Isolation of enzymes

The isolation of enzymes in a pure state is frequently a matter of great difficulty owing to their instability, their low concentrations in plant and animal tissues, and also to their colloidal nature. The methods employed depend upon the physical and chemical nature of the enzyme in question. In the following experiments, no attempt has been made to isolate enzymes in a high slate of purity. 

Use of microorganisms and plant and animal tissues as a biological component of biosensor are also described in the literature (9, 10). The principle is based on the use of the natural bio-reactive systems. They have several advantages over the isolated enzymes and receptors. Isolation of enzymes and receptors are often required to increase the response time. Enzymes and receptors retained in the cells are more stable and have longer lifetimes. Cell-based biosensors are also economical as no purification step is required. 

In the isolation of enzymes, activities were usually discovered by the provision of the substrate to trial organisms, organs, or organelles, which are candidates for possession of the desired activities. The enzyme so identified could be utilized in the intact organism (dead or alive) but could also be exploited in cell free extracts or in pure form. Further improvements in activity were incremental and were obtained through strain selection, strain improvement, alteration of growth conditions, adjustment of pH, or change of temperature (within a limited... 

In the early days of enzyme study research focused on the isolation of enzymes. The studies were laborious and time-consuming. This situation has been greatly improved by innovations such as the HPLC system and microcapillary electrophoresis, which have enabled the purification of a small amount of enzyme effectively. We in general can isolate and purify most enzymes by a combination of gene technology with these methods without difficulty. 

Adapting techniques based on in vitro protein synthesis to the isolation of enzymes requires establishing a link between a nucleic acid-protein complex and product formation. Methods based on binding, analogous to those developed for phage displayed libraries, may be used to enrich catalysts from noncatalysts. In addition, Tawfik and Griffiths (1998) exploited the aqueous core of reverse micelles as artificial compartments... 

Hirose, Y, Production and isolation of enzymes. Enzyme Catcdysis in Organic Synthesis, Vol. 1, pp. 41-66, Drauz, K. and Waldmann, H. (Eds.), Wiley-VCH, Weinheim, 2002. 

For many years salting-out by high concentrations of ammoniiun sulfate has been one of the classical methods of protein separation. There is very little literature on the theoretical basis of the method, particularly as applied to the isolation of enzymes, where it has mainly been used quite empirically. The underlying assumption in most cases seems to have been that the different proteins are precipitated at different fixed ammonium sulfate concentrations, provided the pH and temperature are fixed. For example one may commonly read in instructions for the piuification of an enzyme that the enzyme is precipitated at 65% saturation with ammonium sulfate or that the fraction precipitating between 0.62 and 0.68 saturation should be taken. It is, however, a fairly common experience that when one repeats a published method the enzyme fails to precipitate within the limits given. Furthermore, where the purification of a protein involves more than one salt-fractionation stage, the limits are usually found to be different for the different stages. 

The organomercurial derivative of agarose described by Cuatrecasas [44] has been used in isolation of enzymes and proteins containing free sulphydryl groups in particular good results have been achieved with papain [10]. 

Leaching or solid-liquid extraction are terms that describe the extraction of soluble constituents from a solid or semisolid by means of suitable solvents. The process, which is used whenever tea or coffee is made, is an important stage in the production of many fine chemicals found naturally in animal and vegetable tissue. Examples are found in the extraction of fixed oils from seeds, in the preparation of alkaloids, such as strychnine from Nux vomica beans or quinine from Cinchona bark and in the isolation of enzymes, such as rennin, and hormones, such as insulin, from animal sources. In the past, a wider importance attended the process because the products of simple extraction procedures, known as galenicals, formed the major part of the ingredients used to fulfill a doctor s prescription. 

Of course there are many factors which influence the competitiveness between enzymatic processes and chemical processes, for example, costs of substrates, costs for production/isolation of enzymes, possible space-time yields and costs for... 

One of the most important criteria in the evaluation of a new process is the availability of an enzyme114 (see Chapter 20 Tabular Survey of Commercially Available Enzymes). If the enzymes are not commercially available, their isolation and purification can be expensive and time consuming (see Chapter 2 Production and Isolation of Enzymes). The importance of the product to be synthesized may sometimes justify the additional effort. 

Table II. Isolation of enzymes in aqueous two-phase systems.

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