Biocatalysis isolated enzymes

In recent years ionic liquids have also been employed as media for reactions catalyzed both by isolated enzymes and by whole cells, and excellent reviews on this topic are already available [47]. Biocatalysis has been mainly conducted in those room-temperature ionic liquids that are composed of a 1,3-dialkylimidazolium or N-alkylpyridinium cation and a noncoordinating anion [47aj. 

Some of the industrial biocatalysts are nitrile hydralase (Nitto Chemicals), which has a productivity of 50 g acrylamide per litre per hour penicillin G amidase (Smith Kline Beechem and others), which has a productivity of 1 - 2 tonnes 6-APA per kg of the immobilized enzyme glucose isomerase (Novo Nordisk, etc.), which has a productivity of 20 tonnes of high fmctose syrup per kg of immobilized enzyme (Cheetham, 1998). Wandrey et al. (2000) have given an account of industrial biocatalysis past, present, and future. It appears that more than 100 different biotransformations are carried out in industry. In the case of isolated enzymes the cost of enzyme is expected to drop due to an efficient production with genetically engineered microorganisms or higher cells. Rozzell (1999) has discussed myths and realities... 

Poppe L, Novak L (1992) Selective biocatalysis. A synthetic approach. VCH, Weinheim Roberts SM, Wiggins K, Casy G (1992) Preparative biotransformations. Whole cell and isolated enzymes in organic synthesis. WUey, Chichester Flohe L (1979) CIBA Foundation Symposium 65 95... 

Biocatalysis. Biocatalysis, also termed biotransformation and bioconversion, makes use of natural or modified isolated enzymes, enzyme extracts, or whole-cell systems for the production of small molecules. A starting material is converted by the biocatalyst in the desired product. Enzymes are differentiated from chemical catalysts particularly with regard to stereoselectivity. 

Mention some of the advantages and disadvantages of using isolated enzymes rather than whole cells in biocatalysis. Is it always needed to purify enzymes for a biocatalytic application To what degree should one purify ... 

Most studies of biocatalysis in ionic liquids have been concerned with the use of isolated enzymes. It should not be overlooked, however, that the first report on biocatalysis and ionic liquids involved a whole-cell preparation Rhodococcus R312 in a biphasic [BMIm][PF(s]-water system [7]. It was shown, using a nitrile hydrolysis test reaction, that the microorganism maintained its activity better in ionic liquid than in a biphasic toluene-water system. 

For most enzymes, the CLEC is much more robust than the simple isolated enzyme. CLECs can withstand higher temperatures, they denature more slowly in organic solvents, and they are less susceptible to proteolysis [71]. Moreover, since there is no external support involved, CLECs exhibit a high volumetric productivity. These advantages, together with the tunable particle size (typically 1-100 pm), make CLECs attractive for industrial biocatalysis applications. 

The whole-cell biocatalysis approach is typically used when a specific biotransformation requires multiple enzymes or when it is difficult to isolate the enzyme. A whole-cell system has an advantage over isolated enzymes in that it is not necessary to recycle the cofactors (nonprotein components involved in enzyme catalysis). In addition, it can carry out selective synthesis using cheap and abundant raw materials such as cornstarches. However, whole-cell systems require expensive equipment and tedious work-up because of large volumes, and have low productivity. More importantly, uncontrolled metabolic processes may result in undesirable side reactions during cell growth. The accumulation of these undesirable products as well as desirable products may be toxic to the cell, and these products can be difficult to separate from the rest of the cell culture. Another drawback to whole-cell systems is that the cell membrane may act as a mass transport barrier between the substrates and the enzymes. 

Catalysis is one of the most important and rapidly expanding areas in modem organic chemistry. Catalytic reactions can be achieved by either chemocatalysis or biocatalysis. The former field is dominated by transition metal catalysis, whereas in biocatalysis the use of isolated enzymes dominates over whole-cell transformations. 

Biocatalysis or biotransformation encompasses the use of biological systems, whether whole cells, cellular extracts or isolated enzymes, to catalyze the conversion of one compound to another. 

For cost reasons, if ever possible, whole-cell biocatalysis is used to perform biotransformations. This is possible when the following criteria are met (i) no diffusion limitations for substrate(s) and/or product(s) and (ii) no side or follow-up reactions due to the presence of other cellular enzymes. If these conditions are not fulfilled, the use of isolated enzymes - or in special cases of permeabilized cells - is indicated. 

Biocatalysis is a key route to both natural and non-natural polysaccharide structures. Research in this area is particularly rich and generally involves at least one of the following three synthetic approaches 1) isolated enzyme, 2) whole-cell, and 3) some combination of chemical and enzymatic catalysts (i.e. chemoenzymatic methods) (87-90). Two elegant examples that used cell-fi-ee enzymatic catalysts were described by Makino and Kobayashi (25) and van der Vlist and Loos (27). Indeed, for many years, Kobayashi has pioneered the use of glycosidic hydrolases as catalysts for polymerizations to prepare polysaccharides (88,91). In their paper, Makino and Kobayashi (25) made new monomers and synthesized unnatural hybrid polysaccharides with regio- and stereochemical-control. Van der Vlist and Loos (27) made use of tandem reactions catalyzed by two different enzymes in order to prepare branched amylose. One enzyme catalyzed the synthesis of linear structures (amylose) where the second enzyme introduced branches. In this way, artificial starch can be prepared with controlled quantities of branched regions. 

Both isolated enzymes, either immobilized or not, and whole cells have been used for organic synthesis. As a solvent for biocatalytic reactions, water has been most widely used, of course because the nature developed and evolved the biocatalysts to be used in water however, some reactions have been conducted in organic solvents. In this chapter, characteristics and examples of biocatalysis in water are introduced. 

Solid-gas biocatalysis has not been restricted to the use of isolated enzymes. Whole cells are of particular interest where the in vivo recycling of cofactor can be achieved by addition of a cosubstrate. Dried Saccharomyces cerevisiae cells have been used to catalyze the continuous reduction of hexanal to hexanol with in situ regeneration of NADH via oxidation of ethanol in a solid-gas system [81]. The... 

---