RESEARCH PAPER
Bio-inspired trypsin-chitosan cross-linked enzyme aggregates: a versatile approach for stabilization through carrier-free immobilization
 
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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
 
 
Submission date: 2019-06-09
 
 
Final revision date: 2019-07-19
 
 
Acceptance date: 2019-07-25
 
 
Publication date: 2019-09-26
 
 
BioTechnologia 2019;100(3):301-309
 
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ABSTRACT
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.
eISSN:2353-9461
ISSN:0860-7796
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