What is Enzyme Inactivation?
Enzyme inactivation occurs when an enzyme is incapable of catalyzing a reaction. Enzymes largely consist of proteins, and act as biological catalysts that speed up reactions. The surfaces of enzymes have specific sites that interact with specific substrates in a reaction. Enzyme inactivation occurs when the conformation of the active site changes or is damaged. Temporary (reversible) and permanent (irreversible) inactivation are the two forms of enzyme inactivation. In reversible inactivation, the function of the enzyme is restored once the adverse conditions are eliminated, whereas in irreversible inactivation, the function is permanently lost. The process of enzyme inactivation is significant in controlling the rate of reactions, and in the preservation food.
There are multiple causes of enzyme inactivation. High temperatures can alter the three -dimensional structures of proteins by breaking weak bonds (denaturation). Changes the extreme pH alter the charges of the amino acids composing the enzymes and changes the active site. Enzymes can also be permanently inactivated by certain heavy metals (mercury, lead), some toxins, and irreversible inhibitors. Other forms of inactivation can be caused by prolonged exposure to suboptimal conditions, oxidation, and radiation. In food processes, the heat treatment combines blanching and pasteurization, and intentionally inactivates enzymes to control spoilage during storage.
In biology, medicine, agriculture, and even in industry, the enzyme inactivation is of great importance. In medicine, some drugs help reduce infections, high blood pressure, and even cancer, by helping to inactivate some enzymes involved in these processes. In biotechnology, and in research laboratories, inactivated enzymes help to stop reactions in certain steps and help to provide reliable results in experiments.
In the food industry, enzyme activity can help improve shelf life, flavor, texture, and nutritional value. Inactivated enzymes can even reduce helpful biological activity, but in many biological and industrial activities, enzyme inactivation is important in providing the stability of a product, controlling metabolism, and providing safety and efficacy.
Thermal Enzyme Inactivation
Thermal enzyme inactivation occurs when enzymes lose their catalytic activity after prolonged exposure to high temperatures. Enzymes are protein molecules that facilitate biochemical reactions. Their activity is dependent on a precise 3D structure and if exposed to high temperatures, this structure will begin to change and lose activity. Thermal inactivation of enzymes is very important in food science, biotechnology, and pharmaceuticals and microbiology. Understanding how heat can affect enzymes helps to ensure safety of processing methods
Mechanism of Thermal Enzyme Inactivation
One of the primary mechanisms of thermal enzyme inactivation is protein denaturation. As temperature increases, the kinetic energy of the molecule increases, and the weak bonds that hold the three-dimensional structure of the enzyme (for example, hydrogen bonds, ionic interactions, and van der Waals forces) begin to break. Following the disruption of these weak bonds, the enzyme will begin to enter an extended (denatured) state. The active site of the enzyme will lose its shape, and, without an active site, the enzyme is incapable of catalyzing the reaction. In some instances of mild heating, enzymes may completely refold into the native state, and regain activity. However, severe denaturation renders the enzyme permanently inactive.
Factors Affecting Thermal Enzyme Inactivation
Thermal inactivation of enzymes depends on several factors. Temperature is the most important factor; the higher the temperature, the higher the rate of inactivation. The duration of heating causes greater thermal inactivation due to increased exposure. The pH of the surrounding environment influences the stability of enzymes because extreme acidic or alkaline changes can affect the thermal sensitivity of the enzymes.
The moisture content, and the presence of salts, sugars, and stabilizing compounds can protect enzymes or cause more inactivation. The heat resistance of enzymes varies based on their amino acid composition and molecular structure. For example, enzymes from some heat-loving microorganisms remain active at temperatures above 80°C, while enzymes from plants or animals can become inactive at those temperatures.
Kinetics of Thermal Enzyme Inactivation
The inactivation of enzymes through the application of heat exemplifies first-order reaction kinetics. Here, the extent of inactivation is directly proportional to the concentration of the active enzyme. To facilitate the design of heat processing methods, scientists have developed mathematical models to predict the stability of enzymes to heat. Important parameters of the mathematical models of enzyme inactivation include the D-value and the z-value.
The D-value is defined as the time required to reduce the active enzyme population to ten percent of its original value at a given temperature. The z-value is the temperature increase required to reduce the D-value by a factor of ten. Enzyme denaturation and inactivation is associated with a specific level of energy; this is called the activation energy of inactivation. These kinetic characteristics are extensively utilized by food engineers and food technologists to develop heat processing methods that inactivate enzymes while reducing the impact on food nutrients, texture, and flavor.
Applications in Food Processing
The impact of enzyme activity on food quality and safety has resulted in the widespread use of the inactivation of enzymes through the application of heat in food processing. Browning, discoloration, and loss of nutrients in freshly harvested fruits and vegetables is the result of an enzyme, polyphenol oxidase, and peroxidase. In order to retain flavor and nutrition, fruits and vegetables are heat-treated through blanching prior to freezing and drying.
The inactivation of both enzymes and microorganisms through heat processing of liquid foods is the basis of the extended shelf life of pasteurized milk and fruit juices. The canning process also relies on the permanent inactivation of enzymes that would otherwise lead to food spoilage during storage. The careful control of the application of heat in food processing inactivates enzymes without substantially reducing food processing vitamins or causing undesirable changes in the texture of food.
Industrial and Pharmaceutical Applications
Outside of food processing, thermal enzyme inactivation has a variety of industrial and pharmaceutical uses. In a biotechnology lab, researchers inactivate enzymes by heating them once they have completed a reaction. This allows precise stopping of the experiment at a certain time. In enzyme-based pharmaceutical production, researchers inactivate certain enzymes to avoid unwanted side reactions or to increase the stability of the final product. In industrial-based enzyme fermentation, heat treatment is also used to stop enzyme activity after the product has been formed. In the production of diagnostic kits, vaccines, and biological reagents, thermal inactivation of enzymes is also used to maintain product quality and reliability.
Advantages and Limitations
There are many advantages to the use of thermal enzyme inactivation. It is a very simple and reliable method that is inexpensive and easy to implement at an industrial scale. It can also simultaneously reduce the number of microorganisms present. However, the method does have a number of disadvantages that include altered sensory properties, nutrient loss, destruction of heat labile vitamins, and an increase in energy costs. The method is used in the food industry to inactivate enzymes, but only to the extent of achieving the desired effects. The most recent methods, such as high-temperature short-time (HTST) processing, incorporate this idea by employing the most extreme heating for a very short amount of time.
Enzyme Inactivation at pH Value
Enzyme inactivation at pH describes the permanent loss of an enzyme’s ability to catalyze a chemical reaction due to the change of the operating conditions of an enzyme away from the optimum pH. pH affects the structure of enzymes. Pepsin, an enzyme that catalyzes the breakdown of proteins into smaller peptides, has an optimum pH of 2, while trypsin, another proteolytic enzyme, has an optimum pH of 8. While pH 2 is a very acidic condition and pH 8 is a slightly basic condition, both of these conditions are optimum for the respective enzymes. As the concentration of H⁺ ions increases (very acidic solution) or decreases (very basic solution), the structural activity of an enzyme decreases.
Extreme conditions of pH compromise the integrity of the side chains of the amino acids of the enzyme and disrupts the structure of the enzyme. Denatured enzymes cannot catalyze the reaction because the substrate cannot bind to the enzyme. If the pH is altered beyond the optimum pH for a short period of time, the enzyme may reverse the damage done. However, irreversible inactivation is likely to occur if the enzyme is exposed for a long time to the extreme conditions of pH.
Enzyme inactivation can result from many factors including changes in pH, time and temperature of exposure, and the inherent stability of the specific enzyme. Some enzymes can be relatively stable with changing pH levels, while, for some others, the opposite can be true. To minimize the inactivation of enzymes to changing pH, buffer solutions can be used. Since enzymes play such a crucial role for optimized speed of reactions, controlling pH can minimize and help with inactivation of enzymes. This is important for optimizing reaction speed in biological and industrial processes.
Inactivation of enzymes due to pH changes is common in many industries. In food, controlling pH minimizes the enzymes that cause spoilage. In medicine, pH controls and changes of digestive enzymes affect drug formulation. During fermentation and other enzyme processes in biotechnology, the stability of enzymes and the yield of the processes can be negatively affected. Therefore, for practical and quality considerations in biological and industrial processes, enzyme inactivation due to buffer solutions must be understood.
Inactivation of Enzymes by Water Activity (Aw)
Water activity (Aw) in enzyme inactivation refers to decreased or lost enzyme activity triggered by changes in water availability in food or biological systems. Water activity is a measure of the availability of water (from 0—total absence of water—to 1.0—total water). Enzymes require water to retain their tertiary structures and to facilitate the interaction between the enzyme and the substrate. A decrease in Aw (e.g. drying or freezing or addition of salt/sugar) immobilizes the enzyme and reduces the formation of an enzyme-substrate complex, ultimately decreasing enzyme activity. At low Aw levels, most enzymes will become completely or partially inactive and some may regain activity with the addition of water.
At low water activity, enzyme inactivation occurs as a result of both structural and functional changes. Enzymes rely on water for the formation of stabilizing hydrogen bonds. A reduction in water displaces such stabilizing interactions and causes conformational changes in the enzyme and its active site. Water is also critical for the molecular movement of enzymes.
Reduced movement leads to a lower rate of diffusion of both the substrate and the product, and also decreases the rate of a catalytic reaction. The degree of inactivation is influenced by the water activity level, temperature, pH, duration of exposure, and the specific enzyme. Enzymes of certain microorganisms that live in arid conditions are known to be more tolerant of low water activity. In many instances, the enzyme inactivation at low water activity is a reversible process upon the reestablishment of moisture.
To minimize spoilage and improve shelf life, controlling water activity is a common practice in food preservation. Water activity is lowered by the addition of sugar or salt and food processing methods such as drying and freeze drying. Reducing water activity also helps control the activity of enzymes responsible for the loss of nutritional value and food quality, such as browning and spoilage.
Many dried and powdered food products rely on the low water activity to sustain product quality during food storage. Certain processing methods and the controlled addition of excipients in the biopharmaceutical industry help improve enzyme stability, prolong shelf life, and promote consistent efficacy of the biopharmaceutical product. The control of water activity is critical to the stability of enzymes, preservation of food, and the quality of many biological products.
Enzyme Inactivation by Electrolytes and Ionic Strength
When enzyme activity is lost or diminished due to the presence of certain electrolytes and changes in ionic strength, we say the enzyme is inactivated by these electrolytes or ions. Depending on their chemical nature, electrolytes dissociate in solution to give positive or negative ions. Commonly used sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl2), and magnesium (MgSO4) and phosphate salts (MgSO4).
These ions can alter physical and chemical properties of the medium surrounding the enzyme. Because proteins and hence enzymes are made up of amino acids which can be positive or negative, then ionic or electrolytic changes can affect enzyme structure and hence stability and also its catalytic activity. Hence, for normal biological and industrial processes, the ionic environment around the enzyme must be preserved.
Effect of Ionic Strength on Enzyme Activity
Ionic strength is the measurement of the concentration of total ions in a solution. It influences the catalytic activity of enzymes. Electrolytes in low or moderate ionic strength enhance the stability of the structure of the enzyme. The charged residues in the enzyme are shielded. In very high or very low ionic strength the stability of structure of the enzyme is lost and the shape of the active site is impaired. Enzyme activity relies on the binding between the enzyme and the substrate, which is diminished in this case. Consequently, in high or low ionic strength the enzyme is inactive.
Mechanism of Enzyme Inactivation by Electrolytes
The ability of some electrolytes to denature enzymes by changing the weak forces that hold their tertiary structure together (in 3D) is well known. At high salt concentrations, hydrogen bonds, ionic bonds, and hydrophobic interactions that hold the protein are destabilized, leading to partial or complete denaturation. In addition, metal ions such as mercury (Hg²⁺), lead (Pb²⁺), silver (Ag²⁺), and copper (Cu²⁺) can directly block the -SH, amino, or carboxyl groups of the protein.
This modifies the active site and/or also alters the accessibility of the enzyme to the substrate, causing permanent inactivation. Conversely, some metal ions such as magnesium (Mg²⁺), calcium (Ca²⁺), and zinc (Zn²⁺) can be used as cofactors for the same enzyme to be active. Thus, the concentration and chemical nature of the electrolytes define their respective effects.
Factors Affecting Electrolyte-Induced Inactivation
Many things impact the enzyme inactivation by electrolytes. The type of ion and how ions will react with enzymes. The concentration will determine what kind of effect will occur, a stabilizing effect, or an inhibiting effect. The duration of contact will also impact inactivation, a longer contact time will result in higher losses of activity. The temperature and pH will also affect how ions behave and will most likely make an enzyme more susceptible to disruptions by electrolytes. The presence of supporting compounds like sugar and proteins also can make an enzyme more susceptible to disruptions by electrolytes. All of these factors should be considered and controlled in experiments.
Applications in Food and Biotechnology
Most applications in biotechnology and in food processing rely on manipulating the concentration of electrolytes. The salting of cheese, the salting in the processing of meats, and the controlling of concentrations of salt in food all help control the activity of enzymes and help improve the quality of the food. The preservation of food also relies on controlling salt concentrations to control enzyme activity and reduce food spoilage. Controlled buffer solutions also help maintain the stability of enzymes throughout the processing in biochemistry. The processing of fermentation-based products also relies on controlling the optimum ionic concentration of products. The development of therapeutical enzymes and products also relies on the control of electrolytes to formulate stable products.
Controlling ionic strength and electrolytes has both pros and cons. Keeping the right conditions can help maintain the correct levels of enzyme activity and stability and can help improve the productivity of both lab and industrial processes. It can also help improve the selectivity of enzymes and the rates of enzyme action while minimizing side reactions. However, having too many electrolytes can cause the permanent inactivation of enzymes, the quality of the products can suffer, and biochemical reactions can be damaged. Heavy metal ions can cause irreparable damage to enzyme proteins. Therefore, it is important to keep the right ionic levels to ensure the highest possible activity of enzymes while avoiding inactivation.
The inactivation of enzymes due to varying ionic strength and electrolytes is a major concern in biochemistry, food science and technology, and biotechnology. The presence of different electrolytes can cause changes to the electric interactions of enzymes, affect the conformation of proteins, and also alter the binding of substrates. Moderate ionic strength tends to favor the stability of enzymes, while ionic strengths that are too high or too low can cause unfavorable changes to the conformation of the enzyme and can reduce the overall activity of the enzyme.
Some heavy metal ions can cause permanent inactivation of enzymes due to their binding with the important functional groups, while other metal ions are needed as cofactors by many enzymes. The use of low levels of different electrolytes and manipulation of ionic strength can help engineers and scientists develop methods to process materials and intentionally maintain enzyme activity. In some cases, to improve the safety, quality, and shelf life of products, the enzyme inactivation in the products is useful.
Enzyme Inactivation by Temperature
Loss of an enzyme’s functionality due to temperature is when an enzyme loses activity after being exposed to high temperatures that exceed the enzyme’s optimal range. An increase in temperature can break the weak hydrogen bonds that preserve the three-dimensional structure of the enzyme. Then the protein undergo denaturation and changes to the active site. Due to this, the enzyme can no longer effectively facilitate a reaction because the substrate can no longer bind to it. The degree of enzyme inactivation is dependent on temperature, duration of heating, the pH level, and enzyme stability. Typically, prolonged treatment of harsh conditions causes the permanent inactivation of an enzyme.
Enzymatic Inactivation in Antibiotic Resistance
Enzyme inactivation is chiefly what causes most of the antibiotic resistance that is exhibited by bacteria. In this case, bacteria secrete enzymes that destruct or modify the structure of the antibiotic molecules to prevent them from binding to their target sites. For instance, penicillin’s and cephalosporins that are inactivated due to the breakage of the β-lactam ring by β-lactamase are a good example of this. Other resistance-causing enzymes can modify an antibiotic by reducing its activity. This mechanism of resistance caused by bacteria can reduce the reliability of antibiotic treatment and cause a heightened need for different methods of treatment and different drugs to treat the infection.