Introduction

The goal of enzyme purification is to obtain a substantial amount of a specific enzyme while conserving its function in vivo. Enzyme purification consists of three phases, which can be elaborated into four general steps.

Depending on the physical and chemical properties, downstream application and the source of the target enzyme, each enzyme requires its unique purification strategy. This article discusses the importance of enzyme purification, along with the general steps involved in purifying enzymes and the factors that affect these processes.

The Importance of Enzyme Purification

Enzymes are proteins that serve as natural catalysts, initiating and accelerating cellular non-spontaneous chemical reactions. They have high turnover rates but are also selective and specific in their actions. Most importantly, their catalytic activities are not restricted only to in vivo conditions but can also be initiated in vitro, provided that they are in favorable conditions with all necessary components present.

Before an enzyme is applied industrially, the target enzyme must be isolated so that it can be thoroughly characterized in terms of its catalytic capabilities. It’s also characterized in terms of the necessary components and conditions for its in vitro activity and suitable applications.

Well-characterized enzymes can be valuable as catalysts in manufacturing processes. For example, lipase is used to prepare milk for cheese production and flavor development, while catalase is used in combination with cellulase and xylanase to digest lignin and cellulose in raw materials for papermaking and bioethanol production.

Other enzymes are intrinsically valuable and commercialized as consumer products. For instance, lactase is used in dairy products or as digestive aids for people who are lactose intolerant. Adenosine deaminase and 𝜷-glucocerebrosidase are used in replacement therapies for patients who suffer from Severe Combined Immunodeficiency Disease (SCID) and Gaucher’s disease, respectively.

Since enzymes readily exist in all living organisms, enzymes for functional studies and industrial applications are produced in living cells either by recombinant DNA technology or fermentation of suitable microorganism strains. Once a substantial amount of enzyme is produced, the target enzyme is isolated and refined for its intended use.

General Steps in Enzyme Purification

Enzyme purification starts with removing the target enzyme from the host cells. Then, the isolated enzyme is decontaminated in a series of purification steps so that it is free from other cellular components and non-targeted enzymes.

Nevertheless, enzyme purification generally consists of three main phases: capture, intermediate purification, and polishing. In practice, enzyme purification can be divided into the following steps:

1. Cell Extract Preparation

Also referred to as cell lysis, cell extract preparation is a step in the capture phase. It involves releasing the cellular content from the host cells or tissue into an extraction buffer that conserves and stabilizes the target protein, resulting in the crude extract.

Depending on the characteristics of the host cell, cell lysis can be done mechanically or chemically.

For instance, plant cells, which possess thick cell walls, are typically homogenized using a blender or ground using mortar and pestle. While bacterial cells are lysed using detergent-containing buffers, enzymatic digestion, or sonicators because they have thinner cell walls.

2. Enzyme Clarification

This step is considered the intermediate purification phase. Here, unwanted cellular components are removed from the crude extract.

The resulting clarified extract originates from multiple rounds of ultracentrifugation to remove the undesirable particles based on their size, shape, density, and viscosity in the buffer. Alternatively, cellular debris and unwanted components can be precipitated out to separate them from the target enzyme.

In practice, crude extract can be subjected to sequential or differential precipitation to precipitate unwanted components as a group based on their chemical and physical properties. In differential precipitation, a gradient buffer is used to precipitate molecules based on their solubility in the buffer.

Another method of removing salts and other unwanted small molecules is via dialysis. The clarified extract is treated against a large volume of buffer and diffused through semipermeable membranes based on the rate of diffusion.

3. Enrichment of Clarified Extract

In this step, the protein extract is polished by precipitating and pooling the target enzyme to increase the purity, concentration, and quantity.

Several enrichment techniques exist, including lyophilization or freeze-drying, ultrafiltration, ion-exchange chromatography, and salting-out methods such as precipitation and fractional precipitation.

The choice of enrichment technique is dependent on the characteristics of the target enzyme and its intended application.

4. Final Purification

After the enrichment, the extract is frequently subjected to a chromatography-based separation technique like affinity chromatography to increase the purity of the enzyme.

If the clarified extract is enriched using a chromatography-based technique, another chromatography-based technique is adopted to enhance the purity of the extract.

Factors Affecting Enzyme Purification

Typically, as a quality control measure, the enzyme’s activity and concentration are estimated at the end of each step. How each step is done depends on the downstream applications and several factors.

For instance:

1. Downstream Applications and Intended Use

The intended use of the target enzyme determines the overall purification strategy, including the best way to tackle each purification step.

This is because most downstream applications are typically dictated by technical or financial constraints. For example, characterization techniques such as mass spectrometry, optical spectroscopy and crystallization are sensitive to detergents which are frequently found in the extraction buffer.

Consequently, the extraction buffer used to prepare the cell extract meant for such a technique must contain a minimal amount of detergents, or an additional purification step may be necessary to remove detergents from the purified enzymes.

Industrial enzymes are another example to look out for. Enzymes for pharmaceutical use are subjected to safety regulations and high purification standards, as well as enzymes used in the manufacturing of food and livestock, causing them to go through several rounds of polishing procedures.

On the contrary, enzymes for non-food manufacturing processes do not need to achieve such high standards since the relevant regulations are not as stringent, and their commercial values are generally not as high as products used in animals or human beings.

2. Enzyme Properties

Enzyme functionality is determined by their amino acid composition, which also defines the physical and chemical properties of the target enzyme. The enzyme functionality relies on its catalytic center, also known as the active site where its interaction with the substrate and other relevant factors take place.

The three-dimensional structure of a particular enzyme is dictated by its amino acid composition. The intra– and intermolecular forces between the amino acids and between amino acids and surrounding water molecules give the enzyme its conformation, including the typical pocket-shaped catalytic center. That way, only the electrically and geometrically compatible substrates can gain access, interact with the functional residues, and initiate the catalytic activity of the enzyme.

Since the molecules that surround the enzyme can form intermolecular bonds with the enzyme, the components in the buffer solutions used in the purification process inevitably affect the enzyme, including its solubility. As a result, the purification strategy and conditions such as pH, temperature, ions, and buffer components must be optimized to accommodate the properties of the target enzyme and retain the catalytic activity.

Table 1. Examples of enzymes from various cellular localization that influence the properties of target enzymes, the suggested buffer composition, working condition, and purpose of use (Adapted from Handbook for Protein Purification, Amersham Biosciences).

Properties of the target enzyme, cellular localization Suggested buffer composition Working condition Purpose of use
Soluble proteins Salt 5-100 mM Maintain ionic strength, mimic the physiological condition
Lysosomal membrane proteins Sucrose or glucose 25 mM Stabilize lysosomal proteins
Integral membrane proteins Detergents 0.05-1.5% Solubilize membrane proteins
Metal-dependent proteases Chelators such as EDTA or EGTA 2-10 mM Eliminate contaminating divalent cations, inhibit protease activity

3. Combination of Separation Techniques

While an enzyme is characterized by many properties, one separation technique rarely covers more than one or two characteristics in its principle of separation. Hence, combining many techniques with different separation principles will accommodate many aspects of the target enzyme’s characteristics.

Table 2. Common chromatography-based techniques and the corresponding principle used for separation (Adapted from Vijayaraghavan et al., 2016).

Chromatography Technique Principle of Separation
Ion-exchange chromatography Ionic charge
Hydrophobic-interaction chromatography Hydrophobicity
Gel-filtration chromatography Molecular sieve
Affinity chromatography Affinity constant

4. Target Enzyme Abundance in Host Cells or Tissues

Enzymes are naturally mixed with other enzymes and biomolecules. While protein purification is generally designed to remove undesirable cellular components and non-target enzymes, the abundance of the target enzyme in the host cell and tissue plays a role in the efficiency of enzyme purification.

If the target enzyme is highly concentrated, the proportion of contaminants in the host cells are lower in comparison and less likely to damage the target enzyme.

On the contrary, if the target enzyme is low in the host cells, the proportion of contaminants is higher and more likely to interfere with the purification process. Consequently, it may be necessary to add additives and other components to facilitate the purification process.

For example, trehalose and mannitol can be added to the lysis buffer to help stabilize the enzyme during extraction. Chaotropic salts like calcium chlorides (CaCl2) can interfere with hydrophobic bonds and aid in the purification steps.

These additional components are eventually removed during the enrichment phase, which may lower the yield of the target enzyme.

5. Frequency of Sample Handling

As a rule of thumb, loss of the target enzyme and its activity can occur in every step at any time during purification. For this reason, purification protocols that consist of many steps or frequently require direct contact with the sample are more exposed than the ones that have fewer purification steps or do not require frequent sample handling.

One way to decrease the frequency of sample handling is to simplify the workflow and use an elution buffer that is compatible with all of the selected separation techniques.

In Conclusion

The purification of enzymes for in vitro use revolves around capturing the target enzyme from the source, removing non-target enzymes and other cellular components, and enhancing the concentration and purity of the target enzyme while preserving its activity.

For the most part, successful enzyme purification relies upon leveraging on the enzyme’s characteristics, which can be used as a basis for purification strategies and the selection of separation techniques.

Apart from the enzyme’s inherent properties, the sources from which the target enzyme is extracted and subsequent purification steps also contribute to successful enzyme purification.

References

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