(Magnetic) Cross-Linked Enzyme Aggregates of Cellulase from <i>T. reesei</i>: A Stable and Efficient Biocatalyst

Dušica Ifko; Katja Vasić; Željko Knez; Maja Leitgeb

doi:10.3390/molecules28031305

Molecules (Jan 2023)

(Magnetic) Cross-Linked Enzyme Aggregates of Cellulase from <i>T. reesei</i>: A Stable and Efficient Biocatalyst

Dušica Ifko,
Katja Vasić,
Željko Knez,
Maja Leitgeb

Affiliations

Dušica Ifko: Laboratory for Separation Processes and Product Design, Faculty of Chemistry and Chemical Engineering, University of Maribor, Smetanova Ulica 17, SI-2000 Maribor, Slovenia
Katja Vasić: Laboratory for Separation Processes and Product Design, Faculty of Chemistry and Chemical Engineering, University of Maribor, Smetanova Ulica 17, SI-2000 Maribor, Slovenia
Željko Knez: Laboratory for Separation Processes and Product Design, Faculty of Chemistry and Chemical Engineering, University of Maribor, Smetanova Ulica 17, SI-2000 Maribor, Slovenia
Maja Leitgeb: Laboratory for Separation Processes and Product Design, Faculty of Chemistry and Chemical Engineering, University of Maribor, Smetanova Ulica 17, SI-2000 Maribor, Slovenia

DOI: https://doi.org/10.3390/molecules28031305
Journal volume & issue: Vol. 28, no. 3
p. 1305

Abstract

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Cross-linked enzyme aggregates (CLEAs) represent an effective tool for carrier-free immobilization of enzymes. The present study promotes a successful application of functionalized magnetic nanoparticles (MNPs) for stabilization of cellulase CLEAs. Catalytically active CLEAs and magnetic cross-linked enzyme aggregates (mCLEAs) of cellulase from Trichoderma reesei were prepared using glutaraldehyde (GA) as a cross-linking agent and the catalytic activity and stability of the CLEAs/mCLEAs were investigated. The influence of precipitation agents, cross-linker concentration, concentration of enzyme, addition of bovine serum albumin (BSA), and addition of sodium cyanoborohydride (NaBH3CN) on expressed activity and immobilization yield of CLEAs/mCLEAs was studied. Particularly, reducing the unsaturated Schiff’s base to form irreversible linkages is important and improved the activity of CLEAs (86%) and mCLEAs (91%). For increased applicability of CLEAs/mCLEAs, we enhanced the activity and stability at mild biochemical process conditions. The reusability after 10 cycles of both CLEAs and mCLEAs was investigated, which retained 72% and 65% of the initial activity, respectively. The thermal stability of CLEAs and mCLEAs in comparison with the non-immobilized enzyme was obtained at 30 °C (145.65% and 188.7%, respectively) and 50 °C (185.1% and 141.4%, respectively). Kinetic parameters were determined for CLEAs and mCLEAs, and the KM constant was found at 0.055 ± 0.0102 mM and 0.037 ± 0.0012 mM, respectively. The maximum velocity rate (Vmax) was calculated as 1.12 ± 0.0012 µmol/min for CLEA and 1.17 ± 0.0023 µmol/min for mCLEA. Structural characterization was studied using XRD, SEM, and FT-IR. Catalytical properties of immobilized enzyme were improved with the addition of reducent NaBH3CN by enhancing the activity of CLEAs and with addition of functionalized aminosilane MNPs by enhancing the activity of mCLEAs.

Published in Molecules

ISSN: 1420-3049 (Online)
Publisher: MDPI AG
Country of publisher: Switzerland
LCC subjects: Science: Chemistry: Organic chemistry
Website: http://www.mdpi.com/journal/molecules

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