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Thermal stabilization of trypsin by enzymic modification with beta-cyclodextrin derivatives
Reynaldo Villalonga1, Michael Fernández, Alex Fragoso
1Enzyme Technology Group, Center for Biotechnological Studies, University of Matanzas, Autopista a Varadero km 3 1/2, Matanzas, C.P. 44740, Cuba. reynaldo.villalonga@umcc.cu
Biotechnology and Applied Biochemistry
|February 21, 2003
Summary
Enzymatic modification of bovine trypsin with beta-cyclodextrin derivatives enhanced its esterolytic activity and thermostability. This process also improved enzyme resistance to thermal inactivation and autolysis.
Area of Science:
- Biochemistry
- Enzymology
- Protein Modification
Background:
- Bovine pancreatic trypsin is a crucial enzyme with applications in various biological processes.
- Enhancing enzyme stability and activity is vital for industrial and therapeutic applications.
- Cyclodextrin derivatives offer potential for modifying protein properties.
Purpose of the Study:
- To enzymatically conjugate beta-cyclodextrin derivatives to bovine trypsin.
- To evaluate the impact of this modification on trypsin's catalytic activity and stability.
- To investigate the enzyme's resistance to thermal denaturation and autolysis.
Main Methods:
- Utilized Streptoverticillum sp. transglutaminase as a catalyst.
- Conjugated four different beta-cyclodextrin derivatives to trypsin's glutamine residues.
- Quantified the degree of modification (approx. 3 mol oligosaccharides/mol protein).
Main Results:
- Increased specific esterolytic activity (4-21%) of modified trypsin.
- Reduced K(m) values for cyclodextrin-trypsin complexes (58-87% of native).
- Enhanced optimal temperature (5-10°C increase) and thermostability (16°C increase).
- Improved resistance to thermal inactivation and autolysis at alkaline pH.
Conclusions:
- Enzymatic modification with beta-cyclodextrin derivatives significantly improves trypsin's functional properties.
- The modified trypsin exhibits enhanced activity, stability, and resistance to denaturation.
- This approach offers a promising strategy for enzyme engineering and application development.