Chimeric versus isolated proteins: Biochemical characterization of the NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 fused with the Baeyer-Villiger monooxygenase from Thermobifida fusca.

The expression of multifunctional proteins can facilitate the setup of a biotechnology process that requires multiple functions absolved by different proteins. Herein the functional and conformational characterization of a formate dehydrogenase-monooxygenase chimera enzyme is presented. The fused enzyme (FDH-PAMO) was prepared by linking the C-terminus of the mutant NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 (FDH) to the N-terminus of the NADPH-dependent monooxygenase from Thermobifida fusca (PAMO) through a peptide linker of 9 amino acids (ASGGGGSGT) generating a chimera protein of 107,056 Da. The catalytic properties (e.g., kinetic parameters kcat and Km), stability, fluorescence and circular dichroism spectra showed that the so-obtained chimera enzyme FDH-PAMO retains the same functional and conformational properties of the two parental enzymes. Furthermore, SEC chromatographic analysis indicated that, in solution (pH 7.4), FDH-PAMO assembles to tetramers (up to 4.2 %) due to the propensity of FDH and PAMO to form dimers, up to 96.6 % and 6.2 %, respectively. This study provides valuable insights into the structural stability of a thermostable protein (e.g., PAMO) after increasing its size through fusion with another similarly sized thermostable protein (e.g., FDH).

Changes in the Microbiome Profile in Different Parts of the Intestine in Piglets with Diarrhea.

Determining the taxonomic composition of microbial consortia of the piglet intestine is of great importance for pig production. However, knowledge on the variety of the intestinal microbiome in newborn piglets is limited. Piglet diarrhea is a serious gastrointestinal disease with a high morbidity and mortality that causes great economic damage to the pig industry. In this study, we investigated the microbiome of various sections of the piglet intestine and compared the microbiome composition of healthy and diarrheal piglets using high-throughput sequencing of the 16S rRNA gene. The results showed that bacteria of the Lactobacillus genus were the most common in the ileum, while Fusobacterium and Bacteroides dominated in the rectum. Comparing the microbiome composition of healthy and diarrheal piglets revealed a reduced number of Lactobacillus bacteria as a hallmark of diarrhea, as did an increased content of representatives of the Escherichia-Shigella genus and a reduced number of Bacteroides, which indicates the contribution of these bacteria to the development of diarrhea in piglets. The relative abundance of Enterococcus bacteria was higher in the diarrhea group. Although some bacteria of this genus are commensals, a small number of species may be associated with the development of diarrhea in piglets. Therefore, our results indicate that the gut microbiome may be an important factor in the development of diarrhea in piglets.

Parameters of nonspecific resistance of calves with respiratory pathology before and after treatment.

OBJECTIVE: The research was conducted to study the effect of a complex antimicrobial drug with an anti-inflammatory effect and an antimicrobial drug with an immunostimulating effect on the parameters of nonspecific resistance in calves. MATERIALS AND METHODS: Two groups (n = 5 each) of sick calves with respiratory pathology were selected for this study. For the treatment of the first experimental group, a complex antimicrobial drug Sulfetrisan® was used. The second experimental group of the calves was intramuscularly injected with the experimental drug gentaaminoseleferon (GIA). To assess the cellular component of immunity in the blood before and after treatment, the number of white blood cells, T-lymphocytes, B-lymphocytes, phagocytic activity of leukocytes, phagocytic number, and phagocytic index (PhI) were determined. In addition, for assessing the humoral component, serum complement activity (SCA), serum lysozyme activity, serum bactericidal activity (SBA), circulating immune complexes (CIC), and total immunoglobulins (total Ig) were measured. The results were compared with the baseline parameters of healthy calves of the control group. RESULTS: When studying the parameters of the humoral and cellular components of nonspecific resistance, it was found that in sick animals, compared with healthy ones, respiratory pathology was accompanied by an imbalance in the immune system. In the process of recovery in animals of the experimental groups under the effect of the drugs, positive changes occurred. However, many of the studied parameters did not reach the values of healthy animals. In the group of calves that received GIA, compared with the calves given Sulfetrisan®, a significant increase in PhI (p < 0.05), SBA (p < 0.006), SCA (p < 0.05), total Ig (p < 0.0005), and CIC (p < 0.05) was observed, which indicated an increase in natural resistance due to the immunostimulating action. CONCLUSION: The use of GIA in sick animals added to an increase in the general nonspecific cellular and humoral resistance of calves, which made it possible to increase therapeutic efficacy and shorten their recovery time.

Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity.

Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. were fused to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. The order of the genes in the fusion as well as the linkers that bridge the enzymes were varied. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. We suggest that P450 BM3 undergoes conformational changes upon fusion which resulted in altered properties, however, no NADPH channeling was detected for the fusion constructs.