eISSN: 2353-9461
ISSN: 0860-7796
BioTechnologia
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3/2019
vol. 100
 
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abstract:
RESEARCH PAPERS

Bio-inspired trypsin-chitosan cross-linked enzyme aggregates: a versatile approach for stabilization through carrier-free immobilization

Heidi Abdel Mageed
1, 2
,
Nermeen Abuel Ezz
3
,
Rasha Radwan
4

  1. Molecular Biology Department, Genetic Engineering and Biotechnology Division, National Research Centre, Cairo, Egypt
  2. Faculty of Pharmaceutical Sciences and Pharmaceutical Industries, Future University in Egypt (FUE), Cairo, Egypt
  3. Biochemistry Department, College of Pharmaceutical Sciences and Drug Manufacturing, Misr University for Science and Technology, Cairo, Egypt
  4. Biochemistry and Biotechnology Department, Faculty of Pharmacy and Drug Technology, Heliopolis University, Cairo, Egypt
BioTechnologia vol. 100 (3) C pp. 301–309 C 2019
Online publish date: 2019/09/26
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Enzymes are versatile catalysts for numerous industrial biocatalytic processes. Cross-linked enzyme aggregates (CLEAs) as a carrier free immobilization approach has drawn much attention being simple, cost efficient, capable of preserving high catalytic efficiency and improve enzyme reusability. The aim of this study was to develop a reusable, thermally and operationally stable trypsin CLEAs through co-aggregation with chitosan (CHS). Physicochemical characterization of the prepared CLEAs, including pH and temperature optimum, kinetic parameters, and operational and thermal stability in the absence (CLEA-T), and presence (CLEA-T-CHS) of CHS was carried out. CLEA-T-CHS and CLEA-T were prepared under mild conditions and cross linked using glutaraldehyde with 92% and 31% residual activity, respectively. Immobilized trypsin showed improved pH stability at alkaline pH. At 70EC the immobilized enzyme had 62% residual activity while the free enzyme lost 91% of its initial activity. The kinetic parameters (Km and Vmax) of the immobilized trypsin marginally increased, leading to a decreased catalytic efficiency. Operational and thermal stability were highly improved for CLEA-T-CHS; the half-life (t 1/2) of free trypsin and CLEA-T-CHS were 15 min and 65 min, respectively. Storage stability was highly improved; CLEAT-CHS and the free enzyme had 82% and 21% residual activity, respectively, after storage for 4 weeks. CLEA-TCHS retained 64% residual activity after five consecutive hydrolytic cycles, thus reinforcing its robust potentials. In this study, we successfully prepared a thermally stable and highly active immobilized trypsin through crosslinking in the presence of CHS. Results suggest that CLEA-T-CHS has great potential for industrial applications, including re-use in protein digestion.
keywords:

cross-linked enzyme aggregates (CLEAs), trypsin enzyme, carrier-free immobilization, stability study, chitosan, reusability



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