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C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli
(2010)
Recently, a new alkaline protease named HP70 showing highest homology to extracellular serine proteases of Stenotrophomonas maltophilia and Xanthomonas campestris was found in the course of a metagenome screening for detergent proteases (Niehaus et al., submitted for publication). Attempts to efficiently express the enzyme in common expression hosts had failed. This study reports on the realization of overexpression in Escherichia coli after structural modification of HP70. Modelling of HP70 resulted in a two-domain structure, comprising the catalytic domain and a C-terminal domain which includes about 100 amino acids. On the basis of the modelled structure the enzyme was truncated by deletion of most of the C-terminal domain yielding HP70-C477.
This structural modification allowed effective expression of active enzyme using E. coli BL21-Gold as the host. Specific activity of HP70-C477 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 30 ± 5 U/mg compared to 8 ± 1 U/mg of the native enzyme. HP70-C477 was most active at 40 °C and pH 7–11; these conditions are prerequisite for a potential application as detergent enzyme. Determination of kinetic parameters at 40 °C and pH = 9.5 resulted in KM = 0.23 ± 0.01 mM and kcat = 167.5 ± 3.6 s⁻¹. MS-analysis of peptide fragments obtained from incubation of HP70 and HP70-C477 with insulin B indicated that the C-terminal domain influences the cleavage preferences of the enzyme. Washing experiments confirmed the high potential of HP70-C477 as detergent protease.
The MicroMed DeBakey ventricular assist device is an axial flow pump designed for providing long-term support to end-stage heartfailure patients. Previously, we presented computational analysis of the blood pump flow. From the analysis, we were able to identify regions of high shear and recirculating flow that may cause blood damage, for example, deformation and fragmentation of the red blood cell (RBC). This mechanical hemolysis can be predicted using a tensor-based blood damage model that is based on the physical properties of the RBCs, for example, the relaxation time of the RBC membrane. However, an extensive and detailed analysis was complicated by the fact that the previous method predicts hemolysis along a finite number of pathlines traversed by the RBCs, possibly omitting parts of the flow domain. Furthermore, it is computationally expensive and is not easily parallelizable.
Here, we propose a new method to estimate hemolysis. The method is based on treating the shape of droplet (tensor) as a field variable, like velocity in the Navier-Stokes system. The governing equation for the RBC shape is treated by least-squares finite element method and the volume conservation of the RBC is augmented by Lagrangian multiplier. Unlike the previous method, the proposed method can visualize areas of high RBC strain that is potentially dangerous for mechanical hemolysis. Also, the amount of plasma-free hemoglobin and, consequently, normalized index of hemolysis can be computed as a byproduct. The method is tested in a simple shear flow for validation and an artery graft flow is chosen to show its potential usefulness. Finally, the method is applied to the blood damage estimation for the pump.
pH-sensitive properties of barium strontium titanate (BST) high-k thin films as alternative gate material for field-effect capacitive (bio-)chemical sensors based on an electrolyte-insulator-semiconductor system have been investigated. The BST films of different compositions (Ba0.31Sr0.69TiO3, Ba0.25Sr0.75TiO3 and Mg-doped Ba0.8Sr0.2Mg0.1Ti0.9O3) were deposited by pulsed laser deposition technique from targets fabricated by self-propagating high-temperature synthesis. The realised sensors have been electrochemically characterised by means of impedance-spectroscopy, capacitance–voltage and constant-capacitance method. The sensors possess a Nernstian-like pH sensitivity in the concentration range between pH 3 and 11 with a response time of 5–10 s. An equivalent circuit model for the BST-based capacitive field-effect sensor is discussed.