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Successful nanobiotechnology implementation largely depends on control over the interfaces between inorganic materials and biological molecules. Controlling the orientations of biomolecules and their spatial arrangements on the surface may transform many technologies including sensors, to energy. Here, we demonstrate the self-organization of L-lactate dehydrogenase (LDH), which exhibits enhanced enzymatic activity and stability on a variety of gold surfaces ranging from nanoparticles to electrodes, by incorporating a gold-binding peptide tag (AuBP2) as the fusion partner for Bacillus stearothermophilus LDH (bsLDH). Binding kinetics and enzymatic assays verified orientation control of the enzyme on the gold surface through the genetically incorporated peptide tag. Finally, redox catalysis efficiency of the immobilized enzyme was detected using cyclic voltammetry analysis in enzyme-based biosensors for lactate detection as well as in biofuel cell energy systems as the anodic counterpart. Our results demonstrate that the LDH enzyme can be self-immobilized onto different gold substrates using the short peptide tag under a biologically friendly environment. Depending on the desired inorganic surface, the proposed peptide-mediated path could be extended to any surface to achieve single-step oriented enzyme immobilization for a wide range of applications.

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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.

The Candida methylica (cm) recombinant wild type formate dehydrogenase (FDH) gene has been cloned into the pQE-2 TAGZyme expression vector and the 6xHis-tagged FDH gene has been overexpressed in JM105 cells to purify the FDH protein more efficiently, by the use of exopeptidases, TAGZyme Purification System, which has allowed the complete removal of the small N-terminal His-tag. After the purification procedure, 1.2 mg/mL cmFDH protein of >95% purity was obtained. The kinetic parameters of cmFDH have been determined by observing the oxidation of the nicotinamide coenzyme at 340 nm. The results have also been compared to the yield of standard vs. affinity purification of FDH.

Porous polymers carrying sulfonamide (SAM) groups with interconnected pores were prepared by high internal phase emulsion (HIPE) method. The resulting polymer named polyHIPE (PH) has the disadvantage of low surface area due to the large macropores. This disadvantage has been overcome through the hypercrosslinking reaction by introducing meso-and microporosities to the interconnected framework of hierarchically macroporous PH. The resulting hypercrosslinked polyHIPE (HCLPH) was successfully functionalized with chlorosulfonic acid and tris(hydroxymethyl)aminomethane (tris) in two consecutive steps to obtain a SAM-functionalized (SAM-f) polymer surface. It has been very well established that there is a selective interaction between the SAMs and human serum albumin (HSA). Here, we conduct an adsorption performance comparison study for a number of SAM-f-polymers, which differ from each other in the applied template strategy such as emulsion and suspension (PHs or beads) and/or the hypercrosslinking degree based on the reaction time of (0−5−15−60 min). SAM-f-15 min HCLPH with its unusual performance was found to achieve highly selective (compared to lysozyme and α-amylase) and very fast (80% of its uptake capacity in 2 min) adsorption of HSA. The polymer loaded with HSA can easily be regenerated by treating it with only tris buffer (pH 7) and was reused eight times without losing any significant adsorption capacity. The results of a comprehensive study of isotherms (Langmuir, Freundlich, Dubinin−Radushkevich) and kinetic (pseudo-first-order, pseudo-second-order, intraparticle diffusion) models as well as the thermodynamic analysis were presented for SAM-f-15 min HCLPH. The binding mechanism of HSA/SAM ligand complex was investigated by performing simplified molecular modeling via docking. Hydrophobic interactions and hydrogen bonding were found to be the major forces dominating the adsorption process. These results follow the outcome obtained from the thermodynamic analysis. The fast uptake, easy reuse protocol, and high affinity toward HSA make SAM-f-HCLPHs excellent materials for albumin adsorption.