AqpZ reconstituted proteoliposomes integrated into polyamide matrix using different ultrafiltration support membranes successfully with a performance increase of 12 times.

In NADH regeneration, Candida methylica formate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependent cmFDH from NAD+ to NADP+ and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+ to 5.6 × 104-fold and 5 × 104-fold value, respectively. To increase the thermostability of cmFDH, the conserved residue at position 1 in the catalytic domain of cmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating “smarter libraries” for improving the properties of cmFDH.

No abstract

Aquaporins have high water permeability and selectivity, which make them attractive to use in membrane applications for water purification and desalination. Due to their hydrophobic nature, they have to be embedded in cell membrane-like structures such as liposomes to form proteoliposomes. In this study, the effects of aquaporin Z (AqpZ) addition on liposome characteristics and behavior were investigated by evaluating the adsorption of proteoliposomes onto gold as reference substrate, polysulfone (PS) and polyacrylonitrile (PAN) spin-coated sensor surface as common nanofiltration membrane material by quartz crystal microbalance with dissipation. AqpZ incorporation into liposomes did not significantly change mean liposome diameter and surface charge, but the water permeability was increased up to almost sixfold. The results of viscoelastic modeling showed a stiffer membrane structure for the lipid bilayer of proteoliposomes compared to that of liposomes. Although liposomes and proteoliposomes were found to be adsorbed successfully on negatively charged PS and neutral PAN surfaces, the interaction strength and adsorption rate were found to be different for polymers with different surface properties, indicating the importance of the polymer for the stabilization of proteoliposomes. Findings of this study offer valuable information about the incorporation of proteoliposomes in membrane structures for the fabrication of nanofiltration membranes.