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Background
Aminoacylases are highly promising enzymes for the green synthesis of acyl-amino acids, potentially replacing the environmentally harmful Schotten-Baumann reaction. Long-chain acyl-amino acids can serve as strong surfactants and emulsifiers, with application in cosmetic industries. Heterologous expression of these enzymes, however, is often hampered, limiting their use in industrial processes.
Results
We identified a novel mycobacterial aminoacylase gene from Mycolicibacterium smegmatis MKD 8, cloned and expressed it in Escherichia coli and Vibrio natriegens using the T7 overexpression system. The recombinant enzyme was prone to aggregate as inclusion bodies, and while V. natriegens Vmax™ could produce soluble aminoacylase upon induction with isopropyl β-d-1-thiogalactopyranoside (IPTG), E. coli BL21 (DE3) needed autoinduction with lactose to produce soluble recombinant protein. We successfully conducted a chaperone co-expression study in both organisms to further enhance aminoacylase production and found that overexpression of chaperones GroEL/S enhanced aminoacylase activity in the cell-free extract 1.8-fold in V. natriegens and E. coli. Eventually, E. coli ArcticExpress™ (DE3), which co-expresses cold-adapted chaperonins Cpn60/10 from Oleispira antarctica, cultivated at 12 °C, rendered the most suitable expression system for this aminoacylase and exhibited twice the aminoacylase activity in the cell-free extract compared to E. coli BL21 (DE3) with GroEL/S co-expression at 20 °C. The purified aminoacylase was characterized based on hydrolytic activities, being most stable and active at pH 7.0, with a maximum activity at 70 °C, and stability at 40 °C and pH 7.0 for 5 days. The aminoacylase strongly prefers short-chain acyl-amino acids with smaller, hydrophobic amino acid residues. Several long-chain amino acids were fairly accepted in hydrolysis as well, especially N-lauroyl-L-methionine. To initially evaluate the relevance of this aminoacylase for the synthesis of N-acyl-amino acids, we demonstrated that lauroyl-methionine can be synthesized from lauric acid and methionine in an aqueous system.
Conclusion
Our results suggest that the recombinant enzyme is well suited for synthesis reactions and will thus be further investigated.
Changes in intestinal microflora in rats induced by oral exposure to low lead (II) concentrations
(2015)
This study evaluates neuromechanical control and muscle-tendon interaction during energy storage and dissipation tasks in hypergravity. During parabolic flights, while 17 subjects performed drop jumps (DJs) and drop landings (DLs), electromyography (EMG) of the lower limb muscles was combined with in vivo fascicle dynamics of the gastrocnemius medialis, two-dimensional (2D) kinematics, and kinetics to measure and analyze changes in energy management. Comparisons were made between movement modalities executed in hypergravity (1.8 G) and gravity on ground (1 G). In 1.8 G, ankle dorsiflexion, knee joint flexion, and vertical center of mass (COM) displacement are lower in DJs than in DLs; within each movement modality, joint flexion amplitudes and COM displacement demonstrate higher values in 1.8 G than in 1 G. Concomitantly, negative peak ankle joint power, vertical ground reaction forces, and leg stiffness are similar between both movement modalities (1.8 G). In DJs, EMG activity in 1.8 G is lower during the COM deceleration phase than in 1 G, thus impairing quasi-isometric fascicle behavior. In DLs, EMG activity before and during the COM deceleration phase is higher, and fascicles are stretched less in 1.8 G than in 1 G. Compared with the situation in 1 G, highly task-specific neuromuscular activity is diminished in 1.8 G, resulting in fascicle lengthening in both movement modalities. Specifically, in DJs, a high magnitude of neuromuscular activity is impaired, resulting in altered energy storage. In contrast, in DLs, linear stiffening of the system due to higher neuromuscular activity combined with lower fascicle stretch enhances the buffering function of the tendon, and thus the capacity to safely dissipate energy.
Aneurysmal subarachnoid hemorrhage (aSAH) is associated with early and delayed brain injury due to several underlying and interrelated processes, which include inflammation, oxidative stress, endothelial, and neuronal apoptosis. Treatment with melatonin, a cytoprotective neurohormone with anti-inflammatory, anti-oxidant and anti-apoptotic effects, has been shown to attenuate early brain injury (EBI) and to prevent delayed cerebral vasospasm in experimental aSAH models. Less is known about the role of endogenous melatonin for aSAH outcome and how its production is altered by the pathophysiological cascades initiated during EBI. In the present observational study, we analyzed changes in melatonin levels during the first three weeks after aSAH.