Enzyme process stability

The rate acceleration achieved by enzymes is due to several factors. Particularly important is the ability of the enzyme to stabilize and thus lower the energy of the transition state(s). That is, it s not the ability of the enzyme to bind the substrate that matters but rather its ability to bind and thereby stabilize the transition state. Often, in fact, the enzyme binds the transition structure as much as 1012 times more tightly than it binds the substrate or products. As a result, the transition state is substantially lowered in energy. An energy diagram for an enzyme-catalyzed process might look like that in Figure 26.8. 

The transition state-stabilizing power of the rapid enzymic process can be held up for comparison with a particularly slow nonenzymic reaction that is brought about through addition of a large amount of heating. In particular, comparisons of the rates of enzyme-catalyzed process and spontaneous nonenzymic reaction produce dramatically large ratios. 

Unlike activity, stability of enzymes is often interpreted simplistically as thermal stability, i.e., a temperature beyond which the enzyme loses stability. Although this quantity is important, first every statement of stability at a certain temperature depends on exposure time and thus is often ambiguous and second, for biocatalytic process applications, a more important quantity is the process or operational stability, which is the long-term stability under specified conditions. 

S. R. Weijers and K. van t Riet, Enzyme stability in downstream processing Part 1 Enzyme inactivation, stability and stabilization, Biotech. Adv. 1992a, 10, 237-249 Part 2 Quantification of inactivation, 251-273. 

The term biochemical stabilization refers to the biotic or abiotic production of organic substances that are refractory to decomposition by microorganisms and contribute, through condensation and complex formation, to the stabilization of otherwise easily decomposable substrates such as enzymes. This stabilization process coincides with the process of humification. 

The sol-gel process is the name given to a number of processes in which a solution, or sol, undergoes a sol-gel transition. In this broadest sense, the term sol-gel refers to the preparation of inorganic oxides by wet chemical methods, irrespective of final form product—monolith, crystalline, or amorphous (1). Using sol-gel materials for mechanical entrapment of enzymes permitted stabilization of the proteins, tertiary structure owing to the tight gel network (2). Moreover, the easy insertion of substituent groups into... 

The cost of enzyme purification is a major factor in the expense of most enzyme processes. If a bioreactor is incorporated into the process, then costs associated with bioreactor preparation and regeneration as well as the operational stability of the immobilized enzyme are major factors in the economics of the bioprocess (Swaisgood, 1991). 

The use of mixed catalytic systems with several enzymes can provide multiple benefits in terms of costs, effectiveness of the particular production process, stability of the bio-catalysts, and possible structures. 

Each molecule of MnP was first reported to contain two Ca + ions, which are believed to have a structural role. It has been shown that the thermal stability of the enzyme depends on the presence of these Ca + ions. Thermal inactivation appears to be a two-step process loss of a Ca + decreases the enzyme s stability [89]. If excess Ca + is added to the medium at this stage, the enzyme can be reactivated. However, further inactivation caused by the loss of heme, cannot be reversed. In another study... 

The mineral nutrient elements take part in many processes. Interestingly enough, most elements are involved simultaneously in different reactions of metabolism. Thus, P, K, Mg, Ca, and B are important for the formation of nucleic acids, production of nucleotide phosphates, energy metabolism, and stabilization of membrane structures. Zn is a component of many different enzyme processes, and even Mn and Fe are involved in various reactions. Therefore, no clear assignment of individual elements to distinct areas of metabolism is possible. Indeed, results obtained with different vege-... 

In general, enzymes have a disadvantage in that they are deactivated due to heat-induced structural changes or, in the case of proteolytic enzymes, due to decomposition by themselves. It is therefore desirable to distribute and use enzyme preparations in the form of solids, such as powders and granules, instead of liquids. Although drying itself is a valuable tool in the improvement of the enzyme storage stability, the process step itself often causes a substantial loss of activity and the final product is still susceptible to inactivation. 

Also, a combination of salts as well as a combination of enzymes can be used. The addition of divalent cations is preferred because they provide the best storage and processing stability. Sulfate is preferred as anion because it provides the best drying yield (Table 48.8). 

The work-up of batch processes, run in stirred vessels, had often faced the challenge to efficiently separate and recover the enzyme used. Meanwhile, there is abundant know-how available to immobilise enzymes on different carriers, though some issues need always to be considered maintained activity of the enzyme, its stability towards solvents and the operating temperature used in a reaction. Enzyme immobilisation allows for continuous reactions carried out in columns or in a sequence of continuous stirred-tank reactors. Certain advantages are offered by Degussa s enzyme-membrane-reactor (EMR), where the enzyme is surrounded by a hoUow-fibre membrane, that is permeable to substrate and product. 

The use of different types of membranes for enzyme entrapment is a well-described technique useful for the processes with allosteric enzymes, where the presence of cofactor(s) is required. The main properties of the membrane-bound biocatalysts is the large surface area of the matrix, allowing high amount of enzymes deposited on the surface and in contact with the substrate, allowing to have high substrate conversion rates. However, the main disadvantage is the exposition of the enzyme to the medium where the shear and hydrolytic activities can compromise the enzyme complex stability and activity. 

In the second volume in the series Topics in Enzyme and Fermentation Biotechnology attention is given to various aspects of enzyme biotechnology. An interdisciplinary approach is favoured and subjects covered include the use of immobilized enzymes, the stability problem of enzymes in use and in storage, and the biological treatment of industrial waste water viewed as a fermentation process. It relates also to the utilization of enzymes in many industries, including pharmaceutics, food, textiles, and paper. 

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