Related Experiment Video
Updated: Jul 3, 2026
08:37
Measuring Enzymatic Stability by Isothermal Titration Calorimetry
Published on: March 26, 2019
How do additives affect enzyme activity and stability in nonaqueous media?
A O Triantafyllou1, E Wehtje, P Adlercreutz
1Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, PO Box 124, S-22100 Lund, Sweden. angeliki.oste-triantafyllou@biotek.lu.se
Biotechnology and Bioengineering
|April 5, 1997
Summary
Adding buffer salts or potassium chloride significantly boosts enzyme activity, especially for chymotrypsin transesterification. Additives act as immobilization matrices, improving enzyme performance and stability.
Area of Science:
- Biocatalysis and Enzyme Engineering
- Protein Chemistry and Biochemistry
Background:
- Lyophilized enzymes like alpha-chymotrypsin and Candida antarctica lipase often exhibit limited activity.
- Enzyme immobilization is a key strategy to enhance catalytic efficiency and stability.
Purpose of the Study:
- To investigate the impact of additives (buffer salts, potassium chloride, sorbitol) on the catalytic activity of lyophilized alpha-chymotrypsin and Candida antarctica lipase.
- To explore the role of these additives as immobilization matrices and their effect on enzyme stability.
Main Methods:
- Preparation of enzyme powders with varying concentrations of buffer salts, potassium chloride, and sorbitol.
- Assay of transesterification and peptide synthesis activities of the modified enzyme preparations.
- Thermal stability analysis through denaturation temperature and enthalpy measurements.
Main Results:
- Enzyme activity increased 4-8 fold with buffer salts or KCl; sorbitol showed a modest increase.
- Buffer species were crucial for chymotrypsin transesterification, preventing pH changes and yielding a 100-fold activity increase.
- Immobilization on celite significantly improved protease transesterification activity; buffer salts further enhanced immobilized enzyme activity 3-4 fold.
- Additives decreased thermal stability of chymotrypsin, lowering denaturation temperature and increasing denaturation enthalpy.
Conclusions:
- Buffer salts and potassium chloride act as effective immobilization aids, enhancing enzyme catalytic activity.
- Buffer species are essential for maintaining optimal pH, critical for certain enzymatic reactions like chymotrypsin transesterification.
- While improving activity, additives can decrease thermal stability, suggesting a trade-off between activity enhancement and thermostability.
Related Concept Videos
01:13Introduction to Mechanisms of Enzyme Catalysis
For many years, scientists thought that enzyme-substrate binding took place in a simple "lock-and-key" fashion. This model stated that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a more refined view scientists call induced fit. The induced-fit model expands upon the lock-and-key model by describing a more dynamic interaction between enzyme and substrate. As the enzyme and substrate come together, their interaction causes a mild...
02:34Enzymes
Inside living organisms, enzymes act as catalysts for many biochemical reactions involved in cellular metabolism. The role of enzymes is to reduce the activation energies of biochemical reactions by forming complexes with its substrates. The lowering of activation energies favor an increase in the rates of biochemical reactions.
Enzyme deficiencies can often translate into life-threatening diseases. For example, a genetic abnormality resulting in the deficiency of the enzyme G6PD...
Enzyme deficiencies can often translate into life-threatening diseases. For example, a genetic abnormality resulting in the deficiency of the enzyme G6PD...
01:30Enzyme Inhibition
Inhibitors are molecules that reduce enzyme activity by binding to the enzyme. In a normally functioning cell, enzymes are regulated by a variety of inhibitors. Drugs and other toxins can also inhibit enzymes. Some inhibitors bind to the enzyme’s active site, while others inhibit enzymatic activity by binding to other sites on the protein structure.
01:16Titration in Nonaqueous Solvents
Most acid-base titrations are performed in an aqueous medium. In aqueous titrations, water competes with weaker acids or bases for proton donation or acceptance, leading to ambiguous endpoints in the titration curve. Water also affects the partial ionization of weak acids or bases. For example, water accepts a proton from acetic acid to form hydronium and acetate ions. The hydronium ion formed is a stronger acid than acetic acid, and the acetate ion is a stronger base than water. As a result,...
01:22Introduction to Enzymes
The use of enzymes by humans dates to 7000 BCE. Humans first used enzymes to ferment sugars and produce alcohol without knowing that this was an enzyme-catalyzed reaction. Wilhelm Kuhne coined the term 'enzyme' in 1877 from the Greek words ‘en’ meaning ‘in’ or ‘within’ and ‘zyme’ meaning ‘yeast.’
Most enzymes are proteins that speed up biochemical reactions without being consumed. Enzymes contain one or more active sites that bind the substrates and convert them into products. Many enzymes also...
Most enzymes are proteins that speed up biochemical reactions without being consumed. Enzymes contain one or more active sites that bind the substrates and convert them into products. Many enzymes also...
01:19Enzyme Kinetics
Enzymes speed up reactions by lowering the activation energy of the reactants. The speed at which the enzyme turns reactants into products is called the rate of reaction. Several factors impact the rate of reaction, including the number of available reactants. Enzyme kinetics is the study of how an enzyme changes the rate of a reaction.
Scientists typically study enzyme kinetics with a fixed amount of enzyme in the controlled environment of a test tube. When more reactant, or substrate, is...
Scientists typically study enzyme kinetics with a fixed amount of enzyme in the controlled environment of a test tube. When more reactant, or substrate, is...