An overall framework for the E. coli γ-glutamyltransferase-catalyzed transpeptidation reactions.

γ-Glutamyl derivatives of proteinogenic or modified amino acids raise considerable interest as flavor enhancers or biologically active compounds. However, their supply, on a large scale and at reasonable costs, remains challenging. Enzymatic synthesis has been recognized as a possible affordable alternative with respect to both isolation procedures from natural sources, burdened by low-yield and by the requirement of massive amount of starting material, and chemical synthesis, inconvenient because of the need of protection/deprotection steps. The E. coli γ-glutamyltransferase (Ec-GGT) has already been proposed as a biocatalyst for the synthesis of various γ-glutamyl derivatives. However, enzymatic syntheses using this enzyme usually provide the desired products in limited yield. Hydrolysis and autotranspeptidation of the donor substrate have been identified as the side reactions affecting the final yield of the catalytic process. In addition, experimental conditions need to be specifically adjusted for each acceptor substrate. Substrate specificity and the fine characterization of the activities exerted by the enzyme over time has so far escaped rationalization. In this work, reactions catalyzed by Ec-GGT between the γ-glutamyl donor glutamine and several representative acceptor amino acids have been finely analyzed with the identification of single reaction products over time. This approach allowed to rationalize the effect of donor/acceptor molar ratio on the outcome of the transpeptidation reaction and on the distribution of the different byproducts, inferring a general scheme for Ec-GGT-catalyzed reactions. The propensity to react of the different acceptor substrates is in agreement with recent findings obtained using model substrates and further supported by x-ray crystallography and will contribute to characterize the still elusive acceptor binding site of the enzyme.

Integration of enzymatic data in Bacillus subtilis genome-scale metabolic model improves phenotype predictions and enables in silico design of poly-γ-glutamic acid production strains.

BACKGROUND: Genome-scale metabolic models (GEMs) allow predicting metabolic phenotypes from limited data on uptake and secretion fluxes by defining the space of all the feasible solutions and excluding physio-chemically and biologically unfeasible behaviors. The integration of additional biological information in genome-scale models, e.g., transcriptomic or proteomic profiles, has the potential to improve phenotype prediction accuracy. This is particularly important for metabolic engineering applications where more accurate model predictions can translate to more reliable model-based strain design. RESULTS: Here we present a GEM with Enzymatic Constraints using Kinetic and Omics data (GECKO) model of Bacillus subtilis, which uses publicly available proteomic data and enzyme kinetic parameters for central carbon (CC) metabolic reactions to constrain the flux solution space. This model allows more accurate prediction of the flux distribution and growth rate of wild-type and single-gene/operon deletion strains compared to a standard genome-scale metabolic model. The flux prediction error decreased by 43% and 36% for wild-type and mutants respectively. The model additionally increased the number of correctly predicted essential genes in CC pathways by 2.5-fold and significantly decreased flux variability in more than 80% of the reactions with variable flux. Finally, the model was used to find new gene deletion targets to optimize the flux toward the biosynthesis of poly-γ-glutamic acid (γ-PGA) polymer in engineered B. subtilis. We implemented the single-reaction deletion targets identified by the model experimentally and showed that the new strains have a twofold higher γ-PGA concentration and production rate compared to the ancestral strain. CONCLUSIONS: This work confirms that integration of enzyme constraints is a powerful tool to improve existing genome-scale models, and demonstrates the successful use of enzyme-constrained models in B. subtilis metabolic engineering. We expect that the new model can be used to guide future metabolic engineering efforts in the important industrial production host B. subtilis.

An In Vitro Study on the Role of Cellulases and Xylanases of Bacillus subtilis in Dairy Cattle Nutrition.

The administration of Bacilli to dairy cows exerts beneficial effects on dry matter intake, lactation performance, and milk composition, but the rationale behind their efficacy is still poorly understood. In this work, we sought to establish whether cellulases and xylanases, among the enzymes secreted by B. subtilis, are involved in the positive effect exerted by Bacilli on ruminal performance. We took advantage of two isogenic B. subtilis strains, only differing in the secretion levels of those two enzymes. A multi-factorial study was conducted in which eight feed ingredients were treated in vitro, using ruminal fluid from cannulated cows, with cultures of the two strains conveniently grown in a growth medium based on inexpensive waste. Feed degradability and gas production were assessed. Fiber degradability was 10% higher (p < 0.001) in feeds treated with the enzyme-overexpressing strain than in the untreated control, while the non-overexpressing strain provided a 5% increase. The benefit of the fibrolytic enzymes was maximal for maize silage, the most recalcitrant feed. Gas production also correlated with the amount of enzymes applied (p < 0.05). Our results revealed that B. subtilis cellulases and xylanases effectively contribute to improving forage quality, justifying the use of Bacilli as direct-fed microbials to increase animal productivity.

Silver Doped Magnesium Ferrite Nanoparticles: Physico-Chemical Characterization and Antibacterial Activity.

Spinel phases, with unique and outstanding physical properties, are attracting a great deal of interest in many fields. In particular, MgFe2O4, a partially inverted spinel phase, could find applications in medicine thanks to the remarkable antibacterial properties attributed to the generation of reactive oxygen species. In this paper, undoped and Ag-doped MgFe2-xAgxO4 (x = 0.1 and 0.3) nanoparticles were prepared using microwave-assisted combustion and sol-gel methods. X-ray powder diffraction, with Rietveld structural refinements combined with micro-Raman spectroscopy, allowed to determine sample purity and the inversion degree of the spinel, passing from about 0.4 to 0.7 when Ag was introduced as dopant. The results are discussed in view of the antibacterial activity towards Escherichia coli and Staphylococcus aureus, representative strains of Gram-negative and Gram-positive bacteria. The sol-gel particles were more efficient towards the chosen bacteria, possibly thanks to the nanometric sizes of metallic silver, which were well distributed in the powders and in the spinel phase, with respect to microwave ones, that, however, acquired antibacterial activity after thermal treatment, probably due to the nucleation of hematite, itself displaying well-known antibacterial properties and which could synergistically act with silver and spinel.