Introduction of biocatalysis and modern trends of research on mentioned area in the world. Introduction of research in the field of biocatalysis in the Laboratory for Separation Processes and product design at UM, with emphasis on the preparation of magnetic nanoparticles as potential carriers of biological substances.
B.04 Guest lecture
COBISS.SI-ID: 15456790The research work offers a concise guide of the synthesis of maghemite magnetic nanoparticles, Fe2O3, for immobilization of a biocatalyst. Magneticnanoparticles, that are gaining an exceptionally increased attention as potential enzyme support in the recent years, were synthesized by the coprecipitation technique of ferrous (II, Fe2+) and ferric (III, Fe3+) ions inalkaline medium at harsh stirring and high temperature, respectively. Surface functionalization of magnetic nanoparticles was carried out stepwise and divided into two major steps. Primary functional layer of silica (SiO2) that enhanced the stability of magnetic nanoparticles was synthesized under strictly regulated reaction conditions from sodium silicate. Next, the secondary functional layer formed of organic molecules of amino silane or 3-(2-Aminoethylamino)-propyl-dimethoxymethylsilane in order to achieve higher functionality and reactivity of the surface of magnetic nanoparticles was synthesized in acidic reaction medium. Furthermore, the prepared magnetic nanocomposites were used for the immobilization of a specific biocatalyst cholesterol oxidase (ChOx, EC 1.1.3.6) from Corynebacterium sp. The analysis of the magnetic nanoparticles with immobilized ChOx showed that the nanoparticles adopted a typical spherical shape with a mean diameter of 50,2 nm. The binding of ChOx was successfully confirmed by FT-IR technique. The binding efficiency was 92 % and was maximally achieved at enzyme concentrationof 100 g mL-1. The activity of immobilized ChOx onto magnetic nanoparticles, coated with a thin functional layer of silica with a thickness of 3 nm, was estimated to be 57 % in comparison to its native ChOx. The effects of pH and temperature also indicated the bound ChOx had better pH-tolerance and exhibited higher thermal stability. Furthermore, the immobilized system revealed also good reusable stability. The second part of the PhD work was focused on the synthesis of active enzyme aggregates of peroxidase (HRP, EC 1.11.1.7) from horse radish roots (lat.: Armoracia rusticana or Cochlearia armoracia), further cross-linked with glutaraldehyde in order to obtain the final form of stable cross-linked enzyme aggregates or CLEAs. The procedure to prepare CLEAs was divided into two major steps, that involves first the precipitation of the soluble enzyme with a suitable precipitant such and second the crosslinking step with an appropriate cross-linker. The final suspension of CLEAs obtained was moderately turbid andenzyme particles could be normally observed with an average diameter of 250 nm. The CLEAs of HRP were successfully produced under different testing conditions afterwards. The final recovery activity of the CLEAs attained was 83 % compared to the activity of native enzyme. The synthesised CLEAs were prepared in the presence of egg albumin and functional additive as pentaethylenehexamine (PEHA). The role of albumin in CLEAs preparation plays asignificant role as a stabilizing agent of CLEAs particles, whereas the addition of PEHA was fundamental to obtain fully cross-linked HRP aggregates. For, the HRP possesses only 6 Lys (lysine) amino residues, that substantially impedes the cross-linking of the enzyme to completion, the addition of PEHA increased the free amino groups (-NH2) on the outer surface of the enzyme, making the cross-linking more feasible.
D.09 Tutoring for postgraduate students
COBISS.SI-ID: 14930966