling in marine environments.     

Ladderane phospholipids in anammox bacteria comprise phosphocholine and phosphoethanolamine headgroups

Anammox bacteria present in wastewater treatment systems and marine environments are capable of anaerobically oxidizing ammonium to dinitrogen gas. This anammox metabolism takes place in the anammoxosome which membrane is composed of lipids with peculiar staircase-like 'ladderane' hydrocarbon chains that comprise three or four linearly concatenated cyclobutane structures. Here, we applied high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to elucidate the full identity of these ladderane lipids. This revealed a wide variety of ladderane lipid species with either a phosphocholine or phosphoethanolamine polar headgroup attached to the glycerol backbone. In addition, in silico analysis of genome data gained insight into the machinery for the biosynthesis of the phosphocholine and phosphoethanolamine phospholipids in anammox bacteria.     

Bioenergetics and solute uptake under extreme conditions

The ion and particularly the proton and sodium ion permeabilities of cytoplasmic membranes play crucial roles in the bioenergetics of microorganisms. The proton and sodium permeabilities of membranes increase with temperature. Psychrophilic and mesophilic bacteria and mesophilic, (hyper)thermophilic, and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains constant (homeoproton permeability). Thermophilic bacteria are an exception. They rely on the less permeable sodium ions to generate a sodium motive force, which is subsequently used to drive energy-requiring membrane-bound processes. Transport of solutes across bacterial and archaeal membranes is mainly catalyzed by primary ATP-driven transport systems or by proton- or sodium-motive-force-driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary uptake systems. Several high-affinity ATP-binding cassette (ABC) transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments.     

Membrane composition and ion-permeability in extremophiles

Abstract: Protons and sodium ions are the only used coupling ions in energy transduction in Bacteria and Archaea. At their growth temperature, the permeability of the cytoplasmic membrane of thermophilic bacteria to protons is high as compared to sodium ions. In some thermophiles, therefore, sodium is the sole energy coupling ion. Comparison of the proton- and sodium permeability of the membranes of variety of bacterial and archaeal species that differ in their optimal growth temperature reveals that the permeation processes of protons and sodium ions must occur by different mechanisms. The proton permeability increases with the temperature, and has a comparable value for most species at their respective growth temperatures. The sodium permeability is lower than the proton permeability and increases also with the temperature, but is lipid independent. Therefore, it appears that for most bacteria the physical properties of the cytoplasmic membrane are optimised to ensure a low proton permeability at the respective growth temperature.     

Enrichment and characterization of marine anammox bacteria associated with global nitrogen gas production

Microbiological investigation of anaerobic ammonium oxidizing (anammox) bacteria has until now been restricted to wastewater species. The present study describes the enrichment and characterization of two marine Scalindua species, the anammox genus that dominates almost all natural habitats investigated so far. The species were enriched from a marine sediment in the Gullmar Fjord (Sweden) using a medium based on Red Sea salt. Anammox cells comprised about 90% of the enrichment culture after 10 months. The enriched Scalindua bacteria displayed all typical features known for anammox bacteria, including turnover of hydrazine, the presence of ladderane lipids, and a compartmentalized cellular ultrastructure. The Scalindua species also showed a nitrate-dependent use of formate, acetate and propionate, and performed a formate-dependent reduction of nitrate, Fe(III) and Mn(IV). This versatile metabolism may be the basis for the global distribution and substantial contribution of the marine Scalindua anammox bacteria to the nitrogen loss from oxygen-limited marine ecosystems.     

Specific interaction of central nervous system myelin basic protein with lipids. Specific regions of the protein sequence protected from the proteolytic action of trypsin

The tryptic hydrolysis of the basic protein of central nervous system myelin (A₁ basic protein) and of A₁ basic-lipid complexes was studied. The tryptic digestion was monitored by “finger printing”, column chromatography and amino acid analysis of the resulting pure peptides.Specific regions of the protein sequence were found to be protected from the hydrolytic action of the trypsin only after the protein was recombined with specific lipids. The degree of protection was in the order: cerebroside sulphate > acidic lipid fraction of myelin > phosphatidylsrine = total lipid extract of myelin. The protected Lys-X, Arg-X bonds were all situated in the region amino acid 20 to amino acid 113 of the intact protein. This region contains the (proline)₃ bend in the protein which is stabilized by interaction with lipids and also the encephalitogenic site for monkey and rabbit.From the results reported in this publication we would like to suggest a specific interaction between a region of the A₁ basic protein molecule and cerebroside sulphate. Differences in A₁ basic protein-lipid interaction in different animals arising from differences in lipid composition and fatty acid composition of the different lipid species combined with minor changes in the protein sequence could explain the species variability of the encephalitogenic sites of the A₁ basic protein.