I. Hegedüs(1*), E. Nagy(2), J. Kukolya(3), T. Barna(4), Cs. A. Fekete(5)

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(*) Corresponding Author

Cellulase and hemicellulase enzymes as single molecular nanobiocomposites


Abstract



We have worked out two different stabilization procedures for glycolytic enzymes with significant biotechnology relevance: for the commercial Novozyme cellulase complex (Celluclast) and for recombinant Thermobifida fuscahemicellulases (β-D-xylosidase and β-D-mannosidase). In the applied methods, individual cellulase and hemicellulase enzyme molecules were conjugated with a polymer nano-layer providing nanobiocomposites (single enzyme nanoparticles, SENs) that exihibit an excellent stability under extreme conditions. The first method that we have been successfully used earlier on chymotrypsin [3], creates a polymer layer around the enzymemolecules with trimethoxysilyl (TMS) functionalities in three steps [4]. The second stabilization method is a novel onepot reaction resulting in an acrylamide-bisacrylamide (AA) copolymer layer around the enzyme molecules in two steps. The heat stability of the developed single enzyme nanoparticles with TMS and AA nanolayers were tested at two different temperatures (+4 °C; 80 °C) following the residual activities of the modified enzymes in regular time interval. Upon incubation of the cellulase complex nanoparticles with TMS and the non-modified cellulase at 80 °C, the nanoparticle has kept its 40% of its starting cellulase activity after 6 hours, whereas the non-modified enzyme lost its activity completely in half an hour. The relativeactivity of SENs with AA is about 50% of the initial activity. After 12 hours under 80 °C the activity of NCK is reduced to 14%, while the activity of NCKAis 24% of the activity of the native enzyme at the start of the incubation. The nanoparticles of the Thermobifida fusca hemicellulases (β-D-xylosidase and β-D-mannosidase) obtained from both TMS and AA nanolayers have exhibited concomitant stability increase. TMS nanoparticles of β-xylosidase has lost 60% of its activity after 120 days during an incubation procedure at +4 °C, whereas the non-modified β-xylosidase lost all its activity in 40 days during the same conditions. The 40% of the starting enzyme activity of β-D mannosidase with AA has remained after 6 hours, during an incubation process at 80 °C, the activity of the non-modified enzyme has lost in one hour.

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