Site-directed chemically-modified magnetic enzymes: fabrication, improvements, biotechnological applications and future prospects.

Numerous enzymes of biotechnological importance have been immobilized on magnetic nanoparticles (MNP) via random multipoint attachment, resulting in a heterogeneous protein population with potential reduction in activity due to restriction of substrate access to the active site. Several chemistries are now available, where the modifier can be linked to a single specific amino acid in a protein molecule away from the active-site, thus enabling free access of the substrate. However, rarely these site-selective approaches have been applied to immobilize enzymes on nanoparticles. In this review, for the first time, we illustrate how to adapt site-directed chemical modification (SDCM) methods for immobilizing enzymes on iron-based MNP. These strategies are mainly chemical but may additionally require genetic and enzymatic methods. We critically examine each method and evaluate their scope for simple, quick, efficient, mild and economical immobilization of enzymes on MNP. The improvements in the catalytic properties of few available examples of immobilized enzymes are also discussed. We conclude the review with the applications and future prospects of site-selectively modified magnetic enzymes and potential benefits of this technology in improving enzymes, including cold-adapted homologues, modular enzymes, and CO2-sequestering, as well as non-iron based nanomaterials.

Microwave-assisted digestion followed by parallel electromembrane extraction for trace level perchlorate detection in biological samples.

A simple and parallel electromembrane extraction (pEME) method was developed and used to investigate trace perchlorate ion contamination in seafood. In this method, three different EME units were arranged simultaneously and connected parallel to a single DC power supply. In each unit, the ClO4(-) ions were electro-kinetically extracted from the microwave digested seafood homogenates into 100mM NaOH via a supported liquid membrane (1-Hexanol). Influential extraction parameters were carefully investigated. Under optimized conditions, good linearity with a coefficient of determination (R(2)) of 0.9949 over a concentration range of 1-125µg/g was obtained. The limit of detection (LOD) was 0.04µgg(-1). The methods intraday and inter day precision varied between 4.3-5.6% respectively. Mean recoveries were up to 107% (n=6, RSD=0.7-6.8%). This method was applied to different seafood samples to assess its feasibility for real applications and it exhibited an enhanced sample throughput compatible with both microwave and ion chromatography.