Summary
In this study, the suitability of fumed silica nanoparticles (FNS) and its derivatives (amino‐modified FNS (AFNS), cyanuric chloride‐activated AFNS (CCAFNS) and epoxy‐modified FNS (GFNS)), for covalent immobilisation of two commercial protease preparations Alcalase® and Flavourzyme® was investigated. The highest hydrolytic activities of immobilised preparations were 25 IU g−1 support (Alcalase‐GFNS) and 2.95 IU g−1 support (Flavourzyme‐CCAFNS). Furthermore, the immobilised preparations showed 43% and 20% of initial specific activities of commercial protease preparations, respectively. Flavourzyme‐CCAFNS also exhibited the highest exopeptidase activity of 22.83 L‐pNAU g−1 support. Finally, these two nanobiocatalysts were successfully applied for hydrolysis of sunflower meal protein isolate (SMPI), providing two times higher hydrolysis yields in comparison to free enzymes, justifying the applied immobilisation process. Namely, the highest hydrolysis yield (30%) was gained by the sequential hydrolysis with Alcalase‐GFNS and Flavourzyme‐CCAFNS, which resulted in the formation of small hydrophobic and hydrophilic peptides, ≤5 kDa, confirmed by HPLC analysis and electrophoretic separation.
Cellulases are enzymes which catalyse cellulose hydrolysis and are widely used in various industry branches. Lately, their application in treatment of different agroindustrial waste materials which could serve for fuel production is being extensively explored. In order to increase their stability and cost-effectiveness of their usage, application of their immobilized forms are preferred over free enzymes. Hereby, we tested eight different Lifetech TM immobilization supports differing in polarity, porosity and functional groups as carriers for Asspergillus niger cellulase immobilization. Most promising carrier was methacrylate based, with primary amino groups, C6 "space arm" and pores with diameter of 60-120 nm-Lifetech TM ECR8409F. For this support, most important immobilization parameters were investigated and after 3 h at pH 6 with initial protein concentration of 23.3 mg/g support immobilized cellulase with 406 IU/g (with carboxymethyl cellulose as a substrate) was obtained. This preparation was successfully applied in the hydrolysis of lignocellulosic fraction of sunflower seed meal, which is widely available byproduct of sunflower seed meal fractionation for protein-rich fractions production. Initial reaction rates and yields of reducing sugars were unchanged comparing to free enzyme, indicating that there were no significant diffusion limitations for substrate to approach active sites of A. niger cellulase molecules immobilized onto Lifetech TM ECR8409F support.
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