Prions Biotech

Enzyme Stability and Compatibility in Wastewater Treatment Systems

Wastewater treatment systems rely on microorganisms and enzymes to break down organic compounds in wastewater. This helps to decrease BOD and COD, control filamentous bacteria growth, and reduce odors.

Enzyme activity declines markedly outside its optimal temperature range and many are (permanently) denatured if exposed to excessive heat. Immobilized enzymes are being used to help address these issues.

1. Chemical Stability

Chemical stability refers to a chemical system’s ability to remain in its lowest energy state, or thermodynamic equilibrium, with its environment. A material is considered to be chemically stable if it cannot be altered by any external perturbation, such as heat or pressure. Chemically stable materials are typically less reactive than their more unstable counterparts, and are more resistant to corrosion.

In chemistry, a solution’s pH can significantly affect its electrochemical properties. A low pH can promote the formation of metal carbonate complexes that are more soluble than neutral salts, while a high pH may encourage the dissolution of metallic ions.

A good way to stabilize pH is by using a buffer solution. Buffers contain a strong acid or base, which helps to balance out the concentration of positive and negative charges in the sample. In biochemistry, buffer solutions are often used to stabilize protein structures against reversible denaturation.

There is ongoing interest in the production of carrier-free immobilized enzymes, which are more stable than soluble proteins. One method for immobilization is crosslinking dissolved enzymes with glutaraldehyde, producing crystalline lattices of crosslinked enzyme molecules (CLECs; References 45 and 91). These immobilized enzymes exhibit enhanced thermostability, owing to their chemical stabilization. This type of immobilization also allows for the use of protease inhibitors that would otherwise inhibit enzyme activity.

2. Thermal Stability

When organic substances or plastics are exposed to heat and atmospheric oxygen over a long period of time, they undergo an oxidation reaction. This process affects not only the chemical properties but also the physical ones. A plastic that is thermally stable can be considered to have good mechanical characteristics because it will not deform under high temperatures.

Another important factor is how well a metal alloy retains its physical properties under elevated temperature conditions. In order to assess this, one of the most common tests is the OIT test or Oxidation Induction Time, where a sample is brought to a temperature below its melting point and then gradually heated in an inert atmosphere. Once a calorimetric signal is detected, the inert atmosphere is changed to oxygen and the reaction starts. The rate of the reaction is directly related to the temperature increase, and the OIT value is calculated from this relationship.

Opportunities exist for bioaugmentation of lactose-rich waste streams in dairy processing, as well as other food processing waste streams. However, the stability of immobilized enzyme systems needs to be improved in order to be competitive with existing technologies for waste stream valorization (such as surfactants). In addition, it is important to ensure that the microorganisms used in the augmentation are well matched to the contaminants. For example, microorganisms that produce lipases will be good at digesting fatty contaminants, while those that produce amylase will be well suited to breaking down starches and those that produce urease will be well adapted to breaking down urea.

3. Mechanical Stability

In biological wastewater treatment processes, enzymes are released into the aqueous phase as extracellular polymeric substances (EPS) from microbial cells. Several studies [27-29] demonstrate that shearing forces induced by agitation can disrupt EPS and release hydrolytic enzymes into the aqueous phase for recovery. However, the mechanical stability of these enzymes is often limited by their tethering to the EPS matrix. Enhanced mechanical stability can be achieved by the addition of surfactants, such as Triton X100 or Tween 20, to cell disruption protocols [26,27].

Enzyme immobilization is an important technology for achieving higher efficiency and greater process economics in industrial waste valorization applications. However, it is challenging to optimize the physicochemical properties of immobilized enzymes in order to achieve commercial translatability. This is particularly true for enzymes used in the valorization of food processing waste streams, which typically have conditions that are not ideal for enzyme activity (e.g. elevated temperatures and non-neutral pH).

Molecular simulations and experimental measurements show that the structural stability of immobilized enzymes depends on the number and locations of tethering sites, as well as the degree to which the tethering sites are chemically bound to one another. For example, NfsB enzymes tethered to two tethering sites via positions 423 and 111 exhibit higher thermal stability than NfsB enzymes tethered through only one site.

4. pH Stability

pH is a measure of the concentration of free hydrogen ions in an aqueous solution. The pH scale is a logarithmic one and ranges from 0 to 14. The higher the pH number, the more acidic the solution. The lower the number, the more basic the solution. A neutral pH range of 5 to 9 is optimal for enzyme stability. Beyond this, proteins are more prone to degradation in extreme acidic or alkaline conditions. This is especially the case for enzymes that are formulated in solution dosage forms, such as oral, parenteral, nasal and ophthalmic solutions.

Stability is the degree to which a compound retains, within specified limits, the properties and characteristics that it possessed at the time of manufacture or compounding. This includes its resistance to degradation, which is influenced by pH, temperature, and other factors such as particle size, air, light and solvents.

A protein’s structural integrity is governed by interactions between its folded and unfolded states, which alter the ionization equilibria of its acidic and basic groups. These interactions also result in differential pKa values between the two states. Previous attempts to calculate the pH dependence of protein stability have been limited by a lack of experimental unfolded state pKa data. However, our data set demonstrates that the pH of optimum activity and the pH of optimum stability are correlated.