Purification, enzyme

The crude extract or clarified broth containing the enzyme is then subjected to purification, conceived as a sequence of operations (see Fig. 2.1) aimed to remove all contaminants that can interfere with its intended use. Purification can also serve to the purpose of increasing the specific activity of the biocatalyst in the case of enzyme immobilization. The situation will be radically different for an intracellular than for an extracellular enzyme. In the first case, the enzyme extract is a complex mixture of proteins, nucleic acids and other cell constituents, while in the latter the enzyme concentrate contains only some extracellular proteins and small molecular weight solutes, since the cell membrane acts as a powerful barrier to retain most of the cell constituents aiding powerfully to purification. Many extracellular hydrolases 

Small molecular weight solutes, particularly ions, might interfere with the initial purification steps and in such case they should be previously removed either by diafiltration or size exclusion chromatography. This is not usual, so that removal of ions (desalination) is rather used as a final polishing step. 

Enzyme purification is aimed mainly to the removal of contaminant proteins therefore, enzyme purification is in essence a series of operations of protein fractionation. A compromise exists between purification and yield of recovery. Each operation intended for purification produces an increase in purity (conveniently expressed in terms of specific activity units of activity per unit mass of protein) but inevitably some enzyme activity is lost so that yield of recovery is lower than 100%. Purification factors and yields for a given operation (i) and global values for N consecutive operations are then  

There is clearly a compromise between purification and yield of recovery, since increasing levels of purity require a higher number of operations, each of them having its own yield of recovery. Even if the yield of recovery per operation is high (i.e. 80%), if purification requires several (i.e. five) consecutive operations, global yield of recovery will be low (i.e. only 33%). If average purification factor is 2.5 per operation, after five operations it will be 98. This can be appreciated in Fig. 2.3. 

In principle, any method intended for protein fractionation can be used for enzyme purification. However, the methods applicable for production purposes are restricted to those amenable for scale-up at a reasonable cost. Only those will be reviewed here. Comprehensive reviews on protein purification can be found elsewhere (Asenjo 1990 Janson and Ryd6n 1998 Roe 2001 Hatti-Kaul and Mat-tiasson 2003b Rosenberg 2004) and handbooks on the subject are also available (http //www.biochem.uiowa.edu/donelson/Database%20items/protein purification handbook.pdf). 

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S. P. Colowick u. N. O. Kaplan, Methods in Enzymology, Vol. XXII, Enzyme Purification and Related Techni-... 

The discovery and exploitation of enzymes in aldoxime-nitrile pathway nitrile hydratase, amidase, nitrilase, aldoxime dehydratase, etc., are shown along with the use of methodologies, such as organic chemistry, microbial screening by enrichment and acclimation culture techniques, enzyme purification, gene cloning, molecular screening by polymerase chain reaction (PCR). 

Enzyme purification and characterisation Acetyl esterases were isolated from a Rapidase C-80 preparation according to the scheme shown in Figure 1. The purified acetyl esterases were devoid of relevant side activities, and showed great differences in their specificity towards the different acetylated substrates. 

Downstream Processing Microfiltration plays a significant role in downstream processing of fermentation products in the pharmaceutical and bioprocessing industry. Examples are clarification of fermentation broths, sterile filtration, cell recycle in continuous fermentation, harvesting mammahan cells, cell washing, mycelia recovery, lysate recovery, enzyme purification, vaccines, and so forth. 

Volume XXXIV. Affinity Techniques (Enzyme Purification Part B) Edited by William B. Jakoby and Meir Wilchek... 

Volume 104. Enzyme Purification and Related Techniques (Part C)... 

H. Kuhl, J. Kruip, A. Seidler, A. Krieger-Liszkay, M. Bunker, D. Bald, J.A. Scheidig, M. Rogner (2000) Towards structural determination of the water-splitting enzyme. Purification, crystallization, and preliminary crystallographic studies of photosystem II from a thermophilic cyanobacterium. J. Biol. Chem., 275 20652-20659... 

Enzyme Purification. The purification of the xylanolytic enzymes began with adsorption on a cation exchanger (CM-Sepharose FF) at pH 4.0. The final purification was accomplished by another ion exchange step as described previously for xylanase (24), / -xylosidase (25), a-arabinosidase (26) and acetyl esterase (27). 

Hou, W. C., Chen, H. J., and Lin, Y. H. (2003). Antioxidant peptides with angiotensin converting enzyme inhibitory activities and applications for angiotensin converting enzyme purification. ]. Agric. Food Chem. 51,1706-1709. 

The earliest applications of bioaffinity chromatography involved its use in enzyme purification (see Figure 13.7) [7]. Enzyme purification has continued to be a major application of this technique [57]. Some ligands that are employed for this purpose are enzyme inhibitors, coenzymes, substrates, and cofactors. Examples include methods that use nucleotide mono-, di-, and triphosphates for the... 

FIGURE 13.7 An early example of an enzyme purification performed by affinity chromatography. (Reproduced with permission from Cuatrecasas, P. et ah, Proc. Natl. Acad. Sci. U.S.A., 68, 636, 1968.)... 

Wikstrom, P. and Larsson, P.-O., Affinity fibre—A new support for rapid enzyme purification by high-performance liquid affinity chromatography, J. Chromatogr., 388, 123-134, 1987. 

Very recently, Lazarova and Tonova [53] reported an integrated process for extraction and stripping of a-amylase using RMs in a stirred cell with separated compartments for each process. A comparison between the classical process and the integrated process indicated a 1.27-fold enhancement in the enzyme purification by the latter. This integrated process was operated with 100 ml volume in... 

The ligands used for enzyme purification can be specific to the desired enzyme (substrate, substrate analogue, enzyme inhibitor, antibody), specific for different classes of enzyme (AMP, NAD, PLP) or of limited predefined specificity (dye affinity chromatography withProcion, Cibacron dyes). 

Chen YT, He JK, Ding JH, Brown BI (1987) Glycogen debranching enzyme purification, antibody characterization, and immunoblot analysis of type III glycogen storage disease. Am J Hum Genet 41 1002-1015... 

Mao, Q. Schunk, T. Gerber, B. Erni, B. A string of enzymes, purification and characterization of a fusion protein comprising the four subunits of the glucose phosphotransferase system of Escherichia coli. J. Biol. Chem., 270, 18295-18300 (1995)... 

The development of methods of enzyme purification, the discoveiy and recognition of the importance of coenzymes such as NAD, and the discoveiy of the pivotal metabolic role of ATP and other phosphorylated compounds ail came out of studies of glycolysis. The glycolytic enzymes of many species have long since been purified and thoroughly studied. 

Methods of Enzymatic Analysis, H. Bergmeyer, Editor. Contains methods for enzyme purification and assay, in several volumes. 

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