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Techniques for Enzyme Purification

Adrie H. Westphal1 and Willem J. H. van Berkel1,2

1 Wageningen University & Research, Laboratory of Biochemistry, Stippeneng 4, 6708WE Wageningen, The Netherlands

2 Wageningen University & Research, Laboratory of Food Chemistry, Bornse Weilanden 9, 6708WG, Wageningen, The Netherlands

1.1 Introduction

Biocatalysis is the chemical process through which enzymes or other biological catalysts perform reactions between organic components. Biocatalysis gives an added dimension to synthetic chemistry and offers great opportunities to prepare industrial useful chiral compounds [1, 2]. Depending on the goal of the chemical conversion and the costs involved, biocatalyst-driven reactions are performed using whole cell systems or isolated enzymes, either in free or immobilized form [3-5].

Initially, industrial applications utilizing isolated enzymes were mainly developed with amylases, lipases, and proteases [6-8]. These hydrolytic enzymes were usually applied in a partially purified form, also because crude enzyme preparations are often more stable than the purified ones. However, for obtaining highly pure products, especially in the pharmaceutical industry, the purity of the enzyme preparation can be a critical factor.

Many enzyme purification methods have been developed over the years. Traditional purification procedures make use of the physicochemical properties of the enzyme of interest. These procedures were developed during the twentieth century for elucidating enzyme mechanisms and solving protein three-dimensional structures but also appeared to be valuable for the preparation of highly pure biocatalysts. Yet, progress in the preparation of biocatalysts has been given the biggest boost by the amazing developments in recombinant DNA technology and the accompanying revolutionary changes in enzyme production, enzyme purification, and enzyme engineering [9].

Here, we describe our experiences with the contemporary techniques for enzyme purification. For more information about the practical issues of enzyme purification, the reader is referred to the "Guide to Protein Purification" in Methods in Enzymology 463 [10].

1.2 Traditional Enzyme Purification

Before summarizing the traditional enzyme purification methods, it is important to note that the purification of enzymes is made easier by the fact that they are such specific catalysts. This enables the determination of the amount of a given enzyme in units (where 1 unit [U] of enzyme activity is defined as the amount of enzyme that catalyzes the conversion of 1?µmol substrate per minute) and its specific activity (in U?mg-1) in crude extracts and after each purification step. The specific activity is a good indication of the purity and quality of the enzyme preparation, especially if the specific activity of the pure enzyme under defined conditions is known. During enzyme purification, the improvement in specific activity and the yield of the enzyme after each purification step can be summarized in a purification scheme. The purification factor (specific activity obtained after a purification step divided by that of the starting material) provides an insight into the "efficiency" of each step. If a pure enzyme is obtained, it also indicates the relative amount of that enzyme present in the starting material. A theoretical example of a purification scheme, comprising three purification steps, is shown in Table 1.1.

Enzymes that are used for biocatalysis are typically purified from microbial cells or from culture media after or during growth of microorganisms (in case of excreted proteins). The enzyme purification generally starts with a cleared cell extract in which the enzyme is present in a soluble form. If the enzyme to be purified is excreted into the culture medium, it is usually sufficient to remove the cells from the medium by centrifugation (for small-scale purifications) or by filtration (for large-scale industrial purifications). In the case of an intracellular enzyme, cells should be broken first to release the protein into solution. Depending on the type of cells, different techniques are employed. The microbial cells are first harvested from the culture medium by centrifugation and resuspended in a small amount of buffer. The cells can be broken using a variety of techniques, e.g. by treatment with enzymes that digest cell walls (e.g. lysozyme), followed by osmotic shock, by using lysis buffers containing detergents, by exposure to ultrasound using sonicators, by pushing cells under high pressure through a small orifice using a pressure cell system, or by grinding frozen cells in liquid nitrogen. Extracts thus obtained are cleared from unbroken cells and large, insoluble particles by centrifugation or filtration. To prevent enzyme inactivation during these treatments, and also in the following purification steps, the temperature of the enzyme solution is usually kept around 4?°C. Proteolytic degradation of the enzyme to be purified can be precluded by adding a protease inhibitor cocktail during breaking of the cells.

Table 1.1 Imaginary traditional enzyme purification scheme.

Steps: CE, cell extract; AS, ammonium sulfate fractionation; IEC, ion exchange chromatography; GF, gel filtration.

Once a cell-free extract has been obtained, several methods can be employed for further purification of the desired enzyme. These separation methods can be roughly divided into the following categories: (i) selective precipitation, (ii) separation based on charge, (iii) separation based on molecular size, (iv) separation based on bio-affinity, and (v) separation based on adsorption principles. Except for the first category, all these methods generally make use of column chromatography, with column sizes depending on the scale of the sample volumes and protein concentrations.

The strategy applied during enzyme purification is such that separation methods belonging to different categories are carried out in a logical order until the goal is reached. A good purification results in the recovery of most of the enzyme activity (i.e. a high yield) and in removal of many "contaminating" proteins and other types of (bio)molecules (i.e. a strong increase in specific activity). An often-experienced phenomenon during purification is the inactivation and/or aggregation of the enzyme (Figure 1.1). Because of increased enzyme concentration in the final steps of purification, aggregation can occur. If proteases are still present, the enzyme becomes more and more the only target for the protease, which can lead to proteolysis. In addition, wrong physical conditions (pH, temperature, and ionic strength) can lead to (partly) unfolding, followed by aggregation and/or proteolysis. Changing the type of buffer, pH, and/or ionic strength and the addition of protecting agents may alleviate these processes.

The purity of the final enzyme preparation can be tested in several ways. The most common methods used are sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 1.2), analytical gel filtration, and mass spectrometry [11].

Figure 1.1 Enzyme aggregation and proteolytic degradation processes.

Figure 1.2 Example of an SDS-PAGE gel. (1) Molecular mass markers, (2) cell extract, (3) sample after ammonium sulfate fractionation, (4) sample after ion exchange chromatography, and (5) sample after gel filtration. Mr., relative molecular mass (kDa).

Traditional enzyme purification procedures many times start with an ammonium sulfate fractionation. This type of fractionation makes use of the fact that individual proteins precipitate at different concentration ranges of ammonium sulfate [12].

To make an estimation of the fractionation range, a small-scale pilot experiment can be performed. For such an experiment, different amounts of ammonium sulfate (from 0% to 90% saturation) are added to small samples of cell extract (usually, 1?ml). After dissolving the ammonium sulfate and removal of the formed protein precipitates by centrifugation, enzyme activity of the supernatants is measured (Figure 1.3). Such an analytical pilot experiment tells us at which saturation value the enzyme starts to precipitate (in our pilot, around 30%) and at which degree of saturation precipitation of the enzyme is more or less complete (in our pilot, around 65%). Once these values have been determined, the bulk...