Enzyme Purification

1. Enzyme Purification A variety of enzyme purification services are available at Creative Enzymes. We provide purification and quality analysis in small trial scales or large industrial scales from natural resources or production mixtures, for clinical, therapeutic, research, and chemical industries. Our services also cover the preliminary steps, as well as post-purification recovery and analysis. Although initial characterizations of an enzyme in a mixture or sample matrix is practical, such as activity measurement and preliminary quantification, more fundamental knowledge relies on more advanced studies of the enzyme, which can only performed with pure enzyme samples. Pure enzymes also mean easier assays with less interferences. Some analysis methods, such as crystallography, are sensitive to sample purity and give desired results only with the highest samples purity. In large scale production for industrial applications, enzyme purification is directly related to product quality, in addition to regulatory requirements. Therefore, enzyme purification must be thoroughly considered and cautiously operated for both research and production purposes. However, the task is not straightforward. Many factors could change the efficiency, the yield, and stability of activity during purification, and the effects of these factors vary largely from one enzyme to another. At Creative Enzymes, we depend on the knowledgeable scientists and their years of experiences to design and perform the most suitable purification process for each enzyme. We understand that the customers may have different preferences on the purity, yield, and stability in different cases, and we will further customize the purification process to satisfy these needs. Almost all samples need to be prepared before the actual purification. For the enzymes from cell sources, they need to be fractionated into components before purification. The first step usually involves homogenization of cells, which disrupt the cell wall to release the enzyme into the homogenate, along with other components. Depending on the cell type, the homogenization could be easy as in the case of mammalian tissue without rigid cell wall, or it may need harsher conditions such as abrasion, freezing, and high pressure due to the rigid cell wall of the plant tissue. Sometimes, additional hydrolytic enzymes or detergents are added for better extraction. The mixture is then fractionated by centrifugation, yielding a dense pellet of heavy material at the bottom of the centrifuge tube and a lighter supernatant above (Figure 1). The supernatant is again centrifuged at a greater force to yield yet another pellet and supernatant. The procedure, called differential centrifugation, yields several fractions of

2. decreasing density, each still containing hundreds of different proteins, which are subsequently assayed for the activity being purified. Usually, one fraction will be enriched for such activity, and it then serves as the source of material to which more discriminating purification techniques are applied. The choice of temperature, pH, buffering salt, buffer strength, ionic strength, osmolarity, additives (EDTA, SDS, non-ionic detergents etc.), and homogenization technique are important of the success of purification.