Enzyme stability and activity

In vitro enzymatic polymerizations have the potential for processes that are more regio-selective and stereoselective, proceed under more moderate conditions, and are more benign toward the environment than the traditional chemical processes. However, little of this potential has been realized. A major problem is that the reaction rates are slow compared to non-enzymatic processes. Enzymatic polymerizations are limited to moderate temperatures (often no higher than 50-75°C) because enzymes are denaturated and deactivated at higher temperatures. Also, the effective concentrations of enzymes in many systems are low because the enzymes are not soluble. Research efforts to address these factors include enzyme immobilization to increase enzyme stability and activity, solubilization of enzymes by association with a surfactant or covalent bonding with an appropriate compound, and genetic engineering of enzymes to tailor their catalytic activity to specific applications. 

The choice of surfactant, which is mostly constrained by the choice of the oil and the resulting phase behaviour of the microemulsion, can have different effects on the enzyme stability and activity. In general we have to differentiate between ionic and nonionic surfactant types ... 

In the field of enzymatic oxidations especially, the class of flavoenzymes with bound FAD as cofactor is interesting for synthetic applications. The regeneration of the oxidized FAD within the enzyme can be performed by oxygen. In this case, however, hydrogen peroxide is formed, which drastically diminishes the enzyme stability and activity, rendering it unsuitable for synthesis. 

Many enzymes are stable and catalyze reactions in supercritical fluids, just as they do in other non- or microaqueous environments (7). Enzyme stability and activity may depend on the enzyme species, supercritical fluid, water content of the enzyme/support/reaction mixture, decompression rates, exposure times, and pressure and temperature of the reaction system. 

Proteins from extremophilic organisms, particularly thermophiles, have been the subject of intensive research in recent years. This work has been the subject of numerous reviews (Jaenicke and Bohm, 1998 Russel and Taylor, 1995 Vogt and Argos, 1997 Gerday et al., 1997 Somero, 1995), and we will make no attempt at an in-depth summary. We will confine ourselves to briefly stating the major trends identified thus far. Explaining these trends becomes complicated because the many weak interactions that determine enzyme stability and activity have complex temperature dependencies (see Section II). And evolution injects considerable confusion beyond the laws of physical chemistry. 

Abstract This chapter discusses the potential usefulness of ionic liquids with respect to biocatalysis by illustrating the stability and activity of enzymes in ionic liquids in the presence or absence of water. Ionic liquids are a class of coulombic fluids composed of organic cations like alkyl-substituted imidazolium, pyrrolidin-ium, and tetraalkylammonium ions and anions such as halides, tetrafluoroborates, hexafluorophosphates, tosylates, etc. The possibility of tunable solvent properties by alternation of cations and anions has made ionic liquids attractive to study biocatalysis which warrants an understanding of enzyme stability and activity in ionic liquids. This chapter systematically outlines the recent studies on the stability of enzymes and their reactivity toward a wide range of catalytic reactions. A careful approach has been taken toward analysis of relationship between stabil-ity/activity of enzymes versus chaotropic/kosmotropic nature of cations and anions of ionic liquids. 

Enzyme stability and activity can be enhanced by performing enzyme immobilization in the presence of inert proteins, such as albumin, or polyamines in order to shift the maximum enzyme activity towards blood pH. 

One of the bottlenecks of enzyme technology is enzyme availability. When the biocatalyst is commercial, the price may be too high, but in most cases there is no commercial source available so that the enzyme must be produced by means of an overproducing strain and finally the enzyme should be purified. Enzyme purification (discussed in section 6.3.1) is a time consuming process and may represent up to 80% of the enzyme production cost. The usual procedures for lipase purification are sometimes troublesome, time consuming and result in low final yields (Gupta et al. 2004). Enzyme immobilization overcomes this handicap because it allows its reuse and can also enhance enzyme stability and activity (Sharma et al. 2001) furthermore, enzyme immobilization facilitates bioreactor design and final product downstream from reaction medium (see section 4.1). 

The chemical and physical properties of galleries can be altered as described earlier to accommodate the delicate needs of enzymes, and the local pH can be adjusted to their individual requirements. Chemical characteristics such as hydrophobicity, hydrophilicity, and hydrogen-bonding ability are readily controlled to maximize enzyme stability and activity. Initial results indicate that the enzymes bound in a-ZrP galleries are nearly as active as native enzymes in aqueous media, but higher activity and improved selectivities are also noted. 

Several factors affect enzymatic reactions in SCFs. The solubility of the substrates and the activity and stability of the enzyme are the most common parameters that are usually considered. Pressure and temperature can always be used to change the reaction mixture density and other transport properties, which will significantly affect the solubility of the substrates and products in the flnid (Randolph et al., 1991). On the other hand, it may be difficult to understand the absolute effect of process variables and their ability to enhance enzyme stability and activity. 

Active immobilized enzyme stabilizer and activator of co-immobilized D-glucose oxidase... 

Ionic liquids have been employed as the reaction media for a wide range of (bio)catalytic processes, as catalysts or as (bio)catalyst-supports. These modem solvents have been shown to possess a significant effect on the stabilization of charged intermediates in chemical catalytic processes, as well as on enzymes stability. Some ionic liquids have been shown to be by far the best non-aqueous media for biocatalytic processes due to their positive influences on enzyme stability and activity, as well as on the enantioselectivity of the reactions catalyzed by them (Park Kazlauskas, 2003). When the right ionic liquid is chosen for a given reaction, it can lead not only to enhanced selectivity, yield or reaction rate, but it also improves the result of the work-up, from product separation (extraction/distillation) to recycling of the system ionic liquid/(bio)catalysts (Dyson et al, 2003 Parvulescu Hardacre, 2007 van Rantwijk Sheldon, 2007). Separation of products from ionic liquid and ionic liquid recovery can be accomplished by distillation of product (if the product is sufficiently volatile), extraction with supercritical CO2, or simple phase separation (if the product is immiscible in the ionic liquid) (Cornils, 1999). 

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